Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR TREATMENT OF DISEASE
Priority
This application claims priority from United States Provisional Application
No. 60/166,60, filed November 19, 1999, the contents of which are hereby
incorporated by
reference in their entirety.
Technical Field
The present invention relates to the treatment of disease and infection caused
by non-eukaryotic microorganisms, particularly bacteria and mycoplasma.
Background of the Invention
to Infectious disease remains the largest cause of mortality in the world. A
significant proportion of infectious disease-associated morbidity and
mortality results from
prokaryotic pathogens, particularly bacteria. The process and underlying
mechanisms of the
infectious process have been the subjects of intensive study for several
decades.
Bacteria respond to nutritional stress by the coordinated expression of
different genes. This facilitates their survival in different environments.
Among these
differentially regulated genes are the genes responsible for the expression of
virulence
determinants. The selective expression of these genes in a sensitive or
susceptible host
allows for the establishment and maintenance of infection or disease.
Virulence genes
include those which encode toxins, colonization factors and genes required for
siderophores
production or other factors that promote this process.
The expression of virulence genes in bacteria therefore enables the organism
to invade, colonize and initiate an infection in humans and/or animals. These
genes are not
necessarily expressed constantly (constitutively), however. That is, the
bacterium is not
always orchestrating gene expression patterns to maximize "infectious"
potential. In many
circumstances, the expression of virulence genes is controlled by regulatory
circuitry that
include repressor proteins and a corresponding operon or operator. One class
of repressors
that are activated upon binding to or forming a complex with a transition
metal ion such as
iron, zinc or manganese, is thought to control the expression of a subset of
genes in a
number of Gram positive organisms. When such repressors are activated and
associated
with virulence gene expression in pathogens, they bind to operator sites
thereby preventing
production of virulence determinants.
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Virulence determinants are most often expressed when the bacterial pathogen
is exposed to environmental stress such nutritional restriction. An iron-poor
environment is
an example of such a condition. In many eukaryotes, insufficient free iron is
present to
maintain the repressor in its active state. In the inactive form, the
repressor cannot bind to
target operators. As a result, virulence genes are de-repressed and the
bacterium is able to
initiate, establish, promote or maintain infection.
The expression of these virulence determinants is in many bacterial species
co-regulated by metal ions. In most instances, the metal co-factor that is
involved in vivo is
iron but can include zinc, nickel, manganese and cobalt. In the presence of
iron, the
l0 repressor is activated and virulence gene expression is halted.
This pattern of gene regulation is illustrated by the following example. The
bacterium that causes diphtheria produces one of the most potent toxins known
to man. The
toxin is only produced under conditions of iron deprivation. In the presence
of iron, the
bacterial repressor (which in this species is known as diphtheria toxin
repressor protein,
abbreviated "DtxR") binds iron and undergoes conformational changes that
activate it and
allow it to dimerize and bind a specific DNA sequence called the tox operator.
The tox
operator is a specific DNA sequence found upstream of the gene that produces
the
diphtheria toxin, thereby preventing its expression. Typically, during
infection of a human
host the diphtheria bacillus (or other pathogenic/opporiunistic bacteria)
grows in an
environment that rapidly becomes restricted in several key nutrients.
Paramount among
these essential nutrients is iron, and when iron becomes limiting the
diphtheria bacillus
begins to produce the toxin. Moreover, the constellation of virulence genes
that DtxR
controls become de-repressed and the diphtheria bacillus becomes better
adapted to cause
an infection. In the case of diphtheria, the toxin kills host cells thereby
releasing required
nutrients including iron.
Antibiotic therapy has been the accepted mode of treatment for bacterial
infections and diseases. As a consequence of the widespread use and perhaps
even misuse
of antibacterial drugs, however, strains of drug-resistant pathogens have
emerged.
Antibiotic-resistant bacterial strains have been associated with a variety of
infections,
including tuberculosis, gonorrhea, staphylococcal and pneumococcal infections,
and the
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3
bacteria most commonly associated with pneumonia, ear infections and
meningitis. More
importantly, infectious disease remains the largest cause of mortality in the
world.
The typical response to an ineffective antibiotic has simply been change
antibiotics. Unfortunately, this alternative no longer offers a guarantee of
success. For
example, certain strains of enterococci are resistant to vancomycin -- a drug
formerly
considered as the ultimate weapon against many different types of bacteria.
The World
Health Organization has expressed concern that the development of new drugs is
not
keeping pace with the numbers of antibiotics that become ineffective. World
Health Report
1996: FiQhtin~ Disease, Fostering Development, Executive Summary (World Health
1o Organization 1996). Despite ongoing research, there remains a pressing need
to develop
new antibiotics. There is also a need for anti-bacterial agents that are
effective in treating
disease while not stimulating the emergence of resistant strains.
Summary of the Invention
The present invention is directed to compositions and methods for treating
infection and disease in mammals caused or mediated by non-eucaryotic
pathogenic
microorganisms such as bacteria and mycoplasma. The therapeutic agents
administered to
the mammals promote activation of a protein (such as a repressor protein) that
regulates
virulence gene expression in the pathogen. The activation of the protein
results in
attenuated or reduced infectiousness of the pathogens. The agents of the
present invention
2o are Sarcoma homology domain 3 (SH3) ligands; they bind SH3 domains present
in the
native proteins.
Proteins possessing SH3 domains are common in eucaryotes. They play a
role in controlling the activity of certain enzymes that transmit signals
between the
eucaryotic cell and its external environment. As their name implies, they have
received
considerable interest as potential targets for the development of drugs to
treat malignancies
in humans. SH3 domains were not known to exist in prokaryotic or bacterial
proteins, or
for that matter, to help regulate virulence gene expression in prokaryotic
pathogens. The
therapeutic agents include peptides and non-peptides alike. Known agents that
target the
SH3 domain in eucaryotic proteins may be used in the present methods. Newly
discovered
3o SH3 ligands that contain a proline-rich peptide are also provided.
Brief Description of the Figures
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Fig. 1. (a) Chemical shift deviation (CSD) between the measured H~' chemical
shift (14)
and the random coil H°' value (25). Clusters of positive and negative
values suggest ~-
strands and helical structures, respectively. (b) Backbone 15N-{'H}
heteronuclear NOE for
each residue. NOE values for some residues were not presented because of
resonance
overlap. (c) The number of proton-proton NOES per residue (No. NOE/res). (d)
The
backbone rms deviation per residue of the 20 structures from the average
structure. The
helical (open ellipses) and ~-strand (solid squares) regions of DtxR(130-226)
are depicted at
the top.
Fig. 2. Structure of DtxR(130-226) with the unstructured residues 130-145
omitted.
Stereoview of the backbone superposition of the 20 refined structures
determined as
described in the text. The N terminus of the structure is located at lower
left.
Fig. 3. Nondenaturing polyacrylamide gels indicating the degree of
oligomerization of
DtxR(130-226) (lane A) and DtxR(144-226) (lane B). The reduced electrophoretic
mobility
of DtxR(144-226) monomer compared to DtxR(133-226) monomer reflects the
additional
residues at the N terminus of DtxR(144-226) arising from the expression
construct (see
Materials and Methods).
Fig. 5. Stereoview of the C~ trace of the SH3-like domain of DtxR showing the
residues
implicated in peptide binding. Residues that shifted upon addition of the
peptide are
depicted as red balls.
Best Mode of Carrying out the Invention
SH3 stands for Sarc homology domain 3 as the structure was originally
identified in a protein kinase that when deregulated is associated with the
development of
sarcoma. It is well known that eukaryotic SH3 domains are important regulatory
elements
that function through the recognition of proline-rich motifs that specify
distinct regulatory
pathways important for cell growth, migration, differentiation, and responses
to the external
milieu. In general, the Src homology 3 (SH3) domain is a 50 amino acid modular
element
that is found in a number of eukaryotic non-receptor tyrosine kinases (e.g.,
Src, Fyn, Lyn,
Yes, PI3K, Hck, Itk/Tsk). The SH3 domain has been proposed to provide a
regulatory
function in Src and related tyrosine kinases. In the inactive forms of Src (Xu
et al., 1997),
Hck (Sicheri et al., 1997) and Itk (Andreotti et al., 1997), the SH3 domain
was found to be
bound to an internal, proline-containing region that links the SH2 and
catalytic domains and
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thereby stabilize the inactive form of the kinase. The SH2 domain is composed
of three
antiparallel beta-sheets with two shorter beta sheets, betaA and betaG. SH2
domains bind
phospho-tyrosine-containing peptides having the sequence pTyr-Glu-Glu-Ile. The
SH3
domain is composed of a five stranded up-and-down antiparallel ~~ structure
that is twisted
5 into a barrel such that they form two anti-parallel sheets that pack against
each other. While
some SH3 domains have been shown to contain small regions of secondary
structure, this
fold is common to all known SH3 domains. SH3 domains specifically bind proline-
rich
peptides of approximately 10 amino acid residues in length. Two distinct
classes of
peptides have been described, namely class I (RXXPXXP) and class II (PXXPXR).
l0 Dalgarno & Kaye. These ligands each bind to SH3 domains in one of two
pseudo-
symmetrical orientations. This functional interaction between proline-rich
peptides and
SH3 domains from Src, Fyn, Lyn, Yes, PI3K, Hck, and Itk/Tsk kinases has been
successfully examined by M13 phage display (Bunnell et al., 1996; Rickles et
al., 1995;
Schumacker et al., 1996; Sparks et al., 1995; Feng et al., 1995).
