Note: Descriptions are shown in the official language in which they were submitted.
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CONJUGATES BETWEEN A PEPTIDES AND A NUCLEIC ACID ANALOG, SUCH AS A PNA, LNA OR
A MORPHOLINO
The present invention concerns novel drugs for use in combating infectious
microorganisms, in particular bacteria. More particular the invention concerns
peptide nucleic acid (PNA) sequences, which are modified in order to obtain
novel
PNA molecules with enhanced anti-infective properties.
BACKGROUND OF THE INVENTION
From the discovery of penicillin in the 1940's there has been an ever-growing
search
for new drugs. Many drugs or antibiotics have been discovered or developed
from
already existing drugs. However, over the years many strains of bacteria have
become resistant to one or more of the currently available drugs, which were
effective, drugs in the past. The number of antibiotic drugs currently being
used by
clinicians is more than 100.
Most antibiotics are products of natural microbic populations and resistant
traits
found in these populations can disseminate between species and appear to have
been acquired by pathogens under selective pressure from antibiotics used in
agriculture and medicine (Davis 1994). Antibiotic resistance may be generated
in
bacteria harbouring genes that encode enzymes that either chemically alter or
degrade the antibiotics. Another possibility is that the bacteria encodes
enzymes
that makes the cell wall impervious to antibiotics or encode efflux pumps that
eject
antibiotics from the cells before they can exert their effects.
Because of the emergence of antibiotic resistant bacterial pathogens, there is
an on-
going need for new therapeutic strategies. One strategy to avoid problems
caused
by resistance genes is to develop anti-infective drugs from novel chemical
classes
for which specific resistance traits do not exist.
Antisense agents offer a novel strategy in combating diseases, as well as
opportunities to employ new chemical classes in the drug design.
Oligonucleotides can interact with native DNA and RNA in several ways. One of
these is duplex formation between an oligonucleotide and a single stranded
nucleic
acid. Another is triplex formation between an oligonucleotide and double
stranded
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DNA to form a triplex structure.
Results from basic research have been encouraging, and antisense
oligonucleotide
drug formulations against viral and disease causing human genes are
progressing
through clinical trials. Efficient antisense inhibition of bacterial genes
also could have
wide applications; however, there have been few attempts to extend antisense
technology to bacteria.
Peptide nucleic acids (PNA) are compounds that in certain respects are similar
to
oligonucleotides and their analogs and thus may mimic DNA and RNA. In PNA, the
deoxyribose backbone of oligonucleotides has been replaced by a pseudo-peptide
backbone (Nielsen et al. 1991 (29)) (Fig. 1). Each subunit, or monomer, has a
naturally occurring or non-naturally occurring nucleobase attached to this
backbone.
One such backbone is constructed of repeating units of N-(2-aminoethyl)glycine
linked through amide bonds. PNA hybridises with complementary nucleic acids
through Watson and Crick base pairing and helix formation (Egholm et al. 1993
(30)). The Pseudo-peptide backbone provides superior hybridization properties
(Egholm et al. 1993 (30)), resistance to enzymatic degradation (Demidov et al.
1994
(31)) and access to a variety of chemical modifications (Nielsen and Haaima
1997
(32)).
PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes. The
resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than
corresponding DNA/DNA or DNA/RNA duplexes as determined by Tm's. This high
thermal stability might be attributed to the lack of charge repulsion due to
the neutral
backbone in PNA. In addition to increased affinity, PNA has also been shown to
bind
to DNA with increased specificity. When a PNA/DNA duplex mismatch is melted
relative to the DNA/DNA duplex, there is seen an 8 to 20°C drop in the
Tm.
Furthermore, homopyrimidine PNA oligomers form extremely stable PNAZ-DNA
triplexes with sequence complementary targets in DNA or RNA oligomers.
Finally,
PNA's may bind to double stranded DNA or RNA by helix invasion.
An advantage of PNA compared to oligonucleotides is that the PNA polyamide
backbone (having appropriate nucleobases or other side chain groups attached
thereto) is not recognised by either nucleases or proteases and are thus not
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cleaved. As a result, PNA's are resistant to degradation by enzymes unlike
nucleic
acids and peptides.
For antisense application, target bound PNA can cause steric hindrance of DNA
and
RNA polymerases, reverse transcription, telomerase and the ribosome's (Hanvey
et
al. 1992 (33), Knudsen et a. 1996 (34), Good and Nielsen 1998 (39,40), etc.
A general difficulty when using antisense agents is cell uptake. A variety of
strategies to improve uptake can be envisioned and there are reports of
improved
uptake into eukaryotic cells using lipids (Lewis et al. 1996 (35)),
encapsulation
(Meyer et al. 1998 (36)) and carrier strategies (Nyce and Metzger 1997 (37),
Pooga
et al, 1998 (38)).
WO 99/05302 discloses a PNA conjugate consisting of PNA and the transporter
peptide transportan, which peptide may be used for transport cross a lipid
membrane and for delivery of the PNA into interactive contact with
intracellular
polynucleotides.
US-A-5 777 078 discloses a pore-forming compound which comprises a delivery
agent recognising the target cell and being linked to a pore-forming agent,
such as a
bacterial exotoxin. The compound is administered together with a drug such as
PNA.
As an antisense agent for microorganisms, PNA may have unique advantages. It
has been demonstrated that PNA based antisense agents for bacterial
application
can control cell growth and growth phenotypes when targeted to Escherichia
coli
rRNA and mRNA (Good and Nielsen 1998a,b (39,40) and WO 99/13893).
However, none of these disclosures discuss ways of transporting the PNA across
the bacterial cell wall and membrane.
Furthermore, for bacterial application, poor uptake is expected, because
bacteria
have stringent barriers against foreign molecules and antisense oligomer
containing
nucleobases appear to be too large for efficient uptake. The results obtained
by
Good and Nielsen (1998a,b (39,40)) indicate that PNA oligomers enter bacterial
cells poorly by passive diffusion across the lipid bilayers.
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US-A-5 834 430 discloses the use of potentiating agents, such as short
cationic
peptides in the potentiation of antibiotics. The agent and the antibiotic are
co-
administered.
WO 96/11205 discloses PNA conjugates, wherein a conjugated moiety may be
placed on terminal or non terminal parts of the backbone of PNA in order to
functionalise the PNA. The conjugated moieties may be reporter enzymes or
molecules, steroids, carbohydrate, terpenes, peptides, proteins, etc. It is
suggested
that the conjugates among other properties may possess improved transfer
properties for crossing cellular membranes. However, WO 96/11205 does not
disclose conjugates, which may cross bacterial membranes.
WO 98/52614 discloses a method of enhancing transport over biological
membranes, e.g. a bacterial cell wall. According to this publication,
biological active
agents such as PNA may be conjugated to a transporter polymer in order to
enhance the transmembrane transport. The transporter polymer consists of 6-25
subunits; at least 50% of which contain a guanidino or amidino sidechain
moiety and
wherein at least 6 contiguous subunits contain guanidino and/or amidino
sidechains.
A preferred transporter polymer is a polypeptide containing 9 arginine.
Thus, despite the promising results in the use of the PNA technology obtained
previously, there is a great need of developing new PNA antisense drugs, which
are
effective in combating microorganisms.
SUMMARY OF THE INVENTION
The present invention concerns a new strategy for combating bacteria. It has
previously been shown that antisense PNA can inhibit growth of bacteria.
However,
due to a slow diffusion of the PNA over the bacterial cell wall a practical
application
of the PNA as an antibiotic . has not been possible previously. According to
the
present invention, a practical application in tolerable concentration may be
achieved
by modifying the PNA by linking a peptide or peptide-like sequence, which
enhances
the activity of the PNA.
Surprisingly, it has been found out that by incorporating a peptide, an
enhanced
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anti-infective effect can be observed. The important feature of the modified
PNA
molecules seems to be a pattern comprising in particular positively charged
and
lipophilic amino acids or amino acid analogues. An anti-infective effect is
found with
different orientation of the peptide in relation to the PNA-sequence.
5
Thus, the present invention concerns a modified PNA molecule of formula (I):
Peptide - L - PNA (I)
wherein L is a linker or a bond;
Peptide is any amino acid sequence and
PNA is a Peptide Nucleic Acid, and pharmaceutically acceptable salts thereof.
More particularly, the present invention concerns a modified PNA molecule of
formula (I)
Peptide - L - PNA (I)
wherein Peptide is a cationic peptide or cationic peptide analogue or a
functionally
similar moiety, the peptide or peptide analogue having the formula (II):
C-(B-A)~ D, (II)
Wherein A consists of from 1 to 8 non-charged amino acids and/or amino acid
analogs;
B consists of from 1 to 3 positively charged amino acids and/or amino acid
analogs;
C consists of from 0 to 4 non-charged amino acids and/or amino acid analogs;
D consists of from 0 to 3 positively charged amino acids and/or amino acid
analogs;
n is 1-10; and
the total number of amino acids and/or amino acid analogs is from 3 to 20.
In one embodiment, the Peptide of the present invention contains from 2 to 60
amino acids.
The amino acids can be negatively, non-charged or positively charged naturally
occurring, rearranged or modified amino acids.
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In a preferred embodiment of the invention the peptide contains from 2 to 18
amino
acids, most preferred from 5 to 15 amino acids.
In another preferred embodiment of the invention A in formula (II) consists of
from 1
S to 6 non-charged amino acids and/or amino acid analogs and B consists of 1
or 2
positively charged amino acids and/or amino acid analogs. In another
embodiment,
A consists of from 1 to 4 non-charged amino acids and/or amino acid analogs
and B
consists of 1 or 2 positively charged amino acids and/or amino acid analogs.
In a preferred embodiment of the invention the modified PNA molecules of
formula I
are used in the treatment or prevention of infections caused by Escherichia
coli or
vancomycin-resistant enterococci such as Enterococcus faecalis and
Enterococcus
faecium or infections caused by methicillin-resistant and methicillin-
vancomycin-
resistant Staphylococcus aureus.
The peptide is linked to the PNA sequence via the amino (N-terminal) or
carboxy (C-
terminal) end.
In a preferred embodiment the peptide is linked to the PNA sequence via the
carboxy end.
Within the present invention, the compounds of formula I may be prepared in
the
form of pharmaceutically acceptable salts, especially acid-addition salts,
including
salts of organic acids and mineral acids. Examples of such salts include salts
of
organic acids such as formic acid, fumaric acid, acetic acid, propionic acid,
glycolic
acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid,
tartaric acid,
citric acid, benzoic acid, salicylic acid and the like. Suitable inorganic
acid-addition
salts include salts of hydrochloric, hydrobromic, sulphuric and phosphoric
acids and
the like. Further examples of pharmaceutically acceptable inorganic or organic
acid
addition salts include the pharmaceutically acceptable salts listed in Journal
of
Pharmaceutical Science, 66, 2 (1977) which are known to the skilled artisan.
Also intended as pharmaceutically acceptable acid addition salts are the
hydrates
which the present compounds are able to form.
The acid addition salts may be obtained as the direct products of compound
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synthesis. In the alternative, the free base may be dissolved in a suitable
solvent
containing the appropriate acid, and the salt isolated by evaporating the
solvent or
otherwise separating the salt and solvent.
The compounds of this invention may form solvates with standard low molecular
weight solvents using methods known to the skilled artisan.
In another aspect of the invention the modified PNA molecules are used in the
manufacture of medicaments for the treatment or prevention of infectious
diseases
or for disinfecting non-living objects.
In a further aspect, the invention concerns a composition for treating or
preventing
infectious diseases or disinfecting non-living objects.
In yet another aspect, the invention concerns the treatment or prevention of
infectious diseases or treatment of non-living objects.
In yet a further aspect, the present invention concerns a method of
identifying
specific advantageous antisense PNA sequences which may be used in the
modified PNA molecule according to the invention.
In yet a further aspect, the present invention relates to other antisense
oligonucleotides with the ability to bind to both DNA and RNA.
Oligonucleotide analogues are oligomers having a sequence of nucleotide bases
(nucleobases) and a subunit-to-subunit backbone that allows the oligomer to
hybridize to a target sequence in an mRNA by Watson-Crick base pairing, to
form
an RNA/Oligomer duplex in the target sequence. The oligonucleotide analogue
may
have exact sequence complementarity to the target sequence or near
complementarity, as long as the hybridized duplex structure formed has
sufficient
stability to block or inhibit translation of the mRNA containing target
sequence.
Oligonucleotide analogues of the present invention are selected from the group
consisting of Locked Nucleoside Analogues (LNA) as described in International
PCT
Publication W099/14226, oligonucleotides as described in International PCT
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Publication W098/03533 or antisense oligomers, in particular morpholino
analogues
as described in International PCT Publication W098/32467.
PCT Publication W099/14226, W098/03533 and W098/32467 are all incorporated
by reference.
Thus, further preferred compounds of the invention are modified
oligonucleotides of
the formula (III):
Peptide - L - Oligon (III)
wherein L is a linker or a bond;
Peptide is any amino acid sequence and
Oligon designates an oligonucleotide or analogue thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows the chemical structure of DNA and PNA oligomers.
