WO2008088301A2 - Inhibitors of dnaa activity - Google Patents

Abstract

The present invention concerns a double-stranded oligodeoxynuclcotide (dsODN) molecule capable of specific binding to a bacterial DnaA, wherein said dsODN inhibits binding of DnaA to its natural binding site in the chromosome of a bacterium.

Description

INHIBITORS OF DnaA ACTIVITY

Background of the Invention

Field of the Invention

[0001] The present invention concerns inhibitors of DnaA activity.

Description of the Related Art

[0002] DnaA is DNA binding protein that is required for initiation and replication of both chromosomes and plasmids in bacteria. Binding of DnaA to its DNA binding sites, called DnaA DoXeB₁ in the chromosomal origin of replication (oriC) (s essential for initiation of chromosome replication, The DnaA boxes have the consensus sequence TT( A/T)TNCACA (Messer, FEMS Microbiology Reviews 26:355-374 (2002)), DnaA is also a transcription factor (Messer and Weigel, MoI. Microbiol.24:1-6 (1997)). DnaA boxes arc found in the promoter regions of many genes where they can mediate repression, or transcription. DnaA is also involved in replication of many bacterid plasmids. and these plasmids contain one or several DnaA boxes (Messer, 2002, supra ).

[ 0003 ] Replication of the bacterial chromosome is initiated with the DnaA protein binding to the DnaA boxes (Baker et al, J. Biol. Chem. 262:6877-6885 (1987)) within the oriC (replication origin) region of the chromosome and opens the DNA helix, after which the DnaB helicase and the DnaC protein are loaded at the 13-mer sequence within the origin (Bramhill and Komberb, Cell 54:915-918 (198B)) through interaction with DnaA (Marszalek et al., J. Biol. Chem.271 ;18535-18542 (1996)). The DnaB protein then proceeds to unwind the belix in advance of the replication fork (LeBowitz and McMackcn. J, Biol. Chem, 261 :4738-4748 (1986)), interacting also with the primase, EtoaG (Lu at al,, Proc. Natl. Acad. Sci. USA 93:12902-12907 (1996)) and DNA polymerase ITI (PoIIII) holoenzyme (Kim et at.. Cell 84:643-650 (1996), Miron «r al., Proc. Natl. Acad. Scj. USA 91.-6438-6442 (1994)), and consequently helping to bring other components of the replication machinery to the origin.

[0004| The initiator protein DnaA is founding all eubacteria analyzed so far (Messer et al., 2002, supra). The function of DnaA depends on its ability to bind to a 9 base pair recognition sequence, the dnaA box, which has a consensus sequence 5 '-TTATNCACA- 3' (Messer et at, 2002, supra). DnaA binds to multiple binding sites in the oriC Tho interactions with the 9-mer dnaA box are high affinity interactions whereas the binding to additional 6- mer sequences in other regions outside the origin of replication ere low affinityinteractions. A threshold level of DnaA protein is required for initiation of replication (Meεser el aL, 2002, supra). [0005] Despite the success of antimicrobial agents, infectious disease is now the second leading cause of death worldwide. This is in large part due to increased resistance or pathogens to a variety of antimicrobial agents and is an accelerating trend. It is evident that the dynamic and resilient nature of microbial life, coupled with the profligate use of antibiotics, has selected drug-resistant microbes across a wide spectrum of pathogenic organisms. There is a clear and urgent need for novel antibiotics to help combat this problem.

Summary of the Invention

[0006] The present invention provides double-stranded oligodeoxynucleotide (dsODN) molecules that bind to DnaA and prevent binding of DnaA to its natural binding sites in the original of replication of bacteria thereby blocking bacterial growth. The invention specifically provides double-stranded oligodooxynucleotide (dsODN) molecules that bind to DnaA and prevent binding of DnaA to its natural binding sites in the original of replication of bacteria and plasmids, thereby blocking bacterial growth and blocking replication and spread of plasmids that often carry antibiotic resistance.