In preferred embodiments of the present invention, the therapeutic agents are
administered to mammals to treat caused or mediated by gram positive bacteria
having
virulence gene expression regulated, at least in part, by DtxR or a DtxR
homolog.
Applicants have established that the C-terminal domain of DtxR folds into an
SH3 domain,
and like its eucaryotic counterparts, binds proline-rich peptides. Applicants
have also
established that disruption of normal C-terminal SH3 domain function modulates
DtxR
activation. In other words, SH3 ligands with sufficiently high affinity
promote activation of
DtxR, which in turn, leads to suppression of virulence gene expression.
Applicants have
further established that DtxR homologs also possess SH3 domains and
corresponding
polyproline-rich docking sites.
DtxR is a metal dependent repressor which under limiting concentrations of
metal ions becomes inactivated permitting the derepression of a number of
virulence genes
including diphtheria toxin. The repressor contains a metal binding domain that
binds iron
and subsequently allows the dimerization of DtxR and repression of virulence
gene
expression in vivo. Manabe, et al., Proc. Natl. Acad. Sci. USA 96:12844-12848
(1996).
More specifically, DtxR is a metal iron-dependent DNA-binding protein having a
deduced
molecular weight of 25,316 and which functions as a global regulatory element
for a variety
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of genes on the C. diphtheriae chromosome. See Tao, et al., Proc. Natl. Acad.
Sci. USA
89:5897-5901 (1992); Schmitt, et al., Infect. Immun. 59:1899-1904 (1994). For
example,
DtxR regulates the expression of the diphtheria toxin structural gene (tox) in
a family of
closely related Corynebacteriophages. The DtxR gene has been cloned and
sequenced in E.
coli and its DNA and amino acid sequences have been reported. See Boyd, et
al., Proe.
Natl. Acad. Sci. USA 87:5968-5972 (1990); Schmitt, et al., supra. DtxR is
activated by
divalent transition metal ions (e.g., iron). Once activated, it specifically
binds the diphtheria
tox operator and other related palindromic DNA targets. See Ding, et al.,
Nature Struct.
Biol. 3(4):382-387 (1996); Schiering, et al. Proc. Natl. Acad. Sci. USA
92:9843-9850
to (1995); White, et al., Nature 394:502-506 (1998). DNA sequences encoding
DtxR from
various C. diphtheria strains are defined by accession numbers M80336,M80337,
M80338,
and M34239.
DtxR homologs are prevalentGram-positive bacterial
in species,
particularly those listed
in Table 1. The diseases
caused by the mycobacterial
staphylococcal, and species are larly preferred indications
streptococcal particu for the
purposes of the present Mycobacteriumcause significant disease
invention. that include M.
tuberculosis, M smegmatis
and M. leprae.
TABLE 1
S. pneumoniae S. agalactia S.equisimillis
S. meningitis S. bovis S.anginosus
S. pyogenes S. salivariusS. sanguis
S. suis S. mutans Enterococcus faecalis
Staphylococcus species
S. aureus S. epidermitis
Mycobacterium species
M. tuberculosis M. avium complexM. kansasii
M. leprae M. scrofulaceumM. fortuitum
M. ulcerans M. marinum M. bovis
M. microtii M. africanum
3o Actinomyces species
A. pyogenes A. israelii A. bovis
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A. viscosus A. hordeovulneris A. gerencseriae
A. naeslundii A. odontolyticus
Listeria monocytogenes
Proprionibacterium acnes
Erysipelothrix rhusiopathiea
A collection of accession numbers for sequences that are either homologous
to DtxR or contain a consensus tox O/P is presented in Table 2. See also
htt~://www.ncbi.nlm.nih.~ovBLAST and
httn://www.ncbi.nlm.nih.gov/unfmishedgenomes.html. See also, Altschul, et al.,
J. Mol.
l0 Biol. 215:403-410 (1990); Gish, et al., Nature Genet. 3:266-272 (1993);
Madden, et al.,
Meth. Enzymol. 266:131-141 (1996); Altschul, et al., Nucleic Acids Res.
25:3389-3402
(1997); and Zhang, et al., Genome Res. 7:649-656 (1997). This high degree of
sequence
similarity and homology indicates that the iron regulatory pathway that
employs the DtxR-
family of repressors is conserved in many important human and animal
pathogens.
Table 2
DtxR Homologs and Species with DtxR Binding Sites
Pathogenic Other
Human/Veterintary
Applications
CAA67572 S. epidermitis L35906 C. glutamicum
Gi 1777937T. pallidum 250048 S. pilosus
CAA15583 M. tuberculosis 250049 S. lividans
U14191 M. tuberculosis U14190 M. smegmatis
L78826 M. leprae M50379 M. jannaschi
M80336 C. diphtheriae Gi 2621260 M.thermoautotrophicum
M80337 C. diphtheriae Gi 2622034 M. thermoautotrophicum
M34239 C. diphtheriae 033812 S. xylosus
M80338 C. diphtheriae Q57988 M. jannaschi
AAD18491 C. pneumoniae Gi 264870 A fulgidus
Gi 3328463 C. trachomatis Gi 2648555 A fulgidu
TIGR 1280S. aureus Gi 2650396 A fulgidus
Stanford S. meliloti Gi2650706 A fulgidus
382
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AE001439 H. pylori BAA79503 A. pernix
TIGR 1752 V. cholera CAB49983.1 P. abyssi
TIGR1097 C. tepidum BAA30263 P. horikoshi
OUACGT S. pyogenes AL109974 S. coelicolor
Sanger B. bronchosepticaL35906 B. lactofermentum
518
Sanger 1765 M. bovis AE000657 A. aeolius
Sanger 520 B. pertusis TIGR 920 T. ferrooxidans
WUGSC K. pneumoniea TIGR 76 C. crescentus
TIGR 24 S. putrificacieus
l0 TIGR 1351E. faecalis
AE000783 B. burgdorferi
TIGR1313 S. pneumoniea
Sanger 632 Y. pesos
Table 3 depicts a sequence alignment that illustrates the high degree of
conservation in
DtxR type repressors from a number of clinically important species, including
DtxR from
Brevibacterium lactofermentum (B1), DtxR from Corynebacterium diphtheriae
(Cd); IdeR
from Mycobacterium segmatus (Ms), IdeR from Mycobacterium tuberculosis (Mt);
DesR
from Streptomyces lividans (S1), DesR from Streptomyces pilsous (Sp) and SirR
from
Staphylococcus aureus (Sa). The consensus amino acid sequences between these
members
of the DtxR family of iron-dependent repressors is indicated. *, metal ion
coordination
residues in the Primary site; #, metal ion coordination residues in the
Ancillary site; @, the
single amino acid residue that interacts with a base in the binding of DtxR
dimers to the tox
operator. Grey area is the highly conserved iron and DNA binding domain. The C-
terminal
domains exhibit a high degree of structural homology and exhibit an extremely
high degree
of similarity in the functionally significant polyproline region (shown in
black).
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CONSENSUS M--L-DTTEM YLRTI--LEE EGV-P-RARI AERL-QSGPT VSQTV-RMER DGL--V--DR
* @
70 80 90 100 110 120
B1 DtxR
Cd DtxR
Ms IdeR
Mt IdeR
SI DesR
Sp DesR
Sa SirR
CONSENSUS -L--T--GR- LA--VMR--R LAE-LL-D-I ------VH-E ACRWEHVMS- -VERR----L
# # # * **
150 160 170 180
B1 DtxR DQADEPDSGV RAIDLPLGEN LKARIVQLNE ILQVDLEQFQ
Cd DtxR GNSDAAAPGT RVIDAATSMP RKVRIVQINE IFQVETDQFT
Ms IdeR TPGVNTEDVS LVRLTELPVG MPVAVVVRQL TEHVQGDTDL
Mt IdeR GPEPGADDAN LVRLTELPAG SPVAVVVRQL TEHVQGD)DL
Sl DesR TDGADPFLDE GMVSLADLDP GQEGKTVVVR RIGEPIQTDA
Sp DesR KDGADPFLDE GMVSLAELDP GAEGKTVVVR RIGEPIQTDA
Sa SirR SDAAAPGT SILNFEPGER VTVRRV RRDK TELL
CONSENSUS -----SP-GN PIPGL-EL-
190 200 210 220 230
B1 DtxR ALTDAGVEIG TEVDIINEQG RVVITHNGSS VELIDDLAHA VRVEKVEG
Cd DtxR QLLDADIRVG SEVEIVDRDG HITLSHNGKD VELLDDLAHT )ItIEEL
Ms IdeR IGRLKEAGVV PNARVTVEAN NNGGVMIVIP GHEQVELPHH MAHAVKKKVE KVEKV
Mt IdeR ITRLKDAGVV PNARVTVETT PGGGVTIVIP GHENVTLPHE MAHAVKVEKV
SI DesR QLMYTLRRAG VQPGSWSVT ESAGGVLVGS GGEAAELEAD TASHVFVAKR
Sp DesR QLMYTLRRAG VQPGSWSVT EAAGGGVLVG SSGEAAELET DVASHVFVAK P
Sa SirR VYLSSKDIYI GNTVEIVSKD DTNKVIILKR NDIVTILSYE NAMNIFAEK
CONSENSUS -__-______ __________ _-________ _________ __________ -_-_-
Sequence similarity among DtxR homologs is also reported in Schmitt, et al.,
Infect Immun.
