Note: Descriptions are shown in the official language in which they were submitted.
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91-000110PC
COMPOSITIONS AND METHODS TO REDUCE MUTAGENESIS
CROSS-REFERENCE
[0001] This application claims priority to U.S. Application No. 10/718,002
filed
November 19, 2003, which was converted to a provisional application having
U.S. Ser.
No 60/-,-, on November 18, 2004, which is hereby incorporated herein by
reference in its entirety for all purposes.
BACKGROUND
[0002] Drug resistance is an ever increasing problem in modern medicine
impacting
the treatment of conditions as diverse as bacterial infections, viral
infections, protozoan
infections, fungal infections, and cancer.
[0003] In particular, the worldwide emergence of antibiotic-resistant bacteria
threatens
to undo the dramatic advances in human health that followed the discovery of
these
drugs. Antibiotic drug resistance is especially acute with tuberculosis, which
infects
one-third of all humans, most of whom live in the developing world. The health
care
establishment is countering this challenge by trying to create new antibiotics
and by
limiting the use of those already available. However, this approach has not
yet
produced the desired effect, as the prevalence of resistant strains continues
to increase.
[0004] Drug resistance is also a problem with viruses, including the human
immunodeficiency virus ("HIV"). In fact, HIV drug resistance is rapidly
becoming an
epidemic. One study of HIV infected patients between 1996 and 1999, shows that
about 78% of patients harbored viruses that were resistant to at least one
class of drugs,
51% had viruses that were resistant to two classes of drugs, and 18% had
viruses that
were resistant to three classes of drugs. Thus, HIV drug therapies must
constantly
evolve to keep pace with the evolution of resistance.
[0005] Drug resistance is also a problem during cancer therapy. It is
estimated that
half of all cancer patients are cured, mostly by a combination of surgery,
radiotherapy
and/or chemotherapy. However, some cancers can only be treated by
chemotherapy,
and in those cases, only one in five patients survives long-term. It is
believed that the
overriding reason for this poor result is drug resistance, wherein the tumors
are either
innately resistant to the drugs available, or else are initially sensitive but
evolve
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resistance during treatment and eventually re-grow. Allen JD, et al. Cancer
Research
(2002) 62, 2294-2299.
[0006] Drug resistance also occurs with protozoa such as Plasrnodiuyn spp.,
the genus
of protozoa responsible for malaria. In recent years, drug resistance has
become one of
the most important problems in malaria control. Resistance in vivo has been
reported to
all anti-malaria drugs except artemisinin and its derivatives. This
necessitates the use
of drugs which are more expensive and may have dangerous side effects.
[0007] Thus, there is a great need for compounds that inhibit the mutations
that confer
drug resistance and methods for using such compounds to treat and prevent drug
resistant conditions.
SUMMARY OF THE INVENTION
[0008] The present invention relates to compositions comprising, consisting
essentially
of, or consisting of achaogens. Achaogens are compounds that reduce the rate
of
induced mutagenesis. Achaogens can include nucleic acids, peptide nucleic
acids,
phage, phagemids, polypeptides, peptidomimetics, antibodies, small or large
organic or
inorganic molecules or any combination of the above. Achaogens can be
naturally
occurring or non-naturally occurring (e.g., xecombinant) and are preferably
isolated
and/or purified.
[0009] In preferred embodiments an achaogen interacts with or binds to a gene
product
that increases rate of mutation in a cell or an organism. Examples of such
gene
products include RecA, RecB, RecC, RecD, RecF, Recta, Rec N, LexA, UmuC, UmuD,
PoIB, PoIIV, PoIV, PriA, RuvA, RuvB, RuvC, UmuC, UmuD, UvrA, UvrB, UvrD or
any homologs or analogs thereof.
[0010] In some embodiments, an achaogen interacts with or binds to LexA or any
homolog or analog thereof to reduce the rate of mutation in a cell or an
organism. Such
an achaogen can, for example, interact with or bind to LexA's (or homolog of
LexA's)
cleavage site or active site. In some embodiments, an achaogen interferes with
LexA's
(or a homolog of LexA's) autocleavage, which is required for induced
mutagenesis by
binding to the active site of LexA (or homolog of LexA).
[0011] Such an achaogen can comprise, consist essentially of, or consist of a
polypeptide or peptidomimetic of a polypeptide that binds LexA, thus
preventing
LexA's autoproteolysis activity. Examples of such polypeptide (and
peptidomimetics
thereof) include those comprising, consisting essentially of, or consisting of
dipeptide
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Ala-Ala, tripeptide Val-Ala-Ala, or SEQ m NO: 1, 2, or 3. In some embodiments
wherein the achaogen comprises an Ala-Gly bond, the bond may be modified so
that it
is not cleavable under normal physiological conditions. In some embodiments,
the
polypeptide or peptidomimetic is C-terminally modified, e.g., such that it is
electrophilic.
[0012] In some embodiments, an achaogen of the present invention is one of
Formula I,
R6
[0013] wherein where R1, R2, R3, R4, R5, and R6 are each independently
selected from
the group consisting of -(CHRa)X L-Rb, where x is selected from the group
consisting
of 0, 1, 2, 3, or 4; L is a single bond or -C(O)-, -NHC(O)-, -OC(O)-, -S(O)S,
where j is
0, 1, or 2; Ra is a moiety selected from the group consisting of H, (Cl-
C~)alkyl, halogen,
(C1-C~)fluoroallcyl, (C1-C~)allcoxy, -C(O)OH, -C(O)-NH2, -(C1-C~)alkylamine, -
C(O)-
(C1-C~)alkyl, -C(O)-(C1-C~)fluoroalkyl, -C(O)-(C1-C~)alkylamine, and -C(O)-(CI-
C~)allcoxy; and Rb is H, OH, halogen, NHZ, CN, N3, or a moiety, optionally
substituted
with 1-3 independently selected substituents, selected from the group
consisting of
alkyl, alkenyl, alkoxy, mercaptyl, allcylamine, alkynyl, aryl, cycloalkyl,
cycloalkenyl,
and a heterocycle; in addition, Rl and R2, RZ and R3, R3 and Rø, and RS and
R6, can
optionally form a substituted or unsubstituted ring structure.
[0014] In some embodiments, an achaogen is an isolated and purified serine
protease
inhibitor.
[0015] In some embodiments, an achaogen functions as a negative regulator of
induced
mutagenesis. Such an achaogen can comprise, consist essentially of, or consist
of a
gene product that reduces the rate of mutation in a cell or an organism (e.g.,
PsiB, DinI,
Lon protease, CIpXP protease, a serine protease inhibitor). In some
embodiments, an
achaogen is a phage or a phagemid carrying a recombinant nucleic acid encoding
a
gene product that reduces the rate of mutations in a cell or an organism.
[0016] In some embodiments, an achaogen is a nucleic acid that is
complementary to a
nucleic acid encoding a gene product that increases the rate of induced
mutations (e.g.,
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RecA, RecB, RecC, RecD, RecF, Recta, Rec N, LexA, UmuC, UmuD, PoIB, PollV,
PoIV, PriA, RuvA, ~RuvB, RuvC, UmuC, UmuD, UvrA, UvrB, UvrD, or homologs or
analogs thereof). Such achaogens can be used as antisense nucleic acids, zinc
fingers,
RNAi or ribozymes to hybridize with and reduce the transcription and/or
translation of
such gene products.
[0017] The present invention also relates to pharmaceutical formulations whose
active
ingredient is an achaogen that reduces the rate of mutation in a cell or an
organism.
Such pharmaceutical compositions can be formulated for local or systemic
delivery.
Any of the pharmaceutical formulations herein can include additional
therapeutic
agents) such as, for example, antibiotics, antineoplastic agents, antifungal
agents,
antiprotozoan agents, and antiviral agents.
[0018] The invention herein also relates to methods of treating an organism
suffering
from a condition that may become drug resistant by administering to the
organism an
effective amount of an achaogen. An organism treated by the present invention
can be
an animal (e.g., a domesticated animal such as a cow, pig, horse, or a
chicken, an avian,
or a human) or a plant. The condition treated can be any condition that, when
treated,
results in drug resistance, including, for example, bacterial infections,
viral infections,
protozoan infections, fungal infections, and the abnormal cell growth
associated with
cancer. In particular, the present invention relates to methods of treating an
organism
suffering from a bacterial infection. The bacterial infection is one that may
become
resistant, or is resistant, to one or more antibiotic treatments.
[0019] The present invention also relates to methods for screening a cell, a
group of
cells of an organism, or an entire organism for the acquisition of drug
resistance.
Screening for drug resistance involves detecting mutations in an organism (or
a cell or
group of cells of an organism) in genes associated with induced mutation or
detecting
levels of protein expression of genes associated with induced mutation. For
example in
E. coli, such genes include, but are not limited to, a gene for a 16S rRNA, a
gene for a
23S rRNA, clpXP, ding, dinl, dnaE2, gyrA, gyrB, lcatG, zn7zA, lon protease, a
gene for a
L4 ribosomal methylases, lexA, lon protease, norA, recA, recN, psiB, parC,
parE, polB,
psiB, rpoS, rpoB, sxt, umuC, umuD, uvrA, uvrB, and uvrD. The presence of a
mutation
in such genes and/or the level of gene expression of such genes can be
detected using a
diagnostic tool such as a microarray or by sequencing techniques known in the
art (e.g.,
PCR).
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[0020] The present invention also relates to methods for screening for agents
that
interact with naturally occurring compositions that induce mutagenesis, e.g.,
RecA,
RecB, RecC, RecD, RecF, Recta, Rec N, LexA, UmuC, UmuD, PoIB, PoIIV, PoIV,
PriA, RuvA, RuvB, RuvC, UmuC, UmuD, UvrA, UvrB, UvrD, or a LexA-RecA
complex, or homologs or fragments thereof. Such methods include contacting a
candidate agent from a library of candidate agents with a naturally
composition that
induce mutagenesis e.g., RecA, RecB, RecC, RecD, RecF, Recta, Rec N, LexA,
UmuC,
UmuD, PoIB, PoIIV, PoIV, PriA, RuvA, RuvB, RuvC, UmuC, UmuD, UvrA, UvrB,
UvrD, or a LexA-RecA complex, or homologs, analogs, or fragment thereof; and,
in
this manner, detecting a candidate agent that specifically binds to one or
more of the
compositions that induces mutagenesis. Such candidate agents can then be
further
modified to enhance binding to the naturally occurring composition.
[0021] The present invention also relates to methods for screening agents that
interact
with RecA, RecB, RecC, RecD, RecF, Recta, Rec N, LexA, UmuC, UmuD, PolB,
PoIIV, PoIV, PriA, RuvA, RuvB, RuvC, UmuC, UmuD, UvrA, UvrB, UvrD, LexA-
RecA complex, or any homolog, analog, or fragment thereof. Such methods
include
identifying a crystal complex of RecA, RecB, RecC, RecD, RecF, Recta, Rec N,
LexA,
UmuC, UmuD, PoIB, PoIIV, Poly, PriA, RuvA, RuvB, RuvC, UmuC, UmuD, UvrA,
UvrB, UvrD, the LexA-RecA complex, or any homolog, analog, or fragment
thereof;
obtaining atomic coordinates of the crystal; and using the atomic coordinates
with one
or more molecular modeling techniques to identify an agent that interacts with
the
above molecules.
[0022] The present invention also provides kits. The kits described herein
include at
least one container comprising one or more achaogens that inhibit induced
mutation
along with direction for use. The kit may also include a second container of
another
therapeutic agent (e.g., an antibiotic, an antiviral, an antifungal, an
antineoplastic, or an
antiprotozoan medication). The achaogen and the second therapeutic agent can
be
combined prior to administration or may be administered separately. A lcit can
also
include a diagnostic tool for determining if an organism or a cell or group of
cells is
partially or fully drug resistant.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 illustrates the cellular function of LexA under normal
conditions, under
the condition of cellular stress, due to ciprofloxacin exposure and under the
condition
of cellular stress in the presence of an achaogen.
[0024] Figure 2 illustrates a stressful lifestyle adaptive mutation (SLAM)
assay.
[0025] Figure 3 illustrates mutation rates for different bacterial strains in
the presence
of ciprofloxacin.
[0026] Figures 4A -and 4B illustrate a portion (VAAG) of peptide VAAGEPLLAW of
the LexA substrate loop. Figure 4A illustrates the enzyme's active cleft with
its
substrate. Figure 4B illustrates the active cleft without its substrate.
[0027] Figures 5A and 5B illustrate crystal structures of LexA in two
conformations.
Figure 5A illustrates a crystal structure of the non-cleavable conformation of
LexA.
Figure 5B illustrates the cleavable conformation of LexA.
[0028] Figure 6 illustrates the geometry of the active site of LexA and the
position of
S 119 where it is poised to attack the C=O of Ala-Gly dipeptide.
[0029] Figure 7 illustrates a number of viable ciprofloxacin sensitive cells
remaining
on solid media (LB containing 35 ng/ml ciprofloxacin) as a function of time
for SOS
and polymerase deficient E. coli strains.
[0030] Figure 8 illustrates a comparison of mutation rate of three different
E. coli
strains as measured by comparing their ability to evolve a 'first level'
resistance to
ciprofloxacin (i.e., resistance to 35 ng/ml ciprofloxacin, the level of
resistance
conferred by single point mutations in the gyYA gene). Strain 1 is ATCC 25922;
strain
2 is ATCC 25922-~lacZ; and strain 3 is ATCC 25922-lexA(S119A). Bars represent
total mutation rate (base substitution and codon deletion). Error bars
represent standard
deviation from three independent rate determinations.
[0031] Figure 9 illustrates a mutation rate of two different E. coli strains
that are
already resistant to 35 ng/ml ciprofloxacin and their ability to evolve
resistance to
higher concentrations of ciprofloxacin (i.e., to 650 ng/ml ciprofloxacin). ~
Strain 1 is
ATCC 25922-~lacZ(gyrA(S83L)), and strain 2 is ATCC 25922-
lexA(S 119A)(gyrA(S83L)). Error bars represent standard deviation from two
independent rate determinations.
[0032] Figure 10 illustrates mutation rate to ciprofloxacin resistance of the
following
ten strains: OlacZ [strain 1], ~polB [strain 2], ~difzB [strain 3], DunzuDC
[strain 4],
OpolB l 4dinB [strain 5], 4polB lDumuDC [strain 6], OdinB / ~umuDC [strain 7],
6
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~polB l 4dif2B l DuynuDC [strain 8], lexA(S119A) [strain 9], and ~recD [strain
10];
solid bars represent base substitution mutations and shaded bars represent
codon
deletion. Values represent number of resistant mutants per surviving cell per
day.
Error bars represent standard deviation from three independent rate
determinations.
[0033] Figure 11 illustrates a proposed mechanism for recombination dependent
replication restart in the presence of ciprofloxacin. In this model, RuvAB
acts to
displace the trapped topoisomerase complex from DNA, allowing for the
establishment
of a new replication fork on which PriA may reassemble a processive replisome.
[0034] Figure 12 illustrates mutation and survival of tllacZ and lexA(S 119A)
mutants
of E. coli ATCC 25922 (injection of about 10~ cfu/thigh at 0 hours) in thighs
of
neutropenic mice at 24-hour intervals after starting therapy with
ciprofloxacin
injections of ~10~ cfulthigh at t=0. Open circles and triangles correspond to
the total
CFU/thigh of the ~lacZ and lexA(S 119A) strains, respectively. Solid circles
and
triangles represent the number of ciprofloxacin-resistant OlacZ and lexA(S
119A)
mutants/thigh, respectively. The lower limit of detection was 100 organisms
per thigh.
[0035] Figure 13 illustrates the structure of serine protease inhibitor,
diisopropyl
fluorophosphate.
[0036] Figures 14A-14E illustrate structures of exemplary serine protease
inhibitors A-
E.
[0037] Figures 15A-15D illustrate exemplary non-covalent peptidomimetic
inhibitors
of LexA.
[0038] Figure 16 illustrates exemplary non-covalent peptidomimetic inhibitors
of
LexA.
[0039] Figure 17 illustrates various covalent peptidomimetics inhibitors of
LexA.
[0040] Figure 18 illustrates results from a rifampin resistance experiment on
mouse
thighs infected with E. coli either ATCC 25922-~lacZ or ATCC 25922-lexA(S
119A).
[0041] Figure 19 illustrates oligonucleotide primers used in construction of
disruption
cassettes.
[0042] Figure 20 illustrates inhibition of LexA's autocleavage by three
peptides.
[0043] Figure 21 illustrates a comparison of inhibition of LexA's autocleavage
by
peptide 3 and without peptide 3.
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DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0044] The term "Ac" as used herein is synonymous to the term "acetylated."
[0045] The term "achaogen" as used herein refers to an agent that inhibits the
mutational process. That is, exposure of a cell or an organism to an achaogen
results in
a decrease in mutation frequency. The mutation frequency may be of an entire
multicellular organism, a single celled organism, a population of cells, or
some cells of
an organism (as in the case of cancer). In some preferred embodiments, an
achaogen
reduces the rate of mutation by at least 2-fold, more preferably by at least 4-
fold, more
preferably by at least 6-fold, or more preferably by at least 8-fold order of
magnitude.
In some preferred embodiments, an achaogen reduces resistance to a single
drug, more
preferably, at least two drugs, more preferably, at least 3 drugs, or more
preferably, at
least 4 drugs, or more preferably, at least 5 drugs.
[0046] An "alkoxy" group refers to a (alkyl)O- group, where alkyl is as
defined herein.
[0047] An "alkyl" group refers to an aliphatic hydrocarbon group. The alkyl
moiety
may be a "saturated alkyl" group, which means that it does not contain any
alkene or
alkyne moieties. The alkyl moiety may also be an "unsaturated alkyl" moiety,
which
means that it contains at least one alkene or alkyne moiety. An "allcene"
moiety refers
to a group consisting of at least two carbon atoms and at least one carbon-
carbon
double bond, and an "allcyne" moiety refers to a group consisting of at least
two carbon
atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether
saturated
or unsaturated, may be branched, straight chain, or cyclic.
[0048] The "alkyl" moiety may have 1 to 20 carbon atoms (whenever it appears
herein,
a numerical range such as "1 to 20" refers to each integer in the given range;
e.g., "1 to
20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2
carbon
atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the
present
definition also covers the occurrence of the term "alkyl" where no numerical
range is
designated). The alkyl group could also be a "lower allcyl" having 1 to 8
carbon atoms.
The alleyl group of the compounds described herein may be designated as "Cl-C4
alkyl"
or similar designations. By way of example only, "C1-C4 alkyl" indicates that
there are
one to four carbon atoms in the alkyl chain, i.e., the alleyl chain is
selected from the
group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-
butyl, and
t-butyl. Typical alkyl groups include, but are in no way limited to, methyl,
ethyl,
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propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl,
propenyl,
butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
[0049] The term "alkylamine" refers to the -N(alkyl)XHy group, where x and y
are
selected from the group x=1, y=1 and x=2, y=0. When x=2, the alkyl groups,
taken
together, can optionally form a cyclic ring system.
[0050] The term "allcenyl" refers to a type of alleyl group in which the first
two atoms
of the alkyl group form a double bond that is not part of an aromatic group.
That is, an
alkenyl group begins with the atoms -C(R)=C-R, wherein R refers to the
remaining
portions of the alkenyl group, which may be the same or different. Non-
limiting
examples of an alkenyl group include -CH=CH, -C(CH3)=CH, -CH=CCH3 and -
C(CH3)=CCH3. The alkenyl moiety may be branched, straight chain, or cyclic (in
which case, it would also be known as a "cycloalkenyl" group).
[0051] The term "alkynyl" refers to a type of alkyl group in which the first
two atoms
of the alkyl group form a triple bond. That is, an alkynyl group begins with
the atoms -
C=C-R, wherein R refers to the remaining portions of the alkynyl group, which
may be
the same or different. Non-limiting examples of an alkynyl group include -
C=CH, -
C=CCH3 and -C=CCH2CH3. The "R" portion of the alkynyl moiety may be branched,
straight chain, or cyclic.
[0052] An "amide" is a chemical moiety with formula -C(O)NHR or -NHC(O)R,
where R is selected from the group consisting of alkyl, cycloalkyl, aryl,
heteroaryl
(bonded through a ring carbon) and heteroalicyclic (bonded through a ring
carbon).
[0053] The term "amino acid" or "residue" as used herein includes any one of
the
twenty naturally occurring amino acids, the D-form of any one of the naturally-
occurring amino acids, non-naturally occurring amino acids, and derivatives,
analogs,
and mimetics thereof. Any amino acid, including naturally occurring amino
acids, may
be purchased commercially or synthesized by methods known in the art. Examples
of
non-naturally-occurnng amino acids include norleucine ("Nle"), norvaline
("Nva"), (3-
Alanine, L- or D-naphthalanine, ornithine ("Orn"), homoarginine (homoArg) and
others well known in the peptide art, including those described in M.
Bodanzsky,
"Principles of Peptide Synthesis," 1st and 2nd revised ed., Springer-Verlag,
New York,
N.Y., 1984 and 1993, and Stewart and Young, "Solid Phase Peptide Synthesis,"
2nd
ed., Pierce Chemical Co., Rockford, Ill., 1984, both of which are incorporated
herein by
reference. Common amino acids may be referred to by their full name, standard
single-
letter notation, or standard three-letter notation for example: A, Ala,
alanine; C, Cys,
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cysteine; D, Asp, aspartic; E, Glu, glutamic acid; F, Phe, phenylalanine; G,
Gly,
glycine; H, His, histidine; I, Ile isoleucine; K, Lys, lysine; L, Leu,
leucine; M, Met,
methionine; N, Asn, asparagine; P, Pro, proline; Q, Gln, glutamine; R, Arg,
arginine; S,
Ser, serine; T, Thr, threonine; V, Val, valine; W, Trp, tryptophan; X, Hyp,
hydroxyproline; Y, Tyr, tyrosine. Any and all of the amino acids in the
compositions
herein can be naturally occurring, synthetic, and derivatives or mimetics
thereof. When
the amino acid residues contain one or more chiral centers, any of the D, L,
meso, threo
or erythro (as appropriate) racemates or mixtures thereof, fall within the
scope of this
invention. In general, if it is desired to rely on non-enzymatic means of
hydrolysis, D
isomers should be used. On the other hand, L isomers may be more versatile
since they
can be susceptible to both non-enzymatic as well as potential targeted
enzymatic
hydrolysis, and are more efficiently transported by amino acid or dipeptidyl
transport
systems in the gastrointestinal tract. The amino acids herein can be naturally
occurring
or synthetic.
[0054] The term "analog" as used herein refers to a composition that retains
the same
structure or function (e.g., binding to a receptor) as a polypeptide or
nucleic acid herein.
Examples of analogs include peptidomimetics, peptide nucleic acids, small and
large
organic or inorganic compounds, as well as derivatives and variants of a
polypeptide or
nucleic acid herein. The term "derivative" or "variant" as used herein refers
to a
peptide or nucleic acid that differs from the naturally occurring polypeptide
or nucleic
acid by one or more amino acid or nucleic acid deletions, additions,
substitutions or
side-chain modifications. Amino acid substitutions include alterations in
which an
amino acid is replaced with a different naturally-occurring or a non-
conventional amino
acid residue. Such substitutions may be classified as "conservative", in which
case an
amino acid residue contained in a polypeptide is replaced with another
naturally-
occurnng amino acid of similar character either in relation to polarity, side
chain
functionality or size.
[0055] Substitutions encompassed by the present invention may also be "non-
conservative", in which an amino acid residue which is present in a peptide is
substituted with an amino acid having different properties, such as naturally-
occurring
amino acid from a different group (e.g., substituting a charged or hydrophobic
amino
acid with alanine), or alternatively, in which a naturally-occurnng amino acid
is
substituted with a non-conventional amino acid. Preferably, amino acid
substitutions
are conservative.
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[0056] Amino acid substitutions are typically of single residues, but may be
of multiple
residues, either clustered or dispersed. Additions encompass the addition of
one or
more naturally occurring or non-conventional amino acid residues. Deletion
encompasses the deletion of one or more amino acid residues.
[0057] As stated above peptide derivatives include peptides in which one or
more of
the amino acids has undergone side-chain modifications. Examples of side chain
modifications contemplated by the present invention include modifications of
amino
groups such as by reductive alkylation by reaction with an aldehyde followed
by
reduction with NaBH4 ; amidination with methylacetimidate; acylation with
acetic
anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of
amino
groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino
groups
with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation
of lysine
with pyridoxal-5-phosphate followed by reduction with NaBH4.
[0058] The guanidine group of arginine residues may be modified by the
formation of
heterocyclic condensation products with reagents such as 2,3-butanedione,
phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent derivitisation,
for
example, to a corresponding amide. Sulphydryl groups may be modified by
methods
such as carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid
oxidation to cysteic acid; formation of a mixed disulphides with other thiol
compounds;
reaction with maleimide, malefic anhydride or other substituted maleimide;
formation of
mercurial derivatives using 4-chloromercuribenzoate, 4-
chloromercuriphenylsulphonic
acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other
mercurials;
carbamoylation with cyanate at alkaline pH. Any modification of cysteine
residues
must not affect the ability of the peptide to form the necessary disulphide
bonds. It is
also possible to replace the sulphydryl groups of cysteine with selenium
equivalents
such that the peptide forms a diselenium bond in place of one or more of the
disulphide
bonds.
[0059] Tryptophan residues may be modified by, for example, oxidation with N-
bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl
bromide or sulphenyl halides. Tyrosine residues on the other hand, may be
altered by
nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of
the imidazole ring of a histidine residue may be accomplished by allcylation
with
iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Proline
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residue may be modified by, for example, hydroxylation in the 4-position.