FIGURE 2 shows the principle in conjugation using SMCC
FIGURE 3 shows the nucleotide sequence of the mrcA (ponA) gene encoding
PBP1A. The sequence of the gene (accession number X02164) was obtained from
the EMBL sequence database (Heidelberg, Germany) (Broome-Smith et al. 1985,
Eur J Biochem 147:437-46 (41 )). Two possible start codons have been
identified
(highlighted). Bases 1-2688 are shown (ending with stop codon).
FIGURE 4 shows the nucleotide sequence of the mrdA gene encoding PBP2. The
sequence (accession number AE000168, bases 4051-5952, numbered 1-2000) was
obtained from the E. coli genome database at the NCBI (Genbank, National
Centre
for Biotechnology Information, USA). The start codon is highlighted.
FIGURE 5 shows the chemical structures of the different succinimidyl based
linking
groups used in the conjugation of the Peptide and PNA
DETAILED DESCRIPTION OF THE INVENTION
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Antisense PNA's can inhibit bacterial gene expression with gene and sequence
specificity (Good and Nielsen 1998a,b (39,40) and WO 99/13893). The approach
may prove practical as a tool for functional genomics and as a source for
novel
antimicrobial drugs. However, improvements on standard PNA are required to
increase antisense potencies. The major limit to activity appears to be
cellular entry.
Bacteria effectively exclude the entry of large molecular weight foreign
compounds,
and previous results for in vitro and cellular assays seem to show that the
cell
barrier restricts antisense effects. Accordingly, the present invention
concerns
strategies to improve the activity of antisense potencies.
Without being bound by theory, it is believed that the short cationic peptides
lead to
an improved PNA uptake over the bacterial cell wall. It is believed that the
short
peptides act by penetrating the cell wall, allowing the modified PNA molecule
to
cross the cell wall to get access to structures inside the cell, such as the
genome,
mRNA's, the ribosome, etc. However, an improved accessibility to the nucleic
acid
target or an improved binding of the PNA may also add to the overall effect
observed.
According to the invention, PNA molecules modified with short activity
enhancing
peptides enable specific and efficient inhibition of bacterial genes with
nanomolar
concentrations. Antisense potencies in this concentration are consistent with
practical applications of the technology. It is believed that the present
invention for
the first time demonstrates that peptides with a certain pattern of cationic
and
lipophilic amino acids can be used as carriers to deliver agents and other
compounds into micro-organisms, such as bacteria. Further, the present
invention
has made it possible to administer PNA in an efficient concentration, which is
also
acceptable to the patient.
Accordingly, the present invention concerns novel modified PNA molecules
having
the formula:
Peptide - L - PNA, wherein
L is a linker or a bond;
PNA is a peptide nucleic acid sequence; and
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Peptide is a cationic peptide or peptide analogue or a functionally similar
moiety, the
peptide or peptide analogue having the formula:
C-(B-A)~ D, wherein
5
A consists of from 1 to 8 non-charged amino acids and/or amino acid analogs;
B consists of from 1 to 3 positively charged amino acids and/or amino acid
analogs;
C consists of from 0 to 4 non-charged amino acids and/or amino acid analogs;
D consists of from 0 to 3 positively charged amino acids and/or amino acid
analogs;
n is 1-10; and
the total number of amino acids and/or amino acid analogs is from 3 to 20.
A preferred group of modified Peptide Nucleic Acids (PNA) molecule is the
group
wherein A consists of from 1 to 6 non-charged amino acids and/or amino acid
analogs and B consists of 1 or 2 positively charged amino acids and/or amino
acid
analogs. In another preferred group A consists of from 1 to 4 non-charged
amino
acids and/or amino acid analogs and B consists of 1 or 2 positively charged
amino
acids and/or amino acid analogs.
By the terms "cationic amino acids and amino acid analogues" and "positively
charged amino acids and amino acid analogues" are to be understood any natural
or non-natural occurring amino acid or amino acid analogue which have a
positive
charge at physiological pH. Similarly the term "non-charged amino acids or
amino
acid analogs" is to be understood any natural or non-natural occurring amino
acids
or amino acid analogs which have no charge at physiological pH.
Among the positively charged amino acids and amino acid analogs may be
mentioned lysine (Lys, K), arginine (Arg, R), diamino butyric acid (DAB) and
ornithine (Orn). The skilled person will be aware of further positively
charged amino
acids and amino acid analogs.
Among the non-charged amino acids and amino acid analogs may be mentioned the
natural occurring amino acids alanine (Ala, A), valine (Val, V), leucine (Leu,
L),
isoleucine (11e, I), proline (Pro, P), phenylanaline (Phe, F), tryptophan
(Trp, W),
methionine (Met, M), glycine (Gly, G), serine (Ser, S), threonine (Thr, T),
cysteine
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(Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N) and glutamine (Gln, Q), the
non-
natural occurring amino acids 2-aminobutyric acid, ~i-cyclohexylalanine, 4-
chlorophenylalanine, norleucine and phenylglycine. The skilled person will be
aware
of further non-charged amino acids and amino acid analogs.
Preferably, the non-charged amino acids and amino acid analogs are selected
from
the natural occurring non-polar amino acids Ala, Val, Leu, Ile, Phe, Trp and
Met or
the non-natural occurring non-polar amino acids ~3-cyclohexylalanine, 4-
chlorophenylalanine and norleucine.
The term "functionally similar moiety" is defined as to cover all peptide-like
molecules, which functionally mimic the Peptide as defined above and thus
impart to
the PNA molecule the same advantageous properties as the peptides comprising
natural and non-natural amino acids as defined above.
Examples of preferred modified PNA molecules according to the invention are
(Lys
Phe Phe)3 Lys-L-PNA and any subunits thereof comprising at least three amino
acids. One preferred Peptide is (Lys Phe Phe)3 (SEQ ID NO: 1). Others include
(Lys
Phe Phe)2 Lys Phe (SEQ ID NO: 2), (Lys Phe Phe)2 Lys (SEQ ID NO: 157), (Lys
Phe
Phe)Z (SEQ ID NO: 3), Lys Phe Phe Lys Phe (SEQ ID NO: 4), Lys Phe Phe Lys
(SEQ ID NO: 5) and Lys Phe Phe.
Other preferred Peptides are FFRFFRFFR (SEQ ID NO: 6), LLKLLKLLK (SEQ ID
NO: 7), LLRLLRLLR (SEQ ID NO: 8), LLKKLAKAL (SEQ ID NO: 9),
KRRWPWWPWKK (SEQ ID NO: 10), KFKVKFVVKK (SEQ ID NO: 11), LLKLLLKLLLK
(SEQ ID NO: 12), LLKKLAKALK (SEQ ID NO: 13), and any subunits thereof
comprising at least 3 amino acids whereof at least one amino acid is a
positively
charged amino acid.
A third group Of preferred PeptIdeS IS RRLFPWWWPFRRVC (SEQ ID NO: 14),
GRRWPWWPWKWPLIC (SEQ ID NO: 15), LVKKVATTLKKIFSKWKC (SEQ ID NO: 16),
KKFKVKFVVKKC (SEQ ID NO: 17) and any subunit thereof comprising at least 3
amino acids whereof at least one amino acid is a positively charged amino
acid.
A fourth group of preferred Peptides is magainis (Zasloff, M., Proc. Natl.
Acad. Sci.
USA, 84, p. 5449-5453 (1987)), for instance the synthetic magainin derivative
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GIGKFLHAAKKFAKAFVAEIMNS-NHS (SEQ ID NO: 158) aS WG'll aS ~i-amino-acid
oligomers (~3-peptides) as described by Porter, E.A. et al, Nature, 404, p.
565,
(2000).
The number of amino acids in the peptide may be chosen between 3 and 20. It
appears that at least 3 amino acids; whereof at least one is a positively
charged
amino acid is necessary to obtain the advantageous effect. On the other hand,
the
upper limit only seems to be limited by an upper limit of the overall size of
the PNA
molecule for the purpose of the practical use of said molecule. Preferably,
the total
number of amino acids is 15 or less, more preferable 12 or less and most
preferable
10 or less.
The PNA molecule is connected to the Peptide moiety through a direct binding
or
through a linker. A variety of linking groups can be used to connect the PNA
with the
1 S Peptide.
Linking groups are described in WO 96/11205 and W098/52614, the content of
Which are hereby incorporated by reference.
Some linking groups may be advantageous in connection with specific
combinations
of PNA and Peptide.
Preferred linking groups are ADO (8-amino-3,6-dioxaoctanoic acid), SMCC
(succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) AHEX or AHA (6-
aminohexanoic acid), 4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid,
LCSMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amido-
caproate), MBS (succinimidyl m-maleimido-benzoylate), EMCS (succinimidyl N-s-
maleimido-caproylate), SMPH (succinimidyl 6-((i-maleimido-propionamido)
hexanoate, AMAS (succinimidyl N-(a-maleimido acetate), SMPB (succinimidyl 4-(p-
maleimidophenyl)butyrate), a.ALA (~i-alanine), PHG (Phenylglycine), ACHC (4-
aminocyclohexanoic acid), ~i.CYPR (a-(cyclopropyl) alanine) and ADC (amino
dodecanoic acid).
Any of these groups may be used as a single linking group or together With
more
groups in creating a suitable linker. Further, the different linking groups
may be
combined in any order and number in order to obtain different functionalities
in the
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linker arm.
In a preferred embodiment the linking group is a combination of the (i.ALA
linking
group or the ADO linking group with any of the other above mentioned linking
S groups.
Thus, preferred linkers are -achc-(3.ala-, -achc-ado-, -Icsmcc-~i.ala-, -mbs-
(3.ala-, -
emcs-~i.ala-, -Icsmcc-ado-, -mbs-ado-, -emcs-ado- or-smph-ado-.
Most preferred are the linkers -achc-a.ala-,-Icsmcc-ado- and -mbs-ado-.
In the case SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
)
is used in the process of linking PNA to the peptide, it is necessary to add a
cysteine
(C) or a similar thiol containing moiety to the terminal end of the peptide
(see Fig. 2).
Additionally, amino acids, such as glycine, may be a part of the linker.
The chemical structures of the different succinimidyl based linking groups
used in
the conjugation of the Peptide and PNA is shown in Figure 5.
The Peptide is normally linked to the PNA sequence via the amino or carboxy
end.
However, the PNA sequence may also be linked to an internal part of the
peptide or
the PNA sequence is linked to a peptide via both the amino and the carboxy
end.
The modified PNA molecule according to the present invention comprises a PNA
oligomer of a sequence, which is complementary to at least one target
nucleotide
sequence in a microorganism, such as a bacterium. The target may be a
nucleotide
sequence of any RNA, which is essential for the growth, and/or reproduction of
the
bacteria. Alternatively, the target may be a gene encoding a factor
responsible for
resistance to antibiotics. In a preferred embodiment, the functioning of the
target
nucleotide sequence is essential for the survival of the bacteria and the
functioning
of the target nucleic acid is blocked by the PNA sequence, in an antisense
manner.
The binding of a PNA strand to a DNA or RNA strand can occur in one of finro
orientations, anti-parallel or parallel. As used in the present invention, the
term
complementary as applied to PNA does not in itself specify the orientation
parallel or
anti-parallel. It is significant that the most stable orientation of PNA/DNA
and
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PNA/RNA is anti-parallel. In a preferred embodiment, PNA targeted to single
strand
RNA is complementary in an anti-parallel orientation.
In a another preferred embodiment of the invention a bis-PNA consisting of two
PNA
oligomers covalently linked to each other is targeted to a homopurine sequence
(consisting of only adenine and/or guanine nucleotides) in RNA (or DNA), with
which
it can form a PNAZ-RNA (PNAZ-DNA) triple helix.
In another preferred embodiment of the invention, the PNA contains from 5 to
20
nucleobases, in particular from 7-15 nucleobases, and most particular from 8
to 12
nucleobases.
Peptide Nucleic Acids are described in WO 92/20702 and WO 92/20703, the
content of which is hereby incorporated by reference.
In a preferred embodiment of the PNA the backbone is aminoethylglycine as
shown
in Figure 1.
Potential target genes may be chosen based on the knowledge of bacterial
physiology. A target gene may be found among those involved in one of the
major
process complexes: cell division, cell wall synthesis, protein synthesis
(translation)
and nucleic acid synthesis, fatty acid metabolism and gene regulation. A
target gene
may also be involved in antibiotic resistance.
A further consideration is that some physiological processes are primarily
active in
dividing cells whereas others are running under non-dividing circumstances as
well.
Known target proteins in cell wall biosynthesis are penicillin binding
proteins, PBPs,
the targets of, e.g., the beta-lactam antibiotic penicillin. They are involved
in the final
stages of cross-linking of the murein sacculus.