63(11):4284-4289 (1995); Doukhan, et al., Gene 165(1):67-70 (1995); Oguiza, et
al., J.
Bacteriol. 177(2):465-467 (1995); Giinter, et al., 1. Bacteriol. 175:3295-3302
(1993); and
4o Schmitt, et al., Infect. Immun. 63:4284-4289 (1995).
Diseases caused or mediated by other non-eucaryotic pathogenic
microorganisms, including Gram-negative bacteria and mycoplasma, and which are
also
characterized by SH3 domain-mediated modulation of virulence gene expression,
are
included within the scope of the present invention.
Under physiological conditions in the presence of iron, the DtxR-type
repressors are activated and suppress iron dependent gene regulation. When
iron becomes
limiting both in vitro and in vivo, the repressors surrender iron and undergo
a
conformational shift and deactivation, a process that involves the SH3 domain.
Deactivation of the repressors permits the expression of iron dependent genes
which in
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many human and animal pathogens, encode virulence factors that promote the
establishment, growth and maintenance of infection. Activation of the SH3
domain, and in
turn, suppression of virulence gene expression leading to attenuation of
infectiousness, may
be achieved by displacement of the SH3 domain from its native or endogenous
polyproline
5 docking station, even in iron-poor environments. Therefore, compounds that
mimic the
endogenous polyproline sequence and/or bind the SH3 domain contained in the
repressor
with sufficient affinity to inhibit binding with the native docking station,
are useful as
therapeutic antimicrobial agents. The sequence of the SH3 docking site in DtxR
that
Applicants have identified is as follows: VSRSPSGNPIPGLDELGV.
10 In more preferred embodiments, the therapeutic agents of the present
invention contain a polyproline peptide sequence that reproduce potential
recognition motifs
for the SH3 domains of bacterial repressors. The peptides described below
share common
properties of the expanding library of proline peptides that appear to be
involved in the
regulation of protein associations i.n eukaryotic cells. These characteristics
include the
presence of one or more peptides within a hydrophobic stretch of amino acids
often
containing one or more possible phosphorylation sites, serine or threonine
residues.
The sequences described below have been identified by distinct methods. By
virtue of the techings in example 1, we have cloned and identified a number of
DtxR
homologues. Nucleotide sequence analysis and determination of primary amino
acid
sequence confirms the presence of a conserved proline containing sequence in
region
equivalent to amino acids 126-136 of DtxR. Each of these stretches of amino
acids are
putative SH3 ligands for the development of high affinity competitive agonists
of DtxR or
DtxR homologues. At least two distinct families of sequences can be obtained.
Synthesis of a degenerative library of oligo-nucleotides encoding all possible
derivations of
amino acids or subspecies of this library can be created by in vitro
synthesis. Such a library
is systematically created by randomizing the addition of bases in an oligo-
nucleotide library
and then expressing these peptides in the PSDT system as described below in
Example 3. A
general method of preparing a randomized population of molecules based upon
synthesized
oligonucleotides is described in Park and Raines Nature Biotechnolo~y (2000)
Genetic
Selection of for dissociative inhibitors of designed protein protein
interactions 18 847-851.
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This paper also presents a strategy that could be employed to screen for
additional ligands
that would bind to the DtxR SH3 domain.
Alternatively, a series of parental oligo-nucleotides encoding the conserved
proline sequences in DtxR [or any homologue] may be created and cloned into a
suitable
expression vector such as those described below in Example 3. Alternatively,
the peptide-
encoding mingenes could also be cloned into a vector such as M13KE [New
England
Biolabs] or pSKAN [Mo Bi Tech] which express the peptides via phage display
such as on
the minor coat protein pI)1 of M13. Suitable primers can be prepared from the
flanking
regions to allow the amplification of the intervening peptide encoding nucleic
acid
1o sequence. By employing saturation mutagenesis as described by Vartainian a
PCR
generated library of all possible combinations of peptide minigene is created.
These
minigenes can be used to replace the sequences encoding the native proline
containing
sequences in pRCD, pBADT [a,b,c], pSKAN, or pMl3KE. Functional screening as
described in Example 3. or affinity selection of peptides by phage display
Example 2. can
be employed to generate additional peptides that both bind the C-terminal SH3
domain and
activate this family of repressors.
The PSDT screen described below can also be modified such that any
repressor/operator couple from any species employing a DtxR type repressor can
be used in
functional screening. Using the standard molecular biology techniques as in
Example 1, it
2o is also possible to express and utilize any species specific repressor C-
terminal domain for
phage display and peptide library panning.
We have initially employed PCR approaches using oligo-nucleotide primers
to conserved regions of the N-terminal domains of DtxR to isolate additional
DtxR
repressors from other species. Once cloned we have analyzed the nucleotide and
amino
acid sequence to determine if the putative homologues contain the conserved
proline region
found in DtxR. With the advent of high through put publicly sponsored
sequencing of
microbial genomes it is now possible to scan for DtxR homologues through NCBI.
Identification of the potential SH3 domain- internal proline ligand switches
is readily
completed by performing searches for DtxR homologues and multiple sequence
alignments
to identify internal proline ligands. Below is a list of swequences which we
have obtained
by cloning and sequencing of DtxR homologues [CD/SE/SA/SM/EF] or from the NCBI
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database. The sequences below are from microbes which are associated with
bacterial
disease with the exception of 'BL' Brevibacterium lactofermtum and 'MS'
Mycobacterium
smegmatis. An additional list of internal sequences is presented below the
Group I-3
sequences from recently reported DtxR homologues of predominantly non
pathogenic
bacteria.
List of Internal Sequences broken into three families
Alanine Ala A
Cysteine Cys C
Aspartic
AciD Asp
D
Glutamic
Acid Glu
E
Phenylalanine
Phe F
Glycine Gly G
Histidine His H
Isoleucine Ile I
Lysine Lys K
Leucine Leu L
Methionine Met M
AsparagiNe Asn N
Proline Pro P
2o GlutamineGln Q
ARginine Arg R
Serine Ser S
Threonine Thr T
Valine Val V
TryptophanTrp W
Tyrosine Tyr Y
Hydrophobic AA A/V/F/P/M/1/I.
Charged AA D/E/K/R
Polar AA S/T/Y/H/C/N/Q/W
Group 1.
Where Pro=proline, Gly = glycine, Ile= isoleucine, P*=a polar amino acid which
is thr or
ser, H = a hydrophobic amino acid, [+/-] = a charged amino acid and P= a polar
amino acid.
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Consensus sequence for group 1 have a length of 12-14 amino acids of the
general
sequence:
Pro/P*/P*/P*/Pro/H/Gly/P/Pro/Ile/Pro/Gly/H or [+/-]/[+/-]/H/Gly
Natural examples of Group 1. ligands from DtxR homologues.
B1 DtxR VHRSPFGN PIPGLGEIGL
Cd DtxR VSRSPFGN PIPGLDELGV
to Mt IdeR PTTSPFGN PIPGLVELGV
Ms IdeR PTTSPFGN PIPGLTELAV
MI IdeR PTTSPFGN PIPGLLDLGA
SI DesR PTESPYGN PIPGLEELGE
Mtb SirR PQRDPHGD PIPGADGQVP
Group 2
Consensus sequence for group 2 have a length of 12-14 amino acids of the
general sequence
shown below with the fourth polar residue preferably as cysteine the sixth
polar residue a
histidine:
Pro/[-/+]/P*/P/Pro/P/Gly/Gly/Val/Ile/Pro/[+/-]/P or [+/-]/[+/-]
Natural examples of Group 2. ligands froms DtxR homologues.
Se SirR PKTCPHGG VIPRGNSDAA
Sa SirR PETCPHGG VIPRNNEYKE
Ef PEFCPHGG VIPEDNQPIH
Group 3
Consensus sequence for group 3 have a length of 12-18 amino acids of the
general sequence
shown below with the second amino acid being lysine, the fourth amino acid
preferably
being cysteine,and he sixth polar amino acid preferably being histidine.
Pro/[-/+]/P* or H/H/Pro/P/Gly/Gly/Thr/Ile/Pro/H/P or [+/-]/Gly/[+/-]/H/H
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Natural examples of Group 3. ligands froms DtxR homologues.
Sg PKACPHGG TIPAKGELLV
Sm PKVCPHGG TIPGHGQPLV
Spn PKTCPHGG TIPAKGELLV
Spy PKTCPHGG TIPAKGELLV
*Additional Ligands could be developed by the methods disclosed herein from
the
following internal proline rich regions from the DtxR homologues found in
these species.
l0 The following list presents non pathogens and homologues identified by
partial sequence
analysis in unfinished genomes at NCBI
Methanobacterium thermoautotrophicum D69126 pgecpdekpipacefk
Rhodococcus erythropolis AAF36925ttspygnpipgldqlg
Sulfolobus solfataricus pttcphghpignrikv
CAB57634
Deinococcus radiodurans C75261pthdphgdpiptlege
Thermoplasma acidophilum CAC12001vdrcphgnpipdpegn
Archaeoglobus fulgidus 669497 refcpcgkripevkk
Mycobacterium avium pttspfgnpipglldlgvgpesg
2o Mycobacterium bovis pttapfgnpipglvelgvgpepg
In Example 1, figure 3 displays the polymerization of the C-terminal SH3
domain region of DtxR resolved by PAGE in native and denaturing conditions.