[0007] In one aspect, the invention concerns a double-stranded oligodeoxynucleotide (dsODN) molecule capable of specific binding to a bacterial DnaA, wherein the dsODN inhibits binding of said DnaA to its natural binding site in the chromosome of a bacterium.

[0008] In an embodiment, the dsODN acts as a competitive inhibitor of the natural binding site.

[0009] In another embodiment, the dsODN of mimics the DnaA box in the chromosomal original of replication (oriC) of the bacterium.

[0010] In yet another embodiment, the dsODN inhibits bacterial growth.

[0011] In a still further embodiment, the dsODN blocks replication and spread of a bacterial plasmid.

[0012] In a further embodiment, the dsODN is modified to carry one or more positive charges. [0015] In a still further embodiment, the dsOPN is conjugated to a peptide facilitating its entry into the bacteriυm.

[0014] In an additional embodiment, the daODN is conjugates to a fluorescent dye. [0015] In another aspect, the invention concerns a composition comprising a dsODN as hereinabovc described.

[0016] The composition typically is an antimicrobial composition, e.g. a composition having antibiotic activity,

[0017] If desired, the composition may further comprise at least one further ingredient having antibiotic activity,

[0018] In yet another aspect, the invention concerns a method for the prevention or treatment of a bacterial infection comprising administering to a subject in need an effective amount of a dsODN molecule as hereinabove described.

[0019] In a particular embodiment, the bacterial infection is caused, at least in part, by a drug-resistant bacterium, including multi-drug resistant bacteria.

[0020] The bacterial infection may be systemic or local, and includes, without limitation, pneumonia, tuberculosis (TB) and drug-resistant Staphylococcus infections, often developed during hospitalization.

[0021 ] In a further embodiment, the method includes the administration of a Further antibacterial agent, such as, for example, an antibiotic.

Brief Description of the Drawings

[0022] Figure 1 shows the results of a competition binding assay.

[0023] Figure 2 illustrates that positive charges improve the delivery of dsODN inhibitors to E. coli cells.

[0024] Figure 3 shows the sequences used m the Example. The lower sequences were made both as PO and PS sequences, where PO means that the backbone contains only phosphodiester bonds (native DMA) and PS means that all backbone bonds arc phosphorothioate, where one of the oxygens in the phosphodiester bond is replaced by a sulfur. Detailed Description of the Preferred Embodiment

Definitions

[0025] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al.. Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New Yoτk, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992). provide one skilled in the art with a general guide to many of the terms used in the present application.

[0026] The term "double-stranded" is used to refer to a nucleic add molecule comprising two complementary nucleotide strands connected to each other by Watson-Crick base pairing. The term specifically includes molecules which, in addition to the double- stranded region formed by the two complementary strands, comprise single-stranded overhang(s). In addition, the term specifically includes NF-κB oligodeoxynucleotide decoy molecules in which, in addition to the double-stranded region, the two strands are covalently linked to each other at their 3' and/or 5' end.

[0027] The terms "double-stranded oligodeoxynucleotide/ "dsODN," and "double-stranded oligonucleotide," are used interchangeably, and refer to short nucleic acid molecules comprising a double-stranded region, which bind to and interfere with a biological function of a targeted DNA binding protein, such as DNaA.

[0028] As used herein, the phrase "modified nucleotide" refers to nucleotides or nucleotide triphosphates That differ in composition and/or structure from natural nucleotides and nucleotide triphosphates. These modifications include but are not limited to amino end caps (CH2)x-NH2), modified bases with amino groups (i.e. amine modified A, C and T), cationic phospholipids (including phosphatidylethanolamine, N-Caproylamine- phosphatidylethanolamine), cationic PEG phospholipids, and polyethylene imine.

[0029] As used herein, the terms "live prime" or "5" and "three-prime" or "3" refer to a specific orientation as related to a nucleic acid. Nucleic acids have a distinct chemical orientation such that their two ends are distinguished as either five-prime (5') or three-prime (3')- The 3' end of a nucleic acid contains a free hydroxyl group attached to the 3' carbon of the terminal pentose sugar. The 5' end of a nucleic acid contains a free hydroxyl or phosphate group attached to the 5' carbon of the terminal pentose sugar, [0090] As used herein, the term "overhang" refers Io a double-stranded nucleic acid molecule, which does not have blunt ends, such that the ends of the two strands are not coextensive, and.such that the 5' end of one strand extends beyond the 3' end of the opposing complementary strand. It is possible for a linear nucleic acid molecule to have zero, one, or two, 5' overhangs.