E. coli has 12 PBPs, the high molecular weight PBPs: PBP1 a, PBP1 b, PBP1 c,
PBP2 and PBP3, and seven low molecular weight PBPs, PBP 4-7, DacD, AmpC
and AmpH. Only the high molecular weight PBPs are known to be essential for
growth and have therefore been chosen as targets for PNA antisense.
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Protein biosynthesis is an important process throughout the bacterial cell
cycle.
Therefore, the effect of targeting areas in the field of protein biosynthesis
is not
dependent on cell division.
5 Both DNA and RNA synthesis are target fields for antibiotics. A known target
protein
in DNA synthesis is gyrase. Gyrase acts in replication, transcription, repair
and
restriction. The enzyme consists of two subunits, both of which are candidate
targets
for PNA.
10 Examples of potential targets primarily activated in dividing cells are
rpoD, gyrA,
gyrB, (transcription), mrcA (ponA), mrcB (ponB, pbp~, mrrJA, ftsl (pbpB) (Cell
wall
biosynthesis), ftsQ, ftsA and ftsZ (cell division).
Examples of potential targets also activated in non-dividing cells are infA,
inf8, infC,
15 tufAltufB, tsf, fusA, prfA, prfB, and prfC, (Translation).
Other potential target genes are antibiotic resistance-genes. The skilled
person
would readily know from which genes to choose. Two examples are genes coding
for beta-lactamases inactivating beta-lactam antibiotics, and genes encoding
chloramphenicol acetyl transferase.
PNA's against such resistance genes could be used against resistant bacteria.
A further potential target gene is the acpP gene encoding the acyl carrier
protein of
E. Coli
ACP (acyl carrier protein) is a small and highly soluble protein, which plays
a central
role in type I fatty acid synthase systems. Intermediates of long chain fatty
acids are
covalently bound to ACP by a thioester bond between the carboxyl group of the
fatty
acid and the thiol group of the phosphopanthetheine prosthetic group.
ACP is one of the most abundant proteins in E. coli, constituting 0.25% of the
total
soluble protein (ca 6 x 104 molecules per cell). The cellular concentration of
ACP is
regulated, and overproduction of ACP from an inducible plasmid is lethal to E.
coli
cells.
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Infectious diseases are caused by micro-organisms belonging to a very wide
range
of bacteria, viruses, protozoa, worms and arthropods and from a theoretical
point of
view PNA can be modified and used against all kinds of RNA in such micro-
organisms, sensitive or resistant to antibiotics.
Examples of micro-organisms which may be treated in accordance with the
present
invention are Gram-positive organisms such as Streptococcus, Staphylococcus,
Peptococcus, Bacillus, Listeria, Clostridium, Propionebacteria, Gram-negative
bacteria such as Bacteroides, Fusobacterium, Escherichia, Klebsiella,
Salmonella,
Shigella, Proteus, Pseudomonas, Vibrio, Legionella, Haemophilus, Bordetella,
Brucella, Campylobacter, Neisseria, Branhamella, and organisms which stain
poorly
or not at all with Gram's stain such as Mycobacteria, Treponema, Leptospira,
Borrelia, Mycoplasma, Clamydia, Rickettsia and Coxiella,
The incidence of the multiple antimicrobial resistance of bacteria which cause
infections in hospitals/intensive care units is increasing. These include
methicillin-
resistant and methicillin-vancomycin-resistant Staphylococcus aureus,
vancomycin-
resistant enterococci such as Enterococcus faecalis and Enterococcus faecium,
penicillin-resistant Streptococcus pneumoniae and cephalosporin and quinolone
resistant gram negative rods (coliforms) such as E. coli, Klebsiella
pneumoniae,
Pseudomonas species and Enterobacter species. More recently, pan antibiotic
(including carbapenems) resistant gram negative bacilli have emerged. The
rapidity
of emergence of these multiple antibiotic-resistance is not being reflected by
the
same rate of development of new antibiotics and it is, therefore, conceivable
that
patients with serious infections soon will no longer be treatable with
currently
available antimicrobials (1, 2). Several international reports have
highlighted the
potential problems associated with the emergence of antimicrobial resistance
in
many areas of medicine and also outlined the difficulties in the management of
patients with infections caused by these micro-organisms (3, 20).
A. Gram positive bacteria
Methicillin-resistant S. aureus (MRSA) (4,5), methicillin-vancomycin resistant
S.
aureus (VMRSA) and vancomycin resistant enterococci (VRE) have emerged as
major nosocomial pathogens (3, 6, 7, 18). Vancomycin is currently the most
reliable
treatment for infections caused by MRSA but the potential transfer of
resistance
genes from VRE to MRSA may leave few therapeutic options in the future.VRE ,
as
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well as providing a reservoir of vancomycin resistance genes, can also cause
infections in patients with compromised immunity, which are difficult to
treat, with
some strains showing resistance to all major classes of antibiotic. The
increasing
incidence of VRE strains among clinical isolates of enterococci places them as
S important nosocomial pathogens and in some hospitals in the United States
VRE
are responsible for more than 20% of enterococcal infections (17, 18)
S aureus showing intermediate vancomycin resistance (VISA) as well as VMRSA
have now been reported from several numbers of centres/hospitals worldwide (8,
9).
Of the S. aureus isolates from USA, Europe and Japan 60 -72% were MRSA and
most strains being multi-drug-resistant MRSA are the commonest cause of
surgical
site infection and comprise 61 % of all such S. aureus infections and a major
cause
of increased morbidity and mortality of ICU patients (21, 22, 23,19).
1S Coagulase negative staphylococci (CNS) such as S. epidermidis are an
important
cause of infections associated with prosthetic devices and catheters (13).
Although
they display lower virulence than S .aureus, they have intrinsic low-level
resistance
to many antibiotics including beta-lactams and glycopeptides. In addition many
of
these bacteria produce slime (biofilm) making the treatment of prosthetic
associated
infections difficult and often requires removal of the infected prosthesis or
catheter
(24).
Streptococcus pneumoniae, regarded as fully sensitive to penicillin for many
years,
has now acquired the genes for resistance from oral streptococci. The
prevalence of
2S these resistant strains is increasing rapidly worldwide and this will limit
the
therapeutic options in serious pneumococcal infections, including meningitis
and
pneumonia (10). Streptococcus pneumoniae is the leading cause of infectious
morbidity and mortality worldwide. In USA the pneumococcus is responsible for
an
estimated 50.000 cases of bacteremia, 3000 cases of meningitis, 7 million
cases of
otitis media, and several hundred thousands cases of pneumonia. The overall
yearly
incidence of pneumococcal bacteremia is estimated to be 15 to 35 cases per
100.000. Current immunization of small children and old people have not
addressed
the high incidence of pneumococcal infection ( 27, 28 ). Multi-drug resistant
strains
were isolated in the late 1970's and are now encountered worldwide (10)
3S
B. Gram negative bacteria
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Pseudomonas aeruginosa, Pseudomonads species including Burkholderia cepacia
and Xanthomonas malthophilia, Enterobacteriaceae including E. coli,
Enterobacter
species and Klebsiella species account for the majority of isolates where
resistance
has emerged (25,26, 3)
Cystitis, pneumonia, septicaemi and postoperative sepsis are the commonest
types
of infections. Most of the infections in patients being treated on an
intensive care
unit (ICU) results from the patients own endogenous flora and in addition up
to 50%
of ICU patients will also acquire nosocomial infection, which are associated
with a
relatively high degree of morbidity and mortality (19, 11, 12). Microorganisms
associated with these infections include Enterobacteriaceae 34%, S. aureus
30%, P.
aeruginosa 29%, CNS 19% and fungi 17%.
Selective pressure through the use of broad-spectrum antibiotics has lead to
multidrug resistance in Gram-negative bacteria. Each time a new drug is
introduced,
resistant subclones appear and today the majority of isolates are resistant to
at least
one antimicrobial ( 20, 14, 25, 26 )
The cell envelope of P, aeruginosa with the low permeability differs from that
of E.
coli. 46% of P. aeruginosa isolates from Europe are resistant to one or more
antibiotics and the ability of this bacteria to produce slime (biofilm) and
rapid
development of resistance during treatment often leads to therapy failure.
Multidrug
resistant P, aeruginosa has also become endemic within some specialised ICU's
such as those treating burns patients and cystic fibrosis patients (15, 16)
Several international reports have highlighted the potential problems
associated with
the emergence of antimicrobial resistance in bacteria mentioned above, and it
is,
therefore, conceivable that patients with serious infections soon will no
longer be
treatable with currently available antimicrobials. The increasing incidence of
resistant strains among clinical isolates of S.aureus, S.epidermidis (CNS),
enterococci, Streptococcus pneumoniae, gram negative bacilli (coliforms) such
as
E.coli, Klebsiella pneumoniae, Pseudomonas species and Enterobacter species
make these bacteria major candidates for future PNA design.
METHODS
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The ability of the compounds of the present invention to inhibit bacterial
growth may
be measured in many ways, which should be clear to the skilled person. For the
purpose of exemplifying the present invention, the bacterial growth is
measured by
the use of a microdilution broth method according to NCCLS guidelines. The
present
invention is not limited to this way of detecting inhibition of bacterial
growth.
To illustrate one example of measuring growth and growth inhibition the
following
procedure may be used:
Bacterial strain: E.coli K12 MG1655
Media: 10% Mueller-Hinton broth, diluted with sterile water.
10% LB broth diluted with sterile water.
100% Mueller-Hinton broth.
Trays: 96 well trays, Costar # 3474, Biotech Line AS, Copenhagen. (Extra low
sorbent trays are used in order to prevent / minimize adhesion of PNA to tray
surface).
A logphase culture of E.coli is diluted with fresh preheated medium and
adjusted to
defined OD (here: Optical Density at 600 nm) in order to give a final
concentration of
5x105 and 5x104 bacteria/ml medium in each well, containing 200 u1 of
bacterial
culture. PNA is added to the bacterial culture in the wells in order to give
final
concentrations ranging from 300 nM to 1000 nM. Trays are incubated at
37°C by
shaking in a robot analyzer, PowerWavex, soffinrare KC4~ Kebo.Lab, Copenhagen,
for
16 h and optical densities are measured at 600 nM during the incubation time
in
order to record growth curves. Wells containing bacterial culture without PNA
are
used as controls to ensure correct inoculum size and bacterial growth during
the
incubation. Cultures are tested in order to detect contamination.
The individual peptide-L-PNA constructs have MW between approx. 4200 and 5000
depending on the composition. Therefore all tests were pertormed on a molar
basis
rather than on a weight/volume basis. However, assuming an average MW of the
construct of 4500 a concentration of 500 nM equals 2.25 microgram/ml.
Growth inhibitory effect of PNA-constructs'
The bacterial growth in the wells is described by the lag phase i.e. the
period until
(before) growth starts, the log phase i.e. the period with maximal growth
rate, the
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steady-state phase followed by the death phase. These parameters are used when
evaluating the inhibitory (Minimal Inhibitory Concentration, abbr. MIC) and
bactericidal (Minimal Bactericidal Concentration, abbr. MBC) effect of the PNA
on
the bacterial growth, by comparing growth curves with and without PNA.
5
Total inhibition of bacterial growth is defined as: OD (16h) = OD (Oh) or no
visible
growth according to NCCLS Guidelines
In an initial screening the modified PNA molecules are tested in the sensitive
10%
10 medium assay. Positive results are then run in the 100% medium assay in
order to
verify the inhibitory effect in a more "real" environment (cf. the American
guidelines
(NCCLS)).
In vivo antibacterial efficacy is established by testing a compound of the
invention in
15 the mouse peritonitis/sepsis model as described by N. Frimodt-Moller et al.
1999,
Chap. 14, Handbook of Animal Models of Infection.
For the in vivo efficacy experiment a number of female NMRI mice are
inoculated
with approximately 10' cfu of E. coli ATCC 25922 intraperitoneally. Samples
are
20 drawn from blood and peritoneal fluid at 1, 2, 4 and 6 hrs post infection,
and cfu/ml
counted. 1 hr post infection the animals are treated once in groups with: 1.
Gentamicin (38 mg/kg s.c.); 2. Ampicillin (550 mg/kg s.c.); 3. a compound of
the
invention (50 - 60 mg/kg i.v.); 4. no treatment.
In another aspect of the present invention, the modified PNA molecules can be
used
to identify preferred targets for the PNA. Based upon the known or partly
known
genome of the target micro-organisms, e.g. from genome sequencing or cDNA
libraries, different PNA sequences can be constructed and linked to an
effective
anti-infective enhancing Peptide and thereafter tested for its anti-infective
activity. It
may be advantageous to select PNA sequences shared by as many micro-
organisms as possible or shared by a distinct subset of micro-organisms, such
as
for example Gram-negative or Gram-positive bacteria, or shared by selected
distinct
micro-organisms or specific for a single micro-organism.