Putative
SH3 ligand competitive inhibitors [of internal SH3 ligands] could be screened
by incubation
with the C-terminal domain including residues 120-140 [the internal ligand
residues] and
then analysis by PAGE gel under native conditions. If the peptides or
synthetic compounds
being tested disrupt normal association between the SH3 domain and the
internal ligand the
multimeric complexes observed in the left lane of the gel depicted in figure 3
of Example 1
will not be observed. The C-terminal domain will be resolved essentially as
shown in the
3o right lane of the gel, as a single monomeric form.
Similar to the methods disclosed above any competitive binding assay which
measures the association of labeled internal SH3 ligand based peptide
[residues 125-140 of
DtxR or analogous peptide from a DtxR homolog] to the cognate DtxR or DtxR
homolog
could be used to test for competitive inhibitors of this association. Labeled
peptides can
readily be obtained by I125 labeling peptides or by purchasing fluorescently
labeled
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peptides from vendors. Competition between the SH3 domain and the labeled
peptide by
unlabeled test substances constitutes a method of identifying potential
repressor activators.
To demonstrate that the C-terminal domain of DtxR bound proline
containing ligands we employed phage display to affinity select peptides by
phage display
5 from a random peptide library. The C-terminal domain of DtxR was immobilized
onto a
substrate through a poly-histidine tail placed N-terminal to a GSG space fused
to residue
129. This immobilized SH3 containing domain served as a trap for M13 phage
expressing
epitopes built from a random peptide library. Multiple rounds of panning and
amplification
were performed and a set of affinity purified phage particles was obtained.
Sequence
to analysis of the pill protein [where the random peptides are fused] revealed
the sequences
depicted in Example 2. This technique has been employed extensively in the
study of
eukaryotic SH3 domains and has provided an affinity based approach for
classifying
different SH3 domains and in identifying potential lead compounds targeted at
eukaryotic
proteins modulated by SH3 mediated interactions. The approach is reviewed in
Zarrinpar
15 and Lim (Nature Struct Biol. 2000 7:611-613, and Kay, Williamson and Sudol
FASEB
(2000) 14:231-241) and examples of the technique are presented by Weng et al
(MCB
(1995) 15:5627-5634) and Sparks et al (PNAS (1996) 93:1540-1544 in which
rational
dissection of core and specificity residues are pursued and discussed in
detail. These
approaches can also be used to identify ligands as building blocks for
combinatorial
chemistry efforts to produce compounds which exhibit specificity from
prokaryotic SH3
domains (Dalgarno & Kaye; Parks AB, Adey NB, Quilliam LA, Thorn JM, & Kay BK.
Methods in Enrymology, 1995; 255:498-509., Rickles RJ, Botfield MC, Zhou XM,
Henry
PA, Brugge JS, & Zoller MJ. Proc Natl Acad Sci, USA, 92:10909-12913. ,Kapor,
TM,
Andreotti, AH, and Schrieber, SL (1998) JACS 120(1)., Feng S, Kasahara C,
Rickles RJ, &
Schreiber SL. Proc Natl Acad Sci, USA, 1995; 92:12408-12415.) Thus we have
employed
phage display to demonstrate first that the SH3 domain of DtxR and by analogy
this class of
repressors indeed bind proline peptide ligands and that in the presence of the
internal
polyproline [PIP] sequence that additional [high] affinity proline peptides
can be obtained.
Grouped generically as P=polar AA, P*=Serine or Threonine,
3o H=hydrophobic, G=glycine, - =negatively charged AA, += positively charged
AA,
Pr=proline These peptides have a general sequence of:
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H/P/[+/-]/P/[+/-]/H/Pr H/P/H/G/H/Pr/Pr H/G/[+/-]P/H/Pr/Pr
P*/[+/-]/H/P*/H/Pr/H [+/-]/P[+/-]/H/Pr/P/Pr P/Pr/P/H/H/P/Pr
Other therapeutic agents useful in the present invention, both peptide and
non-peptide alike, may be identified following methods and screening assays
reported in the
literature in connection with eucaryotic SH3 domains. Dalgarno, et al.,
Biopolymers
43:383-400 (1997), for example, reviews the nature of several well-
characterized
to intracellular SH3-ligand interactions found in eucaryotic systems, as well
as current
approaches for design and synthesis of SH3 ligands. One such approach entails
mimicking
the preassembly of the polyproline helices observed in proteins by replacing
the proline-rich
core with a rigid organic moiety (referencing Witter, D., et al., (1997)
presented at 5'h
Chemical Congress of North America, Cancun, Mexico, Poster Presentation 1080).
Another main strategy discussed involves exploration of the specificity pocket
of the SH3
domain binding site using combinatorial chemistry (referencing Combs, et al.
(1996) J. Am.
Chem. Soc. 118, 287-288, and Feng, et al. (1996) Chem. Biol. 3, 661-670).
Dalgarno
further describes a phage display approach using a synthetic D-amino acid Src
SH3 domain
(referencing Schumacher, et al., (1996) Science 271, 1854-1855). The
technique, named
"mirror-image phage display", involves inverting the chirality of the SH3
domain by
producing a D-enantiomic form of the protein from D-amino acids. L-amino acid
phage
libraries are screened with the D-SH3 domain, and are equivalent to screening
a D-amino
acid phage library with the native L-SH3 protein. Kapoor, et al, J. Am. Chem.
Soc. 120:23-
29 (1998), describes the design of non-peptide SH3 ligands using structure-
based, split-pool
synthesis and affinity-based selection.
Other peptides and peptide mimetics targeted to the SH3 domain in this class
of procaryotic repressors may represent a useful approach to developing
antimicrobial
compounds by virtue of their ability to activate DtxR. Peptides can be type I
or type II
eucaryotic ligands or derivatives thereof. References and strategies are
supported by the
3o annotated patents and references, particularly Delgarno, et al. and Nguyen,
et al.
Synthetic organic ligands may also be produced and screened with the screen
previously
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17
described by Sun, et al., 1998. In addition, potential compounds can be
screened for their
ability to inhibit the activation of DtxR [or any homologue] provided that
expressed
fragment contains the putative SH3 domain and the highly conserved poly-
proline sequence
endogenous to that repressor. Fluorescence assays can also be developed in
which
immobilized test compounds can be used to fish out radio- or fluorescent
labeled C-termial
DxtR [or homologue] Sh3 targets.
The peptides of the invention may be provided in the form of
pharmaceutically acceptable salts. Suitable salts include base salts such as
alkali metal salts
(e.g., sodium or potassium salts), ammonium salts, and acid addition salts
such as
l0 hydrochloride and acetate salts. D-Peptides (as opposed to peptides
containing naturally
occurring L-amino acid residues) may also be synthesized as a pure population
in an effort
to produce more stable and effective therapeutics. The peptides may also be
modified to
increase binding specificity using the strategy described by Nguyen, et al.,
[Science 1998],
including cyclization. The active form of the peptides is generally
phosphorylated, but it
may be advantageous to administer a peptide in unphosphorylated form and allow
the
peptide to become phosphorylated inside the body of the patient. Peptides may
be more
easily taken up into cells when unphosphorylated. The therapeutic agents of
the invention
may contain the peptide and at least one non-peptide synthetic moiety.
The peptides of the invention can be synthesized according to standard
2o methods such as those described in Escobedo, J. A., et al., Mol. Cell.
Biol. 11:1125-1132
(1991) or Turck, C. W. Peptide Res. 5:156-160 (1992), for example, using a
protected
prephosphorylated tyrosine residue. In particular, the peptides can be
prepared by liquid or
solid-phase methodologies known to those skilled in the art. (Schroeder, et
al., "The
Peptides", Vol. I, Academic Press 1965, or Bodanszky, et al., "Peptide
Synthesis",
Interscience Publishers, 1966, or McOmie (ed.) "Protective Group in Organic
Chemistry",
Plenum Press, 1973, or Barany et al., "The Peptides: Analysis, Synthesis,
Biology" 2,
Chapter 1, Academic Press, 1980). In the case of solid-phase synthesis any
manual or
automatic peptide synthesizer can be used and the peptides can be assembled in
a stepwise-
manner on a resin support using either Boc or Fmoc strategies.
The mode of administration of the therapeutic agents of the present invention
depends may depend upon the nature and degree of the disease. In general,
these routes are
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18
topical (e.g., cream or ointment), nasal (e.g., aerosol inhaler), parenteral
(e.g., subcutaneous,
intramuscular and intravenous) and ionophoretic. The agents may be conjugated
to another
moiety in order to increase enzymatic stability and cell permeability. The
route of
administration, as well as the dosage amount and frequency of dosing depend
upon
numerous factors including, for example, the purpose of the administration,
the age and
weight of the patient being treated and the condition of the patient. Humans
and animals,
particularly livestock and domestic animals, may be treated in accordance with
the present
invention.
The therapeutic agents may be formulated in a pharmaceutical composition
l0 suitable for any of the described routes of administration using standard
procedures and
ingredients. The pharmaceutical composition also comprises a pharmaceutically
acceptable
carriers or diluents, solubilizers, stabilizers, etc. Aqueous based carriers
are preferred for
the peptide agents. Any appropriate carrier or diluent may be employed,
depending upon
the route of administration. See generally, Remington's Pharmaceutical
Sciences, Mack
Publishers (Easton, PA).