[0031] The term "treatment " refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

[0032] A "subject" is a vertebrate, preferably a mammal, more preferably a human.

[0033] The term "mammal" is used herein to refer to any animal classified as a mammal, including, without limitation, humans, higher primates, rodents, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, doge, horses, cats, cows, etc, Preferably, the mammal herein is human.

[0034] The term "antibiotic" is used in the broadest sense and includes, without limitation, penicillins, cephalosporins, fluroquinolones, tetracyclines, macrolides, aminoglycosides, and the like.

[0035] Further details of the invention are illustrated by the following non- limiting example.

Example dsOND inhibitor design, binding assays (EMSA) and.competition. assays

[0036] Multiple dnaA OND inhibitors were designed based on the consensus sequence as well as various mutant sequences in the literature. These consisted of various core binding regions and flanks. A number of DnaA dsOND inhibitors were labeled with ³²P and incubated with lysates of E. coli for typical EMSA (electromobility shift assays) to determine binding efficiency. The following sequence (S'GTTATCCACAGTGTTATCCACAGTTATCCACAG 3') was determined to bind the most dnaA and was therefore used as the labeled probe for the remaining EMSA assays.

{0037] The DnaA EMSA assays were performed as follows. A double-stranded oligonucleotide containing DnaA binding sites (5'- GTTATCCACAGTGTTATCCΛCAGTTATCCACAG-3' or 5'- GTTATCCACAGTGTTATCCACAO-3') was end-labeled withγ³²P-ATP using T4 Polynucleotide Kinase (Promega). Four μg of a nuclear extract prepared from Electromax DHI OB cells (Invitrogen) was incubated with 35 fmnol of radiolabeled probe in the presence or absence of competing unlabeled DnaA double-stranded oligonucleotidesCdsODN) or scrambled dsODN. The competitors were used at cither 20-fold or 50-fold molar excess over the radiolabeled probe. The incubations were carried out at room temperature for 30 minutos in a 20 μl reaction volume composed of 20mM Hepes-KOH pH 8.0, 4OmM KCl, 2wιM DTT, 1 H)M EDTA, 8% Glycerol, SmM MgCl₂, 0.2% TritonX-100, 0.1% BSA₁ 100μM ATP and 10 ng Poly-dldC. The reactions were loaded onto a 6% polyacrylamide gel, subjected to electrophoresis and dried. The dried gels were imaged and quantitated using a Typhoon 8600 Phosphorimager (Amersham) and ImageQuant software. The bacterial extract WHS prepared using the B-PBH. Protein Extraction Kit (Pierce). The sequences used as competitors included BRll-scram (scrambled), NBR1-2, BRM, EiB-Rl-I, NBRl-I, BR1-2S, BR1-2. HB R 1 -2 and BR 1 -3. Competitors contained either completely phosphodiester or phosphorothiotate backbones.

Uptake of QND inhibitor bv bacteria

[0038] The dnaA dsOND inhibitors must be taken up by the bacteria in order to bind Io dnaA and block plasmid and chromosome replication. A variety of positively and negatively charged modifications of a model dsOND inhibitor (not the dnaA inhibitors used above) were made and tested for uptake using either flow cytometry or a novel real-time PCR-based assay. Briefly, & coll were incubated at 37°C with variouB dsONP inhibitors for 30 minutes, washed five times by PBS₁ and there-suspended E.coli were analyzed by PCR using primers specific for the delivered dsOND. The results are shown in Figure Z. Unmodified dsOND inhibitor 895.895H3 (sense strand sequence: caccagcgtacgtgcctcaggtlo) had an amplification Ct essentially the same as the water control indicating that little to no dsOND was taken up by the cells. In contrast, 895.896H3 modified with a peptide containing 5 positive charges had a Cl that was 2.9 cycles earlier indicating a minimal 7.5-fold increased uptake. 895-896H3 modified with a peptide containing 7 positive charges had a Cl that was 7.4 cycles earlier indicating at least 168 folds increased uptake. The fold of increased uptake could be even more significant if considering all most no unmodified dsOND taken up by the bacteria.