In a further aspect of the present invention, the invention provides a
composition for
use in inhibiting growth or reproduction of infectious micro-organisms
comprising a
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modified PNA molecule according to the present invention. In one embodiment,
the
inhibition of the growth of micro-organisms is obtained through treatment with
either
the modified PNA molecule alone or in combination with antibiotics or other
anti-
infective agents. In another embodiment, the composition comprises two or more
different modified PNA molecules. A second modified PNA molecule can be used
to
target the same bacteria as the first modified PNA molecule or in order to
target
different bacteria. In the latter form, specific combinations of target
bacteria may be
selected to the treatment. Alternatively, the target can be one or more genes,
which
confer resistance to one or more antibiotics to one or more bacteria. In such
a
treatment, the composition or the treatment further comprises the use of said
antibiotic(s).
In another aspect, the present invention includes within its scope
pharmaceutical
compositions comprising, as an active ingredient, at least one of the
compounds of
the general formula I or a pharmaceutically acceptable salt thereof together
with a
pharmaceutically acceptable carrier or diluent.
Pharmaceutical compositions containing a compound of the present invention may
be prepared by conventional techniques, e.g. as described in Remington: The
Science and Practise of Pharmacy 19'" Ed 1995. The compositions may appear in
conventional forms, for example capsules, tablets, aerosols, solutions,
suspensions
or topical applications.
Typical compositions include a compound of formula I or a pharmaceutically
acceptable acid addition salt thereof, associated with a pharmaceutically
acceptable
excipient which may be a carrier or a diluent or be diluted by a carrier, or
enclosed
within a carrier which can be in the form of a capsule, sachet, paper or other
container. In making the compositions, conventional techniques for the
preparation
of pharmaceutical compositions may be used. For example, the active compound
will usually be mixed with a carrier, or diluted by a carrier, or enclosed
within a
carrier which may be in the form of a ampoule, capsule, sachet, paper, or
other
container. When the carrier serves as a diluent, it may be solid, semi-solid,
or liquid
material which acts as a vehicle, excipient, or medium for the active
compound. The
active compound can be adsorbed on a granular solid container for example in a
sachet. Some examples of suitable carriers are water, salt solutions,
alcohols,
polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive
oil, gelatine,
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lactose, terra albs, sucrose, glucose, cyclodextrin, amylose, magnesium
stearate,
talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of
cellulose, silicic
acid, fatty acids, fatty acid amines, fatty acid monoglycerides and
diglycerides,
pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and
polyvinylpyrrolidone. Similarly, the carrier or diluent may include anv
snctainar~
release material known in the art, such as glyceryl monostearate or glyceryl
distearate, alone or mixed with a wax. The formulations may also include
wetting
agents, emulsifying and suspending agents, preserving agents, sweetening
agents,
thickeners or flavouring agents. The formulations of the invention may be
formulated
so as to provide quick, sustained, or delayed release of the active ingredient
after
administration to the patient by employing procedures well known in the art.
The pharmaceutical compositions can be sterilized and mixed, if desired, with
auxiliary agents, emulsifiers, salt for influencing osmotic pressure, buffers
and/or
colouring substances and the like, which do not deleteriously react with the
active
compounds.
The route of administration may be any route, which effectively transports the
active
compound to the appropriate or desired site of action, such as oral, nasal,
rectal,
pulmonary, transdermal or parenteral e.g. depot, subcutaneous, intravenous,
intraurethral, intramuscular, intranasal, ophthalmic solution or an ointment,
the
parenteral or the oral route being preferred.
If a solid carrier is used for oral administration, the preparation may be
tabletted,
placed in a hard gelatin capsule in powder or pellet form or it can be in the
form of a
troche or lozenge. If a liquid carrier is used, the preparation may be in the
form of a
suspension or solution in water or a non-aqueous media, a syrup, emulsion or
soft
gelatin capsules. Thickeners, flavorings, diluents, emulsifiers, dispersing
aids or
binders may be added.
For nasal administration, the preparation may contain a compound of formula I
dissolved or suspended in a liquid carrier, in particular an aqueous carrier,
for
aerosol application. The carrier may contain additives such as solubilizing
agents,
e.g. propylene glycol, surfactants, absorption enhancers such as lecithin
(phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.
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For parenteral application, particularly suitable are injectable solutions or
suspensions, preferably aqueous solutions with the active compound dissolved
in
polyhydroxylated castor oil.
Tablets, dragees, or capsules having talc and/or a carbohydrate carrier or
binder or
the like are particularly suitable for oral application. Preferable carriers
for tablets,
dragees, or capsules include lactose, corn starch, and/or potato starch. A
syrup or
elixir can be used in cases where a sweetened vehicle can be employed.
In formulations for treatment or prevention of infectious diseases in mammals
the
amount of active modified PNA molecules used is determined in accordance with
the specific active drug, organism to be treated and carrier of the organism.
Such mammals include also animals, both domestic animals, e.g. household pets,
and non-domestic animals such as wildlife.
Usually, dosage forms suitable for oral, nasal, pulmonal or transdermal
administration
comprise from about 0.01 mg to about 500 mg, preferably from about 0.01 mg to
about
100 mg of the compounds of formula I admixed with a pharmaceutically
acceptable
carrier or diluent.
In a still further aspect, the present invention relates to the use of one or
more
compounds of the general formula I or pharmaceutically acceptable salts
thereof for
the preparation of a medicament for the treatment and/or prevention of
infectious
diseases.
In yet another aspect of the present invention, the present invention concerns
a
method of treating or preventing infectious diseases, which treatment
comprises
administering to a patient in need of treatment or for prophylactic purposes
an
effective amount of modified PNA according to the invention. Such a treatment
may
be in the form of administering a composition in accordance with the present
invention. In particular, the treatment may be a combination of traditional
antibiotic
treatment and treatment with one or more modified PNA molecules targeting
genes
responsible for resistance to antibiotics.
In yet a further aspect of the present invention, the present invention
concerns the
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use of the modified PNA molecules in disinfecting objects other than living
beings,
such as surgery tools, hospital inventory, dental tools, slaughterhouse
inventory and
tool, dairy inventory and tools, barbers and beauticians tools and the like.
S EXAMPLES
The following examples are merely illustrative of the present invention and
should
not be considered limiting of the scope of the invention in any way. The
principle of
the present invention is shown using E. coli as a test organism. However, as
shown
in Example 19, the advantageous effect applies in the same way to other
bacteria.
The following abbreviations related to reagents are used in the experimental
part:
(The monomers and the PNA sequences are stated in bold)
A monomer N-(2-Boc-aminoethyl)-N-(N6-(benzyloxycarbonyl)adenine-9-
yl-acetyl)glycine
Boc Tert butyloxycarbonyl
Boc-Lys(2-CI-Z)-OHN-a-Boc-N-s-2-chlorobenzyloxycarbonyl-L-lysine
C monomer N-(2-Boc-aminoethyl)-N-(N4-(benzyloxycarbonyl)cytosine-1-
yl-acetyl)glycine
DCM Dichloromethane
DIEA N, N-diisopropylethylamine
DMF N,N-dimethylformamide
DMSO Dimethyl sulfoxide
G monomer N-(2-Boc-aminoethyl)-N-(NZ-(benzyloxycarbonyl)guanine-9-
yl-acetyl)glycine
HATU N-[(1-H-benzotriazole-1-yl)(dimethylamine)methylene)-N-
methylmethanaminiumhexafluorophosphate N-oxide
HBTU 2-(1-H-benzotriazole-1-yl)-1,1,3, 3-tetramethyluronium
hexafluorophosphate
J monomer N-(2-Boc-aminoethyl)-N-(N-2 -(benzyloxycarbonyl)
/nucleobase isocytosine-5-yl-acetyl)glycine
MBHA resin p-methylbenzhydrylamine resin
NMP N-methyl pyrrolidone
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T monomer N-(2-Boc-aminoethyl)-N-(thymine-1-yl-acetyl)glycine
TFA Trifluoroacetic acid
TFSMA Trifluoromethanesulphonic acid
Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol
The following abbreviations relating to linking groups are used in the
experimental
5
part:
(The linking groups as starting materials are indicated with capital letters
whereas
the linking groups in the finished peptide-PNA conjugate are indicated with
small
letters.)
Abbreviation Linker (IUPAC)
SMCC Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
LCSMCC Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-
amido-caproate)
MBS Succinimidyl m-maleimido-benzoylate
EMCS Succinimidyl N-s-maleimido-caproylate
SMPH Succinimidyl 6-(a-maleimido-propionamido)hexanoate
AMAS Succinimidyl N-(a-maleimido acetate)
SMPB Succinimidyl 4-(p- maleimidophenyl)butyrate
(i.ALA a-alanine
PHG Phenylglycine
ACHC 4-aminocyclohexanoic acid
~i.CYPR (i-(cyclopropyl) alanine
AHA, AHEX 6-amino-hexanoic acid
ADO, AEEA-OH ((2-aminoethoxy)ethoxy)acetic acid or 8-amino-3,6-dioxaoctanoic
acid
ADC Amino dodecanoic acid
The linking groups containing a succinimidyl group are shown in Figure 5.
All the linking groups are commercial available.
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The composition of mixtures of solvents is indicates on a volume basis, i.e.
30/2/10
(v/v/v).
Preparative HPLC is performed on a DELTA PAK [Waters ](C18,15 Vim, 300 A,
300x7.8 mm, 3 ml/min) A linear gradient from solvent A: 0.1 % TFA in water to
B:
0.1 % TFA in acetonitrile was used. 0-2 min B 10 %, 2-30 min 40 % B, 30-35 min
100 % B, 35-37 min 100 % B, 37-38 min 10 % B, 37-50 min 10 % B.
Mass Spectrometry was performed on MALDI (Matrix Assisted Laser Desorption
and Ionisation Time of Flight Mass Spectrometry) as HP MALDI-TOF # G2025A
calibrated with peptide nucleic acids of the following weights: Mw~ = 1584.5
g/mol,
Mwz = 3179.0 g/mol and Mw3 = 4605.4 g/mol.
Example 1
Preparation of H-KFFKFFKFFK-ado-TTC AAA CAT AGT-NHS (SEQ ID NO: 18)
The peptide-PNA-Chimera H-KFFKFFKFFK-ado-TTC AAA CAT AGT-NHS (SEQ ID
NO: 18) was synthesized on 50 mg MBHA resin (loading 100 p.mol/g)
(novabiochem) in a 5 ml glass reactor with a D-2 glassfilter. Deprotection was
done
with 2x600 p,L TFA/m-cresol 95/5 followed by washing with DCM, DMF, 5% DIEA in
DCM and DMF. The coupling mixture was 200 pi 0.26 M solution of monomer (Boc-
PNA-T-monomer, Boc-PNA-A-monomer, Boc-PNA-G-monomer, Boc-PNA-C-
monomer, Boc-AEEA-OH (ado) (PE Biosystems Inc.)) in NMP mixed with 200 pi 0.5
M DIEA in pyridine and activated for 1 min with 200 p,1 0.202 M HATU (PE-
biosystems) in NMP. The coupling mixture for the peptide part was 200 p,1 0.52
M
NMP solution of amino acid (Boc-Phe-OH and Boc-Lys(2-CI-Z)-OH (novabiochem))
mixed with 200 ~I 1 M DIEA in NMP and activated for 1 min with 200 pi 0.45 M
HBTU in NMP. After the coupling the resin was washed with DMF, DCM and capped
with 2 x 500 p,1 NMP/pyridine/acetic anhydride 60/35/5. Washing with DCM, DMF
and DCM terminated the synthesis cycle. The oligomer was deprotected and
cleaved from the resin using "low-high" TFMSA. The resin was rotated for 1 h
with 2
ml of TFA/dimethylsulfid/ m-cresol/TFMSA 10/6/2/0.5. The solution was removed
and the resin was washed with 1 ml of TFA and added 1.5 ml of TFMSA/TFA/m
cresol 2/8/1. The mixture was rotated for 1.5 h and the filtrated was
precipitated in 8
ml diethylether.
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The precipitate was washed with 8 ml of diethylether. The crude oligomer was
dissolved in water and purified by HPLC. Preparative HPLC was performed on a
DELTA PAK [Waters ](C18,15 pm, 300 A, 300x7.8 mm, 3 ml/min) A linear gradient
from solvent A: 0.1 % TFA in water to B: 0.1 % TFA in acetonitrile was used. 0-
2 min
B 10 %, 2-30 min 40 % B, 30-35 min 100 % B, 35-37 min 100 % B, 37-38 min 10
B, 37-50 min 10 % B.
Mw calculated: 4791.9 g/mol; found on MALDI: 4791 g/mol.
Example 2
Maleimide activation of PNA
PNA-oligomer ado-TTC AAA CAT AGT-NH= (SEQ ID NO: 19) (purified by HPLC)
(2 mg, 0.589 pmol, Mw 3396.8) was dissolved and stirred for 15 min in NMP:DMSO
8:2 (2 ml). Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)
(PIERCE)(1.1 mg, 3.24 ~mol, 5.5 eq.) dissolved in NMP (50 p,1) and DIEA (34.7
p,1,
198.7 ~mol) was added to the solution: The reaction mixture was stirred for
further
2.5 h. The product was precipitated in diethylether (10 mL). The precipitate
was
washed with ether:NMP; 10:1(3x10mL) and ether (3x10mL).