Other
derivatives contemplated by the present invention include a range of
glycosylation
variants from a completely unglycosylated molecule to a modified glycosylated
molecule. Altered glycosylation patterns may result from expression of
recombinant
molecules in different host cells.
[0060] The term "aromatic" or "aryl" refers to an aromatic group which has at
least one
ring having a conjugated pi electron system and includes both carbocyclic aryl
(e.g.,
phenyl) and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups
(e.g.,
pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings
which
share adjacent pairs of carbon atoms) groups. The term "carbocyclic" refers to
a
compound which contains one or more covalently closed ring structures, and
that the
atoms forming the backbone of the ring are all carbon atoms. The term thus
distinguishes carbocyclic from heterocyclic rings in which the ring backbone
contains
at least one atom which is different from carbon.
[0061] The term "bond" or "single bond" refers to a chemical bond between two
atoms,
or two moieties when the atoms joined by the bond are considered to be part of
larger
substructure.
[0062] The term "complementary" as used herein describes two nucleotides that
can
associate with one another (e.g., form hydrogen bonds with one another). For
example,
adenine is complementary to thymine as they can form two hydrogen bonds.
[0063] The term "covalent" as used herein to describe a bond refers to a
chemical bond
between two species, and may involve single bonds or multiple bonds. The term
"covalent" does not include hydrophobic/hydrophilic interactions, Hydrogen-
bonding,
van der Waals interactions, hydrophobic effect, and ionic interactions, which
are
deemed non-covalent.
[0064] A "cyano" group refers to a -CN group.
[0065] The term "cycloalkyl" refers to a monocyclic or polycyclic radical that
contains
only carbon and hydrogen, and may be saturated, partially unsaturated, or
fully
unsaturated. Cycloalleyl groups include groups having from 3 to 10 ring atoms.
Illustrative examples of cycloalkyl groups include the following moieties:
12
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> >
> > > >
D
' ~~ ,
'
~~ W ~ v
and the like.
[0066] The term "effective amount" as used herein refers to that amount of
composition necessary to achieve the indicated effect.
[0067] The term "ester" refers to a chemical moiety with formula -COOR, where
R is
selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl
(bonded
through a ring carbon) and heteroalicyclic (bonded through a ring carbon).
[0068] The terms "gene(s)" refers to a nucleic acid sequence (DNA, RNA, or
analogs
and/or combinations thereof) that encodes through its template or messenger
RNA a
sequence of amino acids characteristic of a specific peptide. The term "gene"
can
includes intervening, non-coding regions, as well as regulatory regions, and
can include
5' and 3' ends. Examples of genes associated with induced mutations include
but are
not limited to lexA, recA, umuD, umuC, ding, polB, etc., and any homologs,
analogs
or fragments thereof.
[0069] The term "gene product(s)" as used herein refers is meant to include
RNA
transcribed from a gene, or a polypeptide encoded by a gene or translated from
RNA.
Such polypeptides can be unmodified translated polypeptides or post-
translationally
modified polypeptides (e.g., glycosylated, phosphonylated, cleaved, etc.).
Examples of
gene products that are associated with induced mutagenesis include LexA, RecA,
PoIB,
Pol IV, UmuD, UrnuC, and any homologs, analogs and fragments thereof.
[0070] The term "halo" or, alternatively, "halogen" means fluoro, chloro,
bromo or
iodo. Preferred halo groups are fluoro, chloro and bromo.
[0071] The terms "haloalkyl," "haloalkenyl," "haloalkynyl" and "haloalkoxy"
include
alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or
more halo
groups or with combinations thereof. The terms "fluoroalkyl" and
"fluoroalkoxy"
include haloallcyl and haloallcoxy groups, respectively, in which the halo is
fluorine.
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[0072] The terms "heteroalkyl" "heteroalkenyl" and "heteroalkynyl" include
optionally
substituted alkyl, alkenyl and alkynyl radicals and which have one or more
skeletal
chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen,
sulfur,
phosphorus or combinations thereof.
[0073] The terms "heteroaryl" or, alternatively, "heteroaromatic" refers to an
aryl
group that includes one or more ring heteroatoms selected from nitrogen,
oxygen and
sulfur. An N-containing "heteroaromatic" or "heteroaryl" moiety refers to an
aromatic
group in which at least one of the skeletal atoms of the ring is a nitrogen
atom. The
polycyclic heteroaryl group may be fused or non-fused. Illustrative examples
of
heteroaryl groups include the following moieties:
N/\IN N/\IN \ N \ S \ N
N
~/ ' / ~ / ~ / N
N S O N,O N S N,S
~ ~>
, , ~ N , ~ ~ , N , ~ N
NiN NiN ~ N ~ ~ ~ ~N %
\ , 'N , \N , \ , NON
S
N~
S N and the like.
[0074] The term "heterocycle" refers to heteroaromatic and heteroalicyclic
groups
containing one to four heteroatoms each selected from O, S and N, wherein each
heterocyclic group has from 4 to 10 atoms in its ring system, and with the
proviso that
the ring of said group does not contain two adjacent O or S atoms. Non-
aromatic
heterocyclic groups include groups having only 4 atoms in their ring system,
but
aromatic heterocyclic groups must have at least 5 atoms in their ring system.
The
heterocyclic groups include benzo-fused ring systems. An example of a 4-
membered
heterocyclic group is azetidinyl (derived from azetidine). An example of a 5-
membered heterocyclic group is thiazolyl. An example of a 6-membered
heterocyclic
group is pyridyl, and an example of a 10-membered heterocyclic group is
quinolinyl.
Examples of non-aromatic heterocyclic groups are pyrrolidinyl,
tetrahydrofuranyl,
14
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dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,
tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl,
piperazinyl,
azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl,
oxazepinyl,
diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-
pyrrolinyl,
indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl,
dithianyl,
dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl,
imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-
azabicyclo[4.1.0]heptanyl,
3H-indolyl and quinolizinyl. Examples of aromatic heterocyclic groups are
pyridinyl,
imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl,
thienyl,
isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,
isoquinolinyl, indolyl,
benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,
phthalazinyl,
pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,
thiadiazolyl,
furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,
quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing
groups, as
derived from the groups listed above, may be C-attached or N-attached where
such is
possible.. The heterocyclic groups include benzo-fused ring systems and ring
systems
substituted with one or two oxo (=O) moieties such as pyrrolidin-2-one.
[0075] A "heteroalicyclic" group refers to a cycloalkyl group that includes at
least one
heteroatom selected from nitrogen, oxygen and sulfur. The radicals may be
fused with
an aryl or heteroaryl. Illustrative examples of heterocycloalkyl groups
include:
O O O O O O O
~ N
S OI 'O
N N \ N \O
' ~ > > > ~ ~ S '
N N O O N
O
N ' , N > > > N-N '
O O
O S N ~
NI 'O
> > > > > ,
N N N N N
H H H
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O
/~ O
-O N I /
N 'N ~
and the like.
The term heteroalicyclic also includes all ring forms of the carbohydrates,
including but
not limited to the monosaccharides, the disaccharides and the
oligosaccharides.
[0076] The term "homolog" or "homologous" as used herein refers to homology
with
respect to structure and/or function. With respect to sequence homology,
sequences are
homologs if they are at least 50%, preferably at least 60%, more preferably at
least
70%, more preferably at least 80%, more preferably at least 90%, more
preferably at
least 95% identical, more preferably at least 97% identical, or more
preferably at least
99% identical. The term "substantially homologous" refers to sequences that
are at
least 90%, more preferably at least 95% identical, more preferably at least
97%
identical, or more preferably at least 99% identical. Homologous sequences can
be the
same functional gene in different species.
[0077] The term "hybridize" refers to interaction of a nucleotide sequence
with a
second nucleotide sequence. Such interaction can be, e.g., in solution or on a
solid
support, such as cellulose or nitrocellulose. If a nucleic acid sequence binds
to a second
nucleotide sequence with high affinity, it is said to "hybridize" to the
second nucleotide
sequence. The strength of the interaction between the two sequences can be
assessed by
varying the stringency of the hybridization conditions. Under highly stringent
hybridization conditions only highly complementary nucleotide sequences
hybridize.
[0078] The term "inhibition" or "inhibit" when referring to the activity of an
achaogen
refers to prevention or any detectable reduction in mutation rate.
[0079] An "isocyanato" group refers to a -NCO group.
[0080] The term "isolated" as used herein refers to a compound or molecule
(e.g., a
polypeptide or a nucleic acid) that is relatively free of other compounds or
molecules
such as proteins, lipids, nucleic acids or other molecules it normally is
associated with
in a cell. In general, an isolated polypeptide constitutes at least about 75%
by weight of
a sample containing it, more preferably about 90% of a sample containing it,
more
preferably about 95% of the sample containing it, or more preferably about 99%
of a
sample containing it.
[0081] An "isothiocyanato" group refers to a -NCS group.
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WO 2005/056754 PCT/US2004/039064
[0082] The term "membered ring" can embrace any cyclic structure. The term
"membered" is meant to denote the number of skeletal atoms that constitute the
ring.
Thus, for example, cyclohexyl, pyridine, pyran and thiopyran are 6-membered
rings
and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.
[0083] A "mercaptyl" group refers to a (alkyl)S- group.
[0084] The term "moiety" refers to a specific segment or functional group of a
molecule. Chemical moieties are often recognized chemical entities embedded in
or
appended to a molecule.
[0085] The term "nucleic acid" as used herein refers to a ribo- or
deoxyribonucleosides, ribo- or deoxyribonucleotides, ribo- or
deoxyoligonucleotides,
oligonucleotide sequence or polynucleotide sequence, or any variants,
homologs,
fragments, analogues or derivatives thereof. The nucleotide sequence may be
naturally
occurring or synthetic. It may be double-stranded or single-stranded whether
representing the sense or antisense strand.
[0086] The terms "nucleophile" and "electrophile" as used herein have their
usual
meanings familiar to synthetic and/or physical organic chemistry. Carbon
electrophiles
typically comprise one or more alkyl, alkenyl, alkynyl or aromatic (spa, sp2,
or sp
hybridized) carbon atoms substituted with any atom or group having a Pauling
electronegativity greater than that of carbon itself. Examples of carbon
electrophiles
include but are not limited to carbonyls (aldehydes, ketones, esters, amides),
oximes,
hydrazones, epoxides, aziridines, alkyl-, alkenyl-, and aryl halides, acyls,
sulfonates
(aryl, alkyl and the like). Other examples of carbon electrophiles include
unsaturated
carbon atoms electronically conjugated with electron withdrawing groups,
examples
being the 6-carbon in alpha-unsaturated ketones or carbon atoms in fluorine
substituted
aryl groups. Methods of generating carbon electrophiles, especially in ways
which yield
precisely controlled products, are known to those slcilled in the art of
organic synthesis.
[0087] The term "optionally substituted" means that the referenced group may
be
substituted with one or more additional groups) individually and independently
selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy,
alkoxy,
aryloxy, mercapto, allcylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl,
isocyanato,
thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, silyl, and
amino,
including mono- and di-substituted amino groups, and the protected derivatives
thereof.
The protecting groups that may form the protective derivatives of the above
17
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
substituents are known to those of skill in the art and may be found in
references such
as Greene and Wuts, above.
[0088] The term "organism" as used herein includes all living cells including
microorganisms (e.g., viruses, bacteria, protozoa), plants, and animals (e.g.,
humans,
birds, reptiles, amphibians, fish, and domesticated animals, such as cows,
chicken, pigs,
dogs, and goats).
[0089] The term "polypeptide" refers to any composition that includes two or
more
amino acids joined to each other by a peptide bond or peptidomimetic thereof.
The
term includes both short chains, which are also commonly referred to in the
art as
peptides, oligopeptides and oligomers, for example, and to longer chains,
which
generally are referred to in the art as proteins. The term "polypeptide"
includes all
polypeptides as described below. It will be appreciated that polypeptides
often contain
amino acids other than the 20 amino acids commonly referred to as the 20
naturally
occurring amino acids, and that many amino acids, including the terminal amino
acids,
can be modified in a given polypeptide, either by natural processes such as
glycosylation and other post-translational modifications, or by chemical
modification
techniques which are well known in the art. Known modifications which can be
present in polypeptides of the present invention include, but are not limited
to,
acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of
flavin,
covalent attachment of a heme moiety, covalent attachment of a polynucleotide
or
polynucleotide derivative, covalent attachment of a lipid or lipid derivative,
covalent
attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cystine,
formation of pyroglutamate, formylation, gamma-carboxylation, glycation,
glycosylation, GPI anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, proteolytic processing, phosphorylation,
prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated addition of
amino acids
to proteins such as arginylation, and ubiquitination.
[0090] The term "peptidomimetic" as used herein refers to molecules which
mimic an
aspect of a polypeptide structure.
[0091] The term "purified" refers to a material (e.g., compound, molecule, or
structure
of interest) that is relatively free of other materials that it normally is
associated with
and is preferably at least 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%,
18
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WO 2005/056754 PCT/US2004/039064
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of total weight of the
material.
[0092] The term "recombinant" as used herein refers with reference to material
(e.g., a
cell, a nucleic acid, a protein, or a vector) indicates that such material has
been
modified by the introduction of a heterologous material (e.g., a cell, a
nucleic acid, a
protein, or a vector). Thus, for example, recombinant cells express genes that
are not
found within the native (non-recombinant) form of the cell or express native
genes that
are otherwise abnormally expressed, under expressed or not expressed at all.
[0093] A "sulfinyl" group refers to a -S(=O)-R, where R is selected from the
group
consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring
carbon) and
heteroalicyclic (bonded through a ring carbon)
[0094] A "sulfonyl" group refers to a -S(=O)2-R, where R is selected from the
group
consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring
carbon) and
heteroalicyclic (bonded through a ring carbon)
[0095] A "thiocyanato" group refers to a -CNS group.
[0096] The term "treatment" or "treating" as used herein refers to reducing or
alleviating symptoms in a subject, preventing symptoms from worsening or
progressing, or inhibition, elimination, or prevention of the infection,
disorder or
symptoms in a subject who is free therefrom.
II. In General
[0097] The particular mechanism by which a cell modulates its cellular
environment to
control mutation has been studied genetically in E. coli by monitoring
mutation-
mediated reversion or forward mutation of a gene required for cell growth.
Reversion
of a mutation occurs when a second mutation restores the function that was
lost as a
result of the first mutation. The second mutation causes a change in the DNA
that
either reverses the original alteration or compensates for it. The rate at
which the
second mutation occurs reflects the mutation rate of the bacteria under a set
of
conditions and can thus be used to measure induced mutation.
[0098] In E. coli, genes whose effect contributes to induced mutation are part
of the
SOS response system. The SOS response system in bacteria is a programmed
series of
gene derepression events which result in the induction of proteins involved in
DNA
replication, cell division, transposon mobility, lateral gene transfer, error-
prone
transleasion DNA synthesis, etc. which together result in an increased number
of
mutations. See Nickoloff, J. et al., (1998) DNA Damage and Repair (Totowa, New
19
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
Jersey: Humana Press); Huisman, O., Nature (1981) 290, 797-799; Kuan, C., et
al.,
J. Bacteriol. (1992) 174, 6872-6877; and Matic, L, et al., Cell (1995) 80, 507-
515. It is
postulated that the SOS response process in bacteria is activated when a cell
senses that
its replicative polymerase (e.g., Pol III) has stalled.
[0099] The stalling of the replication polymerase Pol III, results in the
accumulation of
ssDNA. It is the ssDNA which binds to and activates RecA. RecA is a
multifunctional
protein known in E. coli to mediate both recombination and the induction of
SOS
responses to stress. Activated RecA binds to and activates the proteolysis of
LexA and
UmuD. See Goodman, MF, Annu. Rev. Biochem. (2002) 71:17-50; Nickoloff, JA and
Hoekstra, MF (eds.) DNA Damage and Repair (Humana Press, Totowa, New Jersey,
1998).
[00100] LexA proteolysis is an autocleavage reaction of the A1a84-G1y85
scissile bond
of LexA. This cleavage bond separates LexA's DNA binding domain from its
dimerization domain, destroying the protein's ability to repress genes that
modulate
induced mutagenesis. (Such genes include error-prone polymerases such as Pol
IV and
both subunits of Pol V as well as genes selected from the group consisting of
16S
rRNA, 23S rRNA, clpXP, ding, dial, dnaE2, gyrA, gyrB, katG, iralrA, lon
protease, I~
ribosomal methylases, lexA, norA, recA. recN, psiB, parC, parE, polB, rpoS,
rpoB, sxt,
urnuC, urrZUD, uvrA, uvrB, and uvrD.) Binding to RecA induces a conformational
change in LexA, from a conformation that cannot undergo the self-cleavage
reaction to
one that can. Upon binding activated RecA, a loop containing A1a84 and G1y85
of
LexA moves into the active site, which is a cleft located on the surface of
the protein
with a catalytic serine-lysine dyad at one end (Figures 5A-5B). The catalytic
residues
(Ser119 and Lys156) catalyze the peptidase reaction in a manner similar to
that of
serine proteases. See Roland, KL., J. Biol. Chem. (1990) 265, 12828-12835; van
Dijl,
et. al., J. Biol. Chem. (1995) 270, 3611-3618; Slilaty, SN., Prot. Engineer-.
(1991) 4,
919-922; and Leung, D., J. Med. CherrZ. (2000) 43, 305-341.
[00101] Figure 1 depicts the state of LexA under normal conditions and under
the
condition of cellular stress due to ciprofloxacin exposure. Under normal
conditions, as
illustrated in Figure 1A, LexA represses genes whose corresponding protein
products
are involved in the cellular response to stress, including gene products that
cause
mutation. See Goodman, MF, Annu. Rev. Biochem. (2002) 71:17-50; Niclcoloff,
JA,
and Hoekstra, MF (eds.), DNA Damage and Repair, (Humana Press, Totowa, New
Jersey, 1998). LexA monomers are bound to DNA, stabilized via interactions
between
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
adjacently bound LexA monomers. Each LexA monomer contains a dimerization
domain that enables LexA to bind DNA cooperatively, as the LexA monomers bind
DNA at adjacent operator sites and thereby stabilize one another's binding via
inter-
protein contacts. The binding of LexA dimers to their cognate binding sites
prevents
access of RNA polymerase to LexA-controlled promoters, keeping the
intracellular
concentrations of SOS response gene products low.
[00102] Figure 1B illustrates what happens when bacteria are exposed to
certain
antibiotics (e.g., ciprofloxacin). See Mamber, SW., Antimicrob. Agents
Chemother.
(1993) 37:213-217; Riesenfeld, C., (1997) 41:2059-2060; Phillips, L, J.
Antimicrob.
Chemother., (1987) 20:631-638; Luo, Y., Cell (2001) 106:585-594; Shinagawa,
H.,
Proc. Nat'l. Acad. Sci. USA (1988) 85:1806-1810; Rehrauer, WM., J. Biol. Chem.
(1996) 271:23865-23873; and Balashov, S., J. Mol. Biol. (2002) 315:513-527.
Such
exposure activates the SOS response via the ssDNA-mediated activation of RecA.
RecA has been shown to form a complex with ssDNA and ATP. This complex
initiates
recombination and also catalyzes the autoproteolysis of LexA.
[00103] Autoproteolysis of LexA results in the cleavage of LexA between its N
and C
terminal domains. After the proteolytic separation of the DNA binding and
dimerization domains, LexA no longer cooperatively binds DNA to repress gene
expression. As a result, SOS gene products are produced. Such SOS gene
products
include, for example, Pol IV and Pol V, which are encoded by ding and urnuDC,
respectively. These gene products (and their analogs and homologs) may have
different names in other organisms. Pol IV and Pol V (a heterodimer of UmuC
and two
copies of UmuD', a product of RecA-mediated cleavage of UmuD) are both error-
prone, mutation-causing polymerases. It is the derepression of these
polymerases
which synthesize DNA with low fidelity, which is responsible for the increased
rate of
mutation at times of stress. Thus, the inhibition of the production or
activity of these
mutation causing polymerases may inhibit the evolution of antibiotic
resistance. See
also Yeiser, B., Proc. Natl. Acad. Sci. USA (2002) 99:8737-3841; McKenzie,
GJ.,
Proc. Natl. Acad. Sci. USA (2000) 97:6646-6651; Goodman, MF., Curr. Opin.
Genet.
Dev. (2000) 10:162-168; Shinagawa, H. in Stress-Inducible Cellular Responses
(eds.
Feige, U., Morimoto, R. L, Yahara, I. & Polla, B.) BirkhSuser Verlag, Basel,
1996;
Sutton, MD., Annu. Rev. Genet. (2000) 34:479-497; Brotcorne-Lannoye, A., Proc.
Natl. Acad. Sci. USA (1986) 83:3904-3908; Bull, HJ., Gefaetics (2000) 154:1427-
1437;
Bull, HJ., Proc. Natl. Acad. Sci. USA, (2001) 98:8334-8341; Kim, B., Cell
(1993)
21
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WO 2005/056754 PCT/US2004/039064
73:1165-1173; Napolitano, R., EMBO (2000) 19:6259-6265; Tang, M., Nature
(2000)
404:1014-1018; and Pralcash, S., Gerzes. Dev. (2002) 16:1872-1883.
[00104] Figure 1C illustrates what happens if an achaogen prevents LexA
cleavage. In
this scenario, the achaogen prevents the proteolysis of LexA, despite the
presence of
the antibiotic. As a result, the bacteria are not able to accelerate their
rate of mutation,
significantly decreasing their ability to evolve antibiotic resistance.
Inhibition of
mutation with an achaogen can be achieved via multiple strategies, including:
(1) the
inhibition of RecA (or any RecA ortholog) activation (2) the inhibition of
RecA (or any
RecA ortholog) binding to LexA or UmuD (or any LexA or UmuD ortholog) or any
other yet to be identified component of the induced mutational response, or
(3) the use
of small molecules to inhibit the proteolysis of LexA or UmuD (or any LexA or
UmuD
ortholog).
[00105] One example of a gene associated with modulating induced mutagenesis
is
dnaE2. d>zaE2 has been implicated in the emergence of drug resistance in
Mycobacterium tuberculosis (MTb). See Boshoff, H., Cell. (2003) Vol. 113, 183-
193.
It has been suggested that MTb contains two functionally redundant replicative
DNA
polymerases: DnaEl and DnaE2. See Boshoff, H., Cell. (2003) Vol. 113, 183-193.
It
has further been shown that mutations conferring resistance to rifampicin
(Rif) in MTb
are mediated primarily by drzaE2 as deletion of the gene prevents the
accumulation of
mutations conferring resistance. Thus, it has been suggested that DnaE2 is an
error-
prone translesion polymerase responsible for mutation conferring resistance to
Rif.
[00106] Currently, MTb is commonly treated with an initial intensive 2-month
regimen
comprising multiple antibiotics: rifampicin (RIF), isoniazid (INH),
pyrazinamide
(PZA), and ethambutol (EMB) or streptomycin (SM), to ensure that mutants
resistant to
a single drug do not emerge and compromise therapy. See MMWR Morb. Mortal
Wkly. Rep. (1993) 42 (RR-7). During the next 4 months, only RIF and INH are
administered. INH and RIF are potent anti-MTb drugs that kill more than 99% of
tubercular bacilli within 2 months of initiation of therapy. See Mitchison DA.
Bulletirz
Interoeatiorzal Urziorz Against Tuberculosis (1985) 65:30-7. Using these drugs
in
conjunction with each other, and possibly other drugs, shortens anti-MTb
therapy from
18 months to 6 months. However, 2% of patients initiating multi-drug therapy
develop
drug resistant MTb by the end of the therapy, even with perfect compliance
with the
prescribed regimen.
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WO 2005/056754 PCT/US2004/039064
III. Achaogens
[00107] In bacterial cells, as the cellular concentration of full-length LexA
decreases due
to LexA auto-proteolysis, the SOS genes are derepressed sequentially, in an
order that
depends on the affinity of their promoters for the LexA repressor. The first
genes that
are derepressed encode direct repair functions, including uvrA, uvrB, and
uvrD. The
cell first attempts to repair the damage, but if the damage persists, the next
genes to be
derepressed include recA, recN, and other genes that mediate more drastic
recombinational repair pathways. Finally, if the damage still persists, the
cell will first
derepress ding (which encodes the error-prone polymerase Pol IV) and then
later
derepress mnuC and urr2uD (which encode the two subunits of the error-prone
polymerase Pol V). In this manner, only when the environment has become
sufficiently lethal does the cell permit elevated rates of mutation.
[00108] An achaogen of the present invention is any agent that inhibits the
mutation
process in a cell, group of cells within an organism, or an entire organism.
In
particular, an achaogen is an agent that inhibits the mutation process, which
is triggered
in response to environmental stress or DNA damage. Examples of environmental
stresses that can induce mutagenesis or DNA damage include: drug treatment, UV
radiation, restricted nutrients, etc.
[00109] A cell or an organism that may be undergoing or affected by induced
mutations
can be prokaryotic or eukaryotic. Examples of prokaryotic cells/organisms
contemplated by the present invention include any of the bacterial strains
disclosed
herein. Examples of eukaryotic cells/organisms contemplated by the present
invention
include mammals, avians, plants, and in particular, humans. Thus, an organism
whose
mutation rate is reduced by an achaogen can be a microorganism (e.g., a virus
or a
bacterium) or a multicellular organism (e.g., a plant or animal).