The invention will be further described by reference to the detailed
examples. These examples are provided for purposes of illustration only, and
are not
intended to be limiting unless otherwise specified.
Example 1
2o The purpose of these experiments was to obtain a more complete
understanding of the function of the intact repressor protein, particularly
the C-terminal
domain. From the sequential assignment of resonances in heteronuclear NMR
spectra of a
recombinant C-terminal domain (residues N130-L226), we have shown that this
isolated
domain contains five ~-strands and three helices (~. Here, we present the
three-
dimensional (3D) structure of DtxR(130-226) determined in solution by using
multidimensional NMR spectroscopy and show that it adopts an SH3-like
conformation. We
also present evidence that this prokaryotic SH3-like domain binds to a proline-
rich segment
that is located in the region linking the N- and C-terminal domains of DtxR
and is conserved
in all known DtxR homologues. NMR chemical shift perturbation studies
demonstrate that a
3o synthetic peptide corresponding to this internal ligand interacts with
specific amino acid
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19
residues in the C-terminal domain. The demonstration of peptide binding by the
C-terminal
domain suggests a mechanism for regulating the activity of the intact
repressor protein.
Materials And Methods
Protein Expression, Purification, and Sample Preparation. The expression
vector for
DbcR(130-226) was constructed by first introducing a unique BamHI restriction
endonuclease site in the dtxR structural gene before N130. The portion of dtxR
cDNA
encoding residues N130-L226 then was excised by digestion with BamHI and
HindllI, and,
after purification by agarose gel electrophoresis, ligated into the BamHI and
HindIlI sites of
the pQE30 expression vector (Qiagen, Chatsworth, CA). The final protein
construct, referred
to as DtxR( 130-226), contains a 13-residue extension at the N terminus that
includes a six-
residue His tag (MRGSHHI-~GSG) to facilitate purification. DtxR(130-226) was
expressed in Escherichia coli strain HMS 174 grown in M9 minimal medium
containing
1 g/liter ~5NH4C1 and 4 g/liter glucose or 2 g/liter 13C6-glucose to produce
uniformly'SN- or
isNy3C- labeled proteins, respectively. Protein expression was induced by
addition of
0.4 mM isopropyl ~-D-thiogalactoside to the culture at an OD6oo of ~.6 and
grown for an
additional 3 h before harvesting by centrifugation. The cell pellet was
resuspended in 20 ml
of lysis buffer (50 mM potassium phosphate, pH 7.5, containing 0.5 M NaCI, 8 M
urea,
5 mM imidazole, and 1 mM PMSF) and Iysed by French press. The clarified lysate
was
chromatographed over a Ni2+-chelating Sepharose Fast Flow column (Amersham
Pharmacia), washed with the lysis buffer (containing no urea or PMSF), and
eluted with a
linear gradient of imidazole (10-600 mM). Fractions containing DtxR(130-226)
(at
approximately 300 mM imidazole) were pooled, dialyzed, and concentrated in a
Centriprep
3 (Amicon) before exchange into phosphate buffer for NMR analysis (50 mM
potassium
phosphate, containing 0.4% NaN3 and 10% D20, pH 6.5). A shorter construct of
the C-
terminal domain, corresponding to residues 144-226 [DtxR(144-226)), was
generated from
DtxR( 130-226) by amplifying the cDNA encoding these residues using PCR,
followed by
ligation into the NdeI and BamHI sites of a pET-15b expression vector
(Novagen). This
construct contains a 21- residue extension at the N terminus of DtxR(144-226),
including a
six-residue His tag and a thrombin cleavage site. DtxR(144-226) uniformly
enriched in 15N
3o was expressed in BL21 (DE3) E. coli grown in M9 minimal medium supplemented
with
lsNH4C1 and purified as described for DtxR(130-226).
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NMR Spectroscopy. NMR spectra were collected at 30°C on a three-channel
500 MHz
Varian Unityplus instrument equipped with waveform generators and three-axis
pulsed field
gradient accessories. A 3D 'SN-separated nuclear Overhauser effect
spectroscopy
(NOESY)-heteronuclear single quantum correlation (HSQC) spectrum (~ was
collected on
5 a uniformly 'SN-enriched DtxR(130-226) sample by using 8,333-, 1,650-, and
6,250-Hz
sweep widths, and digitized as 512, 48, and 128 complex points in the cu3
(1HN), cue (i5N),
and cu, ('H) dimensions, respectively. Two complementary 3D '3C-separated CCH-
NOESY
and HCH-NOESY spectra (~ were collected on uniformly'SN,'3C-labeled DtxR(130-
226)
in the deuterated phosphate buffer by using sweep widths of 2,999.2 and
8,798.8 Hz for'H
l0 and '3C chemical shift dimensions, respectively. A homonuclear two-
dimensional (2D)
NOESY spectrum was collected on a 720-MHz Varian Unityplus spectrometer, by
using
excitation sculpting for solvent suppression (17~. All NOESY spectra were
collected with a
120-ms mixing time. The ~h-dihedral angle restraints were obtained from
analysis of HNHA
(~ and HMQC-J (~ spectra collected on'SN-labeled DtxR(130-226). Slowly
exchanging
15 amide hydrogens were identified from a 2D 'H-'SN HSQC spectrum collected 24
h after
dissolution in the deuterated phosphate buffer. Heteronuclear NOES were
measured as
described (~. All NMR data were processed on Silicon Graphics workstations by
using
NMRPIPE (~ and analyzed with NMKVIEW (~.
Structure Calculation. Structure calculations were performed by using X-PLOR,
version
20 3.843 (~. The interproton NOE peaks of 2D and 3D NOESY spectra were
classified as
1.8-2.8, 1.8-3.5, 1.8-5.0, and 1.8-6.0 ~ corresponding to strong, medium,
weak, and very
weak NOES. Pseudoatom and proton multiplicity corrections were made as
described by
Fletcher et al. (~. Hydrogen bond restraints were added as 2.4-3.5 t~ and 1.5-
2.8 ~ for N-
O and H-O internuclear distances, respectively, in regions where regular
secondary structure
elements were identified in initial structures calculated by using only NOE
restraints. The ~
dihedral angle restraints were applied as -120 ~ 30° for P-strands with
3J> 8 Hz and -
60 t 30° for helical regions with 3J< 5.5 Hz, respectively (~. Hydrogen
bond and
dihedral angle restraints were combined with NOE restraints only in the final
stage of the
structural refinement. A total of 100 structures were calculated, of which 67
showed no
3o restraint violations greater than 0.5 ~ and 5°. From the 67
structures, 20 structures with
lowest total energy were chosen for further refinement by five additional
cycles of simulated
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21
annealing by decreasing the initial temperature by 100 K in each cycle from
900 K to 500 K
(~. The structures were viewed by using INSIGHTII (Molecular Simulations,
Sacramento,
CA), and analyzed by using MOLMOL (~, AQUA, and PROCHECK-NMR software (~.
Peptide Binding Experiments. A 15-residue peptide (RSPFGNPIPGLDELG; residues
8125-
6139 of DtxR) was synthesized by using standard solid-phase methods. The
peptide showed
a single peak on analytical reversed-phase HPLC and gave a mass spectrum
identical to that
expected. Binding experiments were performed by adding aliquots of peptide to
a sample of
uniformly ESN-labeled DtxR( 130-226) or DtxR( 144-226) in the phosphate
buffer, pH 6.5 at
30°C. 2D 1H-t5N HSQC spectra (~ were collected by using 1,024 and 140
complex points
l0 over 8,333.3 and 1,650 Hz spectral widths in the 1H and 15N dimensions,
respectively.
Results And Discussion
Structure Determination. Chemical shift assignments for the backbone and side-
chain ~H,
i3C, and 15N resonances of DtxR(130-226) were obtained by using the standard
suite of
triple-resonance NMR experiments (~. The tHa (Fig. la), t3C", and t3C0
chemical shift
i5 deviations (~ suggested the presence of five ~-strands and three helices,
which
subsequently were confirmed in the final 3D structures (Fig. ~. The structure
of DtxR( 130-
226) was determined from a total of 1,142 NMR restraints in the form of NOE-
derived
interproton distances, 4t dihedral angles, and hydrogen bonds. Structures were
calculated by
using a hybrid distance geometry-simulated annealing protocol (23, ~. A
summary of the
20 structural statistics for the final set of 20 structures is presented in
Table 4. These
20 structures had the lowest total energies, no distance violations greater
than 0.35 ~, and
dihedral angle violations less than 5°. Within this family of
structures, 97.6% of residues
had backbone tV,~ angles located in the allowed regions (~ of the Ramachandran
plot. The
rms deviation for the backbone atoms in all ~-strands superimposed on the
average structure
Table 1. Structure statistics
NOE restraints
Total 1,086
Intraresidue 548
Sequential 263
Medium range 87
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22
Longrange 188
~ dihedral angle restraints 26
Hydrogen bond restraints 30
Deviation from experimental restraints
Distance restraints, ~ 0.014 t 0.003
Dihedral restraints, deg 0.17 t 0.09
Deviation from idealized covalent
geometry
Bonds, ~ 0.002 t 0.000
Angles, deg 0.48 t 0.01
Impropers, deg 0.359 t 0.005
Backbone rms deviation, .~
~SA~ to ~S~l~residues A147-L226 1.45 t 0.16
~SA~ to ~ S~1~a11 ~-strands 0.80 0.08
~SA~ stands for the ensemble of 20 IVMR structures and'~,~~l~is the average
structure of the ensemble
calculated by using X-PLOR. The parameter used to calculate the van der Waals
(vdw) repulsion energy was
0.75 rather than 0.80 (47).