Mw calculated: 3615.8 g/mol; found on MALDI: 3613.5 g/mol.
The product was used without further purification.
Example 3
Coniugation of peptide and maleimide activated PNA
A sOlutlon Of peptide CKFFKFFKFFK (SEQ ID NO: 20) (0.5 mg in 200 ~I degassed
Tris buffer 10mM, pH 7.6 (329 nM)) was added to a solution of the above
activated
product (0.2 mg in 200 p.1 DMF:Water 1:1). The reaction mixture was stirred
over
night. The target compound was purified by HPLC directly from the crude
reaction
mixture. Preparative HPLC was pertormed on a DELTA PAK [Waters ](C18,15 pm,
300 A, 300x7.8 mm, 3 ml/min) A linear gradient from solvent A: 0.1 % TFA in
water
to B: 0.1 % TFA in acetonitrile was used. 0-2 min B 10 %, 2-30 min 40 % B, 30-
35
min 100 % B, 35-37 min 100 % B, 37-38 min 10 % B, 37-50 min 10 % B.
Mw calculated: 5133.0 g/mol; found on MALDI: 5133 g/mol.
Example 4
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28
H-LLKKLAKALKG-ahex-ado-CCATCTAATCCT-NHZ (SEQ ID NO: 21)
Performed in accordance with example 1, however with the use of 6-
aminohexanoic
acid (ahex) as linker together with 8-amino-3,6-dioxaoctanoic acid (ado).
Example 5
Preparation of H-KFFKFFKFF-ado-JTJTJJT-ado-ado-ado-TCCCTCTC-Lys
NH= (SEQ ID NO: 22~
Performed in accordance with example 1, however with the use of PNA oligomer
ado-JTJTJJT-ado-ado-ado-TCCCTCTC-Lys-NHS (SEQ ID NO: 23) 111Stead Of
ado-TTC AAA CAT AGT-NHS (SEQ ID NO: 19). This PNA is a triplex forming bis
PNA in which C (cytosine) in the "Hoogsteen strand" is exchanged with the J
nucleobases (a substitute for protonated C). This substitution assures
efficient
triplex formation at physiological pH (Egholm, M.; Dueholm, K. L.; Buchardt,
O.;
Coull, J.; Nielsen, P. E.; Nucleic Acids Research 1995, 23,217-222 (42)).
Example 6
Preparation of peptide-PNA-chimeras
Different peptide-PNA-chimeras were prepared in the same way as described
above.
1 H-KFFKFFKFFK-ado-CAT AGC TGT TTC-NHZ (SEQ ID NO: 24)
2 H-FFKFFKFFK-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 25)
3 H-FKFFKFFK-ado-CAT AGC TGT TTC-NH_ (SEQ ID NO: 26)
4 H-KFFKFFK-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 27)
5 H-FFKFFK-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 28)
6 H-FKFFK-ado-CAT AGC TGT TTC-NHZ (SEQ ID NO: 29)
7 H-KFFK-ado-CAT AGC TGT TTC-NHZ (SEQ ID NO: 30)
8 H-FFK-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 31)
9 H-FK-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 32)
10 H-K-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 33)
11 H-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 34)
84 H-KFFKFFKFF-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 35)
85 H-FFKFFKFF-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 36)
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29
86 H-FKFFKFF-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 37)
87 H-KFFKFF-ado-CAT AGC TGT TTC-NHz (SEQ ID NO: 38)
88 H-FFKFF-ado-CAT AGC TGT TTC-NHZ (SEQ ID NO: 39)
89 H-FKFF-ado-CAT AGC TGT TTC-NHZ (SEQ ID NO: 40)
90 H-KFF-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 41)
91 H-FF-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 42)
92 H-F-ado-CAT AGC TGT TTC-NHS (SEQ ID NO: 43)
109 H-KFFKFFKFFK-ado-TTC AAA CAT AGT-NHZ (SEQ ID NO: 18)
136 H-KFFKFFKFFK-ado-TGA CTA GAT GAG-NHS (SEQ ID NO: 44)
130 H-KFFKFFKFFK-ado-CCA TCT AAT CCT-NH= (SEQ ID NO: 45)
140 H-KFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH= (SEQ
ID NO:
46)
141 H-FKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NHS (SEQ
ID NO:
47)
142 H-FFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NHS (SEQ
ID
NO: 48)
143 H-KFFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH~(SEQ
ID
NO: 49)
144 H-FKFFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH~(SEQID
NO: 50)
145 H-FFKFFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH=(SEQ
ID NO: 51)
146 H-KFFKFFKFF-ado-JTJTJJT-ado-ado-ado-TCC TCT C-Lys-NH~(SEQ
ID NO: 52)
170 H-FFKFFKFFK-GGC-smcc-ado-TTC AAA CAT AGT-NHS (SEQ ID
NO: 53)
171 H-FFRFFRFFR-GGC-smcc-ado-TTC AAA CAT AGT-NHS (SEQ ID
NO: 54)
172 H-LLKLLKLLK-GGC-smcc-ado-TTC AAA CAT AGT-NH= (SEQ ID
NO: 55)
173 H-LLRLLRLLR-GGC-smcc-ado-TTC AAA CAT AGT-NHS (SEQ ID
NO: 56)
174 H-LLKKLAKALK-GC-smcc-ado-TTC AAA CAT AGT-NHS (SEQ ID
NO: 57)
175 H-KRRWPWWPWKK-C-smcc-ado-TTC AAA CAT AGT-NHZ (SEQ ID
NO: 58)
17 H-KFKVKFVVKK-GC-smcc-ado-TTC AAA CAT AGT-NHS (SEQ ID
6 NO: 59)
177 H-LLKLLLKLLLK-C-smcc-ado-TTC AAA CAT AGT-NHS (SEQ ID
NO: 60)
178 H-FFKFFKFFK-GGC-smcc-ado-TTC AAA CAT AGT-NHS (SEQ ID
NO: 61)
17 H-KFFKFFKFFK-C-smcc-ado-TTC AAA CAT AGT-NHZ (SEQ ID
9 NO: 62)
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218 H-F-ado-CCA TCT AAT CCT-NH= (SEQ ID NO: 63)
219 H-FF-ado-CCA TCT AAT CCT-NHZ (SEQ ID NO: 64)
220 H-KFF-ado-CCA TCT AAT CCT-NHz (SEQ ID NO: 65)
221 H-FKFF-ado-CCA TCT AAT CCT-NHZ (SEQ ID NO: 66)
222 H-FFKFF-ado-CCA TCT AAT CCT-NHS (SEQ ID NO: 67)
223 H-KFFKFF-ado-CCA TCT AAT CCT-NHZ (SEQ ID NO: 68)
224 H-FKFFKFF-ado-CCA TCT AAT CCT-NHS (SEQ ID NO: 69)
225 H-FFKFFKFF-ado-CCA TCT AAT CCT-NHZ (SEQ ID NO: 70)
226 H-KFFKFFKFF-ado-CCA TCT AAT CCT-NHZ (SEQ ID NO: 71)
228 H-LLKKLAKALKG-ahex-ado-CCA TCT AAT CCT-NHS (SEQ ID
NO: 21)
229 H-LLKKLAKALKG-ado-ado-CCA TCT AAT CCT-NH= (SEQ ID NO:
72)
230 H-KFFKFFKFFK-ado-ado-CCA TCT AAT CCT-NH= (SEQ ID NO:
73)
231 H-KFFKFFKFFK-ahex-ado-CCA TCT AAT CCT-NHS (SEQ ID NO:
74)
232 HEN-KFFKFFKFFK-C-smcc-ado-CCA TCT AAT CCT-NHS (SEQ
ID NO: 75)
233 HEN-LLKKLAKALK-GC-smcc-ado-CCA TCT AAT CCT-NHS (SEQ
ID NO:
76)
234 HEN-KFFKFF-C-smcc-ado-CCA TCT AAT CCT-NHS (SEQ ID NO:
77)
249 H-ado-TTC AAA CAT AGT-NHS (SEQ ID NO: 78)
371 H=N-KFFKVKFVVKK-C-~smcc-ado-TTC AAA CAT AGT-NHS (SEQ
ID NO:
79)
381 HEN-KFFKVKFVVKK-C-smcc-ado-TTG TGC CCC GTC-NHS (SEQ
ID NO:
80)
Example 7
5 The peptide-PNA-chimeras in Table I were prepared as described in Example 1
using the linking groups as defined above:
Table I
PA ~ Sequence ~Mw
no.
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31
437 H=N-KKFKVKFVVKKC-achc-(3.ala-TTCAAACATAGT-NHS4808
(SEQ ID NO: 81)
432 H-KFFKFFKFFK-achc-(3. ala-TTCAAACATAGT-NH= 4848
(SEQ ID
NO: 82)
418 HEN-KKFKVKFVVKKC-lcsmcc-ado-TTCAAACATAGT-NHS5203
(SEQ ID NO: 83)
419 H2N-KKFKVKFVVKKC-mbs-ado-TTCAAACATAGT-NHS 5070
(SEQ ID
NO: 84)
420 HEN-KKFKVKFVVKKC-emcs-ado-TTCAAACATAGT-NHS 5064
(SEQ
ID NO: 85)
421 HEN-KKFKVKFVVKKC-smph-ado-TTCAAACATAGT-NHz 5135
(SEQ
ID NO: 86)
422 H~N-KKFKVKFVVKKC-auras-ado-TTCAAACATAGT-NHS 5008
(SEQ
ID NO: 87)
423 H=N-KKFKVKFVVKKC-smpb-ado-TTCAAACATAGT-NHS 5112
(SEQ
ID NO: 88)
446 HEN-KKFKVKFVVKKC-lcsmcc-gly-TTCAAACATAGT-NHS5109
(SEQ ID NO: 89)
447 HEN-KKFKVKFVVKKC-lcsmcc-(3.ala-TTCAAACATAGT-NH~5121
(SEQ ID NO: 90)
448 H=N-KKFKVKFVVKKC-lcsmcc-~i.cypr-TTCAAACATAGT-NH~5147
(SEQ ID NO: 91)
449 HEN-KKFKVKFVVKKC-lcsmcc-aha-TTCAAACATAGT-NHS5163
(SEQ ID NO: 92)
450 H=N-KKFKVKFVVKKC-lcsmcc-adc-TTCAAACATAGT-NHS5247
(SEQ ID NO: 93)
Example 8
The peptide-PNA-chimeras in Table III were prepared as described in Example 1
using the linking groups as defined above.