[00110] The present invention relates to compositions comprising, consisting
essentially
of, and consisting of achaogens. As such a composition of the present
invention can
optionally include a second agent.
[00111] An achaogen can be naturally occurring or non-naturally occurring. An
achaogen of the present invention is preferably isolated and/or purified. An
achaogen
of the present invention can comprise or consist of a nucleic acid, a
polypeptide, a
peptidomimetic, a peptide nucleic acid ("PNA"), an antibody, a phage, a
phagemid, or a
small or large organic or inorganic molecule. Salts, prodrugs, homologs or
analogs of
any of the achaogens herein are also a feature of the invention.
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WO 2005/056754 PCT/US2004/039064
[00112] In some embodiments, an achaogen modulates, reduces, or inhibits the
rate of
mutation in a cell or an organism by interacting with or binding to a gene
product that
increases the rate of mutation either directly or indirectly. Examples of such
gene
products include, but are not limited to, RecA, RecB, RecC, RecD, RecF, Recta,
Rec N,
LexA, UmuC, UmuD, PoIB, PolIV, PoIV, PriA, RuvA, RuvB, RuvC, UmuC, UmuD,
UvrA, UvrB, UvrD, or any homologs, analogs, fragments, or combinations
thereof. Of
course, an achaogen need not bind or interact with all of the above gene
products. In
some embodiments, an achaogen binds or interacts with only 1, 2, 3, 4, 5, 6,
7, 8, 9, 10,
or 11 of the above gene products. For example, in some embodiments an
acachaogen
binds to or interacts with RecA. However, this is but one example of an
achaogen and
therefore in some embodiments, an achaogen does not bind to or interact with
RecA.
In other embodiments, an achaogen does not interact or bind to RecB, RecC,
RecD,
RecF, Recta, Rec N, LexA, UmuC, UmuD, PoIB, PolIV, PoIV, PriA, RuvA, RuvB,
RuvC, UmuC, UmuD, UvrA, UvrB, or UvrD, or a homolog thereof.
[00113] Such modulation or inhibition can be mediated by an achaogen that is a
direct
inhibitor, competitive inhibitor or other form of inhibitor of such gene
products. For
example, an achaogen of the present invention can modulate or inhibit the rate
of
mutation by binding or interacting with a gene product that increases the rate
of
mutation, either covalently or non-covalently. In other examples, an achaogen
can
modulate or inhibit the rate of mutation by binding or interacting with a gene
product
that reduces the rate of mutation, either covalently or non-covalently.
[00114] In some embodiments, an achaogen is an inhibitor of RecA activation or
RecA
binding to ssDNA (e.g., a small molecule or peptidomimetic that interferes
with RecA
binding to ssDNA). This is but one example of an achaogen contemplated by the
present invention, and as such, an achaogen of the present invention can one
other than
an achaogen that interacts with or binds to RecA.
[00115] In some embodiments, an achaogen is an inhibitor of LexA autocleavage
(e.g., a
peptidomimetic that competes with the cleavage site of LexA) or of a homolog
or
LexA. LexA is highly conserved in clinically relevant bacterial species (see
Table 1
below). Thus, an achaogen contemplated by the present invention is one that
interacts
with or binds to LexA or any homolog, analogs, or fragments thereof and which
can
used to reduce induced mutations in a wide spectrum of bacterial infections.
24
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WO 2005/056754 PCT/US2004/039064
TABLE 1
LexA ortholog Identical Similar
Esclzericlzia 100% 100%
coli. K12
Escherichia coli100% 100%
K12 0157
Vibrio cholerae 73% 84%
Haernophilus 67% 83%
irzfluerzzae
Pseudonzonas 43% 62%
s rin ae
Bacillus anthracis3G% 55%
Erzterococcus 35% 50%
faecalis
Sta h lococcus 33% 52%
aureus
Streptorrzyces 35% 51%
coelicolor
[00116] An achaogen interacting with LexA, preferably interacts with the
"cleavage
site" (substrate loop) of LexA or the "active site" of LexA. The "cleavage
site" of
LexA is a peptide sequence of LexA which includes the dipeptide bond A1a84-
G1y85.
In some embodiments, the cleavage site of LexA is preferably less than 50
amino acids
in length, less than 40 amino acids in length, less than 30 amino acids in
length, less
than 20 amino acids in length, more preferably less than 15 amino acids in
length, more
preferably less than 10 amino acids in length, or more preferably less than 6
amino
acids in length.
[00117] In some embodiments, the cleavage site of LexA comprises or consists
of a
polypeptide having amino acid sequence of VAAG (SEQ ID NO: 1), VAAGEPL (SEQ
ID NO: 2) or VAAGEPLLAW (SEQ ID NO: 3), or any homolog or analog thereof.
[0011] The "active site" or substrate loop site of LexA comprises of a peptide
sequence
of LexA which includes Ser119 and Lys156. In some embodiments, the active site
of
LexA is less than 100 amino acids in length, more preferably less than 90
amino acids
in length, more preferably less than 80 amino acids in length, more preferably
less than
70 amino acids in length, more preferably less than 60 amino acids in length,
or more
preferably less than 50 amino acids in length.
[00119] Thus, in some embodiments, an achaogen interacts with either the
cleavage site
or the active site of LexA. Such interactions can be covalent or a non-
covalent. Such
achaogen can be a competitive inhibitor for the active site, a molecular decoy
for LexA,
or a specific protease that cleaves LexA.
[00120] In some embodiments, an achaogen is a peptide fragment of the LexA
cleavage
site or a peptidomimetic that mimics a LexA cleavage site, thereby
competitively
binding to LexA's internal active site preventing autoproteolysis. Examples of
such
achaogens include peptide fragments VAAG (SEQ 117 NO: 1), VAAGEPL (SEQ ll~
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WO 2005/056754 PCT/US2004/039064
NO: 2), VAAGEPLLAW (SEQ 117 NO: 3), or any homologs or analogs thereof.
Preferably such peptides and/or peptidomimetics or analogs thereof are
isolated.
Preferably, such peptides and/or peptidomimetics or analogs thereof include at
least one
non-cleavable bond. More preferably, the Ala-Gly bond is modified to be a non-
cleavable bond.
[00121] Examples of non-cleavable peptidomimetics of the cleavage site of LexA
are
illustrated in Figures 15A-15D. In each of Figures 15A-15D the A1a84-G1y85
scissile
bond was replaced with a non-cleavable analog. Such non-cleavable analogs
include a
keto-moiety (Figure 15A), a trans-olefin moiety (Figure 15B), a reduced amide
moiety
(Figure 15C), and a a-keto moiety (Figure 15D). In more preferred embodiments,
a
peptidomemtic of fewer than 10 amino acids spanning the scissile bond is
constructed.
For example, in Figures 16A-16D, a peptidomimetic of the AAGEPL peptide with a
replacement group replacing the A1a84-G1y85 scissile bond can include a keto-
moiety
(see Figure 16A), trans-olefin moiety (Figure 16B), reduced amide moiety
(Figure
16C), or a-keto moiety (Figure 16D).
[00122] As depicted in Figures 4A and 4B, it is believed that a portion (e.g.,
VAAG) of
LexA's cleavage site becomes situated within LexA's substrate loop during
LexA's
auto-proteolysis reaction. Figure 4A depicts the enzyme's active cleft with
its
substrate. Figure 4B depicts the substrate loop alone. Residues Arg81 to A1a84
of the
cleavage site pack snugly in the cleft of the substrate loop with the side
chains of A1a84
and Va182 hydrophobically packed in the S 1 and S3 sites of the active site
cleft (Figure
4A). Interestingly, while quite distant from the scissile bond, mutation of
G1n92 to
Trp91 dramatically increases the affinity for the corresponding peptide in the
active
site, by increasing favorable interactions.
[00123] The crystal structure of E. coli LexA in both cleavable and non-
cleavable
conformations has been determined to 1.8 angstroms resolution (see Figures 5A-
5B,
respectively). See Luo, Y., Pfuetzner, R.A., Mosimann, S., Paetzel, M., Frey,
E.A.,
Cherney, M., Kim, B., Little, J.W., and Strynadka N.C., Crystal structure of
LexA: a
conformational switch for regulation of self-cleavage. Cell, (2001) Vol. 106,
585-594.
The structure suggests that the P sites located to the N-terminal side of the
scissile bond
make more important contact than the P' site, located on the C-terminal side
of the
scissile bond.
[00124] Figure 6 depicts the LexA self-cleavage complex in which Ser119 (S
119) of
LexA attacks the C=O of the A1a84-G1y85 scissile peptide bond. It is suggested
that
26
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WO 2005/056754 PCT/US2004/039064
Serl 19 serves as the reactive nucleophile and attacks the A1a84-G1y85 peptide
bond in
manner analogous to a serine protease, while the uncharged Lys 156 activates
the
Ser119 hydroxyl group. See Rolan KI, et al., J. Biol. Chem., (1990) 265,
22:12828-
12835. Thus, the present invention contemplates a mutated LexA, having
mutations in
either or both the cleavage site or active site to disrupt substrate binding
without
disrupting the dimer formation of LexA. Such an achaogen can be a mutated LexA
polypeptide (or homolog, analog, or fragment thereof). Such an achaogen can
also be a
nucleic acid encoding the mutated LexA polypeptide (or a homolog, analog, or
fragment thereof).
[00125] Thus, in some embodiments, the present invention contemplates an
achaogen
that binds to the active site nucleotphile Serl 19. Preferably such an
achaogen binds to
Ser119 covalently. This is but one example of an achaogen of the present
invention,
and in some embodiments, an achaogen is one other than an achaogen that binds
to the
active site nucleophile Ser119.
[00126] In some embodiments, such an achaogen comprises a peptide sequence
located
immediately to the N-terminal of the LexA scissile bond, or any homolog or
analog
thereof. Such achaogens comprise, consisting essentially of, or consist of a
dipeptide
Ala-Ala, a tripeptide Val-Ala-Ala or polypeptide Arg-Val-Ala-Ala, or any
homolog or
analog thereof. Such polypeptides, peptidomimetics, or analogs are preferably
C-
terminally modified to enhance its binding to the nucleophilic Ser119. These
achaogens are one example of the achaogens contemplated herein, and as such,
in some
embodiments, an achaogen is one other than those comprising, consisting
essentially of,
or consisting of a dipeptide Ala-Ala, a tripeptide Val-Ala-Ala or polypeptide
Arg-Val-
Ala-Ala, or any homolog or analog thereof.
[00127] In some embodiments, such an achaogen comprises a peptide sequence
located
immediately to the C-terminus of the LexA scissile bond, or any homolog or
analog
thereof. Such peptides can include a dipeptide Gly-Glu, a tripeptide Gly-Glu-
Pro or a
peptide sequence of Gly-Glu-Pro-Leu, or any homolog or analog thereof. Such
polypeptide, peptidomimetic, or analog thereof is preferably N-terminally
modified to
enhance its binding to the nucleophilic Ser119. These achaogens are one
example of
the achaogens contemplated herein, and as such, in some embodiments, an
achaogen is
one other than those comprising, consisting essentially of, or consisting of a
dipeptide
Gly-Glu, a tripeptide Gly-Glu-Pro or a peptide sequence of Gly-Glu-Pro-Leu, or
any
homolog or analog thereof.
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WO 2005/056754 PCT/US2004/039064
[00128] Preferred C- and N-terminal modification that enhance binding of the
peptide/peptidomimetic/analog to Ser119 are electrophile modifications.
Examples of
electrophile modifications include peptide aldehydes, trifluoroketones,
chloromethyl
ketones, and alpha-keto heterocycles. Examples of aldehydes and chlormethyl
ketone
analogs of the Ala-Ala dipeptide to the N-terminal of the scissile bond are
illustrated in
Figure 17, compounds 1 and 2. Examples of the aldehydes and chloromethyl
ketone
analogs of the Val-Ala-Ala tripeptide to the N-terminal of the scissile bond
are
illustrated in Figure 17, compounds 3 and 4.
[00129] Achaogens that interact with LexA are but one example of the achaogens
contemplated herein, and as such, an achaogen of the present invention can be
an
achaogen other than one that interacts with or binds to LexA. In some
embodiments,
an achaogen is one other than that which interacts with the LexA cleavage site
or the
LexA active site.
[00130] In some embodiments, an achaogen is an inhibitor of RecA/LexA complex
formation (e.g., a small molecule or peptidomimetic that interferes with the
RecA/LexA
complex formation). Again, this is but one example of an achaogen, and as
such, an
achaogen of the present invention can be an achaogen other than one that
inhibits the
RecA/LexA.
[00131] Any of the achaogen peptides or peptidomimetics herein can be further
modified for slower release or degradation (e.g., using D-amino acid residues,
PEG-
terminus, etc.).
[00132] Synthesis of polypeptides and analogs thereof is known by those
spilled in the
art. In any of the embodiments herein, it is preferable that a peptide or a
peptidomimetic of the present inventions fit within the substrate binding
site.
Therefore, a peptide or peptidomimetic of the present invention is preferably
less than
about 60 Angstroms, more preferably less than about 45 Angstroms, more
preferably
less than about 30 Angstroms, or more preferably less than about 15 Angstroms.
[00133] In some embodiments, an achaogen is a protease inhibitor, or more
preferably a
serine protease inhibitor. It is believed that LexA and UmuD are serine-lysine
diad
proteases that undergo proteolysis reactions that are critical for the
induction of
mutation in multiple bacterial species. See Roland, KL, et al. J. Biol. Chem.
(1990)
265:12828-12835; Little, JW. J. Bacteriol. (1993) 175:4943-4950; Kim, B, et
al. Cell
1993, 73:1165-1173; and Slilaty, SN, Prot. Engineer. (1991) 4:919-922. The
catalytic
residues of LexA (Ser119 and Lysl56) catalyze the peptidase reaction in a
manner
28
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WO 2005/056754 PCT/US2004/039064
similar to that of serine proteases. See Roland, K.L., J. Biol. Chem. (1990)
265, 12828-
12835. Moreover, it has been shown that the serine protease inhibitor,
diisopropyl
fluorophosphates (DFP), inhibits auto-cleavage of LexA and that Ser119 was the
only
serine residue to react with DFP. See Roland, KL., J. of Biol. Chem., (1990)
265(22):12828-12835. (Structure of DFP is illustrated in Figure 13.). However,
these
are but a few examples of achaogens and in some embodiments an achaogen is one
other than a protease inhibitor or a serine protease inhibitor.
[00134] Thus, in some embodiments, an achaogen that comprises, consists
essentially
of, or consists of a protease inhibitor or a serine protease inhibitor or
analog thereof
preferably reduces the rate of induced mutations by at least a significant
amount, e.g., at
least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Methods of
detecting levels of induced mutations are disclosed herein or are known in the
art.
[00135] Examples of serine protease inhibitors that may be achaogens include
DFP
(diisopropyl fluorophosphates, a small molecule), AEBSF (aminoethyl-benzene
sulfonyl fluoride), aprotinin (trypsin inhibitor from bovine lung), antipain,
antithrombin
III (e.g., from human plasma), (alpha)1-antitrypsin, APMSF (4-amidinophenyl-
methane
sulfonyl-fluoride), chymostatin, leupeptin-hemisulfate, Pefabloc SC (4-(2-
aminoethyl)-
benzenesulfonyl fluoride), PMSF (phenylmethyl sulfonyl fluoride),
phosphoramidon,
TLCK (1-chloro-3-tosylamido-7-amino-2-heptanone), TPCK (1-chloro-3-tosylamido-
4-phenyl-2-butanone), and trypsin inhibitor from soybean. Additional examples
of
serine protease inhibitors contemplated by the present invention are depicted
in Figures
14A-14E.
[00136] Achaogens that are serine protease inhibitors can be optimized using
methods
similar to those used to successfully design inhibitors of thrombin, factor
Xa, elastase,
tryptase, complement convertase, and hepatitis C-NS3 protease. See Vacca, J.P.
Annu.
Rep. Med. Chem. (1998) 33, 81-90; Verstraete, M. Haemostasis (1996) 26;
Morishima,
Y., Thromb. Haemost. (1997) 78, 1366-1371; Edwards, P.D., Med. Res. Rev.
(1994)
14; and Rice, K.D., Curr. Pharm. Des. (1998) 4, 381-396; Oda, M., Jpn. J.
Plza~nacol.
(1990) 52, 23-34; Steinkuhler, C., Biochemistry (1998) 37, 8899-8905; and
Linas-
Brunet, M., et al. Bioorg. Med. Clzefzz. Lett. (1998) 8, 1713-1718.
[00137] Achaogens that comprise, consist essentially of, or consist of a
protease
inhibitor or serine protease inhibitor can be naturally occurring or
synthetic. In one
embodiment, an achaogen comprises, consists essentially of, or consists of an
organic
molecule that is a protease inhibitor. In particular, in some embodiments an
achaogen
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WO 2005/056754 PCT/US2004/039064
can include a heterocyclic molecules that can act as protease inhibitors. By
way of
example only, an achaogen-of the present invention can include a heterocyclic
compounds having the general structure of Formula (I):
R
R, Rs
(I),
[00138] where Rl, R2, R3, R4, R5, and R~ are each independently selected from
the group
consisting of -(CHRa)X L-Rb, where x is selected from the group consisting of
0, 1, 2,
3, or 4; L is a single bond or -C(O)-, -NHC(O)-, -OC(O)-, -S(O)S, where j is
0, 1, or 2;
Ra is a moiety selected from the group consisting of H, (C1-C6)alkyl, halogen,
(Cl-
C6)fluoroalkyl, (Cl-C~)alkoxy, -C(O)OH, -C(O)-NH2, -(C1-C6)alkylamine, -C(O)-
(Cl-
C~)alkyl, -C(O)-(C1-C6)fluoroalkyl, -C(O)-(C1-C6)alkylamine, and -C(O)-(C1-
CG)alkoxy; and Rb is H, OH, halogen, NH2, CN, N3, or a moiety, optionally
substituted
with 1-3 independently selected substituents, selected from the group
consisting of
alkyl, alkenyl, alkoxy, mercaptyl, alkylamine, alkynyl, aryl, cycloalkyl,
cycloalkenyl,
and a heterocycle; in addition, Rl and R2, R2 and R3, R3 and R4, and RS and
R6, can
optionally form a substituted or unsubstituted ring structure. Compounds
having the
structure of Formula (I) are also known as isocoumarins. The ability of
compounds to
act as achaogens, including compounds having the structure of Formula (I), as
well as
other heterocyclic protease inhibitors, other protease inhibitors, and other
organic
compounds, can be ascertained using the methods and techniques described
herein.
[00139] Compounds of Formula (I) may be synthesized using standard synthetic
techniques known to those of skill in the art or using methods known in the
art in
combination with methods described herein. In addition, several of the
compounds of
Formula (I) may be purchased from various commercial suppliers. As a further
guide
the following synthetic methods may be utilized.
[00140] Selected examples of covalent linkage products and precursor
functional groups
(i.e., a nucleophile and an electrophile) which yield them are given in the
Table entitled
"Examples of Covalent Linkages and Precursors Thereof." Precursor functional
groups
are shown as electrophilic groups and nucleophilic groups. The functional
group on the
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WO 2005/056754 PCT/US2004/039064
organic substance may be attached directly, or attached via any useful spacer
or linker.
Thus, Table 2 may be utilized to synthesize or add further functionality to a
precursor
compound in order to synthesize additional compounds having the structure of
Formula
(I). For example, (a) an isocoumarin bearing an alkyl halide group can react
with
another compound having a thiol group in order to form an isocoumarin having a
thioether group; (b) an isocoumarin having an amine group can react with a
compound
having an aryl halide group to form an isocoumarin having an aryl amine group;
or (c)
an isocoumarin having a carboxylic acid group can react with a compound having
a
hydrazide group to form an isocoumarin having a hydrazine group.
TABLE 2: Examples of Covalent Linkages and Precursors Thereof
Covalent Linkage Electrophile y Nucleophile
Product
Carboxamides Activated esters amines/anilines
Carboxamides acyl azides amines/anilines
Carboxamides acyl halides amines/anilines
Esters acyl halides alcohols/phenols
Esters acyl nitriles alcohols/phenols
Carboxamides acyl nitriles amines/anilines
Imines Aldehydes amines/anilines
Hydrazones aldehydes or ketones Hydrazines
Oximes aldehydes or ketones Hydroxylamines
Alkyl amines alkyl halides amines/anilines
Esters alkyl halides carboxylic acids
Thioethers alkyl halides Thiols
Ethers alkyl halides alcohols/phenols
Thioethers alkyl sulfonates Thiols
Esters alkyl sulfonates carboxylic acids
Ethers alkyl sulfonates alcohols/phenols
Esters Anhydrides alcohols/phenols
Carboxamides Anhydrides amines/anilines
Thiophenols aryl halides Thiols
Aryl amines aryl halides Amines
Thioethers Azindines Thiols
Boronate esters Boronates Glycols
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Covalent linkage E.lectrophile Nuoleophile
Product
Carboxamides carboxylic acids amines/anilines
Esters carboxylic acids Alcohols
Hydrazines Hydrazides carboxylic acids
N-acylureas or Anhydridescarbodiimides carboxylic acids
Esters diazoalkanes carboxylic acids
Thioethers Epoxides Thiols
Thioethers haloacetamides Thiols
Ammotriazines halotriazines amines/anilines
Triazinyl ethers halotriazines alcohols/phenols
Amidines imido esters , amines/anilines
Ureas Isocyanates amines/anilines
Urethanes Isocyanates alcohols/phenols
Thioureas isothiocyanates amines/anilines
Thioethers Maleimides Thiols
Phosphite esters phosphoramidites Alcohols
Silyl ethers silyl halides Alcohols
Alkyl amines sulfonate esters amines/anilines
Thioethers sulfonate esters Thiols
Esters sulfonate esters carboxylic acids
Ethers sulfonate esters Alcohols
Sulfonamides sulfonyl halides amines/anilines
Sulfonate esters sulfonyl halides phenols/alcohols
[00141] In some embodiments, a compound of Formula (I) can include a
protecting
group. The term "protecting group" refers to chemical moieties that block at
least some
reactive moieties and prevent such groups from participating in chemical
reactions until
the protective group is removed (or "cleaved"). In one aspect, a particular
reagent
bears at least three different functional groups and the desired product is
synthesized by
reacting only one of those three functional groups. Such a desired product may
be made
by protecting the two functional groups that are not supposed to be modified,
thus
leaving the third functional group available for further reaction. Once this
further
reaction has occurred, the other two functional groups may be restored by
cleaving the
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WO 2005/056754 PCT/US2004/039064
protecting groups. The resulting compound has thus been modified at only one
of the
three potential sites.
[00142] Protective groups that are cleaved under disparate reaction conditions
fulfill the
requirement of differential removal. Protective groups can be removed by acid,
base,
and hydrogenolysis. Groups such as trityl, dimethoxytrityl, acetal and t-
butyldimethylsilyl are acid labile and may be used to protect carboxy and
hydroxy
reactive moieties in the presence of amino groups protected with Cbz groups,
which are
removable by hydrogenolysis, and Fmoc groups, which are base labile.
Carboxylic acid
and hydroxy reactive moieties may be blocked with base labile groups such as,
without
limitation, methyl, ethyl, and acetyl in the presence of amines blocked with
acid labile
groups such as t-butyl carbamate or with carbamates that are both acid and
base stable
but hydrolytically removable.
[00143] Carboxylic acid and hydroxy reactive moieties may also be blocked with
hydrolytically removable protective groups such as the benzyl group, while
amine
groups capable of hydrogen bonding with acids may be blocked with base labile
groups
such as Fmoc. Carboxylic acid reactive moieties may be protected by conversion
to
simple ester derivatives as exemplified herein, or they may be blocked with
oxidatively-removable protective groups such as 2,4-dimethoxybenzyl, while co-
existing amino groups may be blocked with fluoride labile silyl carbamates.
[00144] Allyl blocking groups are useful in then presence of acid- and base-
protecting
groups since the former are stable and can be subsequently removed by metal or
pi-acid
catalysts. For example, an allyl-blocked carboxylic acid can be deprotected
with a Pd0-
catalyzed reaction in the presence of acid labile t-butyl carbamate or base-
labile acetate
amine protecting groups. Yet another form of protecting group is a resin to
which a
compound or intermediate may be attached. As long as the residue is attached
to the
resin, that functional group is blocked and cannot react. Once released from
the resin,
the functional group is available to react.
33
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
[00145] Examples of blocking/protecting groups may be selected from:
H2 H O
z
H C2 ~ C~ ~ CEO ~C~ .O
H2C~ C H3Ci
H2C H H2
2
ally) Bn Cbz allot Me
H2 H3C~ CH3 H OII
H3C~C~ ~H3C)3C~ (H3C)3C~SI~ ~CH3)3C~S ~O~
Et t-butyl TBDMS Teoc
O
_ Ha
O H2C
OH3~3C/O~ ~ O6H5~3C- H3C
H3C0
Boc pMBn trityl acetyl
Fmoc
[00146] Other protecting groups are described in Greene and Wuts, Protective
Groups in
Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is
incorporated herein by reference in its entirety.
[00147] Any of the achaogens presented herein may possess one or more chiral
centers
and each center may exist in the R or S configuration. The achaogens presented
herein
include all diastereomeric, enantiomeric, and epimeric forms as well as the
appropriate
mixtures thereof. Stereoisomers may be obtained, if desired, by methods known
in the
art as, for example, the separation of stereoisomers by chiral chromatographic
columns.
It ill be appreciated that the invention herein is not limited to any given
compound
herein and that, in certain embodiments, the achaogen of interest is a
molecule other
than a compound of Formula I as described herein.