Description of NMR Structure. The structure of DtxR(130-226) consists of a
disordered N-
terminal region (residues N130-A146) followed by a folded domain (residues
A147-L226)
(Fig. ~. The five ~-strands identified in the final ensemble of structures
include residues
V163-Q167 (~1), V193-8198 (~2), H201-H206 (~3), K209-V211 (~4), and 8222-E225
(p5).
These strands are organized into a ~-barrel formed by two partially orthogonal
antiparallel ~-
sheets, with strand ~2 shared by the two sheets. Sheet 1 contains strands ~1,
fZ' (V 193-I195),
and ~5, while sheet 2 is formed by strands ~2" (V196-R198), ~3, and ~4.
Preceding ~1 in the
to folded domain, the polypeptide chain forms two short, extended ~-like
structures (T150-
8151 and S158-P160) that are separated by a single-turn 3to helix [residues
V152-A155
(H1)]. The ~-like structures of these two short segments are indicated by down-
field H°'
chemical shifts (Fig. la) and by long-range NOE contacts from residues T150-
8151 to ~5
and from residues S158-P160 to ~'2". Strands ~l and ~2' are connected by a
long loop
(residues I168-6190) containing the single a-helix [residues D177-A185 (H2)].
A short 3to
helix [residues D215-A218 (H3)] is formed between strands ~4 and ~5, while
strands ~2"-~3
and f3-~4 are connected by tight turns. Many of the hydrophobic residues in
helices H1, H2,
and H3 (V 152, I153, A155, L182, L183, A185, and A218) showed NOE contacts
with the ~-
barrel, forming the hydrophobic core.
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23
To obtain insight into protein chain mobility, we measured steady-state
backbone'SN-{ IH} heteronuclear NOE values. Heteronuclear NOEs for a limited
number of
residues could not be determined because of spectral overlap. Residues
preceding A147
have negative heteronuclear NOES (Fig. 1b), indicating high mobility (~. In
contrast,
residues A147-L226 have positive heteronuclear NOES, indicating lower overall
mobility
and that these residues tumble in solution as a single folded domain. The
slightly lower
heteronuclear NOEs observed for residues I168-E175 suggest an increased
mobility for
these loop residues compared with other residues in the folded domain. The
polypeptide
chain mobility deduced from the heteronuclear NOE data correlated well with
the number of
1o proton-proton NOES and the rms deviation per residue (Fig. 1 c and d),
indicating that the
limited number of intetproton NOES and low structural precision of the linker
and the loop
regions in the final family of structures reflect the internal motions of the
polypeptide
chains.
The C-terminal domain of DtxR adopts a similar fold in the crystal (~ and in
solution, with a 2.6-~ rms deviation obtained when superimposing the C" atoms
of the two
structures (residues P148-8198 and H201-L226). The largest difference between
the two
structures was found in residues I168-6190, consistent with their location in
a long loop and
their increased mobility in solution. Residues 6141-A147, which were not
traced in
previous x-ray structures, were also highly mobile in solution and were poorly
defined by
2o the NMR data.
The C-Terminal Domain of DtxR Binds a Proline-Rich Peptide. During
purification and
characterization of DtxR(130-226), it was observed that highly purified
protein ran as a
series of bands in nondenaturing polyacrylamide gels that correlated in mass
to multiples of
the monomeric protein molecular weight (Fig. 3). As seen in Fig. 3, the
monomeric and
trimeric forms were predominant, with lower amounts of dimer and higher
aggregates
observed. The formation of oligomers was not altered upon incubation with EDTA
or by
addition of 10 mM Ni2+, suggesting that the oligomerization was not induced by
residues of
the His tag binding to metal ions leached during purification. In contrast, a
single molecular
weight band corresponding to monomeric DtxR(130-226) was observed in
denaturing
3o PAGE gels (not shown). As noted previously (~, the structure of residues
P160-L226 is
homologous to eukaryotic SH3 domains. SH3 domains bind peptides with the
consensus
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24
sequence PpXP, where P is a strictly conserved proline, p is generally a
proline, and X is a
hydrophobic residue 3( 2137). Following the His tag and additional residues
associated with
the cloning sites (see Materials and Methods), the DtxR(130-226) sequence
begins as
NPIPGL. We reasoned that the oligomers may result from DtxR(130-226) binding
this
proline-containing segment. To test this hypothesis, DtxR(144-226), in which
this internal
ligand is removed, was created. NMR spectra of DtxR(144-226) showed that the
protein
adopted the same fold as DtxR(130-226). However, in contrast to DtxR(130-226),
DtxR(144-226) migrated as a single band corresponding to monomer molecular
weight in
nondenaturingpolyacrylamide gels (Fig. 3).
1o The possible binding interaction between the SH3 C-terminal domain of
DtxR and the internal proline-rich sequence was further investigated by using
a synthetic
peptide having the sequence RSPFGNPIPGLDELG, which corresponds to residues
R125-
G139 of full-length DtxR. Aliquots of this peptide were added to DtxR(130-
226), and 2D
HSQC spectra were collected. Because chemical shifts are extremely sensitive
reporters of
the local magnetic environment, ligand binding generally changes the chemical
shifts of
backbone and side-chain resonances. This approach is sensitive to weak binding
(into the
millimolar range; ref. ~ and has been used previously to demonstrate binding
between
proline-rich peptides and eukaryotic SH3 domains (~. When a stoichiometric
amount of
this peptide was added to DtxR(130-226), a limited number of protein 1H and/or
ESN
resonances exhibited line broadening or resonance frequency changes in 2D HSQC
spectra
(residues V 174, I187, E192, L204, H206, D215, D216, L217, H219, and T220),
but no
additional resonances appeared. At approximately a 5:1 peptide/protein molar
ratio,
additional residues in strands NG, ~3, ~5, and helix H3 were shifted in an
HSQC spectrum
(Fig. ~. The chemical shift perturbation data demonstrate weak binding of the
peptide by
the SH3-like domain, in fast exchange on the NMR time scale. The perturbed
residues
generate a putative peptide-binding surface located between the long loop and
the ~-barrel
(Fig. ~. The presence of an internal partial ligand that competes with the
external peptide
complicates a quantitative analysis of the binding affinity for the peptide.
By using the
existing NMR data we estimate an apparent dissociation constant in the 100 pM-
1 mM
range, which is slightly higher than that obtained for eukaryotic SH3 domains
binding
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optimized peptide ligands 3( 237). DtxR(144-226) also binds the 8125-6139
peptide, with
the same residues being shifted upon binding.
Except for NOEs observed between side-chain protons of A 146 and I187 that
were consistent with oligomer formation, no NOES were observed between
residues at the N
5 terminus and the folded domain of DtxR( 130-226), although some residues at
the N
terminus of DtxR(130-226) shifted after addition of the 8125-6139 peptide.
These
intermolecular NOES disappeared upon dilution of the DtxR(130-226) sample. The
absence
of NOES from the tail to the folded domain of DtxR(130-226) may be attributed
to the high
flexibility of the N terminus in the monomeric species (Fig. 1b) and to the
variety and low
10 concentration of oligomeric species in solution.
A Proposed Functional Role for Peptide Binding. A working model for
transcriptional
regulation by DtxR is that micromolar concentrations of Fe2+ or other divalent
metals
trigger the formation of the metal-bound dimeric state, which then binds to
the tox and irp
operators 3( 9-42). In the absence of divalent metal ligand, DtxR is thought
to exist as an
15 inactive, monomeric apo-protein that is incapable of binding DNA. Residues
8125-6139
make numerous contacts with the three helices that constitute the dimerization
interface in
the N-terminal domain, thereby contributing to the stabilization of the
dimeric form of DtxR
(~. In the current work, we found that residues 8125-6139 also can interact
with the C-
terminal domain of DtxR. If residues 8125-6139 were to dissociate from the N-
terminal
20 domain, the dimeric structure might be destabilized and dissociate into
monomers. Although
not int4ending to be bound by theory, we propose that the C-terminal domain
binds residues
8125-6139 in the monomeric state, thereby altering the monomer-dimer
equilibrium and
effectively stabilizing the monomeric, inactive form. Our data is consistent
with either an
inter- or intramolecular binding. This model for the regulation of dimer
formation by the
25 SH3-like C-terminal domain is consistent with the weakly cooperative
activation of DtxR by
metal ions (4~ and with the existing C-terminal domain mutants that alter
repressor activity
( 12, ~.
Eukaryotic SH3 domains in Hck (~, Src (~, and Itk (~ regulate tyrosine
kinase activities in signal transduction cascades by weak binding to an
internal proline-
containing peptide whose sequence differs from the high-affinity peptide
sequences that
CA 02389566 2002-05-21
WO 01/35981 PCT/US00/31721
26
activate the kinase. Here, we have postulated that binding to an internal
proline-containing
region by the SH3-like domain of this prokaryotic protein has significance in
regulating the
repressor activity of intact DtxR. According to our model, the C-terminal
domain plays no
direct role in the structure or function of the dimeric form of the repressor
and must be
flexibly linked to the N-terminal domain. This intrinsic flexibility may
explain the low
averaged electron density found for this domain in the existing crystal
structures (6-11).