Table III
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32
PA no. Mw Sequence
S 201 4943,30 H-KFFKFFKFFK-ado-ado-TTCAAACATAGT-NHS
(SEQ
ID NO: 94)
S 202 4841,40 H-KFFKFFKFFK-ado-Gly-TTCAAACATAGT-NHS
(SEQ
ID NO: 95)
S 203 4881,40 H-KFFKFFKFFK-ado-P-TTCAAACATAGT-NH= (SEQ
ID
NO: 96)
S 204 4897,50 H-KFFKFFKFFK-ado-aha-TTCAAACATAGT-NHS
(SEQ
ID NO: 97)
S 205 4855,40 H-KFFKFFKFFK-ado-~i.ala-TTCAAACATAGT-NHL
(SEQ ID NO: 98)
S 206 4909,50 H-KFFKFFKFFK-ado-achc-TTCAAACATAGT-NH~(SEQ
ID NO: 99)
S 207 4841,40 H-KFFKFFKFFK-Gly-ado-TTCAAACATAGT-NH=
(SEQ
ID NO: 100)
S 208 4765,40 H-KFFKFFKFFK-Gly-Gly-TTCAAACATAGT-NHS
(SEQ
ID NO: 101)
S 209 4805,50 H-KFFKFFKFFK-Gly-P-TTCAAACATAGT-NHS (SEQ
ID
NO: 102)
S 210 4821,50 H-KFFKFFKFFK-Gly-aha-TTCAAACATAGT-NH=
(SEQ
ID NO: 103)
S 211 4779,40 H-KFFKFFKFFK-Gly-(3.ala-TTCAAACATAGT-NHS
(SEQ ID NO: 104)
S 212 4833,50 H-KFFKFFKFFK-Gly-achc-TTCAAACATAGT-NHz(SEQ
ID NO: 105)
S 213 4881,40 H-KFFKFFKFFK-P-ado-TTCAAACATAGT-NH_ (SEQ
ID
N O: 106)
S 214 4805,50 H-KFFKFFKFFK-P-Gly-TTCAAACATAGT-NH_ (SEQ
ID
NO: 107)
S 215 4845,50 H-KFFKFFKFFK-P-P-TTCAAACATAGT-NH~(SEQID
NO:
108)
S 216 4861,60 H-KFFKFFKFFK-P-aha-TTCAAACATAGT-NHS (SEQ
ID
NO: 109)
S 217 4819, 50 H-KFFKFFKFFK-P-(3. ala-TTCAAACATAGT-NH~
(SEQ
ID NO: 110)
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33
S 218 4873,60 H-KFFKFFKFFK-P-achc-TTCAAACATAGT-NH~(SEQID
NO: 111)
S 219 4897,50 H-KFFKFFKFFK-aha-ado-TTCAAACATAGT-NHS
(SEQ
ID NO: 112)
S 220 4821,50 H-KFFKFFKFFK-aha-Gly-TTCAAACATAGT-NHS
(SEQ
ID NO: 113)
S 221 4861,60 H-KFFKFFKFFK-aha-P-TTCAAACATAGT-NHS (SEQ
ID
NO: 114)
S 222 4877,60 H-KFFKFFKFFK-aha-aha-TTCAAACATAGT-NHS
(SEQ
ID NO: 115)
S 223 4835,50 H-KFFKFFKFFK-aha-(3.ala-TTCAAACATAGT-NHS
(SEQ ID NO: 116)
S 224 4889,70 H-KFFKFFKFFK-aha-achc-TTCAAACATAGT-NH=(SEQ
ID NO: 117)
S 225 4855,40 H-KFFKFFKFFK-(3.ala-ado-TTCAAACATAGT-NH~
(SEQ ID NO: 118)
S 226 4779,40 H-KFFKFFKFFK-~i.ala-Gly-TTCAAACATAGT-NH=
(SEQ ID NO: 119)
S 227 4819, 50 H-KFFKFFKFFK-(3. ala-P-TTCAAACATAGT-NHS
(SEQ
ID NO: 120)
S 228 4835,50 H-KFFKFFKFFK-(3.ala-aha-TTCAAACATAGT-NH2
(SEQ ID NO: 121)
S 229 4793,50 H-KFFKFFKFFK-(3.ala-(3.ala-TTCAAACATAGT-NHS
(SEQ ID NO: 122)
S 230 4847,60 H-KFFKFFKFFK-(3.ala-achc-TTCAAACATAGT-NH=
(SEQ ID NO: 123)
S 231 4845,50 H-KFFKFFKFFK-P-p-TTCAAACATAGT-NH~(SEQID
NO:
124)
S 232 4845,50 H-KFFKFFKFFK-P-P-TTCAAACATAGT-NH~(SEQID
NO:
125)
S 233 4907,70 H-KFFKFFKFFK-K-K-TTCAAACATAGT-NH~(SEQID
NO:
126)
S 234 4945,70 H-KFFKFFKFFK-F-F-TTCAAACATAGT-NH~(SEQID
NO:
127)
S 235 4926, 60 H-KFFKFFKFFK-F-K-TTCAAACATAGT-NHS (SEQ
ID NO:
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34
128)
S 236 4926, 60 H-KFFKFFKFFK-K-F-TTCAAACATAGT-NH? (SEQ
ID NO:
129)
S 237 4917,50 H-KFFKFFKFFK-phg-ado-TTCAAACATAGT-NHS
(SEQ
ID NO: 130)
S 238 4841,50 H-KFFKFFKFFK-phg-Gly-TTCAAACATAGT-NHS
(SEQ
ID NO: 131)
S 239 4881,60 H-KFFKFFKFFK-phg-P-TTCAAACATAGT-NHS (SEQ
ID
NO: 132)
S 240 4897,60 H-KFFKFFKFFK-phg-aha-TTCAAACATAGT-NHS
(SEQ
ID NO: 133)
S 241 4855,50 H-KFFKFFKFFK-phg-(3.ala-TTCAAACATAGT-NHS
(SEQ ID NO: 134)
S 242 4909,60 H-KFFKFFKFFK-phg-achc-TTCAAACATAGT-NH~(SEQ
ID NO: 135)
S 243 4909,50 H-KFFKFFKFFK-achc-ado-TTCAAACATAGT-NH~(SEQ
ID NO: 136)
S 244 4833,50 H-KFFKFFKFFK-achc-Gly-TTCAAACATAGT-NHZ(SEQ
ID NO: 137)
S 245 4873,60 H-KFFKFFKFFK-achc-P-TTCAAACATAGT-NH~(SEQID
NO: 138)
S 246 4889,60 H-KFFKFFKFFK-achc-aha-TTCAAACATAGT-NH~(SEQ
ID NO: 139)
S 247 4847,60 H-KFFKFFKFFK-achc-(3.ala-TTCAAACATAGT-NHS
(SEQ ID NO: 140)
S 248 4901,70 H-KFFKFFKFFK-achc-achc-TTCAAACATAGT-NH~
(SEQ ID NO: 141)
Example 9
Description of a primary screen
The bacterial growth assay is designed to identify modified PNA molecules that
inhibit or completely abolish bacterial growth. Growth inhibition results from
antisense binding of PNA to mRNA of the targeted gene. The compound tested is
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present during the whole assay.
Components
The experimental bacterial strain for the protocol is Escherichia coli K12
MG1655
5 (E, coli Genentic Stock Center, Yale University, New Haven). The medium for
growth is 10% sterile LB (Lures Bertani) medium.
E. coli test cells are pre-cultured in LB medium at 37 °C over night
(over night
culture). The screen is performed in 96-well microtiter plates at 37 °C
under constant
10 shaking.
PNA's are dissolved in H20 as a 40x concentrated stock solution.
Assav conditions
15 From an over night culture a fresh culture (test culture) is grown to mid-
log-phase
(ODsoo = 0.1 corresponding to 10' cells/ml) at 37 °C. The test culture
is diluted
stepwise in the range 105 to 10' with 10% LB medium. 195 ~I of diluted
cultures plus
5 ~,I of a 40x concentrated PNA stock solution are added to each test well.
20 96-well microtiter plates are incubated in a microplate scanning
spectrophotometer
at 37 °C under constant shaking. OD6oo measurements are performed
automatically
every 3.19 minutes and recorded simultaneously.
Target genes:
Penicillin binding proteins (PBPs)
PBPs act in biosynthesis of murein (peptidoglycan), which is part of the
envelope of
Gram-positive and Gram-negative bacteria. By binding of penicillin, which acts
as
substrate analogue, PBP's are inhibited, and subsequently, hydrolytic enzymes
are
activated by the accumulation of peptidoglycan intermediates, thus hydrolysing
the
peptidoglycan layer and causing lysis.
E.coli has 7-9 PBPs, the high molecular weight PBPs, PBP1A and PBP1 B, PBP2
and PBP3, and the low molecular weight PBPs, PBP 4-9. The high molecular
weight
PBPs are essential for growth, whereas the low molecular weight PBPs are not
essential.
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36
S
PNA design no. 1
PNA26 has been designed according to the sequence of the mrcA (ponA) gene of
E. coli, encoding PBP1A. The sequence of the mrcA gene (accession number
X02164) was obtained from the EMBL sequence database (Heidelberg, Germany)
(Broome-Smith et al. 1985, EurJ Biochem 147:437-46 (41)). The sequence of the
mrcA gene is shown in Figure 3.
The target region of PNA26 is the following:
sense 5' AATGGGAAATTTCCAGTGAAGTTCGTAAAG 3' (SEQ ID NO: 142)
121 -________+_________+_________+ 150
antisense 3' TTACCCTTTAAAGGTCACTTCAAGCATTTC 5' (SEQ ID NO: 143)
Both the coding and the non-coding (antisense) strand of the GTG start codon
region are shown.
The sequence of the GTG start codon region of the antisense strand and PNA26
are
shown in the 5' to 3' orientation:
antisense 5' CTTTACGAACTTCACTGGAAATTTCCCATT 3' (SEQ ID NO: 143)
PNA2 6 H-KFFKFFKFFK-ado-CACTGGAAATTT-Lys-NHZ (SEQ ID NO: 144)
PNA26 is a 12mer PNA molecule (shown in bold) coupled to a 10 amino acid
peptide.
Growth assay with PNA26
The assay was performed as follows:
Dilutions of the test culture corresponding to 105, 10°, 103, 102 and
10' cells/ml
containing PNA26 at a final concentration of 1.5, 2.0, 2.5, 3.0 and 3.5 ~M are
incubated at 37°C for 16 hours with constant shaking. Total inhibition
of growth can
be seen in cultures with 104-10' cells/ml and a PNA concentration of at least
2.5~M
(Table 1 ).
PNA design no. 2
PNA14 has been designed according to the sequence of the mrdA gene encoding
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37
PBP2. The sequence (accession number AE000168, bases 4051-5952) was
obtained from the E. coli genome database at the NCBI (Genbank, National
Centre
for Biotechnology Information, USA).
The sequence of the mrdA gene is shown in Figure 4
The target region of PNA14 is the following:
sense 5' GAGTAGAAAACGCAGCGGATGAAACTACAGAAC 3' (SEQ ID NO: 145)
99 _________+_________+_________+___ 131
antisense 3' CTCATCTTTTGCGTCGCCTACTTTGATGTCTTG 5' (SEQ ID NO: 146)
Both the coding (sense) and the non-coding (antisense) strand of the GTG start
codon region are shown.
In the following sequence of the ATG start codon region of the antisense
strand and
PNA26 are shown in the 5' to 3' orientation:
antisense 5' GTTCTGTAGTTTCATCCGCTGCGTTTTCTACTC 3' (SEQ ID NO: 146)
PNA14 HKFFKFFKFFK-ado-TTTCATCCGCTG-Lys-NHZ (SEQ ID NO: 147)
PNA14 is a 12mer PNA molecule (shown in bold) coupled to a 10 amino acid
peptide.
Growth assay with PNA14
The assay was performed as follows:
Dilutions of the test culture corresponding to 105, 104, 103, 102 and 10'
cells/ml
containing PNA14 at a final concentration of 1.3, 1.4 and 1.5 ~M are incubated
at
37°C for 16 hours with constant shaking. Total inhibition of growth can
be seen in
cultures with 104-10' cells/ml and a PNA concentration of at least 1.4wM
(Table 2).
Example 10
Bacterial 4rowth inhibition with PNA against the LacZ Gene
Peptides are truncated versions of the KFF-motif. The basic peptide sequence
is
KFFKFFKFFK (SEQ ID NO: 148) (PNA 1). PNA 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 all
contain peptides which are truncated from the C-terminal end. PNA 84, 85, 86,
87,
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38
88, 89, 90, 91 and 92 all contain peptides which are truncated from the N-
terminal
end. The PNA against the LacZ-gene has been synthesized with and without an -
NHZ terminal lysine.
The assay was performed as follows:
Dilutions of the test culture E. coli K12 corresponding to, 5x105 and 5x104,
cells/ml
containing truncated versions of the KFF-motif of the PNA's against the LacZ
gene
at a final concentration of 100, 300, 750 and 1500 nM are incubated in M9
minimal
broth with lactose as the sole carbon source (minimal media 9, Bie & Berntsen
Cph)
at 37°C for 16 hours with constant shaking.
Total inhibition of growth can be seen in cultures with 5x104-105 cells/ml and
a PNA
concentration of at least 300nM (see Table 3). The results show that the basic
KFF
motif 10-mer as well as truncated peptides thereof (4, 5, 6, and 9mer) may be
used
to enhance the inhibitory effect of PNA.
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39
r-
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CA 02388991 2002-04-12
WO 01/27261 PCT/DK00/00580
0
X a a a a a a a a a aa a a a a a a a a a
~n Z Z Z Z Z Z Z Z Z ZZ Z Z Z Z Z Z Z Z Z
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:.-_
O
C
L
X w.
1 I 1 I I , I 1 I II I 1 1 1 , I 1 I
O '"
~
'-'
O O .
.C
N j
I 1 I I I 1 1 1 I 11 I 1 I 1 I I I I 1
O Q C
N N O ..C
~
L (~ i~
~a
N
~ J t t 1 t I t 1 t I t t t t ~ a
I 1 1 I t 1 t .~
C
O .~
N N L O O
L L L
L L L L L LL L L L L L L L L L
r ~ ~ ~ ~ r~~ ~~ ~ ~ ~ ~ ~
r r r r r rr r r r r
O
L
. w a I 1 I 1 1 I I I I1 1 1 1 1 I 1 1 1 I
O
V ~ O 0000f'~ Cp(pInn <t~ M M N N m - O W :,_ ..C
f I O
C 'C
M f
~U
do .~
O
U ~
Z O Z
Z ~ ~ ~ ( O I ~ QO O O ~ O N ~ Q y . .
~ N 0 M 0 ~ 0 n 0 O0 ~ 0 00O O p ~ y - ~ t t 1
0 0 0 I 0 C 0I 0
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41
Example 11
Bacterial Growth inhibition with PNA against the infA Gene of E coli (seauence
as
PNA 130).
The PNA130 and 218-226 against the infA-gene have been synthesized with
peptides as truncated versions of the KFF-motif.