[00148] The methods and formulations described herein can include the use of N-
oxides,
crystalline forms (also known as polymorphs), or pharmaceutically acceptable
salts of
achaogens having the structure of Formula (I), as well as active metabolites
of these
achaogens having the same type of activity. All tautomers are included within
the
scope of the achaogens presented herein. In addition, the achaogens described
herein
can exist in unsolvated as well as solvated forms with pharmaceutically
acceptable
solvents such as water, ethanol, and the like. The solvated forms of the
compounds
presented herein are also considered to be disclosed herein.
34
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
[00149] In some embodiments, an achaogen is other than a protease inhibitor,
other than
a serine protease inhibitor, other than a synthetic protease inhibitor, other
than a
naturally occurring protease inhibitor, other than a synthetic serine protease
inhibitor,
or other than a naturally occurring protease inhibitor.
[00150] The development of inhibitors against proteases similar to LexA and
UmuD has
been accomplished using established drug design methods. See Vacca, JP., Annu.
Rep.
Med. Cherra. (1998) 33:81-90; Verstraete, M, Haemostasis (1996) 26 Suppl. 4:
70-77;
Morishima, Y., Thronab. Haemost. (1997) 78:1366-1371; Edwards, PD, et al. Med.
Res.
Rev. (1994) 14(2): 127-94; Rice, KD, et al. Curr-. Pharm. Des. (1998) 4:381-
396; Oda,
M, et al. Jpn. J. Pharmacol. (1990) 52:23-34; Steinkuhler, C., Bioclaenzistry,
(1998)
37:8899-8905; Llinas-Brunet, M., Bioorg. Med. Chem. Lett. (1998) 8:1713-1718;
and
Bogey, DL., Bioorg. Med. Chem. Lett., (2001) 11:1517-1520.
[00151] In some embodiments, an achaogen can be a naturally occurring agent or
an
analog of a naturally occurring agent that is a negative regulator of induced
mutations.
Examples of naturally occurring agents that are negative regulators of induced
mutations include, but are not limited to, DinI, PsiB, CIpXP, Lon protease,
and any
fragments, homologs, or analogs thereof. These are but just a few examples of
achaogens and in some embodiments; an achaogen is other than an isolated
and/or
purified DinI, PsiB, CIpXP, or a Lon protease. Also, in some embodiments, an
achaogen is one other than a naturally occurring agent or an analog of a
naturally
occurring agent that acts as a negative regulator of induced mutations.
[00152] It is believed that chromosomally encoded DinI and F-Plasmid encoded
PsiB
bind to RecA and inhibit LexA auto-cleavage and/or UmuD cleavage. See
McKenzie,
G., Proc. Natl. Aced. Sci. USA (2000) 97, 6646-6651; Bagadasarian, M., et al.,
Mol.
Microbiol. (1992) 6, 885-893; Yasuda, T., EMBO J. (1998) 17, 3207-3216.
Experiments show that when released from repression by lexA deletion (in a
sulA
deletion background, required for survival) PsiB is capable of completely
suppressing
induced mutation even though the SOS response is fully induced. If under the
same
circumstances, psiB is also deleted, the hypermutable state is fully
manifested.
Furthermore, it is known that PsiB competes with both LexA and UmuD for
binding to
RecA, while DinI competes only with UmuD. See Yasuda, T., et al., EMBO J 1998,
17:3207-3216; Yasuda, T., EMBO (2001) 20:1192-1202; and Bagadasarian, M., Mol
Microbiol. (1992) 6:885-893.
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
[00153] Furthermore, induced mutation may be under negative regulation by
proteases.
For example, in bacteria induced mutation is negatively regulated or inhibited
by one or
more proteases, e.g., CIpXP and Lon proteases. See Frank, E, G., et al., Proc.
Ned.
Aced. Sci. USA (1996) 93, 10291-10296. Lon protease degrades UmuDa and UmuC
proteins, while CIpXP specifically proteolyzes UmuD' of a UmuD'/LTmuD
heterodimer. These proteases are thought to ensure rapid exit from the
hypermutable
state once suitable mutations have been acquired.
[00154] Thus, in some embodiments an achaogen of the present invention rnay be
an
isolated and/or purified DinI, PsiB, ClpXP, Lon protease, or fragments,
homologs, or
analogs thereof. In particular, the present invention contemplates the use of
peptidomimetics of DinI, PsiB, CIpXP, Lon protease, or any fragments, homologs
or
analogs thereof to reduce the rate of mutation in a cell or an organism. In
other
embodiments, an achaogen of the present invention is other than isolated
andlor
purified DinI, PsiB, CIpXP, Lon protease, or fragments, homologs, or analogs
thereof.
[00155] In any of the embodiments herein, achaogens can be proteins or
peptidomimetics of gene products that inhibit induced mutation (e.g., PsiB,
DinI,
ClpXP protease, Lon protease, and homologs thereof) modified for increased
affinity
for their target protein (e.g., RecA. LexA, or UmuD) by rational design or
library-based
selections or screens (for example, phage display or high-throughput
screening).
Achaogen peptide mimics may be designed based on the amino acid sequence of
appropriate proteins or peptide fragments, modified for improved function,
including
improved target binding (for example, RecA or LexA in E. coli) or improved
pharmacokinetics (for example, improved stability, cell permeability, or
target
specificity).
IV. Nucleic Acids
[00156] In some embodiments, an achaogen comprises or consists of a nucleic
acid
encoding a negative regulator of induced mutations. Examples of negative
regulators
of induced mutations include DinI, PsiB, CIpXP, Lon protease, and any
fragments,
homologs or analogs thereof. In preferred embodiments, such nucleic acids are
isolated. These are but a few examples of the achaogens herein and in some
embodiments an achaogen is one other than an isolated nucleic acid encoding
DinI,
PsiB, CIpXP, or Lon protease.
36
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
L~~15~/J In some embodiments the present invention contemplates an achaogen
comprising a phage particle wherein the phage particle's genome comprises of a
nucleic acid encoding a negative regulator of induced mutations. For example,
an
achaogen of the present invention may be a bacteriophage whose genome encodes
DinI, PsiB, CIpXP, Lon protease, or any fragments, homologs or analogs
thereof.
Preferably such phage is isolated. In addition, nucleic acid molecules that
enhance the
transcription and/or translation of PsiB or DinI are also contemplated by the
invention
herein. Phage and phagemids are but one example of the achaogens contemplated
herein and in some embodiments an achaogen is one that does not include phage
or a
phagemid as described above.
[00158] In some embodiments, the present invention contemplates achaogens that
bind
to genes or regulators of genes that increase the rate of induced mutation
(e.g., difzB,
lexA, recA, recB, recC, recD, recF, recta, recN, polB, priA, ruvA, ruvB, ruvC,
umuC,
ufnuD, uvrA, uvrB, and uvrD). Such achaogens can include, for example, an
antisense
nucleic acid, a ribozyme, a zinc finger, an RNAi, or a triple helix nucleic
acid the bind
to or interact with a gene product that increases rate of mutation in a cell
or an
organism (e.g., RecA, RecB, RecC, RecD, RecF, Recta, RecN, LexA, UmuC, UmuD,
PoIB, PolIV, PoIV, PriA, RuvA, RuvB, RuvC, UmuC, UmuD, UvrA, UvrB, UvrD, and
any homolog or analogs thereof). When such an achaogen is introduced into a
cell or
an organism susceptible to or experiencing induced mutations, it can inhibit
the rate of
such induced mutations. The above embodiment is but one example of an achaogen
and in some embodiments an achaogen is one other than a nucleic acid that
specifically
binds to a gene that increases the rate of induced mutation, or is one other
than a
nucleic acid that specifically binds to a nucleic acid encoding RecA, RecB,
RecC,
RecD, RecF, Recta, RecN, LexA, UmuC, UmuD, PolB, PoIIV, PoIV, PriA, RuvA,
RuvB, RuvC, UmuC, UmuD, UvrA, UvrB, or UvrD or a nucleic acid complementary
thereof.
[00159] In some embodiments, an achaogen can include, for example, a nucleic
acid that
encodes a negative regulator of the induced mutation system (e.g., psiB, dinI,
lon, or
clpXP protease) or any homolog, analog or negative regulator fragment thereof.
This is
but one example of an achaogen herein and.in some embodiments an achaogen does
not
include a nucleic acid that encodes a negative regulator of induced
mutagenesis or one
or more of psiB, dinl, lora, and clpXP protease.
37
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
[00160] Any of the nucleic acids can be cloned (e.g., from a cDNA library),
and inserted
into a vector. Vectors may be constructed using methods, such as those
disclosed in
Sambrook, J. et al. "Molecular Cloning, A Laboratory Manual," (Cold Spring
Harbor
Press, Plainview, N.Y. 1989), and Ausubel, F. M. et al. "Current Protocols in
Molecular
Biology", (John Wiley & Sons, New York, N.Y., 1989), are incorporated herein
by
reference for all purposes. Vectors may be used to produce desired gene
products)
(e.g., PsiB, DinI, Lon protease, and CIpXP protease) by inserting an
expression cassette
into the vector, which includes a promoter andlor start codon andlor a
regulatory
sequence. Expression cassettes and regulatory sequences may be selected based
on the
host cell. An expression vector of the present invention may be used to
transfer a host
cell. The host cell is then maintained under appropriate condition that will
allow for the
expression of the nucleic acids (e.g. psiB, dinI, lon, or clpXP).
[00161] The vectors herein may also be used for phage therapy. Methods for
phage
therapy are disclosed in U.S. Patent No. 6,054,312, which is incorporated
herein by
reference for all purposes. In particular, the present invention contemplates
the use of
phage therapy as a means of importing and integrating exogenous nucleic acids
into a
bacterial cell. In some embodiments, a nucleic acid that encodes for a
negative
regulator of an induced mutation response (e.g., DinI, PsiB, CIpXP protease,
or Lon
protease, or any homolog thereof, or fragment thereof) is inserted into a
phage plasmid,
also known as phagemid. Phagemids combine features of plasmids and phages.
Phagemids contain an origin of replication and packaging signal of the
filamentous
phage, as well as a plasmid origin of replication. Other elements that are
useful for
cloning and/or expression of foreign nucleic acid molecules are generally also
present.
Such elements include, without limitation, selectable genes, multiple cloning
site,
primer sequences. The phagemids may be packaged into phage particles upon
rescue
by a helper phage. As used herein, "phage particles" refers to particles
containing
either a phage genome or a phagemid genome. The particles may contain other
molecules in addition to the phage genome and capsid proteins.
[00162] Many phage vectors and phagemids are commercially available. For
example,
the pEGFP vector series (Clontech; Palo Alto, Calif.), Ml3mp vectors
(Pharmacia
Biotech, Sweden), pCANTAB 5E (Pharnacia Biotech), pBluescript series
(Stratagene
Cloning Systems, La Jolla, Calif.) and others may be used. Other vectors are
available
in the scientific community (see, e.g., Smith, in Vectors: A Survey of
Molecular
Cloning Vectors and their Uses, Rodriquez and Denhardt, eds., Butterworth,
Boston, pp
38
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
61-84, 1988) or may be constructed using standard methods (Sambrook et al:,
Molecular Biology: A Laboratory Approach, Cold Spring Harbor, N.Y., 1989;
Ausubel
et al., Current Protocols in Molecular Biology, Greene Publishing, N.Y., 1994)
guided
by the principles discussed below
[00163] In preferred embodiments, phage particles are used to deliver nucleic
acids that
encode gene products that inhibit induced mutation in a cell or an organism.
For
example, the phage can be administered to any organism affected by or
susceptible to a
bacterial infection. The use of phage technology to target bacterial cells is
often
referred to as phage therapy. One of the benefits of phage therapy is that
phage are
bacteria specific. Thus, while the bacteria are transfected with the exogenous
nucleic
acids, surrounding mammalian cells are not affected. Furthermore, phage is
able to
withstand harsh environments and conditions such as those common to the
mammalian
digestive tract; thus, they are suited for oral/systemic formulations and
administration.
[00164] In any of the embodiments herein, nucleic acids herein can be inserted
into
vectors that can replicate in eukaryotic cells (e.g., mammalian cells). In
preferred
embodiments, such constructs include a transcription terminator sequence, a
polyadenylation sequence, a splice donor and acceptor sites, and an enhancer.
Other
elements useful for expression and maintenance of the construct in mammalian
cells or
other eukaryotic cells may also be incorporated. Because portions of the
constructs are
produced in bacterial cells, elements that are necessary or enable propagation
in
bacteria are incorporated.
[00165] The promoter that controls expression of the transgene should be
active or
activatable in the targeted cell. The targeted cell may be mammalian, avian,
plant, or
the like. Applications of the present invention will involve transfection of
mammalian
cells, including human, canine, feline, equine, or the like. The choice of the
promoter
will depend in part upon the targeted cell type and the degree or type of
control desired.
Promoters that are suitable within the context of the present invention
include, without
limitation, constitutive, inducible, tissue specific, cell type specific,
temporal specific,
or event-specific.
[00166] Examples of constitutive or nonspecific promoters include the SV40
early
promoter (U.S. Pat. No. 5,118,627), the SV40 late promoter (U.S. Pat. No.
5,118,627),
CMV early gene promoter (U.S. Pat. No. 5,168,062), bovine papilloma virus
promoter,
and adenovirus promoter. In addition to viral promoters, cellular promoters
are also
amenable within the context of this invention. In particular, cellular
promoters for the
39
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
so-called 'housekeeping' genes are useful (e.g., beta-actin). Viral promoters
are often
stronger promoters than cellular promoters.
[00167] In any of the embodiments herein, a nucleic acid achaogen may be
inserted into
a viral vector. Viral vectors have been used in the prior art to introduce
genes into a
wide variety of different target cells. Typically the vectors are exposed to
the target
cells so that transformation can take place in a sufficient proportion of the
cells to
provide a useful therapeutic or prophylactic effect from the expression of the
desired
polypeptide. The transfected nucleic acids (oligonucleotides) may be
administered
locally or systemically such that they are permanently incorporated into the
genome of
the targeted cells, (e.g., tumor cells). Alternatively the treatment may have
to be
repeated periodically. A variety of vectors, both viral vectors and plasmid
vectors are
known in the art, see U.S. Pat. No. 5,252,479 and WO 93/0722. In particular, a
number of viruses have been used as gene transfer vectors, including
papovaviruses,
such as SV40, vaccine virus, adenovirus, herpes viruses including HSV and EBV,
and
retroviruses. Many gene therapy protocols in the prior art have employed
disabled
murine retroviruses.
V. Antibodies
[00165] An achaogen of the present invention can also include an antibody that
specifically binds to and inactivates a gene product that increases the rate
of mutation in
a cell or an organism. Examples of such gene products include but are not
limited to,
LexA, Pol II, Pol IV, Pol V, RecA, RecN, UmuC, UmuD, UvrA, UvrB, UvrD, and any
homologs, analogs and fragments thereof. In some embodiments, an antibody of
the
present invention specifically binds to LexA in its cleavable conformation. In
some
embodiments, an antibody of the present invention binds to RecA, RecB, RecC,
RecD,
RecF, Recta, Rec N, LexA, UmuC, UmuD, PoIB, PoIIV, PoIV, PriA, RuvA, RuvB,
RuvC, UmuC, UmuD, UvrA, UvrB, UvrD in its activated conformation.
[00169] The antibodies useful herein can be whole antibodies, single-chain
antibodies,
and antigen-binding fragments thereof. Preferably the antibodies include, but
are not
limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain
antibodies,
disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
An
antibody herein can be polyclonal, monoclonal, chimeric, or humanized. The
antibodies herein can be derived from any animal including birds and mammals.
Preferably, an antibody__herein is derived from a murine, rabbit, goat, guinea
pig, camel,
horse, or chicken. More preferably, an antibody is from a human or has been
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
humanized. The antibodies herein may be used to reduce mutation rate in a cell
or an
organism (whether prokaryotic or eukaryotic).
[00170] In some embodiments, an antibody of the present invention is generated
using
an epitope-bearing polypeptide wherein the epitope-bearing polypeptide
comprises or
consists of the active site or the cleavage site of LexA. For example, an
epitope-
bearing polypeptide can comprise or consist of SEQ ID NO: 1, 2, or 3 (VAAG,
VAAGEPL, or VAAGEPLLAW). Generally speaking, an epitope-bearing polypeptide
of the present invention is about 1-50 amino acids in length, more preferably
about 2-
40 amino acids in length, more preferably about 3-30 amino acids in length,
more
preferably about 4-25 amino acids in length, or more preferably 5-10 amino
acids in
length. In some embodiments, an epitope-bearing polypeptide of the present
invention
comprises a peptide sequence of LexA including Ser119 and/or Lys156 of LexA.
[00171] The antibodies herein can be prepared by any suitable method known in
the art.
For example, Jones et al., Nature (1986) 321: 522-525 discloses replacing the
CDRs of
a human antibody with those from a mouse antibody. Marx, Science (1985)
229:455-
456 discusses chimeric antibodies having mouse variable regions and human
constant
regions. Rodwell, Nature (1989) 342:99-100 discusses lower molecular weight
recognition elements derived from antibody CDR information. Clackson, Br. J.
Rheumatol. (1991) 3052:36-39 discusses genetically engineered monoclonal
antibodies, including Fv fragment derivatives single chain antibodies, fusion
proteins
chimeric antibodies and humanized rodent antibodies. Reichman et al., Nature
(1988)
332:323-327 discloses a human antibody on which rat hypervariable regions have
been
grafted. Verhoeyen, et al., Science (1988) 239:1534-1536 describes grafting of
a mouse
antigen binding site onto a human antibody.
[00172] In yet another embodiment, antibodies able to withstand expression in
bacterial
cells are introduced into bacteria using phage. Such antibodies could bind to
and
inactivate the function of bacterial genes required for induced mutation
(e.g., RecA,
RecB, RecC, RecD, RecF, Recta, Rec N, LexA, UmuC, UmuD, PoIB, PolIV, Poly,
PriA, RuvA, RuvB, RuvC, UmuC, UmuD, UvrA, UvrB, UvrD, and any homologs,
analogs and fragments thereof).
[00173] In addition, those skilled in the art are enabled to design and
produce
peptidomimetics having binding characteristics similar or superior to the
complementary determining region of the antibodies herein. Horwell et al.,
Bioorg.
Med. Chem. (1996) 4: 1573; Liskamp et al., Recl. Trav. Chim. Pays- Basl (1994)
113;
41
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
Gante et al., A~gew. Claetn. lut. Ed. E~zgl. (1994) 33:1699; 5eebach et al.,
Helv. Chim.
Acta (1996) 79:913). Accordingly, the present invention also contemplates
analogs and
peptidomimetics of the antibodies herein.
VI. Polymerise Inhibitors as Achao~ens
[00174] In any of the embodiments herein, an achaogen can be used to modulate
biochemical pathways) that induce mutations. Such biochemical pathways)
comprise
proteases, DNA binding proteins, helicases, DNA polymerises, as well as other
proteins. See Goodman, MF: Error-prone repair DNA polymerises in prokaryotes
and
eukaryotes. Annu Rev Bioclzem 2002, 71:17-50; Nickoloff, JA, Hoekstra, MF
(eds.)
DNA Damage and Repair (Humana Press, Totowa, New Jersey, 1998). Thus, in some
embodiments, an achaogen comprises a composition that specifically binds to
and
inhibits an activity of gene products that induces mutation, such as a gyrase,
helicase,
error prone DNA polymerise, etc. Examples of such gene products include: RecA,
RecB, RecC, RecD, RecF, Recta, Rec N, LexA, UmuC, UmuD, PolB, PoIIV, PoIV,
PriA, RuvA, RuvB, RuvC, UmuC, UmuD, UvrA, UvrB, UvrD, and~any homologs,
analogs, or mutation inducing fragments thereof.
[00175] The evolution of antibiotic resistance can also be prevented via the
inhibition of
the function of the inducible, non-replicative, mutation-causing polymerises.
Such
polymerises include Pol II, Pol IV and Pol V in E. coli or their functional
analogs in
other species (e.g., DnaE2 in MTb). In essence, the invention herein attempts
to force
bacteria into using a 'high fidelity' means of re-initiating DNA replication
at a stalled
replication fork. For example, methods that force the bacterium to use a 'high
fidelity'
replication pathways would reduce mutability and thus disfavor the evolution
of
antibiotic resistance. In E. coli, inactivation of Pol II, Pol IV or Pol V via
gene
disruption has been shown to result in a significant decrease in the emergence
of
ciprofloxacin resistance.
[00176] Inactivation of a single mutation-causing polymerise (i.e., DnaE2) in
MTb
weakens the induced mutational response required for the evolution of
resistance to
rifampicin in vitro and ih vivo. See Boshoff, HIM, Reed, MB., Cell (2003)
113:183-
193. However, it is likely that other mutation-causing polymerises continue to
function
to facilitate resistance. Hence, in a preferred embodiment, the function of
many or all
mutation-causing polymerises is inhibited simultaneously, either by inhibiting
their
production (at the level of gene regulation as described above via the
inhibition of
LexA proteolysis) or their function (at the level of polymerise enzymatic
activity).
42
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
Due to the relaxed selectivity within the mutation-causing polymerase active
sites, and
further due to the inability of these error-prone enzymes to remove nucleotide
miss-
pairs via an enzymatic 3' to 5' exonuclease activity, these polymerases will
recognize,
incorporate, and not remove nucleoside analogs rejected by the replicative
polymerases.
Therefore, nucleoside analogs (e.g., dideoxy nucleosides with modified
nucleobases or
sugar rings) could selectively inhibit these mutation-causing polymerases
while not
inhibiting higher fidelity polymerases. In one embodiment, a single inhibitor
that
inhibits multiple mutation-causing polymerases can be used, resulting in a far
stronger
suppression of mutation than could be achieved via the inhibition of a single
error-
prone polymerase.
[00177] Thus, the present invention relates to compositions and methods that
inhibit
DNA polymerases, more preferably inducible DNA polymerase II, IV, andlor V, or
more preferably inducible DNA polymerase IV and/or V. Such compositions (e.g.,
achaogens) include small molecules, antisense nucleic acids, polypeptides,
glycopeptides, lipids, dideoxynucleotides, and mimetics, derivatives or
variants thereof
that can bind the above polymerases, thus inhibiting their enzymatic activity,
bind to
the above polymerase transcript, thus blocking translation.
[00178] In any of the embodiments herein, an achaogen reduces the rate of
mutation in a
cell or an organism by at least 2 fold, more preferably by at least 3 fold,
more
preferably by at least 4 fold, or more preferably by at least 5 fold. In any
of the
embodiments herein, an achaogen reduces the rate of mutation in a cell or an
organism
by at least 2%, 5%, 10%, 20%, 50%, 60%, 70%, 80%, 90%, or a greater percent
than
the rate of mutation without the achaogen.
[00179] Also, in some embodiments, an achaogen inhibits the acquisition of at
least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 substitution
mutations. In
some embodiments, an achaogen inhibits the occurance of at least 1, 2, 3, 4,
5, 6, 7, 8,
9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 deletionlinsertion mutations.
VII. Pharmaceutical Formulations
[00180] The present invention contemplates pharmaceutical formulations
comprising an
achaogen in an effective amount to achieve a therapeutic or prophylactic
effect and a
pharmaceutically effective carrier.
[00181] The actual effective amount will depend upon the condition being
treated, the
route of administration, the drug treatment used to treat the condition, and
the medical
history of the patient. Determination of the effective amount is well within
the
43
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
capabilities of those skilled in the art. The effective amount for use in
humans can be
determined from animal models. For example, a dose for humans can be
formulated to
achieve circulating concentrations that have been found to be effective in
animals. The
effective amount of an achaogen can vary if the achaogen is coformulated with
another
therapeutic agent (e.g., an antibiotic, an antineoplastic agent, an antiviral
agent, an
antiprotozoan agent, etc.). It is contemplated that lower dosages will be
needed in such
cases as a result of a synergistic effect of both active ingredients.
[00182] Preferably, an effective amount of an active ingredient (e.g., an
achaogen or
second therapeutic agent) is from about 0.0001 mg to about 500 mg active agent
per
kilogram body weight of a patient, more preferably from about 0.001 to about
250 mg
active agent per kilogram body weight of the patient, still more preferably
from about
0.01 mg to about 100 mg active agent per kilogram body weight of the patient,
yet still
more preferably from about 0.5 mg to about 50 mg active agent per kilogram
body
weight of the patient, and most preferably from about 1 mg to about 15 mg
active agent
per kilogram body weight of the patient. In terms of weight percentage, a
pharmaceutical formulation of an active agent (e.g., an achaogen or second
therapeutic
agent) preferably comprises of an amount from about 0.0001 wt. % to about 10
wt. %,
more preferably from about 0.001 wt. % to about 1 wt. %, and more preferably
from
about 0.01 wt. % to about 0.5 wt. %.
[00183] In any of the formulations herein, the achaogen can be formulated as a
salt, a
prodrug, or a metabolite. Such formulations can also include an additional
therapeutic
agents) such as an antibiotic, an antiviral agent, an antifungal agent, an
antiprotozoan
agent, and/or an antineoplastic agent.
[00184] Examples of antibiotics that may be coformulated with an achaogen
include
aminoglycosides, carbapenems, cephalosporins, cephems, glycopeptides,
fluoroquinolones/quinolones, macrolides, oxazolidinones, penicillins,
streptogramins,
sulfonamides, and tetracyclines.