[0039] In addition, 153/154H3 conjugated with the positive or negative fluorescent dyes were incubated with E. coli for 2 hours, washed 3 times and their fluorescence analyzed by flow cytometry. Once again, the model dsOND inhibitors bearing a positively charged modification showed increased uptake by the bacteria.

[0040] Results are shown in Figure 1 , indicating that various sequences bind dnaA and can compete for binding with the optimal probe as defined above.

Uptake of OND inhibitor by bacteria

[0041 ] dnaA dsOND inhibitors must be taken up by the bacteria in order to bind io dnaA and block plasmid and chromosome replication. A variety of positively and negatively charged modifications of a model dsOND inhibitor (not the dnaA inhibitor used above) were made and tested for uptake using a novel PCR-based assay, Briefly, E. coli were incubated with various dsOND inhibitors for 30 minutes, washed five times, lyscd. and the lysates were analyzed using PCR using primers specific for the delivered OND. Unmodified OND inhibitor 895.895H3 had an amplification Ct essentially the same as the water control indicating that little to no OND was taken up by the cells. In contrast, 895.896H3 modified with a peptide containing 5 positive charges had a Ct that was 2.9 cycles earlier indicating a 7.5 fold increased uptake, 895.896H3 modified with a peptide containing 7 positive charges had a Ct that was 7.5 cycles earlier indicating a 181 fold increased uptake

[0042] All references cited throughout this disclosure are hereby expressly incorporated by reference.

[0043] Although the present invention is illustrated with reference to certain specific embodiments, it iβ not so limited. Modifications and variations are possible without diverting from the idea of the invention, and will be apparent to those skilled in the art. All such modifications and variations are specifically within the scope herein.

Claims

WHAT IS CLAIMED IS:

1. A double-stranded oligodeoxynucleotide (dsODN) molecule capable of specific binding to a bacterial DnaA, wherein said dsODN inhibits binding of said DnaA to its natural binding site in the chromosome of a bacterium.

2. The dsODN of claim 1 which acts as a competitive inhibitor of said natural binding site.

3. The dsODN of claim 1 which mimics the DnaA box in the chromosomal original of replication ((oriC) of said bacterium.

4. The dsODN of claim 1 which inhibits bacterial growth.

5. The dsODN of claim 1 which blocks replication and spread of a bacterial plasmid.

6. The dsODN of claim 1 which is modified to carry one or more positive charges.

7. The dsODN of claim 1 which is conjugated to a peptide facilitating its entry into the bacterium.

8. The dsODN of claim 1 which is conjugates to a fluorescent dye.

9. A composition comprising a dsODN of any one of claims 1-8.

10. The composition of claim 9 which is an antimicrobial composition.

1 1. The composition of claim t0 which has antibiotic activity,

12. The composition of claim 1 1 further comprising at least one furthur ingredient having antibiotic activity.

13. A method for the prevention or treatment of a bacterial infection comprising administering to a subject in need an effective amount of a dsODN molecule of any one of claims 1-8.

14. The method of claim 13 wherein said bacterial infection is caused at least in part, by a drug-resistant bacterium.

15. The method of claim 14 wherein said bacterial infection is caused, at least in part, by a multi-drug resistant bacterium.

16. The method of claim 13 wherein said bacterial infection is systemic.

17. The method of claim 13 wherein said bacterial infection is pneumonia or tuberculosis (TB).

18. The method of claim 13 wherein said bacterial infection is drug-resistant Staphylococcus infection.

19. The method of claim 13 further comprising the adramistration of a further antibactorial agent.

20. The method of claim 19 wherein said further antibacterial agent is an antibiotic