Growth assay with PNA130
The assay was performed as follows:
Dilutions of the test culture E. coli K12 corresponding to, 2x104 and 4x104,
cells/ml
containing truncated versions of the KFF-motif of the PNA's against the infA-
gene at
a final concentration of 200, 400, 600 800 and 1000 nM are incubated in 10%
Mueller-Hinton broth at 37°C for 16 hours with constant shaking.
Total inhibition of growth can be seen in cultures with 4x104-2x104 cells/ml
and a
PNA concentration of at least 600nM (Table 4). The results show that the basic
KFF
motif 10-mer as well as truncated peptides thereof (6 and 9mer) may be used to
enhance the inhibitory effect of PNA.
Example 12
Bacterial growth inhibition with PNA agiainst the a-sarcine loop of ribosomal
RNA.
The PNA's 140-146 against the a-sarcine loop of ribosomal RNA has been
synthesized with peptides as truncated versions of the KFF-motif.
Growth assay
The assay was performed as follows:
Dilutions of the test culture E, coli K12 corresponding to, 2x104 and 4x104,
cells/ml
containing truncated versions of the KFF-motif of the PNA's against a-sarcine
loop
of ribosomal RNA at a final concentration of 200, 400, 600, 800 and 1000 nM
are
incubated in 10% Mueller-Hinton broth at 37°C for 16 hours with
constant shaking.
Total inhibition of growth can be seen in cultures with 5x105-5x104 cells/ml
and a
PNA concentration of at least 200nM (Table 5). The results show that the basic
KFF
motif 10-mer as well as all truncated peptides thereof comprising at least 3
amino
CA 02388991 2002-04-12
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42
acids may be used to enhance the inhibitory effect of PNA.
Example 13
S Bacterial growth inhibition with PNA against the FtsZ Gene of E coli K12.
Growth assay with PNA170-179 and 109
The assay was performed as follows:
Dilutions of the test culture E. coli K12 corresponding to, 700 and 350
cells/ml
containing variations of amphipathic 10, 11 and 12-mer structures with smcc-
linker
of the PNA's against the FtsZ-gene at a final concentration of 200, 300, 400,
500,
600, 800 and 1000 nM are incubated in 100% Mueller-Hinton broth at 37°C
for 16
hours with constant shaking.
Total inhibition of growth can be seen in cultures with 350-700 cells/ml and a
PNA
concentration of at least 300nM (Table 6). When comparing 109 with 179, the
smcc
linker appears to add some advantages to the molecule. Further, sequence 174
shows promising results.
CA 02388991 2002-04-12
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43
0
x
N ~ . ~ W . ~ ~ + t
O p
O c-
O X ~ ~
v
O
X
N ~ ~ ~ ~ ~ '. ~ ~ + +
c
O O
O X
00~f'~ ~ ~ ~ y. ~ . ~ +
c
O
X
N
v
O
O N
+.
N
v >
O :,.
X C
N ~ ~ ~ . ~
N
Q O
O E
O
C O ~ ~ >
O X
e-
N
_~n ~ N C
O ~ ~ L
O w
.C _ _
V X ~ ~ . ~ ~ ~ ~ ~ ~ . ~. ca
N V -p
O _~ O
O
O C
Z _ ~ ~ . ~ . ~ . ~ O ~ C
N X ~ C N O
tt ~ >,
C O O
N _ L C U ~ C
L L L L L L L L . = L 'a
. 'N ~ C CZ
~ E ~ ~ E ~ O ~ ca
N E o (O J z
C7~' ~ CflI~00 O H
U
N
Q O 00 O O r- N M ~ ~ (p O J
Z O r-
d Z N N N N N N N N N
I- f '. ~ C
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44
Q
0
x
~n'. + + + + + c
0 0
0
~n.+.-+ + + + + c
Q
0
~n~ + + + + + +
0 0
ao ~n~ + + + + + +
Q
0
r-
x
~n. + + + + + +
~
o
o x
co ~n. + ~ + + + +
a~
w
o c
ca
~n. + + + + + +
a~
'o
~
a~
p X + ~ + + -, + ~
'. ~ ~ ~ >_
Q L
3 0 .~u c~
,C
V ~ + ~. + ~ ~. v. ~ O 'O
C
O O N
O + ~ C ~
r-~ ~ + . O x
O x ~. >, O
CV ~ _. .
C N
O
C U ~ C
O ~ w ' ~ w w ~ w O
N C ~ 'O
w O O ~.
H (J) J Z
a ' M v ui c~~ 00 0~
U
fB In
W
Q "- J
O ~ r ~ r r ~ ~
Z O
d Z - + v ~y C
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0
m ~ ...
M , ~ , , t , t , , t
C
O
O O
O O
I~ , t , , t , t , , t
O
tn ~ .-.
M , t , , t , ~ , , t
C
O O
O O ~
00 I~ , t , , + , , , t
O
M , . , , t , , , , t ,
_
C
O O
O O
(O I~ , , , , t , , , , t ,
O
M , , , , t , , , , t ,
C
O O
O O
f~ , . . , t , , , , t ,
O
M , , , , t , , , ,
O
N
O O >
O O ~ 'v=
, , , , . . , , '., C
cQ
L
O
M , . , , t , , , , t , ~ w.
L ~ p
C p O 4j "-
O , , , , , , , , , t , O cU C
d
M ~
p
+.. tn
U -p N
M , , , . , , , , , , , L ~ N
-p
O
X C
C C
O , , , , , , , , , , , O N
O
N I~ = C N
Q C V
Z L L L L L L L L L L L
O
C a N O N N O N N O N O p H (!) J Z
0 ~ . , , . , , , , , , ,
U
...
Cfl
L ~
_
C C O Q O c-N M d' ~ (O 1~ d0 O O
O O O Z ~ f~I~ I~ I~ I~I~ I~ I~ I~O
U U z a ~ ~ ~- r- ,- <-
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46
Example 14
Bacterial growth inhibition with PNA with different kinds of linker and
peptide
against the gene encoding
S IF-1 of E. coli.
E. coli K12 in 100% Mueller-Hinton broth.
For the 7 PNA's in this set-up, the sequence of the nucleobases is the same as
the
sequence in PNA 130, but the linking groups and the peptides varies.
PNA Linker Peptide
PNA228 ahex-ado G-KLAKALKKLL (SEQ ID NO: 149)
PNA229 ado-ado G-KLAKALKKLL (SEQ~ID NO: 150)
PNA230 ado-ado KFFKFFKFF (SEQ ID NO: 151)
PNA231 ahex-ado KFFKFFKFF (SEQ ID NO: 152)
PNA232 smcc-ado H-C-KFFKFFKFFK-NHS (SEQ ID NO:
153)
PNA233 smcc-ado H-CG-KLAKALKKLL-NHS (SEQ ID NO:
154)
PNA234 smcc-ado H-C-FFKFFK-NHS (SEQ ID NO: 155)
Experimental set-up corresponds to the set-up as described in Example 13.
1 S As can be seen from Table 7a and 7b, in the present combination of PNA and
Peptide, the smcc-ado linker seems to be the superior linker showing total
inhibition
of growth in cultures with 1.6x103-8x102 cells/ml and a PNA concentration of
at least
600nM.
Example 15
Bacterial growth inhibition with 9 mer pe~~tide
In order to test the effect of the Peptide without the PNA, the peptide no.
2339 with
the sequence: H-KFFKFFKFF-OH (SEQ ID NO: 1) was added to E. coli K12 in 10%
and 100% medium (Mueller-Hinton broth).
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Growth assay of the peptide no. 2339
The assay was performed as follows:
Dilutions of the test culture corresponding to 105, 104, and 103 cells/ml
containing the
peptide no. 2339 at a final concentration of 100 to 20.000 nM, are incubated
at 37°C
for 16 hours with constant shaking. Total inhibition of growth can be seen in
cultures
with 7.9x103 cells/ml and a peptide concentration of at least 20.000 nM,
minimal
signs of inhibition of growth can be detected at concentrations from 5000 nM
(10%
medium: Table 8; 100% medium: Table 9). Conclusions: Peptides are active alone
but only at very high concentrations and above the range used for PNA growth
assays.
Example 16
Bacterial Growth inhibition with 9 mer peptide and non-sense PNA
Growth assay of the peptide no. 2339 together with nonsense PNA 136
The assay was performed as follows:
Dilutions of the test culture corresponding to 105, 104, and 103 cells/ml
containing
PNA 136 alone or PNA 136 and the peptide no. 2339 in equal amounts at a final
concentration of 400 to 1000 nM, are incubated at 37°C for 16 hours
with constant
shaking. No inhibition of growth was detected in any of the concentrations
(Table
10). Conclusions: nonsense PNA is not active in the chosen range.
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48
0
, . , + + ,
0
, , , . + + ,
0 0
0 0
o m
r- ~ , , , , + + ,
0
, . , , + + ,
0
, , , , + +
0
0 0
o m
ao ~ , . , , + + ,
0
, , , . + + ,
0
, , . , + + ,
ca ~
a
o ~ a~
c'u E
o v~~n _.
co cv~ . , , . + + ,
c >
o V=
,~ c
a~o ~
, , , ~ , L
0 r' W.
O E
v-.
O ~ L ~ O
O ~ ~ ~ >
f~ . , , . ~ , ~ w
O cC C
C O t N
p O Q
U ~ .~ .-. '~ (p V7
, , , , , N
~ ~ V ~ p
~ N
O = L_ ~ O
O , , , , , , , ~O..N.C
N ~ \ X
~ c-
O ~ O_ ;t. ~ N
C .' ~ L ~ N O
.- O f7 , , , , , , , O C U
U ~ O L ._ v- L O
C U I~ ~ ~ C Q 'O
O ~ I~U .-
U o ~
Q ~ , , , , , , , , ~ F- (n J
d NO Z r Z
W
J
Z N N N N N N N m .. ..
I- + ~ , C
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49
0
+ + + +
Q
o
r- ~- '. + +
a~
0
o o
1 I + + '
c
s
'. '. + +
0
E
0
1 -~ + + ai
~+./
s
a
o ~, cv
0 0
I . + +
c
a~
0
r 1 I + +
c
U O
<nO _ 'a
!'
I , + + _
c
Q
Z
L
0 ~ ~ + ~ C
1 I +
O
I 1 + v
U E
w
O v ~ ~ L 7
O I I
~ V W
O
ca
O O O I + I .-.
U
C r' 1
O /
Z
N ch O C
O I I I I
N
C N_ r0 :O O
O I I I I
~ C ~C
F- J
a N . I I I
Q J
40 O O r- N w, C M .. C ~'w. C "
N N M M M O O M O O M O O Q .
N N N N N C 'a N C 'a N C 'O ~- + ,
CA 02388991 2002-04-12
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x
o 00
'o ~o +
0
o X~ o
. ~n . +
o o
v~ ~
x~
00 p ~
v o +
,- ~ .
.' ~..
oi~~ xm
o ~o
~ . ~
0
x o ~
o ~
r . ~ ~
Op x ~ U
O ~' N
O w
N
O
O O ~
f~ , I~ ~ V
r- ~
p N ~ L
O
O ~ O O N O + ca
O O
~ Q ~~ . ~nQ ~
x ~ X cU E
,,.> ~
O O m _
o o 3
c vt . c o . cv a~.
c
_
U X X O
U M N M ~ C 7
p O p
O O
~ L
_O _
O O xv O O Xv O ~ U N
O U ~ ~ U O U O ~ E
O O
~ V' . N
~- w0
O w= w
O C O . w N
~ C ~ (~ C C
~
m L co c6
x V X O L
Q~
r~ ~
p p U ..
r~ N m
~ I~ ~ 0 1~ ~ ~ ~ ~ ~ cNn tin
~ ~
~ N
'O ' .
_C _C O O _C O fU ~
O fl
cn0 tn Ln~ ~ t Wn c O N
M ~ , O , ~ _'
~ ~ C C
N L ~ L O O
X O O
C O C ~ ~ O
~ O O C O
U p ,(~~' + ~ C N
~
C ~ C ~ C ~ L N tn tn
X C
V ~ O OU ~ ~~~ V U O 'C w L L
O 0 O
O IB Q Q 'a
~ ~ ~ O L ~ L ~ + ~
I c- I ~- ~
, ~ O o
O w O O O
_
X ~_.O O X :~.OO ~ H (n J J
O O O O M ~ ~ O Z
O
O O ~ O
O
a ,-z ~,-, a ~ z ~ . a z ~r-
' w
Q M Q p
M ~
O . .-:
~
~
H
n1 a N ,
+ '.~
C
CA 02388991 2002-04-12
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51
..
U
N
N
N
O
U
L
O
N ~
~ 3
a~ -
c
ca ~
" vi ~n
~ a~ a~
o' E ~
~ :_, ~.
t -- a~ a~
> >
c~ c c
Cn L ~ N
f~
~ N
U ~ ~
f~
C O O
O ~ C C
O O X X
>, O O
.C N N 4i O
C U to (U C
.- ~ L L O
'C Q Q.'O
r ~ m ~w
O .- fa IB O
H (~ J J z
W
J
~v~C
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52
0
1 1
M
O
r
I 1
N
O O
O
1 1
O O
r r
1 1 ~ 1 1
O O
r r
1 1 ~ 1 1
w
U
O O p N
O r I 1 O r _O I I
O LL O ~
o v o ~ U
In O $ p L
I 1 ~ ~ I I
L w
.. ~ N p
.