[00185] Aminoglycosides are a group of antibiotics found to be effective
against gram-
negative. Aminoglycosides are used to treat complicated urinary tract
infections,
septicemia, peritonitis and other severe intra-abdominal infections, severe
pelvic
inflammatory disease, endocarditis, mycobacterium infections, neonatal sepsis,
and
various ocular infections. They are also frequently used in combination with
penicillins
and cephalosporins to treat both gram-positive and gram-negative bacteria.
Examples
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of aminoglycosides include amikacin, gentamycin, tobramycin, netromycin,
streptomycin, kanamycin, paromomycin, and neomycin.
[00186] Carbapenems are a class of broad spectrum antibiotics that are used to
fight
gram-positive, gram-negative, and anaerobic microorganisms. Carbapenems are
available for intravenous administration, and as such are used for serious
infections
which oral drugs are unable to adequately address. For example, carbapenems
are
often used to treat serious single or mixed bacterial infections, such as
lower respiratory
tract infections, urinary tract infections, intra-abdominal infections,
gynecological and
postpartum infections, septicemia, bone and joint infections, skin and skin
structure
infections, and meningitis. Examples of carbapenems include
imipenem/cilastatin
sodium, meropenem, ertapenem, and panipenem/betamipron.
[00187] Cephalosporins and cephems are broad spectrum antibiotics used to
treat gram-
positive, gram-negative, and spirochaetal infections. Cephems are considered
the next
generation cephalosporins with newer drugs being stronger against gram
negative and
older drugs better against gram-positive. Cephalosporins and cephems are
commonly
substituted for penicillin allergies and can be used to treat common urinary
tract
infections and upper respiratory infections (e.g., pharyngitis and
tonsillitis).
Cephalosporins and cephems are also used to treat otitis media, some skin
infections,
bronchitis, lower respiratory infections (pneumonia), and bone infection
(certain
members), and are a preferred antibiotic for surgical prophylaxis. Examples of
cephalosporins include cefixime, cefpodoxime, ceftibuten, cefdinir, cefaclor,
cefprozil,
loracarbef, cefadroxil, cephalexin, and cephradineze. Examples of cephems
include
cefepime, cefpirome, cefataxidime pentahydrate, ceftazidime, ceftriaxone,
ceftazidime,
cefotaxime, cefteram, cefotiam, cefuroxime, cefamandole, cefuroxime axetil,
cefotetan,
cefazolin sodium, cefazolin, cefalexin.
[00188] Fluroquinolones/quinolones are antibiotics used to treat gram-negative
infections, though some newer agents have activity against gram-positive
bacteria and
anaerobes. Fluroquinolones/quinolones are often used to treat conditions such
as
urinary tract infections, sexually transmitted diseases (e.g., gonorrhea,
chlamydial
urethritis/cervicitis, pelvic inflammatory disease), gram-negative
gastrointestinal
infections, soft tissue infections, pphthalmic infections, dermatological
infections,
sinusitis, and respiratory tract infections (e.g., bronchitis, pneumonia, and
tuberculosis).
Fluroquinolones/quinolones are used in combination with other antibiotics to
treat
conditions, such as multi-drug resistant tuberculosis, neutropenic cancer
patients with
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fever, and potentially anthrax. Examples of fluoroquinolones/quinolones
include
ciproflaxacin, levofloxacin, and ofloxacin, gatifloxacin, norfloxacin,
lomefloxacin,
trovafloxacin, moxifloxacin, sparfloxacin, gemifloxacin, and pazufloxacin.
[00189] Glycopeptides and streptogramins represent antibiotics that are used
to treat
bacteria that are resistant to other antibiotics, such as methicillin-
resistant
staphylococcus aureus (MRSA). They are also be used for patients who are
allergic to
penicillin. Examples of glycopeptides include vancomycin, teicoplanin, and
daptomycin.
[00190] Macrolides are broad spectrum antibiotics and are an important
alternative to
penicillins and cephalosporins. Macrolides are often used to treat respiratory
tract
infections (e.g., otitis media, chronic sinusitis, bronchitis, pharyngitis,
pneumonia,
tonsillitis, and strep throat), sexually transmitted diseases (e.g., nfections
of the cervix
and urinary tract, genital ulcer disease in men, syphilis), and opportunistic
infections
(e.g., pneumonia~and mycobacterium avium complex (MAC) infection). Examples of
macrolides include erythromycin, clarithromycin, azithromycin, axithromycin,
dirithromycin, troleandomycin, oleandomycin, roxithromycin, and telitrhomycin.
[00191] Oxazolidinones are commonly admisntered to treat gram-positive
infections.
Carbapenems are used to treat gram-positive, gram-negative, and/or anaerobes.
Oxazolidinones are commonly used as an alternative to other antibiotic classes
for
bacteria that have developed resistance. Examples of oxazolidinones include
linezolid.
[00192] Penicillins are broad spectrum used to treat gram-positive, gram-
negative, and
spirochaetal infections. Conditions that are often treated with penicillins
include
pneumococcal and meningococcal meningitis, dermatological infections, ear
infections,
respiratory infections, urinary tract infections, acute sinusitis, pneumonia,
and lyme
disease. Examples of penicillins include penicillin, amoxicillin, amoxicillin-
clavulanate, ampicillin, ticarcillin, piperacillin-tazobactam, carbenicillin,
piperacillin,
mezocillin, benzathin penicillin G, penicillin V potassium, methicillin,
nafcillin,
oxacillin, cloxacillin, and dicloxacillin.
[00193] Streptogramins are antibiotics developed in response to bacterial
resistance that
diminished effectiveness of existing antibiotics. Streptogramins are a very
small class
of drugs and are currently very expensive. Examples of streptogramins include
quinupristin/dafopristin and pristinamycin.
[00194] Sulphonamides are broad spectrum antibiotics that have had reduced
usage due
to increase in bacterial resistance to them. Suphonamides are commonly used to
treat
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recurrent attacks of rheumatic fever, urinary tract infections, prevention of
infections of
the throat and chest, traveler's diarrhea, whooping cough, meningococcal
disease,
sexually transmitted diseases, toxoplasmosis, and rhinitis. Examples of
sulfonamides
include co-trimoxazole, sulfamethoxazole trimethoprim, sulfadiazine,
sulfadoxine, and
trimethoprim.
[00195] Tetracyclines are broad spectrum antibiotics that are often used to
treat gram-
positive, gram-negative, and/or spirochaetal infections. Tetracyclines are
often used to
treat mixed infections, such as chronic bronchitis and peritonitis, urinary
tract
infections, rickets, chlamydia, gonorrhea, lyme disease, and periodontal
disease.
Tetracyclines are an alternative therapy to penicillin in syphilis treatment
and are also
used to treat acne and anthrax. Examples of tetracyclines include
tetracycline,
demeclocycline, minocycline, and doxycycline.
[00196] Other antibiotics contemplated herein (some of which may be redundant
with
the list above) include abrifam; acrofloxacin; aptecin, amoxicillin plus
clavulonic acid;
amikacin; apalcillin; apramycin; astromicin; arbekacin; aspoxicillin;
azidozillin;
azithromycin; azlocillin; aztreonam; bacitracin; benzathine penicillin;
benzylpenicillin;
clarithromycin, carbencillin; cefaclor; cefadroxil; cefalexin; cefamandole;
cefaparin;
cefatrizine; cefazolin; cefbuperazone; cefcapene; cefdinir; cefditoren;
cefepime;
cefetamet; cefixime; cefmetazole; cefminox; cefoperazone; ceforanide;
cefotaxime;
cefotetan; cefotiam; cefoxitin; cefpimizole; cefpiramide; cefpodoxime;
cefprozil;
cefradine; cefroxadine; cefsulodin; ceftazidime; ceftriaxone; cefuroxime;
cephalexin;
chloramphenicol; chlortetracycline; ciclacillin; cinoxacin;
ciprofloxacinfloxacin;
clarithromycin; clemizole penicillin; cleocin, cleocin-T, clindamycin;
cloxacillin;
corifam; daptomycin; daptomycin; demeclocycline; desquinolone; dibekacin;
dicloxacillin; dirithromycin; doxycycline; enoxacin; epicillin; erthromycin;
ethambutol;
gemifloxacin; fenampicin; finamicina; fleroxacin; flomoxef; flucloxacillin;
flumequine;
flurithromycin; fosfomycin; fosmidomycin; fusidic acid; gatifloxacin;
gemifloxaxin;
gentamicin; imipenem; imipenem plus cilistatin combination; isepamicin;
isoniazid;
josamycin; kanamycin; kasugamycin; lcitasamycin; kalrifam, latamoxef;
levofloxacin,
levofloxacin; lincomycin; linezolid; lomefloxacin; loracarbaf; lymecycline;
mecillinam;
meropenem; methacycline; methicillin; metronidazole; mezlocillin; midecamycin;
minocycline; miokamycin; moxifloxacin; nafcillin; nafcillin; nalidixic acid;
neomycin;
netilmicin; norfloxacin; novobiocin; oflaxacin; oleandomycin; oxacillin;
oxolinic acid;
oxytetracycline; paromycin; pazufloxacin; pefloxacin; penicillin g; penicillin
v;
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phenethicillin; phenoxymethyl penicillin; pipemidic acid; piperacillin;
piperacillin and
tazobactam combination; piromidic acid; procaine penicillin; propicillin;
pyrimethamine; rifadin; rifabutin; rifamide; rifampin; rifamycin sv;
rifapentene;
rifomycin; rimactane, rofact; rokitamycin; rolitetracycline; roxithromycin;
rufloxacin;
sitafloxacin; sparfloxacin; spectinomycin; spiramycin; sulfadiazine;
sulfadoxine;
sulfamethoxazole; sisomicin; streptomycin; sulfamethoxazole; sulfisoxazole;
quinupristan-dalfopristan; teicoplanin; telithromycin; temocillin;
gatifloxacin;
tetracycline; tetroxoprim; telithromycin; thiamphenicol; ticarcillin;
tigecycline;
tobramycin; tosufloxacin; trimethoprim; trimetrexate; trovafloxacin;
vancomycin;
verdamicin; azithromycin; and linezolid.
[00197] Examples of antineoplastic agents that may be coformulated with an
achaogen
include: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;
aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide;
amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine;
azetepa;
azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride;
bisnafide
dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine;
busulfan;
cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine;
carubicin
hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin;
cladribine;
crisnatol mesylate; cyclophosphamide ; cytarabine; dacarbazine; dactinomycin;
daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine;
dezaguanine
mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;
droloxifene;
droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;
eflornithine;
hydrochloride; elsamitrucin; enloplatinenpromate; epipropidine; epirubicin
hydrochloride; erbulozole; esorubicin hydrochloride; estramustine;
estramustine
phosphate sodium; etanidazole; ethiodized oil; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine;
fludarabine
phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium;
gemcitabine;
gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
imofosine; interferon alpha-2a; interferon alpha-2b ; interferon alpha-nl;
interferon
alpha-n3; interferon beta-Ia; interferon gamma-Ib; iproplatin; irinotecan
hydrochloride;
lanreotide acetate; letrozole; leuprolide acetate liarozole hydrochloride;
lometrexol
sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine;
mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;
melphalan;
menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;
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meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;
mitomycin;
mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole;
nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;
piroxantrone
hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin;
prednimustine;
procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine; rogletimide; safingol; safingol hydrochloride; semustine;
simtrazene;
sparfosate sodium; sparsomycinl; spirogermanium hydrochloride; spiromustine;
spiroplatin; streptonigrin; streptozocin; strontium chloride sr 89; sulofenur;
talisomycin;
taxane; taxoid; tecogalan sodium; tegafur; teloxantrone hydrochloride;
temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa;
tiazofurin;
tirapazamine; topotecan hydrochloride; toremifene citrate; trestolone acetate;
triciribine
phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole
hydrochloride;
uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;
vincristine sulfate;
vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;
vinleurosine
sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate;
vorozole;
zeniplatin; zinostatin; and zorubicin hydrochloride. Additional antineoplastic
agents
that are disclosed herein or known in the art are also contemplated by the
present
invention.
[0019] A "pharmaceutical acceptable carrier" is a pharmaceutically acceptable
solvent,
suspending agent or vehicle for delivering an achaogen of the present
invention to an
animal or human. The carrier may be, for example, gaseous, liquid or solid and
is
selected with the planned manner of administration in mind.
[00199] Examples of pharmaceutically acceptable carriers for oral
pharmaceutical
formulations include: lactose, sucrose, gelatin, agar and bulk powders.
Examples of
suitable liquid carriers include water, pharmaceutically acceptable fats and
oils,
alcohols or other organic solvents, including esters, emulsions, syrups or
elixirs,
suspensions, solutions and/or suspensions, and solution and or suspensions
reconstituted from non-effervescent granules and effervescent preparations
reconstituted from effervescent granules. Such liquid carriers may contain,
for example,
suitable solvents, preservatives, emulsifying agents, suspending agents,
diluents,
sweeteners, thickeners, and melting agents. Preferred carriers are edible
oils, for
example, corn or canola oils. Polyethylene glycols, e.g. PEG, are also
preferred
carriers.
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(00200] Examples of pharmaceutically acceptable carriers for topical
formulations
include: ointments,~cream, suspensions, lotions, powder, solutions, pastes,
gels, spray,
aerosol or oil. Alternately, a formulation may comprise a transdermal patch or
dressing
such as a bandage impregnated with an active ingredient (e.g., achaogen and/or
second
therapeutic agent) and optionally one or more. carriers or diluents. The
topical
formulations may include a compound that enhances absorption or penetration of
the
active ingredient through the skin or other affected areas. Examples of such
dermal
penetration enhancers include dimethylsulfoxide and related analogues.
[00201] To be administered in the form of a transdermal delivery system, the
dosage
administration will be continuous rather than intermittent throughout the
dosage
regimen.
[00202] Formulations suitable for parenteral administration include aqueous
and non-
aqueous formulations isotonic with the blood of the intended recipient; and
aqueous
and non-aqueous sterile suspensions which may include suspending systems
designed
to target the compound to blood components or one or more organs. The
formulations
may be presented in unit-dose or mufti-dose sealed containers, for example,
ampoules
or vials. Extemporaneous injection solutions and suspensions may be prepared
from
sterile powders, granules and tablets of the kind previously described.
Parenteral and
intravenous formulation may include minerals and other materials to make them
compatible with the type of injection or delivery system chosen.
[00203] Commonly used pharmaceutically acceptable carriers for parenteral
administration includes, water, a suitable oil, saline, aqueous dextrose
(glucose), or
related sugar solutions and glycols such as propylene glycol or polyethylene
glycols.
Solutions for parenteral administration preferably contain a water soluble
salt of the
active ingredient, suitable stabilizing agents and, if necessary, buffer
substances.
antioxidizing agents, such as sodium bisulfate, sodium sulfite, or ascorbic
acid, either
alone or combined, are suitable stabilizing agents. Citric acid salts and
sodium EDTA
may also be used as carriers. In addition, parenteral solutions may contain
preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, or
chlorobutanol. Suitable pharmaceutical carriers are described in Remington,
cited
above.
(00204] The present invention additionally contemplates achaogens formulated
for
veterinary administration by methods conventional in the art.
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~UU~US~ The achaogens described herein can also be formulated for industrial
applications with, for example, a cleaning product, such as soap, laundry
detergent,
shampoo, dishwashing soap, toothpaste, and other house cleaning detergents.
VIII. Administration
[00206] The compositions and pharmaceutical formulation herein can be
administered to
an organism by any means known in the art. Routes for administering the
compositions
and pharmaceutical formulations herein to an animal, such as a human, include
parenterally, intravenously, intramuscularly, orally, by inhalation,
topically, vaginally,
rectally, nasally, buccally, transdermally, or by an implated reservoir
external pump or
catheter. When administered to a plan, such means can be by spray or via
irrigation.
[00207] Although any route of administration may be used, parenteral
administration,
i.e., administration by injection, is preferred. Injectable formulations can
be prepared
in conventional forms, either as liquid solutions or suspensions; as solid
forms suitable
for solubilization or suspension in liquid prior to injection; or as
emulsions. Preferably,
sterile injectable suspensions are formulated according to techniques known in
the art
using suitable pharmaceutically acceptable carriers and other optional
components as
discussed above.
[00208] Parenteral administration may be carried out in any number of ways,
but it is
preferred that the use of a syringe, catheter, or similar device, be used to
effect
parenteral administration of the formulations described herein. The
formulation may be
injected systemically such that the active agent travels substantially
throughout the
entire bloodstream.
[00209] Also, the formulation may also be injected locally to a target site,
e.g., injected
to a specific portion of the body for which inhibition of mutagenesis is
desired. An
advantage of local administration via injection is that it limits or avoids
exposure of the
entire body to the active agents) (e.g., achaogens and/or other therapeutic
agents). It
must be noted that in the present context, the term local administration
includes
regional administration, e.g., administration of a formulation directed to a
portion of the
body through delivery to a blood vessel serving that body zone. Local delivery
may be
direct, e.g., intratumoral. Local delivery may also be nearly direct, i.e.,
intralesional or
intraperitoneal, that is, to an area that is sufficiently close to a tumor or
site of infection
so that the active agents) exhibit the desired pharmacological activity. Thus,
when
local delivery is desired, the pharmaceutical formulations are preferably
delivered
intralesionally, intratumorally, or intraperitoneally.
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[00210] It is intended that, by local delivery of the presently described
pharmaceutical
formulations, a higher concentration of the active agent may be directed to
the target
site. There.are several advantages to having high concentrations delivered
directly at
the target site. First, since the active agent is more localized, there is
less potential for
toxicity to the patient since minimal systemic exposure occurs. Second, drug
efficacy
is improved since the target site is exposed to higher concentrations of the
drug. Third,
relatively fast delivery minimizes solubility and stability liabilities of the
active agent
before reaching its target site.
[00211] Preferably the pharmaceutical compositions are in unit dosage form. In
such
form, the composition is divided into unit doses containing appropriate
quantities of the
active component. The unit dosage form can be a packaged preparation, the
package
containing discrete quantities of the preparations, for example, packeted
tablets,
capsules, and powders in vials or ampoules. The unit dosage form can also be a
capsule, cachet, or tablet, or it can be the appropriate number of any of
these packaged
forms.
[00212] Useful pharmaceutical dosage formulations for administration of the
compounds of the present invention are illustrated as follows:
[00213] Capsules: A large number of unit capsules are prepared by filling
standard two-
piece hard gelatin capsules each with 1-100 milligrams of powdered active
ingredient,
150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligrams
magnesium
stearate.
[00214] Soft Gelatin Capsules: A mixture of active ingredient in a digestible
oil such as
soybean oil, cottonseed oil or olive oil is prepared and injected by means of
a positive
displacement pump into gelatin to form soft gelatin capsules containing 1-100
milligrams of the active ingredient. The capsules are washed and dried.
[00215] Tablets: A large number of tablets are prepared by conventional
procedures so
that the dosage unit was 1-100 milligrams of active ingredient, 0.2 milligrams
of
colloidal silicon dioxide, 5 - 6 milligrams of magnesium stearate, 275
milligrams of
microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of
lactose.
Appropriate coatings can be applied to increase palatability or delay
absorption.
[00216] Injectable: A parenteral composition suitable for administration by
injection is
prepared by stirring 0.5-1.5% by weight of active ingredient in 10% by volume
propylene glycol and water. The solution is made isotonic with sodium chloride
and
sterilized.
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[00217] Suspension: An aqueous suspension is prepared for oral administration
so that
each 5 ml contains 1-100 mg of finely divided active ingredient, 200 mg of
sodium
carboxymethyl cellulose, 5 mg of sodium benzoate, 1 g of sorbitol solution,
U.S.P., and
0.02 ml of vanillin.
[00218] Achaogens of the present invention may be administered in the form of
liposome delivery systems, such as small unilamellar vesicles, large
unilamellar
vesicles, and multilamellar vesicles. Liposomes can be formed from a variety
of
phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
[00219] Achaogens of the present invention may be coupled with soluble
polymers as
targetable drug carriers. Such polymers can include polyvinylpyrrolidone,
pyran
copolymer, polyhydroxylpropylmethacrylamide-phenol,
polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysine substituted
with
palmitoyl residues. Furthermore, the compounds of the present invention can be
coupled to a class of biodegradable polymers useful in achieving controlled
release of
the drug, for example, polylactic acid, polyglycolic acid, copolymers of
polylactic and
polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters,
polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or
amphipathic
block copolymers of hydrogels.
[00220] Achaogens can be administered to any organism (eukaryotic or
prokaryotic) to
prevent or treat drug resistance. Achaogens can also be administered to a
first
organism in order to target a second organism associated with the first
organism. For
example, an achaogen can be administered to a mammal infected by bacteria or
to a
plant infected by a fungus.
[00221] Achaogens can be administered as a monotherapy or in combination with
a
second therapeutic agent (e.g., antibiotic, antiviral, antifungal,
antiprotozoan,
antineoplastic agent). When administered as part of a combination therapy, the
achaogens herein can be administered serially or simultaneously with the
second agent.
In some embodiments, an achaogen is administered prior to the administration
of a
second therapeutic agent. In other embodiments, an achaogen is administered
after the
administration of a second therapeutic agent.
[00222] For example, for prophylactic benefit, an achaogen and antibiotic may
be co-
administered to a patient at risk of developing a bacterial infection that
could become
antibiotic resistant. In some embodiments, the achaogen is administered prior
to the
administration of the antibiotic.
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IX. Screening To Identify Gene Products Associated With Mutation
[00223] If a gene product is involved in induced mutation, its function
increases a cell's
ability to mutate. Inactivation of that gene product decreases a cell's
ability to mutate.
These principles can be used to determine if a given gene product is indeed
mutation-
causing and thus a potential drug target, the inhibition of which suppresses
induced
mutation and the development of drug (e.g., antibiotic) resistance.
[00224] In one embodiment, a test gene is genetically inactivated using known
gene
disruption techniques. After such a disruption event, the locus that encoded
the
putatively mutation-causing target would now be unable to produce the gene
product
and the cell would lack the function of that gene product. Various known
'mutability'
assays are used to assess the effect of the gene disruption event on a cell's
mutability.
See Friedberg, EC, Walker, GC, Siede, W. DNA Repair arzd Mutagezzesis (ed.
Friedberg, E. C.) American Society of Microbiology, Washington DC, 1995. For
example, an adaptation of the so-called 'Stressful Lifestyle Associated
Mutation' (or
SLAM) assay (as described in example #2 and Figure 2) wherein the evolution of
resistance to an antibiotic of choice is measured) or a forward mutation or
reversion
assay can be used. See Bull, HJ, Lombardo, MJ, Rosenberg, SM: Stationary-phase
mutation in the bacterial chromosome: recombination protein and DNA polymerase
IV
dependence. Bull HJ., Proc. Natl. Acad. Sci. TISA (2001) 98:8334-8341;
Friedberg, EC.
et al. DNA Repair and Mutageneszs (ed. Friedberg, E. C.) American Society of
Microbiology, Washington DC, 1995; Crouse, GF: Methods (2000) 22:116-119;
Rosenberg, SM Nature. Rev. Genet. (2001) 2:504-515; Rosche, WA., Methods
(2000)
20:4-17; and Foster, PL:, BioEssays (2000) 22:1067-1074.
[00225] In another embodiment, a bacterial strain with an inactivated test
gene and a
non-functional reporter gene is used. Examples of reporter genes that can be
used
include the lacZ gene, green fluorescent protein gene, red fluorescent protein
gene, and
yellow fluorescent protein gene. The frequency at which the reporter gene is
made
functional (via a compensation mutation) in the presence of a wild-type test
gene or an
inactivated test gene is determined. A decrease in the frequency of
restoration of
function of the reporter gene in a cell containing an inactivated test gene
indicates that
the test gene has mutation-causing activity.
[00226] In yet another embodiment, bacterial cells with an inactivated test
gene or a
wild-type test gene are exposed to an antibiotic. The number of cells that
develop
resistance to the antibiotic is quantified in both cells with the inactive
test gene and
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WO 2005/056754 PCT/US2004/039064
cells with the wild-type test gene. A decrease in the number of cells that
develop
antibiotic resistance in the case of the inactive test gene suggests that the
test gene has
potential mutation-causing activity.
[00227] Numerous techniques are known in the art to inactivate genes, many of
which
could be used to inactivate a test gene of interest. These techniques include
the direct
inactivation of the test gene, for example via mutation of the test gene via
homologous
recombination. Another useful technique is the indirect activation of the test
gene, for
example via mutation of a gene whose gene product modulates the activity of
the test
gene.
[00228] Typically, the test gene is inactivated via one or more mutations such
that the
resulting protein encoded by the test gene is inactive. Alternatively, the
entire gene (or
a large portion of the gene's open reading frame) is deleted from the genome.
Mutation
of the test gene may be carried out using numerous mutagenesis techniques
known in
the art. At the genetic level, the mutants ordinarily are prepared by site-
directed
mutagenesis of the DNA encoding the gene. The mutants can be substitution
mutants,
deletion mutants, or insertion mutants.
[00229] In some embodiments, an achaogen is an inhibitor or binding agent of a
gene
product that increases the rate of mutation in a cell or an organism (e.g.,
RecA, RecN,
LexA, Ding, PolII, Pol IV, UmuC, UmuD, UvrA, UvrB, and UvrD). In particular,
the
present invention contemplates an achaogen that is an inhibitor and/or binding
agent of
LexA, RecA, or both. Methods for identifying binding agents are known in the
art and
include yeast two hybrid systems, etc.