Residues L120-L226 were not traced in a crystal structure of DtxR(C102D)
complexed with
a 33-by DNA sequence (11~, so the structure of the proline-containing region
and the C-
terminal domain in this state of the repressor is uncertain.
l0 The N-terminal domain of DtxR shows strong homology with the other
members of Gram-positive toxin gene repressor proteins. A recent crystal
structure of the
DtxR homologue from M. tuberculosis, IdeR, shows the proteins are structurally
homologous as well (~. Similarly, the sequence homology of the C-terminal
domains in
the family of DtxR homologues suggests that they will adopt SH3-like folds.
Residues
S 126-6139 are highly conserved in all known DtxR homologues, therefore we
also believe
that the regulatory mechanism proposed here for DtxR is applicable to the
entire family of
virulence-gene repressor proteins in the Gram-positive bacteria.
A recent study by Goranson-Siekierke et al. (~ has demonstrated that
single alanine substitutions for residues R80, S126, and N130 caused severely
decreased
DtxR activity. Crystallographic analyses of dimeric metal complexes of the
native protein
show that these residues coordinate an oxyanion, which has been identified as
a possible co-
corepressor (9, ~. In dilute solutions, the dimeric form of the protein is
stabilized by low
concentrations of Fe2+ or other divalent transition metal cations, but
dimerization also is
favored in the absence of the metal ions at high protein concentration under
crystallizing
conditions. High-resolution analyses of crystals of the metal-free DtxR (~
show a dimeric
structure very similar to the metal-bound form, in which the segment including
the
conserved sequence S 126-6139 is folded in an ordered conformation contacting
the helices
of the N-terminal domain involved in dimer formation; these polar interactions
among the
residues R80, S126, and N130 together with water and/or anion evidently
contribute to the
stability of the dimer interface. Our results demonstrate that the proline-
rich segment,
including residues S126 and N130, binds to the isolated C-terminal SH3-like
domain of
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WO 01/35981 PCT/US00/31721
27
DtxR in a manner similar to the peptide binding by eukaryotic SH3 domains (43-
45).
According to our model for the regulation of the DtxR activity, binding of the
proline-rich
segment to the C-terminal SH3-like domain should stabilize the inactive
monomeric form of
the repressor. Because replacement of the polar residues R80, S 126, and N130
with alanines
will weaken the interaction between the S126-L138 segment and the N-terminal
dimerization domain, we interpret the recent results reported by Goranson-
Siekierke et al.
(~ to indicate that destabilization of the proline-rich segment in the N-
terminal domain of
the dimer consequently should favor binding of this segment to the C-terminal
SH3-like
domain in the inactive monomer, even in the presence of activating metal ions.
Thus, the
l0 sequence S 126-6139 may function as an internal molecular switch, either
associated with
the N-terminal domain, thereby contributing to the stability of the active,
metal-bound
dimeric form of the repressor, or alternatively bound to the C-terminal
domain, favoring the
inactive monomeric form.
Abbreviations
SH3, Src homology 3; DtxR, diphtheria toxin repressor; 3D, three-
dimensional; NOE(SY), nuclear Overhauser effect (spectroscopy); HSQC,
heteronuclear
single quantum correlation; 2D, two-dimensional.
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1114-1118
10 Example 2
Phage Display
To demonstrate that the C-terminal domain of DtxR bound proline
containing ligands we employed phage display to affinity select peptides by
phage display
15 from a random peptide library. The C-terminal domain of DtxR was
immobilized onto a
substrate through a polyhisitidine tail placed N-terminal to a GSG space fused
to residue
129. This immobilized SH3 containing domain served as a trap for M13 phage
expressing
epitopes built from a random peptide library. Multiple rounds of panning and
amplification
were performed and a set of affinity purified pahge particles was obtained.
Sequence
20 analysis of the pBI protein [where the random peptides are fused] revealed
the sequences
depicted in Example 2 below. This technique has been employed extensively in
the study
of eukan~otic SH3 domains and has provided an affinity based approach for
classifying
different SH3 domains and in identifying potential lead compounds targeted at
eukaryotic
proteins modulated by SH3 mediated interactions. The approach is reviewed in
Zarnnpar
25 and Lim (Nature Struct Biol. 2000 7:611-613, and Kay, Williamson and Sudol
FASEB
(2000) 14:231-241) and examples of the technique are presented by Weng et al
(MCB
(1995) 15:5627-5634) and Sparks et al (PNAS (1996) 93:1540-1544 in which
rational
dissection of core and specificity residues are pursued and discussed in
detail. These
approaches can also be used to identify ligands as building blocks for
combinatorial
30 chemistry efforts to produce compounds which exhibit specificity from
prokaryotic SH3
domains (Dalgarno and Kaye; Parks AB, Adey NB, Quilliam LA, Thorn JM, & Kay
BK.
Methods in Enzymology, 1995; 255:498-509., Rickles RJ, Botfield MC, Zhou XM,
Henry
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31
PA, Brugge JS, & Zoller MJ. Proc Natl Acad Sci, USA, 92:10909-12913. ,Kapor,
TM,
Andreotti, AH, and Schrieber, SL (1998) JACS 120(1)., Feng S, Kasahara C,
Rickles RJ, &
Schreiber SL. Proc Natl Acad Sci, USA, 1995; 92:12408-12415.) Thus we have
employed
phage display to demonstrate first that the SH3 domain of DtxR and by analogy
this class of
repressors indeed bind proline peptide ligands and that in the presence of the
internal
polyproline [PIP] sequence that additional [high] affinity proline peptides
can be obtained.
Grouped generically as P=polar AA, P*=Serine or Threonine,
H=hydrophobic, G=glycine, - =negatively charged AA, += positively charged AA,
to Pr=proline These peptides have a general sequence of:
H/P/[+/-]/P/[+/-]/H/Pr H/P/H/G/H/Pr/Pr H/G/[+/-]P/H/Pr/Pr
P*/[+/-]/H/P*/H/Pr/H [+/-]/P[+/-]/H/Pr/P/Pr P/Pr/P/H/H/P/Pr
20
Example 2
Identification of Repressor SH3 ligands from random peptide libraries
The X-ray and NMR analysis demonstrate that the C-terminal domains of
DtxR and DtxR like repressor IdeR can fold into an SH3-like structure. The
internal
polyproline motif of DtxR employed in Example 1 suggests a functional
consequence of
internal peptide binding. These studies only utilized the C-terminal 136 amino
acids of a
mufti-domain 226 amino acid protein, however. In support of these findings are
the
polyproline rich internal ligands which we have identified by cloning and
sequence analysis
of homologous DtxR repressors from species of clinical interest. The high
degree of amino
acid homology in the proline rich linker region within the DtxR family of
repressors (S 126-
L138; Fig. 2) suggests that these SH3-like domains may share a common
mechanism of
action. Furthermore, to develop lead drug candidates, the SH3 like domain
should be able
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32
to select and bind exogenous polyproline peptidic ligands. Candidate
polyproline ligands of
higher affinity for the unique DtxR SH3 like domain should bind the SH3 domain
in the
presence of the endogenous peptide. As a first step, we employed random
peptide phage
display libraries to determine if affinity selected peptides could be
identified that would
interact with the DtxR SH3 like domain. The peptides selected are disclosed
below.
Screening of phage displayed combinatorial libraries
Affinity selection of targets for receptors, transcription factor and protein
interaction surfaces in which large numbers of random molecules are screened
for their
ability to interact, label or activate the protein of interest is a widely
employed technique.
l0 Phage display of small random peptides having lengths of between 7-25 amino
acids has
provided the ability to rapidly screen a random yet representative universe of
all possible
combinations of amino acids. Random peptide libraries have been widely used
for epitope
mapping (Scott & Smith, 1990), the identification of peptide mimics of non-
peptide ligands
(Scott et al., 1992), and mapping protein-protein contacts (Hong & Boulanger,
1995). In
general, M13 phage display is a selection technique in which a peptide, or
peptide library, is
genetically fused to a bacteriophage coat protein. Following phage assembly,
the peptide
library is then presented on the surface of the virion. Most importantly, this
method allows
the physical linkage between each individual peptide sequence with the DNA
encoding that
sequence. Phage display 7-mer and 12-mer peptide libraries are commercially
available and
2o will be initially employed in these studies (New England Biolabs{Beverly,
MA}; cat #8100,
#8110 and Mo Bi Tech IrLC, Marco Island, FL). After multiple rounds of
affinity selection
and amplification, phage were plated, and individual clones were isolated and
characterized
by DNA sequence analysis. To demonstrate that the C-terminal domain of DtxR
bound
proline containing ligands we employed phage display to affinity select
peptides by phage
display from a random peptide library. This technique has been employed
extensively in
the study of eukaryotic SH3 domains and has provided an affinity based
approach for
classifying different SH3 domains and in identifying potential lead compounds
targeted at
eukaryotic proteins modulated by SH3 mediated interactions.