O
~ o .3 M
1 1 cv o I 1 +, '3
LL ~ ~ >
U U s1=. v7
L N ~ L N C
p O I I O 1H I I
~ ~ O O "'' v~ ~n
~ O O LL ~ N N
w N ~ Ct
w
3 O O O Q L N N
~ ~ 1 1 3 ~n I 1 > >
c ~ o c ~c o ~ ~ :,_ ".-.
.- U ~- ~ fp C C
C O LL O L
V w O ' N ~
O U O 'C(pNt~
M ~- ~ N v1
N >_ p N w "' V 'a
O 1 1 'Q ~ ~ 1 I fB O -p -p
a. ~ ~ Q U LL L C O N
d. ~ O C O X C C
C
Q O O O ~ O O C N N
~ I 1 Q '' 1 I O O X X
V _ _ p O O ~' >' O O
D tL Z ~
~ O LJ LL = ~ N N N
C U fa ffl C
- i,~ L t
a a
N ~ N m_
FO- (/) J J Z
O
W
J
..
C
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53
Example 17
Bacterial Growth inhibition with PNA (without peptide) against the gene
encoding
FtsZ of E, coli and a peptide
E. coli K12 in 100% Mueller-Hinton broth.
PNA 249 is equal with PNA 109, without the peptide but still with the ado-
linker.
The Peptide of PNA 250 has the sequence: H-CG-KLAKALKKLL-NHS (SEQ ID NO:
156). The peptide is also used for PNA 174.
In the wells with both PNA and peptide there is equal amount PNA and peptide.
As can be seen in Table 11, neither 249 nor 250 alone nor 249 and 250 together
show any useful effect in the low concentration end. Only the peptide alone in
concentrations above 2500 nM may show growth inhibition effect.
Example 18
Bacterial growth inhibition with PNA against the gene encoding IF-1 of E coli
E. coli K12 in 10% Mueller-Hinton broth. Peptides are versions of the KFF-
motif
placed C- or N-terminal to the PNA.
From Table 12 it can be seen that the orientation of the Peptide is not so
important.
However, for specific combinations of PNA and Peptide, one of the orientations
may
be preferred.
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ao , aoc +
,
~a
, ~c +
,
o o
o a
, N ~C +
,
O , 00C + p
,
O
I~-a O I'
'
r , c-C +
,
OO +
O
O O O Oa
r- c- , r- s-C + N
,
00 , 00C + 00,
C
O
N
O
a Q ~a
O
, c ~c ~ ~ ~ ~ ,
m ~ +
m cv
co o m o ca
c 0
o
o .- a ~n o ~a + ~0 ~
, c ~c ., .-
.
. a~
0 0 o a
V O , V p, , V p ,
,
O
O O >
p p) p
, , ~ ~, ,
3 3 + O .. in
O O
O C O O C O p V O O
C :_
O O I~ ~ I~ O ~ .
In ~ N O ~ O t ~
~ , , , , ~~ ~-, N
C N
O ~ ~ ~ ~ ~ ~ C
L O
a N ~ a v~ ~ L
00 C ~ ~ 00, C ~ ~ pp, _
, N
c ~ 3 ca 3 ca
~L C ~L C ~L U a
.
U ~ a (~ V I~ a UO
C ~
, O ~ , C CO ~ ~ , ~ C a
O
_ L O~ L ~ C
Q O O O Q~ O O Q+ O O O X
Z '
~ O ~ a ZO O f~ a Zp O ~ w >. N
a N f c- C ~N Z ~-, C ~InZ ~ ~ C O
Z ,
, L N
~ C
C
.Q '~ a . :~ Q
a
Q
w-' ~. ~ C p~
O r..
J
Z
N
W
J
N a N L N H + '. '
C
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x
N ~ .- .
f
X
O O f + ~
N ~
O X
O N O
+ f + , ,
X M
N O
t
X
O O
N r- + + + , , +
X
O N O + + + , , ~
00 l0 '.
c-
X
~nN
O t
Q
v
O t t t , , ~
N ~-
C
O X
p tnN ~ t .-., , ,
CflO tn . ~
c-
O
V X v
N + t , , , _E
.
O
C X ~ >
C O ~ t + , ,
N ~ ~
U
C X N
V O , , ~ , O
~ u7 . .
~
_C f~U L " N
X aj
E N O . , , . , , O N w
~ ~ v~ ~ ccv
c .c~
._
o X '~ ~
n
U U (O :) N
C O , , , , , , U 'O
O ~ ~ N
N ~
C
0 O CV , , , , , , O ~ C
N Z 0 p O
~ c-
w
O
O ~
Z U U U Z Z t
~~ :~ s o
w c
- ~ a~ .-
~ ~ o cv o
N N . . I- (/) J Z
' ~ ~ O O O O
Q.
0 o E E ~ E
G7 (O (fl~ -
W
J
Z MO c~f-~ ~ M (flm
N N N N N H f ~ ~,~ C
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56
Example 19
Inhibition of bacterial growth by PNA-peptide with specificity for the
ribosomal a
sarine loop
In order to show that the present invention may be used in a treatment of many
micro-organisms, a selection of Gram-negative and Gram-positive bacteria were
treated under the same assay conditions as used in example 12. The modified
PNA
molecule used is PNA 146.
Gram-neoative organisms Inhibition of growth
Escherichia coli +
Klebsiella pneumonia +
Pseudomonas aeruginosa +
Salmonella typhimurium +
Gram-positive organisms
Staphylococcus aureus +
Enterococcus faecium +
Micrococcos luteus +
Conclusions: All of the bacterial isolates were inhibited. Using the same
assay
conditions used for testing of E. coli K.12, we have demonstrated growth
inhibition of
different Gram-negative and Gram-positive organisms.
Example 20
Preparation of peptide-PNA-chimeras
A peptide-PNA-chimera was prepared in the same way as described in Example 1:
H=N-SILAPLGTTLVKKVATTLKKIFSKWKC-smcc-Ado-TTCTAACATTTA-NHS (SEQ
ID NO: 159).
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57
Example 21
Gene target selection and Bacterial Growth inhibition with PNA
a. Gene target selection in E. faecalislE. faecium
The annotated E. faecium genome is, alongside with 250 other genomes,
commercially available from Integrated Genomics, Chicago.
Single annotated genes from both organisms are also available in Genbank.
b. In vitro experiments.
The ability of PNA conjugates to inhibit bacterial growth is measured by the
use of a
microdilution broth method using 100% Mueller-Hinton broth, according to NCCLS
Guidelines.
A logphase culture of E. faecium is diluted with fresh prewarmed medium and
adjusted to defined OD (here: Optical Density at 600 nm) in order to give a
final
concentration of 1x10° bacteria/ml medium in each well, containing 195
~,I of
bacterial culture. PNA is added to the bacterial culture in the wells in order
to give
final concentrations ranging from 450 nM to 1500 nM. Trays (e.g. Costar #3474)
are
incubated at 35°C by shaking in a robot analyzer (96 well microtiter
format),
PowerWavex, software KC°' Kebo.Lab, Copenhagen, for 16 h and optical
densities
are measured at 600 nm at short intervals during the incubation time in order
to
record growth curves. All cultures are tested in order to detect
contaminations.
MIC and MBC:
In addition experiments were carried out to evaluate the relationship between
MIC's
and MBC's (Minimal Bactericidal Concentration) of the PNA.
The studies were performed on 3 strains of Enterococcus faecium obtained from
American Type Culture Collection (ATCC). These strains served as initial
indicators
of possible interference from known in vivo selected vancomycin resistance
mechanisms. The table below summarizes the characteristics of the strains.
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58
E, facium Strain Description
8803 susceptible to vancomycin ,ciprofloxacin, gentamycin, rifampin,
teicoplanin
ATCC 51550 Multidrugresistant (ampicillin, ciprofloxacin, gentamycin,
rifampin, teicoplanin, vancomycin)
ATCC 700221 resistant to vancomycin
Experimental setup
S MIC's was detected as previously described. Trays were incubated at 35
° C for
further 24 h in order to analyze regrowth of inhibited bacteria (MBC's).
PNA conjugate from Example 20:
Bacterial strains: 8803, 51550, 700221
PNA concentration in wells: 400, 800 and 1600 nM
Results
The Minimal Inhibitory Concentrations (MIC~s) of the PNA conjugate were as
follows:
E.facium Strain MIC MBC
~g/ml nM
8803 < 400 < 400
ATCC 51550 _< 400 _< 400
ATCC 700221 _< 400 < 400
Peptide control: Seq. of the peptide
of > 5000 > 5000
conjugate from Example 20
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Example 22
Preparation of peptide-PNA-chimeras
A peptide-PNA-chimera was prepared in the same way as described in Example 1:
H=N-KKFKVKFWKKC-smcc-Ado-ACTTTGTCGCCC-NH2 (SEQ ID NO: 160) .
Example 23
Gene target selection and Bacterial girowth inhibition with PNA
The selection of potential gene targets and testing of ensuing PNA constructs
have
been performed with Staphylococcus aureus NCTC 8325. This strain was obtained
from Prof. J. landolo, University of Oklahoma Health Sciences Center,
Department
of Microbiology and Immunology. S.aureus NCTC 8325 is being sequenced in the
S.
aureus Genome Sequencing Project at the University of Oklahoma's Advanced
Center for Genome Technology (OU-ACGT).
The genome is not completely sequenced. The genome size is 2.80 Mb, of which a
total of 2,581,379 by has been sequenced. Annotated gene sequences are
available
from Genbank for a number of putative targets.
a. Target selection approach
The basic approach used was similar to that used in the previous example.
Potential
target genes were retrieved from the unfinished genome sequences of S. aureus
at
the OU-ACGT as well as Genbank. The presence of homologous genes and target
sequences in bacterial genomes were tested by using the BLAST 2.0 programs at
the NCBI (National Center for Biotechnology Information) www BLAST server.
The antibacterial PNA conjugate prepared in Example 22 was used for the
following
experiments:
b. In vitro experiments
The ability of PNA to inhibit bacterial growth is measured by the use of a
microdilution broth method using 100% Mueller-Hinton broth, according to NCCLS
Guidelines.
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A logphase culture of S aureus is diluted with fresh pre warmed medium and
adjusted to defined OD (here: Optical Density at 600 nm) in order to give a
final
concentration of 1x104 bacteria/ml medium in each well, containing 195 ~,I of
5 bacterial culture. PNA is added to the bacterial culture in the wells in
order to give
final concentrations ranging from 450 nM to 1500 nM. Trays (e.g. Costar #3474)
are
incubated at 35°C by shaking in a robot analyzer (96 well microtiter
format),
PowerWaveX, software KC4~ Kebo.Lab, Copenhagen, for 16 h and optical densities
are measured at 600 nm at short intervals during the incubation time in order
to
10 record growth curves. All cultures are tested in order to detect
contaminations.
MIC and MBC:
In addition experiments were carried out to evaluate the relationship between
MIC's
( Minimal Inhibitory Concentration) and MBC's (Minimal Bactericidal
Concentration)
1 S of the PNA's.
The studies were performed on the reference strain Staphylococcus aureus NCTC
8325 obtained from Prof. J. landolo, University of Oklahoma Health Sciences
Center, Department of Microbiology and Immunology. In addition we included two
20 vancomycin resistant isolates of S.aureus obtained from American Type
Culture
Collection. These strains served as initial indicators of possible
interference from
known in vivo selected vancomycin resistance mechanisms. The table below
summarizes the characteristics of the strains.
S.aureus Strain Description Vancomycin MIC
(~.g/ml)
8325 Susceptible to methicillin, vancomycin < 0.5
ATCC 700698 Intermediate vancomycin resistant. 2
Resistant to methicillin
ATCC 7006988 highly vancomycin resistant subclone of 11
ATCC 700698
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Experimental setup
MIC's were detected as described above. Trays were incubated at 35 ° C
for further
24 h in order to analyze regrowth of inhibited bacteria (MBC's).
PNA from Example 22:
Bacterial strains: 8325, 700698, 7006988
PNA concentration in wells: 400, 800 and 1600 nM
Results
The Minimal Inhibitory Concentrations (MIC) were as follows:
S.aureus Strain MIC MBC
~.g/ml NM
8325 800/1600 1600
ATCC 700698 800/1600 1600
ATCC 7006988 800/1600 > 1600
Peptide control: Seq. of the peptide
of > 5000 > 5000
conjugate from Example 22
Example 24
A compound of the invention was tested for antibacterial effect in vivo
according to
the test described by N. Frimodt-Moller.
Untreated animals developed fulminant clinical signs of infection. At all time
points
the compound of the invention suppressed the E. coli cfu/ml compared to non-
treated controls and was as efficient as the two positive controls.
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