X. Screening For Small Molecules That Inhibit Mutation
[00230] Achaogens can be identified by a number of methods including screening
libraries of chemical compounds. Combinatorial libraries and methods for
searching
such libraries are known in the art and include: biological libraries, natural
products
libraries, spatially addressable parallel solid phase or solution phase
libraries, synthetic
library methods requiring deconvolution, the 'one-bead one-compound' library
method,
and synthetic library methods using affinity chromatography selection. The
biological
library approach is largely limited to polypeptide libraries, while the other
four
approaches are applicable to polypeptide, non-peptide oligomer or small
molecule
libraries of compounds. See Lam, K.S. (1997) Araticarzcer Drug Des. 12:145.
[00231] In one embodiment, achaogens are screened using Automated Ligand
Identification System (referred to herein as "ALIS"). See, e.g., U.S. Pat.
Nos.
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WO 2005/056754 PCT/US2004/039064
6,721,665, 6714,875, 6,694,267, 6,691,046, 6,581,013, 6,207,861, and
6,147,344,
which are incorporated herein by reference for all intended purposes. ALIS is
a high-
throughput technique for the identification of small molecules that bind to
proteins of
interest (e.g., RecA or LexA). Small molecules found to bind tightly to a
protein can
then be tested for their ability to inhibit the biochemical activity of that
protein.
[00232] Thus, in some embodiments, a target protein (e.g., RecA, LexA, or Pol
V) is
mixed with pools of small molecules. Preferably, more than 1,000 pools are
used, more
preferably more than 2,000 pools are used, more preferably more than 3,000
pools are
used, or more preferably, more than 10,000 pools are used. Each pool contains
approximately, 1,000 compounds, more preferably approximately 2,500 compounds,
or
more preferably approximately 5,000 compounds that are 'mass encoded,' meaning
that
their precise molecular structure can be determined using only their mass and
knowledge of the chemical library.
[00233] The small molecules and proteins are mixed together and allowed to
come to
equilibrium (they are incubated together for 30 minutes at room temperature).
The
mixture is rapidly cooled to trap bound complexes and subject to rapid size
exclusion
chromatography (SEC). Small molecules that bind tightly to the protein of
interest will
be co-excluded with the protein during SEC. Mass spectroscopic analysis is
performed
to determine the masses of all small molecules found to bind the protein.
Measurement
of these masses allows for the rapid determination of the molecular structures
of the
small molecules.
[00234] Compounds that bind to a target protein (e.g., LexA and/or filamented
RecA) in
ALIS can then be tested for their ability to inhibit RecA-induced LexA
proteolysis ifz
vitro. Molecules with potent irz vitro inhibitory properties can be tested
using a
modified Stressful Lifestyle Adaptation and Mutation (referred to herein as
"SLAM")
assays, described in Example #2 below, for achaogen activity (i.e., the
ability to inhibit
the emergence of ciprofloxacin resistant E. coli grown on ciprofloxacin-
containing (35
nglml) LB agar growth media). Molecules with detectable achaogen activity in
SLAM
assays can be tested for achaogen activity in mouse thigh infection models.
[00235] In one embodiment, a chemical collection of compounds is screened in a
format
similar to the SLAM assay (from example #2 below) to identify molecules that
decrease mutability. Bacterial cells are exposed to either one test compound
or a
library of compounds and the number of mutant cells generated over a period of
time is
determined in the presence and absence of the test compound. A decrease in the
ratio
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of resistant cells to total cells indicates the achaogen activity of the test
compound. The
number of resistant cells generated is determined both before and after
bacteria are
exposed to the drug. The number of mutant cells is quantified using known
assays, for
example forward mutation, gene reversion, or SLAM assays. See Friedberg, EC,
Walker, GC, Siede, W. DNA Repair and Mutagenesis (ed. Friedberg, E. C.)
(American
Society of Microbiology, Washington DC, 1995); Crouse, GF., Methods (2000)
22:116-119. In yet another embodiment, the bacterial cells are exposed to a
mutation-
causing environment and the number of mutant cells generated is quantified in
the
presence and absence of the test compound. For example, a variation of the
SLAM
assay is used to proactively stress the bacteria (for example via exposure to
UV
radiation or chemical mutagens) so as to elevate mutation rates in bacteria.
Such stress
aids in the detection of achaogens that inhibit mutation due to the increased
frequency
of bacterial mutations.
[00236] In another example of a method to screen for achaogens, purified LexA
protein,
purified RecA protein, and ssDNA are exposed to test compounds. In the
presence of
ssDNA and nucleoside triphosphate, RecA is activated and binds to LexA and
facilitates LexA's self-cleavage reaction. In the presence of an achaogen, the
activation
of RecA, its binding to LexA, or LexA's cleavage reaction will be inhibited.
The
decreased activation of RecA, the decreased binding of RecA to LexA, or the
chemical
inhibition of LexA's self-cleavage reaction can be evaluated by measuring the
cleavage
of LexA (e.g., by gel mobility assay, chromogenic assay, mass spectrometry, or
cell-
based GFP reporter assay). The inhibition of LexA cleavage indicates that the
test
compound is a potential achaogen.
[00237] A similar assay can also be designed with the purified UmuD gene
product in
order to find inhibitors of its cleavage that thus prevent production of
functional Pol V.
Similar assay can also be designed with purified RecB, RecC, RecD, RecF,
Recta,
RecN, UmuC, PoIB, PolIV, PoIV, PriA, RuvA, RuvB, RuvC, UmuC, UmuD, UvrA,
UvrB, UvrD, and any homologs or fragments thereof.
[00238] The inhibition of different mutation-causing polymerases by potential
achaogens can be quantified using standard methods. See Ogawa, AK,. JAm
Clzefzz Soc
(2000) 122:3274-3287. For example, the rate of DNA synthesis with a given
polymerase may be measured in the presence and absence of the potential
achaogen
using 5'-radiolabeled oligonucleotide primers resolved after the reaction by
polyacrylamide gel electrophoresis and quantification by standard methods.
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Alternatively, high-throughput assays can be used to screen through large
compound
libraries to identify potential achaogens. Such assays rely on arraying the
reaction
mixtures in 96-well plates, where each well also contains a different
achaogen.
Fluorophore labeled nucleoside triphosphates or oligonucleotide primers or
templates
can be used in conjunction with standard plate handling and visualization
procedures to
determine which molecules effectively inhibited the activity of a given
polymerise. In
one embodiment, libraries can be screened in the presence of one or more of
the
inducible polymerises in order to identify achaogens that would most
efficiently
prevent mutation by inhibiting one or more polymerises simultaneously (for
example,
Pol IV and/or Pol V in E. coli).
[00239] Other methods of screening libraries of compounds for achaogens
include
screening for helicase inhibitors (e.g., RuvA, RuvB, RuvC, RecB, RecC, RecD
inhibitors or combinations thereof, such as RuvABC and RecBC), inhibitors of
reporter
genes such as GFP or luciferase under the control of LexA regulated
propomoters, or
inhibitors that reduce the rate of evolution of drug resistance (or more
preferably
antibiotic resistance) in a SLAM assay as described herein.
XI. Structure-Based Design Methods to Create Small Molecule Achaogens
[00240] In some embodiments, one can use molecular modeling software tools to
create
realistic 3-D models of how molecules are shaped. Such methods include the use
of,
for example, molecular graphics (i.e., 3D representations) and computational
chemistry
(e.g., calculations of the physical and chemical properties).
[00241] Using molecular modeling, rational drug design programs can predict
which of
a collection of different drug like compounds may fit into the active site of
an enzyme,
and by computationally adjusting their bound conformation, decide which
compounds
actually might fit the active site well. See William Bains, Biotechnology from
A to Z,
2nd edition, Oxford University Press, 1998, at 259.
[00242] For basic information on molecular modeling, see, e.g., M. Schlecht,
Molecular
Modeling on the PC, 1998, John Wiley & Sons; Gans et al., Fundamental
Principals of
Molecular Modeling, 1996, Plenum Pub. Corp.; N. C. Cohen (editor), Guidebook
on
Molecular Modeling in Drug Design, 1996, Academic Press; and W. B. Smith,
Introduction to Theoretical Organic Chemistry and Molecular Modeling, 1996.
U.S.
Patents which provide detailed information on molecular modeling include U.S.
Pat.
Nos. 6,093,573; 6,080,576; 5,612,894; 5,583,973; 5,030,103; 4,906,122; and
4,812,12.
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[00243] The present invention permits the use of molecular and computer
modeling
techniques to design, and select compounds (e.g., achaogens) that bind to LexA
(in its
cleavable or non-cleavable form), RecA, Pol IV, Pol V or other gene products
that
increase the rate of induced mutagenesis. Thus, the invention enables the use
of atomic
coordinates deposited at the RCSB Protein Data Bank with the accession number
PDB
ID: 1AA, 1LEA, 1LEB to design compounds that interact with such gene products
(e.g., LexA and/or RecA). For example, this invention enables the design of
compounds that act as competitive inhibitors of LexA by binding to, all or a
portion of,
the active site involved in LexA self-cleavage or the RecA-LexA binding
interface.
[00244] This invention also enables the design of compounds that act as
uncompetitive
inhibitors of RecA-induced LexA proteolysis. These inhibitors may bind to, all
or a
portion of, the active site of RecA and/or LexA. Similarly, non-competitive
inhibitors
that bind to either RecA andlor LexA and inhibit RecA and/or LexA (whether or
not
bound to another chemical entity) may be designed using the atomic coordinates
of
RecA and/or LexA of this invention.
[00245] Alternatively, the atomic coordinates provided by the present
invention are
useful in designing improved analogues of known gene products that inhibit
induced
mutation (e.g., DinI, PsiB, homologs thereof, and fragments thereof) or to
design novel
classes of inhibitors based on the LexA-RecA-binding complex. This provides a
novel
route for designing potent and selective inhibitors.
[00246] The availability of both protein crystals and of atomic coordinates
determined
by X-ray diffraction studies enables 'soaking' experiments with RecA and/or
LexA
crystals with molecules composed of a variety of different chemical entities
to identify
potential sites for interaction between candidate inhibitors and RecA and/or
LexA. For
example, high resolution X-ray diffraction data collected from crystals
saturated with
solvent allows the determination of where each type of solvent molecule binds
the
protein. Small molecules that bind tightly to those sites can then be tested
for their
ability to inhibit induced mutation (Travis, J., Science (1993) 262: 1374).
[00247] Moreover, the present invention enables computational screening of
small
molecule databases for chemical entities, agents, or compounds that can bind
in whole,
or in part, to RecA and/or LexA and, thereby prevent RecA-induced LexA
proteolysis.
In this screening technique, the quality of fit of such entities or compounds
to the
binding site may be judged either by shape complementarity or by estimated
interaction
energy. See Meng, E. C. et al., J. Coma. Chem., 13: 505-524 (1992).
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[00248] The design of compounds that bind to or inhibit RecA and/or LexA
according to
this invention generally involves consideration of two factors. First, the
compound
must be capable of physically associating with RecA, LexA, Pol IV, Pol V, or
other
protein required for induced mutation. Non-covalent molecular interactions
important
in the association of compounds with RecA, LexA, Pol IV, Pol V, or other
protein
required for induced mutation, include hydrogen bonding, van der Waals and
hydrophobic interactions. Second, the compound must be able to assume a
conformation that allows it to associate with RecA, LexA, Pol IV, Pol V, or
other
protein required for induced mutation. Although certain portions of the
compound will
not directly participate in this association with RecA, LexA, Pol IV, Pol V,
or other
protein required for induced mutation, those portions may still influence the
overall
conformation of the molecule. This, in turn, may have a significant impact on
potency.
Such conformational requirements include the overall three-dimensional
structure and
orientation of the chemical entity or compound in relation to all or a portion
of the
active site of RecA, LexA, Pol IV, Pol V, or other protein required for
induced
mutation or the spacing between functional groups of a compound comprising
several
chemical entities that directly interact with RecA, LexA, Pol IV, Pol V, or
other protein
required for induced mutation.
[00249] The potential inhibitory or binding effect of a chemical compound on
induced
mutation may be analyzed prior to its actual synthesis and by the use of
computer
modeling techniques. If the theoretical structure of the given compound
precludes any
potential association between it and RecA, LexA, Pol IV, PoIV, or other
protein
required for induced mutation, synthesis and testing of the compound is
obviated.
However, if computer modeling suggests a strong interaction is possible, the
molecule
may then be synthesized and tested for its ability to interact with RecA,
LexA, Pol IV,
PoIV, or other protein required for induced mutation and to thereby inhibit
induced
mutation. In.this manner, synthesis of inactive compounds may be avoided.
[00250] One slulled in the art may use one of several methods to screen
chemical
entities fragments, compounds, or agents for their ability to associate with
RecA, LexA,
Pol IV, PoIV, or other protein required for induced mutation and more
particularly with
the individual binding pockets of RecA, LexA, Pol IV, Pol V, or other protein
required
for induced mutation. This process may begin by visual inspection of, for
example, the
active site on the computer screen based on the RecA, LexA, Pol IV, Pol V, or
other
protein required for induced mutation coordinates deposited in the RCSB
Protein Data
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
Bank with the accession number PDB ID: 1AA3, ILEA, and ILEB. Selected chemical
entities, compounds, or agents may then be positioned in a variety of
orientations, or
docked, within an individual binding pocket of RecA, LexA, Pol IV, Pol V, or
other
protein required for induced mutation as defined above. Docking may be
accomplished
using software such as Quanta and Sybyl, followed by energy minimization and
molecular dynamics with standard molecular mechanics force fields, such as
CHARMM or AMBER.
[00251] Specialized computer programs also assist in the process of selecting
chemical
entities. These include but are not limited to GRID (Goodford, P.J., J. Med.
Cheni.,
(1985) 28, 849-857). GRID is available from Oxford University, Oxford, UK;
MCSS
(Miranker, A. et al., Structure, Function and Genetics, (1991) Vol. 11, 29-
34), MCSS is
available from Molecular Simulations, Burlington, Mass, AUTODOCK (Goodsell,
D.S.
and A.J. Olsen, "Automated Docking of Substrates to Proteins by Simulated
Annealing" Proteins: StructuYe. Function, and Genetics, 8, 195-202 (1990)).
AUTODOCK is available from Scripps Research Institute, La Jolla, Calif.; DOCK
(Kuntz, I. D. et al., "A Geometric Approach to Macromolecule-Ligand
Interactions" J.
Mol. Biol., (1982) 161, 269-288). DOCK is available from University of
California,
San Francisco, Calif.
[00252] Once suitable chemical entities, compounds, or agents have been
selected, they
can be assembled into a single compound or inhibitor. Assembly may proceed by
visual
inspection of the relationship of the fragments to each other on the three-
dimensional
image displayed on a computer screen in relation to the atomic coordinates of
RecA,
LexA, Pol IV, Pol V, or other protein required for induced mutation. This
would be
followed by manual model building using software such as Quanta or Sybyl.
[00253] Useful programs to aid one of skill in the art in connecting the
individual
chemical entities, compounds, or agents include but are not limited to CAVEAT
(Bartlett, P. A. et al, "CAVEAT: A Program to Facilitate the Structure-Derived
Design
of Biologically Active Molecules". In Molecular Recognition in Chemical and
Biological Problems", Special Pub., Royal Chem. Soc., 78, pp. 82-196 (1989)).
CAVEAT is available from the University of California, Berkeley, Calif.; 3D
Database
systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif.). This
area is reviewed in Martin, Y. C., "3D Database Searching in Drug Design", J.
Med.
Chem., 35, pp. 2145-2154 (1992); also HOOK (available from Molecular
Simulations,
Burlington, Mass.).
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[00254] Instead of designing an inhibitor of a RecA, LexA, Pol IV, Pol V, or
other
protein required for induced mutation in a step-wise fashion one chemical
moiety at a
time as described above, inhibitors of RecA, LexA, Pol IV, Pol V may be
designed as a
whole or "de novo" using either an empty binding site or optionally including
some
portions) of known inhibitor(s). These methods include LUDI (Bohm, H.-J., "The
Computer Program LUDI: A New Method for the De Novo Design of Enzyme
Inhibitors", J. CoTnR. Aid. Molec. Design, (1992) 6, 61-78). LUDI is available
from
Biosym Technologies, San Diego, Calif. and LEGEND (Nishibata, Y. and A. Itai,
Tetr-ahedroh, (1991) 47, p. 8985). LEGEND is available from Molecular
Simulations,
Burlington, Mass.; and LeapFrog (available from Tripos Associates, St. Louis,
Mo.).
[00255] Other molecular modeling techniques may also be employed in accordance
with
this invention. See, e.g., Cohen, N. C. et al., "Molecular Modeling Software
and
Methods for Medicinal Chemistry," J. Med. Chern., (1990) 33, 883-894. See
also,
Navia, M.A. and M.A. Murcko, "The Use of Structural Information in Drug
Design"
Current Opifaiohs ifz Structural Biology, (1992) 2, 202-210.
[00256] Once a compound has been designed or selected by the above methods,
the
efficiency with which that compound may bind to RecA, LexA, Pol IV, Pol V, or
other
protein required for induced mutation may be tested and optimized by
computational
evaluation. An effective inhibitor of mutation must preferably demonstrate a
relatively
small difference in energy between its bound and free states (i.e., a small
deformation
energy of binding). Thus, the most efficient RecA, LexA, Pol IV, Pol V, or
other
protein inhibitors should preferably be designed with deformation energy of
binding of
not greater than about 10 kcal/mole, or more preferably, not greater than 7
kcal/mole.
[00257] Inhibitors of RecA, LexA, Pol IV, Pol V, or other protein required for
induced
mutation may interact with their target in more than one conformation that is
similar in
overall binding energy. In those cases, the deformation energy of binding is
taken to be
the difference between the energy of the free compound and the average energy
of the
conformations observed when the inhibitor binds to the LexA and/or RecA.
[00258] A compound designed or selected, as binding to LexA and/or RecA can be
further computationally optimized so that in its bound state it would
preferably lack
repulsive electrostatic interaction with the target. Such non-complementary
(e.g.,
electrostatic) interactions include repulsive charge-charge, dipole-dipole and
charged
dipole interactions. Specifically, the sum of all electrostatic interactions
between the
inhibitor and the enzyme when the inhibitor is bound to its target (e.g.,
RecA, LexA,
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Pol IV, Pol V, or other protein required for induced mutation), preferably
make a
neutral or favorable contribution to the enthalpy of binding.
[00259] Specific computer software is available in the art to evaluate
compound
deformation energy and electrostatic interaction. Examples of programs
designed for
such uses include: Gaussian 92, revision C (M. J. Frisch, Gaussian, Inc.,
Pittsburgh,
Pa., 1992); AMBER, version 4.0 (P. A. Kollman, University of California at San
Francisco, 1994); QUANTA/CHARMM (Molecular Simulations, Inc., Burlington,
Mass. 1994); and Insight II/Discover (Biosysm Technologies Inc., San Diego,
Calif.,
1994). These programs may be implemented, for instance, using a Silicon
Graphics
workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550. Other hardware
systems and software packages will be known to those spilled in the art.
[00260] Once an inhibitor of RecA, LexA, Pol IV, Pol V, or other protein
required for
induced mutation has been optimally selected or designed, as described above,
substitutions may then be made in some of its atoms or side groups to improve
or
modify its binding properties. Generally, initial substitutions are
conservative, e.g., the
replacement group will have approximately the same size, shape, hydrophobicity
and
charge as the original group. It should, of course, be understood that
components
known in the art to alter conformation should be avoided. Such substituted
chemical
compounds may then be analyzed for efficiency of fit into the 3-D structures
of RecA,
LexA, Pol IV, Pol V, or other protein required for induced mutation by the
same
computer methods described in detail, above.
[00261] The compounds designed by any of the above methods are useful for
inhibiting
induced mutagenesis and thus are useful as therapeutic agents to reduce drug
resistance.
XII. Uses of Achao _dens
[00262] The achaogens described herein can be used to modulate the rate of
induced
mutations or more preferably to inhibit the rate of induced mutations in a
cell, group of
cells, or a multi-cellular organism. Such induced mutations can result from a
drug
treatment, UV radiation, inadequate nutrients, etc. Examples of drug
treatments that
may result in induced mutations include treatments with an antineoplastic
agent, an
antibacterial agent, an antiviral agent, an antiprotozoan agent, and/or an
antifungal
agent. Such induced mutations can lead to drug resistance or other
undesireable
mutations. Thus, in some embodiments, an achaogen is used to inhibit drug
resistance
to a drug selected from the group consisting of: an antineoplastic agent, an
antibacterial
agent, an antiviral agent, an antiprotozoan agent, and/or an antifungal agent.
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[00263] For example, an achaogen of the present invention can be used to
inhibit
resistance to any antibiotic disclosed herein or otherwise known in the art.
In preferred
embodiments, an achaogen is used to inhibit resistance to rifampin,
oxazolidinones
(e.g., linezolid), fluoroquinolones (e.g., ciprofloxacin, levofloxacin,
moxifloxacin,
gatifloxacin, gemifloxacin), macrolides (e.g., azithromycin and
clarithromycin), and
later generation cephalosporins (e.g., cefaclor, cefadroxil, cefazolin,
cefixime,
cefoxitin, cefprozil, ceftazidime, cefuroxime, and cephalexin).
[00264] In some embodiments, the achaogens herein can be used to reduce the
rate of
mutation in bacteria. Mutation rate may be reduced in either or both gram-
positive or
gram-negative bacteria, whether such bacteria are cocci (spherical), rods,
vibrio
(comma shaped), or spiral.
[00265] Of the cocci bacteria, rnicrococcus and staphylococcus species are
commonly
associated with the skin, and Streptococcus species are commonly associated
with tooth
enamel and contribute to tooth decay. Of the rods family, bacteria Bacillus
species
produce endospores seen in various stages of development in the photograph and
B.
cereus cause a relatively mild food poisoning, especially due to reheated
fried food. Of
the vibrio species, V. cholerae is the most common bacteria and causes
cholera, a
severe diarrhoeal disease resulting from a toxin produced by bacterial growth
in the gut.
Of the spiral bacteria, rhodospirillum and Treponenza pallidum are the common
species
to cause infection (e.g., Treporzema pallidum causes syphilis). Spiral
bacteria typically
grow in shallow anaerobic conditions and can photosyntheize to obtain energy
from
sunlight.
[00266] Moreover, the present invention relates to achaogens that can be used
to reduce
the rate of mutation in either gram positive, gram negative, or mixed flora
bacteria.
Such bacteria include, but are not limited to, Baciccis Antracis; Euterococcus
faecalis;
Coryuebacterzunz; diphtheriae; Escherichia coli; Streptococcus coelicolor;
Streptococcus pyogetzes; Streptobacillus trzoyzilafos7nas; Streptococcus
agalactiae;
Streptococcus pneunzoniae; Salmofzella typlzi; Salfrzorzella paratyphi;
Salmonella
schottfrzulleri; Salmofzella hirshfeldii; Staphylococcus epidermidis;
Staphylococcus
aureus; Klebsiella przeumouvae; Legionella pneunzophila; Helicobacter pylori;
Mycoplasma pneunzorzia; Mycobacterium tuberculosis; Mycobacterium leprae;
Yersinia enterocolitica; Yersinia pestis; Vibrio clzolerae; Vibrio
parahaemolyticus;
Rickettsia prowazekai; Rickettsia rickettsii; Rzckettsia akari; Clostradaunz
dafficile;
Clostridiufrz tetatzi; Clostridium perfr ingefzs; Clostridium novyii;
Clostridium septicum;
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Clostridium botulinurn; Legionella pneumoplzila; Hemoplzilus influenzae;
Hernophilus
parainfluenZae; Herzzophilus aegyptus; Clzlamydia psittaci; Chlamydia
trachonzatis;
Bordetella pertusis; Slaegella spp.; Canzpylobacter jejune; Proteus spp.;
Citrobacter
spp.; Enterobacter spp.; Pseudonzonas aeruginosa; Propionibacterium spp.;
Bacillus
anthracis; Pseudomonas syringae; Spirrilum minus; Neesseria meningitides;
Listeria
nzonocytogerzes; Neisseria gonorrheae; Treponema pallidum; Francisella
tularensis;
Brucella spp.; Borrelia recurrentis; Borrelia laermsii; Borrelia turicatae;
Borr~elia
burgdorferi; Mycobacterium aveum; Mycobacterium sznegmatis; Methicillirz-
resistant
Staphyloccus aureus; Vanomycin-resistant enterococcus; and multi-drug
resistant
bacteria (e.g., bacteria that are resistant to more than 1, more than 2, more
than 3, or
more than 4 different cli-ugs).
[00267] In some embodiments, an achaogen herein is used to treat an already
drug
resistant bacterial strain such as Methicillin-resistant Staphylococcus aureus
(MRSA)
or Vancomycin-resistant enterococcus (VRE) by exploiting unusual aspects of
rifampicin resistance. Rifampicin has fallen out of common clinical use,
because
rifampicin resistance emerges within 24 hours from initiation of treatment.
Because the
mutations that confer rifampicin resistance impose a significant growth
disadvantage on
bacteria, resistant bacterial populations promptly revert to rifampicin
sensitivity within
a few weeks of cessation of treatment. As such, nearly all MRSA and VRE
strains
encountered are initially rifampicin sensitive. Therefore, the present
invention
contemplates the use of rifampicin in combination with an achaogen to treat
against
MRSA and VRE: The present invention also contemplates the use of achaogens in
combinations with other antibiotics to fight Gram-positive bacteria that
cannot maintain
resistance to certain drugs.