Protein Expression, Purification, and Assay Plate Preparation. The expression
vector
for DtxR(130-226) was constructed by first introducing a unique BamHI
restriction
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33
endonuclease site in the dtxR structural gene before N130. The portion of dtxR
cDNA
encoding residues N 130-L226 then was excised by digestion with BamHI and
HindIlT, and,
after purification by agarose gel electrophoresis, ligated into the BamHI and
HindBI sites of
the pQE30 expression vector (Qiagen, Chatsworth, CA). The final protein
construct, referred
to as DtxR(130-226), contains a 13-residue extension at the N terminus that
includes a six-
residue His tag (MRGSiHGSG) to facilitate purification. DtxR(130-226) was
expressed in Escherichia coli strain HMS 174 grown in M9 minimal medium.
Protein
expression was induced by addition of 0.4 mM isopropyl ~-D-thiogalactoside to
the culture
at an OD6~ of ~0.6 and grown for an additional 3 h before harvesting by
centrifugation.
to The cell pellet was resuspended in 20 ml of lysis buffer (50 mM potassium
phosphate, pH
7.5, containing 0.5 M NaCI, 8 M urea, 5 mM imidazole, and 1 mM PMSF) and lysed
by
French press. The clarified lysate was chromatographed over a Ni2+-chelating
Sepharose
Fast Flow column (Amersham Pharmacia), washed with the lysis buffer
(containing no urea
or PMSF), and eluted with a linear gradient of imidazole (10-600 mM).
Fractions containing
DtxR(130-226) (at approximately 300 mM imidazole) were pooled, dialyzed, and
concentrated in a Centriprep 3 (Amicon). This protein was used to determine
conditions for
maximal binding to Ni+ affinity micro titer plates (Qiagen, Chatsworth, CA)
and wells were
prepared under saturating conditions. These wells washed to remove free C-
terminal
domain and then used in affinity selection of random phage by biopanning.
2o The 7-mer peptide library has been reported to carry 2 x 109 independent
clones, a number which is sufficiently large to represent a significant
fraction of the 20'
possible sequences. In contrast, the 12-mer library has been reported to also
contain
approximately 2 x 109 independent clones, which in this instance is only a
small fraction of
the 2012 possible sequences. Phage were incubated in SH3 coated microtiter
wells for
between 2 and 12 hrs after which unbound phage were removed and washed away.
Specifically bound pahge were removed in step imidazole washes fractions were
amplified
by preparing new stocks of enriched M13 phage.
The selected and amplified phage were reprocessed through additional
rounds of selection until a population enriched phage is derived [4-5 rounds].
After selction
3o random phage were picked from a PFU assay and used to prepare template DNA
for
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34
sequenceing. Sequencing reactions were carned out by a vendor and alignment of
random
peptide in pIII and the amino acid sequence of the random peptides determined.
Results
Since Example 1 shows that DtxR(130-226) binds the proline rich peptide
8125-L135, it was not surprising to identify proline containing peptides by
affinity
selection using phage display. However, with the use of phage display one must
be
concerned with the complexity of the library, peptide degradation, specificity
of binding,
and (perhaps most importantly) assignment of function.
Using a commercially available library, we have utilized phage display to
isolate and characterize a number of phage isolates after 4 rounds of affinity
selection on the
DtxR C-terminal domain peptide, DtxR(130-226). It is noteworthy that this
target contains
the C-terminal half of the internal proline sequence of DtxR including PIP.
The construct is
fused to a poly histidine tag by small linker region [GSG]. This means that
there is the
potential for the SH3 to SH3 domain interactions depicted in Example 1 above
in this assay
suggesting that the phage isolated to date have a higher affinity for the DtxR
SH3 domain
than the internal PIP containing ligand. To further expand this set of ligands
affinity
selection with coupled in vitro mutagenesis and affinity selection to define a
consensus
sequence amongst potential proline containing peptides could be performed.
Affinity
purification can also be sensitive to selection conditions, therefore by
adjusting the pH or
salt concentration during affinity purification it is possible to more readily
define or
differentiate ligand specificity and expand the set peptide ligands.
Poly Proline Peptides Mono Proline Peptides Proline Free Peptides
SMPITPP GDNAPP VPASVKS SDGEVWE
AHLGFPP DHRLPSP FTNRLLP WRAMRAG
YPHAMQP
Example 3
Identification Of Random Sh3 Ligands Which Activate Dtxr
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A critical step is to provide evidence that one could isolate peptides that
activate DtxR and contain a polyproline motif. In a recently published article
(Sun et al.,
1998), the assay selection system ("PSDT") was used to isolate and
characterize the first
hyper-repressor mutants of DtxR. In this study, PCR mutagenesis was employed
to
5 generate a library of variant DtxR genes which were then screened in the
PSDT system. In
this system, only those variants which maintained functional DtxR: aox
operator interaction
in the presence of the iron chelator 2,2"-dipyridyl were selected on medium
supplemented
with chloramphenicol. We have employed a similar approach to screen random
peptide
libraries. The results of these studies yielded a polyproline containing
peptide that is
to capable of activating DtxR in the presence of 2,2"-dipyridyl.
To identify polyproline peptides capable of activating DtxR and DtxR
homologues we employed the PSDT system. The approach utilized a minigene
library
created from bacterial gDNA inserted into an expression vector carrying a copy
of the DtxR
repressor gene. Expression of DtxR in this system results in active repression
of a the Tet
15 repressor which is carried on a second plasmid under the control of the tox
operator. A
chromosomal insertion in the host strain of E. coli carries the
chloramphenical acetyl-
transferase [CAT] gene under the conrtol of the tet repressor. When iron
becomes limiting
the DtxR in the cells becomes inactivated thereby de-repressing TetR and in so
doing
repressing CAT. The host cells switch from a chloramphenicol resistant to a
2o chloramphenicol senstive phenotype. To screen for peptide activators of
DtxR inserted
random gene fragments under the control of a constitutive promoter and
selected colonies
on chloramphenicol in the presence of the iron chelator 2'-2 dipyridyl. Only
cells co-
expressing the repressor and a peptide fragment capable of activating the
repressor are
selected.
25 As illustrated in the published PCT Application No. US99/22770, the PSDT
system consists of a lysogenic E. coli TOP10 host strain which carries the
reporter gene cat
(chloramphenicol acetyltransferase, Cat) on an integrated lambda phage,
7~RS65T, and a set
of detector plasmids. In this system, expression of cat from 7~RS65T is
controlled by the
tetA promoter / operator (tetAPO). In the absence of the tetracycline
repressor (TetR), the
3o expression of Cat is constitutive in E. coli TOP10/~,RS65T and as a result
this strain is
resistant to chloramphenicol (CmR). The detector plasmid, pSC6, carries the
tetR gene
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36
under the control of the diphtheria toxPO. When pSC6 is transformed into E.
coli
TOP10/~,RS65T, this host strain becomes Cm sensitive (Cms) by virtue of the
constitutive
expression of tetR. In this instance, TetR recognizes and binds to the tetAPO
and represses
cat gene expression. However, if either a functional dtxR allele or homolog is
introduced
into the bacterial host on a second compatible plasmid (e.g., pRCD or PBADT-
A,B,C), the
interaction between DtxR and the tox0 will repress the expression of tetR and
the bacterial
host, E. coli TOP10/~,RS65T/pSC6/pRDA, will then regain its CmR phenotype.
Several classes of peptides were identified; however, most striking was a
peptide with the following sequence:
to MITPSAQLTLTKGNKSWVPGPPSRSTVSISLISNSSSVPL.
When expressed in the beta-galactosidase reporter strain in the presence of 2'-
2 dipyridyl
followed by ONPG based beta galactosidase assay the clones carrying this
construct failed
to display beta-galactosidase activity whereas clones carrying a copy of DtxR
alone yielded
activity indicating that the DtxR repressor had been inactivated.
Repressor Peptide Gene BetaGal Fe + BetaGal Fe -/DPl
DxtR None -- ++
DtxR SH3 Activator Peptide -- --
The central core of this 40 amino acid peptide contains a polyproline stretch
which is
analogous to class I SH3 ligands employed by eukaryotic systems. Moreover,
this proline
rich region is related to the DtxR proline rich region that is positioned
between the N- and
C-terminal [AA 125-139] of the repressor and compares favorably to the
peptides identified
by phage display.
Dissection of Peptide Activators minimal sequences: Synthetic minigenes
splitting the
peptide into two overlapping sequences can encode (1) the N-terminus to
residue 25, (2)
from the C-terminus in 25 residues to the central polyproline rich core. Each
of these
peptides can be used in the PSDT screen to identify additional SH3 ligands.
The Minigene
can also be subject to PCR mediated saturation mutagenesis in addition to 5'
and 3'
deletions to derive additional peptide activators. Derivatives of the peptide
core can also be
constructed by oligo-nucleotide assembly of a minigene and tested in the PSDT
system.
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WO 01/35981 PCT/US00/31721
37
1. MITPSAQLTLTKGNKSWVPGPPSRS
2. NKSWVPGPPSRS TVSISLISNSSSVPL
3. XGPP
4. PGXP
5. PGPX
6. XGPX
7. PGXX
8. XGXP
9. PGPPSX
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38
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Industrial Applicability
The present invention is useful in the treatment of diseases and infection.
All publications mentioned in this specification are indicative of the level
of skill of persons
skilled in the art to which this invention pertains. All these publications
are herein
incorporated by reference to the same extent as if each individual publication
was
specifically and individually indicated as being incorporated by reference.