[00268] As such, the achaogens herein may be used to treat a bacterial
infection
condition such as urinary tract infections, ear infections, sinus infections,
bacterial
infections of the skin, bacterial infections of the lungs, sexually
transmitted diseases,
tuberculosis, pneumonia, lyme disease, and Legionnaire's disease. Thus any of
the
above conditions and other conditions resulting from bacterial infections may
be
prevented or treated by the compositions herein.
[00269] In another example, an achaogen is used to inhibit resistance to an
antiviral
agent selected from the group consisting of: AZT; Ganciclovir; valacyclovir
hydrochloride (ValtrexTM); Beta Interferon; Cidofovir; AmpligenTM; penciclovir
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(DenavirTM), foscarnet (FoscavirTM), famciclovir (FamvirTM), and acyclovir
(ZoviraxTM).
[00270] Examples of viruses whose mutations rate may be inhibited by an
achaogen
include but are not limited to, human immunodeficiency virus (HIV); influenza;
avian
influenza; ebola; chickenpox; polio; smallpox; rabies; respiratory syncytial
virus
(RSV); herpes simplex virus (HSV); common cold virus; severe acute respiratory
syndrome (SARS); Lassa fever (Arenaviridae family), Ebola hemorrhagic fever
(Filovirzdae family), hantavirus pulmonary syndrome (Bunyaviridae family), and
pandemic influenza (Orthomyxoviridae family).
[00271] In another example, an achaogen is used to inhibit resistance to an
antiprotozoan agent selected from the group consisting of: Chloroquine;
Pyrimethamine; Mefloquine Hydroxychloroquine; Metronidazole; Atovaquone;
Imidocarb; MalaroneTM; Febendazole; Metronidazole; IvomecTM; Iodoquinol;
Diloxanide Furoate; and Ronidazole.
[00272] Examples of protozoan organisms whose mutation rate may be inhibited
by an
achaogen include but are not limited to, Acantlzameba; ActizZOplZrys; Amoeba;
Anisonema; Anthoplzysa; Ascaris lumbricoides; Bic~soeca; Blastocystis
honzinis;
Codozzella; Coleps; Cotlzurina; Cryptosporidia Difflugia; Entamoeba
histolytica (a
cause of amebiasis and amebic dysentery); Entosiplzon; Epalxis; Epistylis;
Euglypha;
Flukes; Giardia lambia; Hookworm Leishznania spp.; Mayorella; Monosiga;
Naegleria
Hartmannella; Paragoninzus westernzani; Paruroleptus; Plasmodium spp. (a cause
of
Malaria) (e.g., Plasmodium falczparum; Plasnzodiuzn malariae; Plasmodium vivax
and
Plasmodium ovals); Pneumocystis carinii (a common cause of pneumonia in
immunodeficient persons); rnicrofilariae; Podophrya; Raphidiophzys;
Rhynchomonas;
Salpingoeca; Sclzistosoma japonicum; Schistosoma haematobiunz; Sclzistosoma
nzansoni; Stentor; Strongyloides; Stylonychia; Tapeworms; Trichonzonas spp.
(e.g.,
Trichuz-is trichiuris and Triclaomonas vaginalis (a cause of vaginal
infection));
Typaszosoma spp.; and Vorticella.
[00273] In another example, an achaogen is used to inhibit resistance to an
antifungal
agent selected from the group consisting of: imidazoles (e.g., clotrimazole,
miconazole;
econazole, ketonazole, oxiconazole, sulconazole), ciclopiroz, butenafine, and
allylamines.
[00274] Examples of fungus infections whose mutation rate may be inhibited by
an
achaogen include but are not limited to, tinea; athlete's foot; jock itch; and
candida.
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2005/056754
[00275]
In
particular,
the
present
invention
contemplates
the
prevention
and
treatment
of
infectious which have re-emerged with increased
diseases
identified
in
Table
3
resistance
to
medications:
TABLE
3
-
Examples
of
Infectious
Diseases
With
Increased
Resistance
to
Medications.
Cryptosporidiosis Cryptosporidium parvum (protozoan)
Diphtheria Coryuebacterium diptheriae
(bacterium)
Malaria ' Plasmodium species (protozoan)
meningitis, necrotizing fasciitisGroup A Streptococcus (bacterium)
(flesh-
eating disease), toxic-shock
syndrome, and
other diseases
rpertussis (whooping cough) Bordetella pertussis (bacterium)
Rabies Rlzabdovirus group (virus)
rubeola (measles) Morbillivirus genus (virus)
Schistosomiasis~~ Schistosoma species (helminth)
Tuberculosis Mycobacterium tuberculosis
(bacterium)
yellow fever Flavivirus group (virus)
HIV ~ staphylococcus
[00276] The inducible mutation pathways discussed herein are also known to
exist in
eukaryotic cells, though they are expected to be quite different in
mechanistic detail.
See Diaz, M., et. al. Mol Cancer Res., (2003) 1:836-847; Zhang, Y., et al.,
Nucleic
Acids Res., (2000) 28:4147-4156. Thus, achaogens can be used to inhibit
mutation in
eukaryotic cells. In one embodiment, achaogens are used as an adjuvant or
supplement
to therapies in which therapeutic outcomes are compromised by mutations. These
therapies include, but are not limited to cancer chemotherapy. In another
embodiment,
an achaogen is used as a prophylactic to prevent mutations, for example to
prevent
tumorgenesis and carcinogenesis. The achaogens are suitable to prevent both
benign
and malignant tumors.
[00277] Examples of cancers that may be treatable or preventable by the
present
invention include, but are not limited to, breast cancer; skin cancer; bone
cancer;
prostate cancer; liver cancer; lung cancer; brain cancer; cancer of the
larynx;
gallbladder; pancreas; rectum; parathyroid; thyroid; adrenal; neural tissue;
head and
neck; colon; stomach; bronchi; kidneys; basal cell carcinoma; squamous cell
carcinoma
of both ulcerating and papillary type; metastatic skin carcinoma; osteo
sarcoma;
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Ewing's sarcoma; veticulum cell sarcoma; myeloma; giant cell tumor; small-cell
lung
tumor; gallstones; islet cell tumor; primary brain tumor; acute and chronic
lymphocytic
and granulocytic tumors; hairy-cell leukemia; adenoma; hyperplasia; medullary
carcinoma; pheochromocytoma; mucosal neuronms; intestinal ganglioneuromas;
hyperplastic corneal nerve tumor; marfanoid habitus tumor; Wilm's tumor;
seminoma;
ovarian tumor; leiomyomater tumor; cervical dysplasia and in situ carcinoma;
neuroblastoma; retinoblastoma; soft tissue sarcoma; malignant carcinoid;
topical skin
lesion; mycosis fungoide; rhabdomyosarcoma; Kaposi's sarcoma; osteogenic and
other
sarcoma; malignant hypercalcemia; renal cell tumor; polycythermia vera;
adenocarcinoma; glioblastoma multiforme; leukemias (including acute
myelogenous
leukemia); lymphomas; malignant melanomas; epidermoid carcinomas; chronic
myleoid lymphoma; gastrointestinal stromal tumors; and melanoma.
[00278] In particular, the methods and compositions herein are useful for
inhibiting the
development of resistance to anti-cancer (antineoplastic) medications
including, but are
not limited to, Gleevec; antineoplastic drugs; including alkylating agents
such as alkyl
sulfonates (busulfan; improsulfan; piposulfan); aziridines (benzodepa;
carboquone;
meturedepa; uredepa); ethylenimines and methylmelamines (altretamine;
triethylenemelamine; triethylenephosphoramide; triethylenethiophosphoramide;
trimethylolmelamine); nitrogen mustards (chlorambucil; chlornaphazine;
cyclophosphamide; estramustine; ifosfamide; mechlorethamine; mechlorethamine
oxide hydrochloride; melphalan; novembichin; phenesterine; prednimustine;
trofosfamide; uracil mustard); nitrosoureas (carmustine; chlorozotocin;
fotenmustine;
lomustine; nimustine; ranimustine); dacarbazine; mannomustine; mitobranitol;
mitolactol; pipobroman; doxorubicin; and cisplatin (including derivatives).
[00279] Additional anticancer medications that may benefit from co-
administration with
an achaogen include, but are not limited to, Acivicin; Aclarubicin; Acodazole
Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin;
Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin;
Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat;
Benzodepa;
Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;
Bleomycin
Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;
Caracemide; Carbetirner; Carboplatin; Carmustine; Carubicin Hydrochloride;
Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine;
Crisnatol
Mesylate; Cyclophosphamide ; Cytarabine; Dacarbazine; Dactinomycin;
Daunorubicin
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Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate;
Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene;
Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate;
Eflornithine; Hydrochloride; Elsamitrucin; Enloplatin; Enpromate;
Epipropidine;
Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine;
Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil; Etoposide;
Etoposide
Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide;
Floxuridine;
Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin
Sodium;
Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride;
Ifosfamide; Imofosine; Interferon Alfa-2a; Interferon Alfa-2b ; Interferon
Alfa-nl;
Interferon Alfa-n3; Interferon Beta-Ia; Interferon Gamma-Ib; Iproplatin;
Irinotecari
Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate Liarozole
Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride;
Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate;
Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate;
Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin;
Mitocromin;
Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone
Hydrochloride;
Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel;
Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide;
Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane;
Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride;
Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide;
Safingol;
Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium;
Sparsomycinl;
Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin;
Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane;
Taxoid;
Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide;
Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin;
Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate;
Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;
Tubulozole
Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine
Sulfate;
Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate;
Vinglycinate
Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;
Vinzolidine
Sulfate; Vorozole; Zeniplatin; Zinostatin; and Zorubicin Hydrochloride.
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[00280] In particular, the present invention contemplates the use of an
achaogen to
prevent the development of drug resistance, wherein drug resistance results
from at
least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, '~0, 30, 40 or 50 mutations. The
achaogen can also be
used to prevent the development of drug resistance, wherein the drug
resistance results
from at least one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50
deletion/insertion mutations.
[00281] Such an achaogen can be administered independently or in combination
with
another therapeutic agent (e.g., any of the antibodies, antifungal agents,
antiviral
agents, antiprotozoan agents, and antineoplastic agents herein).
[00282] In addition to the therapeutic uses described above, the achaogens
described
herein are also useful in numerous industrial applications. In particular,
achaogens are
useful in industrial processes that are hindered due to the development of
mutations in
the organisms used in the processes. Suitable applications include the
prevention of
mutations in yeast used in breweries and other biotechnology applications.
Another
suitable use is to prevent mutations in bacteria (or eukaryotic cells) that
are used for the
synthesis of proteins, like antibodies, etc. Industrial applications include
the improved
utility of cleaning products, such as soap, toothpaste, and house-cleaning
products.
Others suitable uses will be apparent to one of skill in the art based on the
disclosure
herein.
XIII. Screening For Resistance
[00283] In any of the embodiments herein, an organism or patient can be first
tested for
drug resistance prior to the administration of an achaogen. A test to detect
drug
resistance or susceptibility to drug resistance may involve taking a biopsy or
sample
from the patient. Samples can be obtained from microorganisms (e.g., viruses,
bacteria,
fungi, protozoans) or larger organisms (e.g., human, monkey, cows, pigs,
horses, sheep,
dogs and cats). The samples can come from tissues or tissue homogenates or
fluids of
an organism and cells or cell cultures. For example, samples can be obtained
from
whole blood, serum, semen, vaginal fluid, ear wax, nasal drips, saliva, tears,
urine, fecal
material, sweat, buccal, skin, spinal fluid, tissue biopsy or necropsy, and
hair. Samples
can also be derived from ex vivo cell cultures, including the growth medium,
recombinant cells and cell components. In some embodiments, samples can be
obtained from diseased cells and from non-diseased cells.
[00284] Several techniques are known in the art for determining whether a
particular
strain of bacteria has developed resistance to an antibiotic. For example, if
the
administration of an antibiotic at a dose equivalent to its ED50 (the dose at
which 50%
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of patients being treated respond) to a patient suffering from a bacterial
infection does
not result in a therapeutic benefit, the bacteria is considered to be
resistant to the
antibiotic.
[00285] In some embodiments, a sample obtained from an organism is assayed to
detect
one or more mutations in a gene that modulates induced mutation. Examples of
such
genes include but are not limited to 16S rRNA, 23S rRNA, clpXP, ding, dinl,
dfzaE2,
gyrA, gyrB, katG, inhA, lon protease, L4 ribosomal methylases, lexA, norA,
recA. psiB,
parC, parE, polB, rpoS, rpoB, sxt, umuC, and unzuD. Generally, mutations in
psiB,
dial, clpXP, and/or lon are associated with an increased rate of mutagenesis
or
susceptibility to induced mutations. On the other hand, mutations in 16S rRNA,
23S
rRNA, ding, dnaE2, gyrA, gyrB, katG, inhA umuC, urnuD, lexA, norA, recA, I~
ribosomal methylases, parC, parE, rpoS, rpoB, sxt, and polB are associated
with
resistance to induced mutations or lack of induced mutagenesis.
[00286] Examples of mutations that may be used as a diagnosis for drug
resistance or
susceptibility thereto include: mutations in lexA that affects its ability to
auto-cleave
(e.g., S119A and S141A); mutations in recA and its ability to bind ssDNA
and/or
interact with LexA; mutations in gyrA and gyrB genes, which encode gyrase;
mutations
in parC and parE genes, which encode subunits of topoisomerase IV; mutations
in
genes that affect outer membrane permeability or export through an active
efflux
system (see e.g. Poole, I~., 44(10): 2595-2599 (2000)).; mutations in dnaE2;
mutations
in the rpoS and rpoB associated with resistance to Rifampin (see, e.g.,
Boshoff, H.,
Cell, (2002) 113, 183-193); mutations in the 23S rRNA genes or L4 ribosomal
methylases associated with resistance to linezolid, erythromycin, and other
macrolides;
mutations in the katG or znlzA genes associated with resistance to isoniazid;
mutations
in the 16S rRNA genes associated with resistance to streptomycin; and
mutations in
gene and gene products that modulate induced mutagenesis in non-bacterial
organisms.
Other examples of genes whose mutations may be associated with drug resistance
are
disclosed in herein.
[00287] Assays that test the level of expression of gene products that enhance
or
suppress induced mutagenesis may be used as a diagnostic for drug resistance
or
susceptibility to drug resistance. Such expression can be detected by
measuring the
level of gene transcripts or gene products of such genes. Examples of genes
whose
overexpression may be used as a diagnosis for laclc of drug resistance or
susceptibility
thereto include, but are not limited to, psiB, dznl, clpXP, and lon. Examples
of genes
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whose overexpression may be used as a diagnosis of drug resistance or
susceptibility
thereto include, but are not limited to,16S rRNA, 23S rRNA, ding, dnaE2, gyrA,
gyrB,
katG, inhA unzuC, unzuD, lexA, norA, recA, I/~ ribosomal nzetlzylases, parC,
parE,
rpoS, rpoB, sxt, and polB.
[00288] Detection of level of expression can be made using any method known in
the
art. In preferred embodiments, expression levels are detected using a
microanay. For
example, a sample can be obtained from an organism being tested. The sample
can be
assayed to detect a level of expression of, for example, a cancerous cell,
bacterial
infection, viral infection. This level of expression can then be compared with
a level of
expression in a control. If the level of expression of the above genes is
greater in the
sample than in a control - drug resistance or susceptibility to drug
resistance is likely to
have occurred or to occur. If the level of expression in an organism is less
than a level
of expression in a control - drug resistance or susceptibility to drug
resistance is not
predicted to have occurred or to occur.
[00289] After screening for drug resistance, a patient having detectable
levels of drug
resistance can be administered an achaogen and one or more therapeutic agents
disclosed herein.
XIV. Kits
[00290] The present invention also contemplates kits comprising one or more
vials,
wherein at least one vial comprises an achaogen of the present invention. The
kits also
contain a set of written instructions for use of the compositions therein. For
example,
instructions can direct an individual as to the specific achaogen to be used,
dosages to
be applied, frequency and duration of use, and methods of adminsteration. The
kits can
also include additional agents to be co-adminstered, e.g., any drug (e.g.,
antibiotic,
antiviral, anticancer, etc.) as well a instructions for the coadministration
of the
achaogen and the additional agent(s).
[00291] Preferably, a vial comprises an achaogen in a pharmaceutical
formulation. In
some embodiments, a kit comprises one or more vials of an achaogen forumated
for
local or system adminsterations. In some embodiments, an achaogen in a vial
may
coformulated with a second therapeutic agent (e.g., antiprotozoan, antiviral,
antibiotic,
antifungal, or an antineoplastic agent).
[00292] The lut can also include one or more containers with additional
achaogens
and/or therapeutic agents.
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[00293] The lcit can also include a diagnostic tool to detect the presence,
absence, and/or
susceptibility to drug resistance. A diagnostic tool of the present invention
can include
nucleic acid primers, probes, antibodies, microarrays, microfluidic devices,
etc. A
diagnostic tool of the present invention can detect level of gene expression,
SNPs, or
rate of induced mutation. In preferred embodiments, the diagnostic tool is a
microarray.
[00294] In some embodiments, a diagnostic tool detects one or more mutations
in a
genes) associated with induced mutations, such as 16S rRNA, 23S rRNA, clpXP,
ding,
dial, dnaE2, gyrA, gyrB, katG, inhA, lon protease, I~ ribosomal methylases,
lexA, lon
protease, norA, recA. recN, psiB, parC, parE, polB, psiB, rpoS, rpoB, sxt,
umuC,
umuD, uvrA, uvrB, and uvrD.
[00295] In some embodiments, a diagnostic tool detects level of expression of
a genes)
associated with induced mutagenesis. Examples of such genes include, but are
not
limited to,16S rRNA, 23S rRNA, clpXP, ding, dial, draaE2, gyrA, gyrB, katG,
inhA, lon
protease, I~ ribosomal methylases, lexA, lon protease, norA, recA, recN, psiB,
parC,
parE, polB, psiB, rpoS, rpoB, sxt, umuC, unauD, uvrA, uvrB, and uvrD.
EXAMPLES
Example 1
[00296] To demonstrate that the administration of ciprofloxacin induces
mutation, we
examined the evolution of resistance of wild type MG1655 E. coli on solid
media plates
containing 35 ng/mL ciprofloxacin. First we differentiated colonies that arose
during
the exponential growth phase, prior to plating, and those that arose after
plating. This
was done by isolating colonies, noting the day on which they appeared, and
then
regrowing the colonies and determining the time it took for a colony to
appear. For
example, if a colony appeared on day three, but then required three days to re-
grow, the
founding cell was assumed to have obtained a resistance mutation during the
exponential growth phase, prior to exposure to antibiotic. If a colony is
isolated on day
five and then found to require only one day to re-grow, the founding cell was
assumed
to have mutated on day four after exposure to the antibiotic.
[00297] At least four observations were immediately apparent from inspection
of the
data. First, the number of mutations per viable cell per day increased by at
least four-
orders of magnitude, and possibly at least five orders of magnitude (from 3.4
(+/- 2.0) x
10-1° to 2.9(+/- 0.41) x 10-5) after exposure to the antibiotic
(similar to results reported
earlier in Riesenfeld, C, Antimicrob. Agents Chenzother. (1997) 41:2059-2060).
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Second, the rates of mutation prior to drug exposure were virtually
independent of the
gene deletions described below in Example 2, whereas the mutation rate after
drug
exposure was strongly dependent (being both increased or reduced upon deletion
of
certain genes, see below). Fourth, the spectrum of mutations occurring in the
presence
of the antibiotic differed significantly from those occurring during
exponential growth.
The isolated growth-dependent mutations were strictly substitutions, while the
induced
mutations were both substitutions and small, in frame deletions (Figure 11).
The type
of mutation (base substitution versus deletion) that arose before drug
exposure was
independent of gene deletion, whereas those that arose after exposure to
ciprofloxacin
depended strongly on gene deletion. This data implies that a mutational system
is
induced upon exposure to ciprofloxacin and that this system is both
mechanistically
distinct from any system conferring mutations during exponential growth and
responsible for the majority of the mutations that give rise to ciprofloxacin
resistance.
[0029] To further understand the effects of induced mutation resulting from
exposure
to antibiotics, we determined whether or not pre-exposure to rifampicin, an
unrelated
antibiotic, could induce mutations that bestowed the bacteria with
ciprofloxacin
resistance, E. coli MG1655 were incubated in PBS containing 0, 4, 12, and 36
mg/mL
rifampicin for four days at 37°C, and then plated on agar containing
Luria Broth (LB)
with 35 ng/mL ciprofloxacin. Incubation in the presence of 0 or 4 mg/mL had no
effect
on the number of ciprofloxacin resistant colonies present in the culture.
However,
incubation in the presence of more rifampicin (12 or 36 mg/mL) resulted in a
dramatic
increase in the number of ciprofloxacin resistant cells. This effect was
essentially
completely lost in the umuDC deletion stain, demonstrating that the resistance
in the
wild type strain results from induced mutation mediated at least in part by
Pol V.
Apparently, pre-incubation with a suitable concentration of rifampicin induces
the
mutation system (for example, during repair synthesis).
Example 2
[00299] To determine whether the evolution of resistance to ciprofloxacin is
under
genetic control, and if so, to determine which genes are involved, we
constructed a
series of isogenic loss of function strains of E. coli K-12 (MG1655) (Table
5). We
have selected the E. coli strain MG1655 as the genetic background, as this K-
12 strain
was used in the E. coli genome sequencing project. Strains listed in the
accompanying
Table 4 were constructed using PCR-mediated gene replacement. See Daiguan Yu,
et
74
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
al., Proc. Natl. Acid. Sci., May 2000; 97: 5978 - 5983. PCR reactions were
performed
using Platinum pfx DNA polymerise from Invitrogen, with standard cycling
parameters. Genomic template DNA was prepared from a fresh bacterial overnight
culture using the DNeasy lcit (Qiagen).
[00300] Mutation cassettes were constructed using 3-way PCR, as described by
Murphy
et al. Gene (2000) 246:321-330. Gene specific components were annealed to an
antibiotic resistance marker by combining the three fragments in a PCR
reaction, in
equal volume. Conditions for this PCR reaction were standard, with the
exception that
the proximal primers were used in limiting amounts.
[00301] Deletion cassettes consisted of approximately 500 base pair regions
upstream
and downstream to the gene deleted, including the first and last 2-10 codons
of the
gene, on either side of an antibiotic resistance cassette in reverse
orientation to the gene
being deleted. To construct the lexA point mutant, the lexA gene was amplified
from
genomic _MG1655 DNA by PCR using primers lexA NF-SphI and lexA_OrfR-NdeI,
digested with SphI, NdeI and ligated into SphI, NdeI digested pUCl8 vector
(5). The
S 119A mutation (TCG->GCG) was introduced in the resulting plasmid using the
Quikchange Site-directed Mutagenesis kit (Stratagene, La Jolla, CA) and
primers
LexA_S 119A_QCF and LexA_S 119A_QCR. The resulting allele was confirmed by
sequencing, digested with SphI, NdeI and ligated to a DNA fragment containing
an
antibiotic resistance cassette and 500 by of sequence downstream to the lexA
gene.
Therefore, the final cassette contains 500 by of upstream DNA, the mutated
ORF, an
NdeI site attaching an antibiotic resistance marker in reverse orientation,
and
approximately 500 by of downstream DNA.
[00302] The antibiotic resistance markers were amplified as follows. The
kanamycin
(Km) resistance cassette was amplified from pUC4K using primers 5'-GGA AAG CCA
CGT TGT GTC TC and 5'-CGA TTT ATT CAA CAA AGC CGC. Similarly, the
spectinomycin (Spec) resistance cassette was amplified from pOmega, and the
chloramphenicol (Cm) resistance cassette from pSUl8. All oligonucleotide
primers
used in the construction of the disruption cassettes are listed in Figure 19.
[00303] Generation of the genomic deletions in MG1655 proceeded in two steps:
(i)
genomic insertion into strain PS6275 and (ii) P1-mediated transfer of the
deletion
cassette to MG1655. In the first step, the linear DNA fragments (PCR products)
were
electroporated into the hyper-recombinational E. coli strain PS6275 [Yu, D, et
al. Proc
Natl Acad Sci USA (2000) 97:5978-5983], a derivative of MG1655 which carries
the
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lambda phage red genes. This strain accepted the linear PCR product and
recombined
it into the genome with high efficiency. Recombination genes were activated by
growing DY329 at 42°C and the competent cells stored at -80°C.
The competent cells
were transformed with the desired cassettes and transformants selected at
30°C on LB
supplemented with the appropriate antibiotic (kanamycin 30 -g / mL,
chloramphenicol
25 _g / mL, or spectinomycin 100 _g / mL), and grown at 30 °C. Although
MG-
DY329 was engineered such that the lambda _phage red genes could be easily
removed
to return the cell to a non-hyper-recombinational background, we used P1
transduction
to move the gene-specific disruption from PS6275 into MG1655. MG1655 provides
a
more 'wild-type' background than MG-DY329, and thus simplifies the
interpretation of
the results. Gene deletions were verified by PCR; the lexA(S 119A) strain was
confirmed by PCR followed by sequencing.
TABLE 4
Table of strains used.
Parent Mutation
MG1655 -
ATCC25922 -
MG1655 DY329 (nadA::RED)
MG1655 lacZ~: : kan
MG1655 polBO: : kan
MG1655 polB~: : spc
MG1655 dinB~: : kan
MG1655 urnuDC~: : kan
MG1655 umuDC~: : cat
MG1655 polB~: : Spc, dinB~: : kan
MG1655 polB~: : Spc, umuDCO: : kan
MG1655 dinBO: : kan umuDCO:: cat
MG1655 polB~: : spc dinB~: : kan,
UrnuDC: : Cat
MG1655 LexA(SI19A): : kan
MG1655 recAO: : kara
MG1655 recB~: : kan
MG1655 recD~: : kafz
MG1655 recFO: : kan
MG1655 recG~: : kan
MG1655 ruvBO: : kan
MG1655 ruvCO: : kan
MG1655 sulAd:: kan
MG1655 priA~:: kan
ATCC25922 lacZO: : kan
ATCC25922 LexA(S119A): : kan
ATCC25922 recF4: : kan
[00304] With the isogenic loss of function strains in hand, mutation in
response to
ciprofloxacin (obtained from U.S. Biologicals) was determined using a protocol
based
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on the Stressful Lifestyle Adaptive Mutation (SLAM) assay as illustrated in
Figure 6.
Five colonies of each strain, selected from 30 ug/mL kan plates, were grown
for 24
hours in LB at 37 °C. Dilutions of each culture were made in duplicate
and plated on
LB plates to determine the number of viable cells.
[00305] To assay for mutation, 150 ~,L of each culture was plated twice on LB
plates
containing 35 ng/mL ciprofloxacin. Also, two 150 ~.L cultures from each strain
were
plated on five additional plates for use in 'survival' experiments (see
below). The
concentration of ciprofloxacin used was chosen based on trial experiments with
the
MG1655 parent strain which indicated that 35 ng/mL ciprofloxacin maximized
mutation-dependent growth. Every twenty-four hours for thirteen days post-
plating,
colonies were counted and marked and up to 10 representative colonies per
strain were
stocked in 15% glycerol and stored at -~0 °C, for use in the
reconstruction experiments
(see below). Also, to determine the number of ciprofloxacin susceptible cells
remaining on the plates, parallel 'survival' experiments were performed. The
'survival'
experiment plates were treated exactly as the SLAM plates, except at specified
time
points, representative plates were sacrificed by excising all visible
colonies, recovering
the remaining agar in 9 mg/mL saline, and plating dilutions of the resulting
solution on
LB to determine the number of viable cells.
[00306] After thirteen days, a reconstruction experiment was performed to
determine
which of the resistant colonies isolated had evolved resistance via induced
mutation
after exposure to the antibiotic. The stocked colony suspensions isolated
during the
original experiment were used to inoculate 1 mL of LB and grown overnight at
37 °C.
The resulting cultures were then diluted and duplicate plated on LB and LB
containing
35 ng/mL ciprofloxacin and the time elapsed to colony formation was recorded
and
compared to the original experiment. Only those colonies that grew in a
shorter time
during the reconstruction experiment than in the original experiment were
considered to
have acquired an induced mutation, i.e. occurred after exposure to the
antibiotic. Using
the colony counts of induced mutants on the ciprofloxacin containing SLAM
plates and
the viable cell counts from the 'survival' experiments, an induced mutation
rate was
calculated per viable cell.
[00307] The data on rates of mutation to Ciprofloxacin resistance are shown in
Figures 3
and 10. As shown, resistance was found to be significantly reduced in several
strains,
including polB_ (Pol II deletion strain); dinB_ (Pol IV deletion strain);
umuDC_ (Pol V
deletion stain), and lexA(Ind-) (which cannot under autocleavage and thus
malees the
77
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
strain uninducible). The largest effect from any single mutation was seen for
the
LexA(Ind-) strain which was more than two orders of magnitude less able to
evolve
resistance to ciprofloxacin (the precise amount depending on the antibiotic
concentration). The observed effect is remarkably large when considered in the
context
of clinical resistance. Clinically relevant high resistance requires multiple
independent
mutations. See Drlica, K, et al. Microbiol Mol. Biol. Rev. (1997) 61:377-392;
Gibreel,
A, et al. Antimicrob. Agents Chemother. (1998) 42:3276-3278; Kaatz, GW,
Antimicrob
Agents Chemother. (1993) 37:1086-1094; Yoshida, H, et al. J. Bacteriol.,
(1990)
172:6942-6949; Poole, K., Antimicrob. Agents Chemother. (2000) 44:2233-2241;
Kern, WV, Antimicrob. Agents Chemother. (2000) 44:814-820; Fukuda, H,
Antimicrob. Agents Chemother. (1998) 42:1917-1922, whereas resistance in these
experiments requires a single mutation (in the gyrA gene, confirmed by
sequencing).
[00308] Sequencing the gyrA gene revealed an interesting pattern. In the wild
type
stain, induced mutants (arose after day 4) showed an approximately ~2:1 ratio
of point
mutation to codon deletion. Deletion of any of the three polymerases resulted
in 100%
codon deletion, implying a major mutational sub-branch that depends on the
activity of
all three polymerases is required for base substitution mutation. The codon
deletion
pathway, however, can function with any one of the induced mutation-causing
polymerase genes deleted.
[00309] TABLE 5 - Strain growth, ciprofloxacin sensitivity, and mutation
spectra
Post-ciprofloxacin
Exposure
ciprofloxacin Exponential Day
MIC Growth 5-13
Mutation
(n Mutation S
/ S ectra
ml) ectra
% %
Relative Base % Base ~ %
Doubling WT gyrA gyrA % Substi-Codon % Substi-Codon
Strain Time rA 5830 S83L WT tution0 WT tution0
~lacZ 1.0 (0.01)35.0 250.0450 16.783.3 0.0 22.2 61.2 16.7
OpoIB 1.1 (0.10)30.0 250.0450 28.671.4 0.0 0.0 0.0 100.0
~dinB 1.0 (0.03)35.0 250.0450 16.783.3 0.0 0.0 0.0 100.0
~urnuDC1.0 (0.01)35.0 250.0450 25.075.0 0.0 33.3 0.0 66.7
~polB, 1.0 (0.12)25.0 250.0450 50.050.0 0.0 83.3 0.0 16.7
OdinB
~polB, 1.1 (0.19)25.0 250.0450 66.733.3 0.0 0.0 0.0 100.0
~urnuDC
~dinB, 1.1 (0.08)35.0 250.0450 16.783.3 0.0 33.3 0.0 66.7
0unauDC
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Post-ciprofloxacin
Exposure
ciprofloxacin Exponential Day
MIC Growth 5-13
Mutation
(n Mutation S
/ S ectra
ml) ectra
% %
Relative Base % Base %
Doubling WT gyrA gyrA % Substi-Codon % Substi-Codon
Strain Time rA 583 S83L WT tutionO WT tutionO
dpolB, 1.2 (0.17)25.0 250.0450 42.9 57.1 0.0 0.0 0.0 100.0
~dinB,
~umuDC
lexA 1.0 (0.03)30.0 250.0350 16.7 83.3 0.0 0.0 0.0 100.0
(S119A)
OrecD 1.0 (0.10)35.0 250.0350 0.0 100.0 0.0 ~ ~ 80.020.0
~ ~ ~ 0.0 ~
[00310] In addition to the genes listed in Table 5 and Figures 3 and 10, our
model
predicts that strains deficient in replication restart (Figure 11) should also
have reduced
rates of mutatiom to quinolone resistance when under selection by quinolones.
More
specifically, our model predicts that inhibitors of the proteins encoded by
recA, recB,
recta, ruvA, ruvB, ruvC and priA may have achaogenic properties as they would
inhibit
access to the error prone repair of DNA by PoIIV and PoIV.
Exa~aple 3
[00311] It was determined if blocking the ability of LexA (the DNA binding
protein that
represses the expression of SOS gene products) to undergo its proteolytic self-
cleavage
reaction would cripple the ability of E. coli cells to become resistant to
ciprofloxacin, as
well-defined point mutations are required for ciprofloxacin resistance. Figure
8 shows
compares three E. coli strains for their ability to evolve 'first level'
resistance to
ciprofloxacin (i.e., the ability to grow in the presence of 35 ng/ml
ciprofloxacin). Strain
(1) is ATCC 25922; strain (2) is ATCC 25922-~lacZ; and strain (3) is ATCC
25922-
lexA(S 119A). The LexA protein in strain (3) cannot undergo self-cleavage,
because its
nucleophillic serine has been replaced with an alanine. Because it contains a
non-
cleavable LexA rather than a wild-type version of the LexA protein, Strain (3)
is
approximately 100-fold less able to acquire the single point mutation in gyrA
conferring
resistance to 35 ng/ml ciprofloxacin. While a single point mutation in the
gyrA gene
allows cells to grow at low ciprofloxacin concentrations (35 ng/ml), clinical
resistance
is far higher (approximately 100 fold higher, >15 ug/ml) and requires 3 to 5
independent point mutations.
[00312] To determine if ATCC 25922-lexA(S 119A) is similarly crippled in its
ability to
evolve the next 'tier' of ciprofloxacin resistance (e.g., an additional
mutation, this time
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CA 02544018 2006-04-27
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in the ParC gene, which would allow E. coli cells to grow in the presence of
650 ng/ml
ciprofloxacin) ATCC 25922-~lacZ and ATCC 25922-lexA(S 119A) clones that had
already acquired low level resistance to ciprofloxacin (35 ng/ml) via single
point
mutations in the gyrA gene and were used to measure their ability to evolve
resistance
to 650 ng/ml ciprofloxacin.
[00313] Figure 9 compares the ability of two already resistant strains (to 35
ng/ml
ciprofloxacin) to evolve resistance to 650 ng/ml ciprofloxacin. This 'second
step'
mutation rate was 1.9 (~ 0.21) x 10-~ mutants/(cell day) in the wild-type
strain and 5.5
(~ 4.9) x 10-7 mutants/(cell day) in the lexA(S 119A) strain. Thus, combining
the
relative rates for both steps, the lexA mutant evolves resistance to 650 ng/mL
ciprofloxacin with a rate more than 104 times reduced relative to wild-type.
[00314] Considering that clinical resistance requires three to five
independent mutations,
the data implies that the lexA(S 119A) mutant should evolve clinical
resistance at least
10~ times more slowly. These results demonstrate that LexA cleavage-mediated
derepression of one or more genes, possibly the inducible polymerases, is not
important
for survival in the presence of the antibiotic at these concentrations, but is
critical for
mutation and the evolution of resistance.
Example 4
[00315] Ifz vivo experiments were performed in mice to predict the effect on
the
emergence of drug resistance in an infection context with the cleavage of LexA
effectively inhibited.
[00316] Infections were established in mice thigh muscle with one of the
following two
bacterial strains - an essentially wild type 25922 (a pathogenic strain of E.
coli) where
the lacZ gene was replaced with the kan marker (as described above) or a
variant of
25922 modified as described above to possess the lexA gene product was LexA(S
119A)
gene instead of lexA wild type gene. These strains are referred to as 'LacZ'
or as
'ATCC 25922-~lacZ' and 'LexA(S 119A)' or 'ATCC 25922-lexA(S 119A),
respectively. Ciprofloxacin was administered to the mice at a drug dose that
was
approximately cytostatic. Mice were killed, their legs homogenized, total cell
counts
were taken from each thigh, and then the thighs were plated on ciprofloxacin
containing
plates to count ciprofloxacin resistance colonies. The data from this
experiment are
presented in Table 6 and in Figure 12.
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TABLE 6 - Colonies isolated on plates containing 20/80 ng/mL ciprofloxacin
ays post
DLacZ LexA(S119A)
infection
1 93/65 3/1
2 28/23 2/0
[00317] As can be seen from Table 6, very few ciprofloxacin resistant bacteria
were
observed in the mutant LexA(S 119A) mutant strain compared to the wild type
bacteria.
[00318] In Figure 12, open circles and triangles correspond to the total
colony forming
units (CFU)/thigh of the ATCC 25922-OlacZ and ATCC 25922 lexA(S 119A) strains,
respectively. Solid circles and triangles represent the number of
ciprofloxacin-resistant
~lacZ and lexA(S 119A) mutants/thigh, respectively. Clearly, in the context of
an actual
infection, ATCC 25922-lexA(S 119A) was unable to evolve resistance to
ciprofloxacin,
despite the permissive drug concentration in the animal (35 ng/ml
ciprofloxacin).
Materials and methods for the experiments in this section are detailed in
Cirz, R. and
Romesberg E. F., submitted.
[00319] While the feasibility experiments shown here examined the effect on
the
emergence of ciprofloxacin resistant E. coli, similar experiments were
performed
similar experiments looking at the emergence of erythromycin (a macrolide)
resistance
in E. coli and have observed similar results. Boshoff et al. Cell, (2003) 113,
183-193
have performed similar experiments in which they observe that crippling the
ability of
RecA to activate LexA self-cleavage prevents Myobacteria from evolving
resistance to
rifampicin in mouse lung infection models. Collectively, these results
strongly suggest
that crippling the SOS response severely restricts the ability of multiple
bacterial
species to induce mutation and thereby evolve resistance to multiple classes
of widely-
used antibiotics.
Example 5
[00320] To examine the role of each individual LexA-repressed polymerases in
the
induction of resistance-conferring mutations, the ~pol B, ~dinB, and DumuDC
strains
were constructed as described above (See Example 4, Table 5). Deletion of polB
resulted in a very slight sensitivity to the antibiotic, but deletion of
either ding or
umuDC had no detectable effect on antibiotic sensitivity. Consistent with this
observation, ~polBddinB, ~polB~urnuDC, and the triple mutant,
dpolB~difzBOumuDC,
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were slight but reproducibly more sensitive to the antibiotic, while the
OdinB~umuDC
double mutant exhibited wild-type sensitivity. These data imply that Pol II
may play a
role in replication restart in response to ciprofloxacin, unlike Pol IV and
Pol V, which
are not required.
[00321] Figures 3 and 10 depicts mutation rate of the following ten strains:
~lacZ [strain
1], ~polB [strain 2], ~dirzB [strain 3], ~umuDC [strain 4], ~polB l OdinB
[strain 5],
~polB ldumuDC [strain 6], ~dirzB l DumuDC [strain 7], OpolB l ~dinB l DumuDC
[strain 8], lexA(S 119A) [strain 9], and 4recD [strain 10]; solid bars
represent base
substitution mutations and shaded bars represent codon deletion. Values
represent
number of resistant mutants per surviving cell per day. Error bars represent
standard
deviation from three independent rate determinations. The above data implies
that
induced mutation requiring point mutations (such as those involved in
ciprofloxacin
resistance) will be largely dependent upon Pol V, as opposed to those
involving
deletion/insertion mutation, which requires Pol IV.
[00322] In contrast to the minor changes in sensitivity to ciprofloxacin, as
shown in
Figure 3 and 10, deletion of any one of the three inducible polymerises has a
pronounced effect on mutation rate in the presence of the drug. Sequencing
revealed
that the reduced mutation rates in each deletion strain resulted from the
complete
absence of substitution mutations (solid portion of bars in Figure 10). In
contrast, the
rate of codon deletion remained virtually unchanged (hatched portion of bars
in
Figure 10). This implies that the three polymerises are all required for
substitution
mutation but not for deletion mutation
[00323] To further examine the role of each polymerise in codon deletion, the
double
mutants and triple polymerise mutants were examined. As shown in Fig. 15,
during
days 5 to 8, all deletion strains show low levels of codon deletion. However,
by days 9
to 13 each single mutant shows codon deletion rates indistinguishable from
wild type
cells, while the rates in the double and triple mutants remain low. This
implies that
codon deletion is mediated by a process involving multiple polymerises that
become
increasingly less efficient, although not absent, in the single, double, and
triple
polymerise mutants. As shown in Fig. 15, despite their persistence, these
deletion
mutants are unlikely to contribute to the evolution of clinically relevant
drug resistance,
as they remained significantly more sensitive to ciprofloxacin than the
substitution
derived mutants. This is consistent with the fact that clinically isolated
resistant strains
always contain substitution mutations. The evolution of clinically significant
antibiotic
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resistance appears to require substitution mutation, and thus all three LexA
repressed-
polymerases.
Example 6
[00324] Two naturally occurring inhibitors of the SOS response (DinI and PsiB)
were
found which serve as models of such inhibitors.
[00325] Two bacterial strains were constructed. One strain harbored a plasmid
over
producing DinI and another strain harboring a plasmid over producing PsiB.
Over
production of either protein crippled the ability of these strains to evolve
gyrA
mutation-mediated resistance to ciprofloxacin (35 ng/ml) by approximately 40-
fold.
Because clinical ciprofloxacin resistance (15 uglml) requires 3 - 5
independent point
mutations, we suspect that an in traps inhibitor of RecA-mediated LexA
proteolysis
would hinder the emergence of clinical ciprofloxacin resistance by at least
64,000-fold.
Example 7
[00326] Two groups of 10 neutropenic Swiss ICR mice were infected in their
thigh with
either E. coli ATCC 25922-OlacZ and ATCC 25922 lexA(S 119A) strains. (The LacZ
strain had a LacZ knock-out and was replaced with Kan marker as described
above; the
LexA strain had a LexA gene substituted with LexA S 119A as described above)
[00327] Two hours after infection the mice received rifampin at 100 mg/kg
(subcutaneous) every 12 hours. The mice continued the rifampin regimen until
they
were sacrificed. Two mice were sacrificed at 0, 24, 48, 72 and 96 hours. The
thighs
were removed, homogenized, 1:10 serial diluted, and plated for CFU
determination.
Homogenates were plated on MH agar (MHA) to quantify the entire population of
organisms and on rifampin containing MHA plates (16 mg/L) to quantify the
population of organisms with a rifampin resistance phenotype. Data from the
experiment are presented in Table 7 below.
TABLE 7
CFU/Thi
h
Time ZexA Strain ZacZ Strain
Media Media + Media Media +
Rif Rif
0 7.790.10 <2.0 7.750.09 <2.0
24 6.950.88 5.431.0 6.130.47 5.880.25
48 6.650.89 5.530.70 7.332.0 5.590.90
72 7.930.96 5.790.50 7.701.2 7.161.28
96 7.071.15 5.391.1 mice dead.
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[0032] In vitro susceptibility testing of strains was preformed using the
NCCLS
method. (See National Committee for Clinical Laboratory Standards. 1997.
Reference
method for broth dilution anti-fungal susceptibility testing of yeasts.
Approved standard
M27-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.) Eight
colonies from each group at the 72 hr time point from MHA plate with and
without
rifampin were obtained. Minimum inhibitory concentrations ("MICs") were
performed
in duplicate on three occasions on each colony. Minimum inhibitory
concentrations
concentration ranges tested were 0.03 to 16,384 mg/L as illustrated in Table 8
below.
Minimum inhibitory concentrations of both the LexA and LacZ strains pre-
treatment =
8.0 mg/L.
TABLE 8 - Minimum Inhibitory Concentrations
Strains Pre Post
LexA 8.0 mg/L 16-32 mg/L
LacZ 8.0 mglL >16,384 mg/L
Exafnple 8
[00329] In another experiment similar to the one preformed above, thigh high
infection
was produced in neutropenic CDl mice with either E. coli ATCC 25922-OlacZ and
ATCC 25922 lexA(S 119A) strains as described above. Two hours after infection
mice
were treated with rifampin 100 mg/kg twice daily via the subcutaneous route.
Groups
of two mice were sacrificed at the started of therapy (zero hour) and every 24
h for a
period of 72 h. After euthanasia, thigh were removed, homogenized, diluted,
and
plated for CFU enumeration (lower limit of detection is 2 log cfu/thigh).
Homogenate
dilutions were plated on MHA with and without rifampin (128 mg/L). Results are
illustrated in Figure 18 and demonstrate that LexA controls the SOS response
pathway
and that this pathway can be activated by various drugs, not just
ciprofloxacin. It also
demonstrates that at certain rifampin concentrations (e.g., about 128 mg/L),
growth of
parent strains (e.g., S 119A) would be inhibited.
[00330] In vitro susceptibility testing was performed with rifampin on the
parent strains
and on cells isolated from the agar plates after therapy.
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[00331] In the animal experiment with ATCC 25922 lexA(S 119A), no colonies
grew on
rifampin containing plates over the 72 h study period. A large number of E.
coli ATCC
25922-OlacZ colonies grew on rifampin containing plates after 72 h of therapy.
[00332] MICs of both parent strains was 8.0 mg/L. MICs of ATCC 25922 lexA(S
119A)
colonies after therapy remained 8.0 mg/L. MICs of E. coli ATCC 25922-~lacZ
colonies isolated from the rifampin containing plates were >256 mg/L.
Example 9
[00333] RecA protein (12.5 uM, Sigma) was activated by incubation for 30 min
on ice
in a solution containing 20 mM Tris pH 7.4, 4 mM MgCl2, 2 mM ATPgS, 1 mM DTT
and 60 ug/mL l8mer ssDNA (sequence: 5'-TTG TTG TTG TTG TTG TTG-3'). A
solution of LexA (5 uM) containing 20 mM Tris pH 7.4, 5 mM MgCl2, 1 mM ATPgS,
2 mM DTT and no peptide or the indicated peptide (at 1.2 mM for peptide 1 (SEQ
ID
NO: 4), 1.8 mM for peptide 2 (S~Q ID NO: 5), 1.5 mM for peptide 3 (SEQ ID NO:
2))
was incubated at 37 degrees for 5 minutes. Cleavage of LexA was initiated by
the
addition of 0.25 uM activated RecA. The mixture was incubated at 37 degrees
for 30
min. Aliquots were removed and quenched in aqueous acetonitrile containing 0.1
%
TFA.
[00334] The cleavage of LexA protein was monitored by HPLC using a gradient of
30 to
90% buffer B (9.9%H20, 90%CH3CN, 0.1%TFA) in buffer A(98%H20,
1.9%CH3CN, 0.1%TFA) and recording absorbance at 214 nm. Results are
illustrated
in Figure 20. Error bars represent one standard deviation of three separate
experiments.
Figure 21 illustrates a comparison of LexA autocleavage inhibition by peptide
3 over a
30 minute period of time.
Exa»aple 10
[00335] Despite stringent hygiene practices, hospitals and elder-care
facilities remain
two of the major breeding grounds for antibiotic resistant bacteria, as well
as ideal
environments for their transmission. MRSA (Methicillin-resistant
Staphylococcus
aureus), VRE (Vancomycin-resistant Enterococcus), and MDR (mufti-drug
resistant)
pneumonia are only a few of a spectrum of "hospital infections" that could be
curbed
with the aid of a resistance-suppressing agent.
[00336] It is quite common for already drug resistant bacterial strains (e.g.,
MRSA or
VRE) to infect patients who initially come to a hospital free of infection.
The present
CA 02544018 2006-04-27
WO 2005/056754 PCT/US2004/039064
invention contemplates the use of an achaogen to kill dangerous mufti-drug-
resistant
strains such as MRSA and VRE.
[00337] In particular, rifampin has fallen out of common clinical use, because
rifampin
resistance emerges within 24 hours from the initiation of treatment. See
Example 10
above. Because the mutations that confer rifampin resistance impose a
significant
growth disadvantage on bacteria, resistant bacterial populations promptly
revert to
rifampin sensitivity within a few weeks of cessation of treatment. As such,
nearly all
MRSA and VRE strains encountered in hospitals are initially rifampin
sensitive. The
present invention contemplates the use of an achaogen in combination with
rifampin to
kill dangerous gram-positive organisms resistant to one or more other
antibiotics, but,
initially sensitive to rifampin. Preferably, an achaogen and rifampin are co-
formulated
to create an 'evergreen' drug to which resistance could not be maintained.
[00338] Example 11
[00339] Bacteria can be classified as gram-positive or gram-negative by the
following
Gram test steps. Bacterial cells are dried onto a glass slide and stained with
crystal
violet, then washed briefly in water. Iodine solution is added so that the
iodine forms a
complex with crystal violet in the cells. Alcohol or acetone is added to
solubilise the
crystal violet - iodine complex. The cells are counterstained with safranin,
then rinsed
and dried for microscopy. Gram-positive bacteria retain the crystal violet-
iodine
complex and thus appear purple (shown for Bacillus cereus in the left-hand
image
below). Gram-negative bacteria are decolourised by the alcohol or acetone
treatment,
but are then stained with safranin so they appear pinlc (shown for Pseudomonas
aeruginosa in the right-hand image below). Thus, the essential difference
between
Gram-positive and Gram-negative cells is their ability to retain the crystal
violet-iodine
complex when treated with a solvent.
[00340] Examples of gram-negative bacilli include E. coli, (causes UTI and
other
infections) Eraterobacter (causes UTI and other infections), Psea~domonas
aueruginosa
(UTI, pneumonia and bacteremia), Salmonella (causes typhoid fever, paratyphoid
fever, bacteremia, and acute gastroenteritis), Shigella (acute
gastroenteritis),
Campylobacter (causes enteritis, bacteremia, endocarditis, and meningitis),
Vibrio
cholerae (causes cholera).
[00341] Examples of gram-positive include B. anthracis which causes anthrax
and
pneumonia
86
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WO 2005/056754 PCT/US2004/039064
[00342] Examples of gram negative cocci include Neisseria, which causes
gonorrhea
and meningitis.
[00343] Examples of gram positive cocci include Staphylococci, which causes
abscesses, bacteremia, endocarditis, pneumonia, osteomyelitis, an dcellulitis.
[00344] All publications and patent applications mentioned in this
specification are
herein incorporated by reference to the same extent as if each individual
publication or
patent application was specifically and individually indicated to be
incorporated by
reference.
[00345] It will be apparent to one of ordinary skill in the art that many
changes and
modifications can be made thereto without departing from the spirit or scope
of the
appended claims.
[00346] All publications and patent applications mentioned in this
specification are
herein incorporated by reference to the same extent as if each individual
publication or
patent application was specifically and individually indicated to be
incorporated by
reference. It will be apparent to one of ordinary skill in the art that many
changes and
modifications can be made thereto without departing from the spirit or scope
of the
appended claims.
87
