WO2008025152A1 - Antibacterial agents - Google Patents

Antibacterial agents Download PDF

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WO2008025152A1
WO2008025152A1 PCT/CA2007/001522 CA2007001522W WO2008025152A1 WO 2008025152 A1 WO2008025152 A1 WO 2008025152A1 CA 2007001522 W CA2007001522 W CA 2007001522W WO 2008025152 A1 WO2008025152 A1 WO 2008025152A1
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compound
group
gene
substituted
antibacterial
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PCT/CA2007/001522
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French (fr)
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Eric Brown
Ranjana Pathania
Fred Capretta
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Mcmaster University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/08—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

Definitions

  • the present invention relates to a new class of antibiotics that are effective against Gram-negative bacteria.
  • Multidrug resistant bacteria are a major problem both in hospital and community settings. Some strains of pathogens, e.g., Pseudomonas aeruginosa, Staphylococcus aureus and Mycobacterium tuberculosis, are highly resistant to most antibiotics. Remarkably, in the past forty years, only two new chemical classes of antibiotics, the oxazolidinone linezolid (Ford et al. 2001 ) and the lipopeptide daptomycin (LaPlante and Rybak 2004) classes, have reached the clinic. Furthermore, existing antibiotics are directed to a small number of targets, in particular cell wall, DNA and protein biosynthesis. It has been estimated that fewer than 30 proteins have been exploited commercially as targets for antibacterial drugs (Hahneck et al. 2002). Multidrug resistance among bacterial pathogens exists, in large part, to the limited repertoire of antibacterial mechanisms that are known.
  • pathogens e.g., Pseudomonas a
  • the present invention addresses the problems of the prior art.
  • the invention is based on the discovery of a new lead series of molecules for the treatment of Gram-negative infections.
  • An entirely new chemical class of antibiotics as demonstrated by four exemplary structurally related compounds is provided.
  • Cell-based small molecule screening was used to search for growth-inhibitory molecules with a variety of antibacterial mechanisms.
  • Secondary screens made use of newly available, genome-scale clone sets for the model bacterium £. coli to conduct highly parallel suppression screens and provide insights into the mechanism of action of molecules with antibacterial activity.
  • the novel antibacterials of the present invention also have an entirely novel mechanism of action, namely bacterial lipoprotein targeting.
  • the lead series provides a basis for the establishment of synthetic molecules based on structure and activity relationships. These synthetic compounds are encompassed within the present invention.
  • an antibacterial compound that targets lipoprotein is provided.
  • the present invention provides compounds of the formula (I)
  • the core of the molecule consists of a 1 ,2,3,4- tetrahydro[1 ,3,5]triazine core that may be substituted at one or more positions.
  • iii) m is an interger varying between 0 and 12; and iv) n is an integer varying between 0 and 12.
  • an antibacterial compound having the general structure as shown below is provided:
  • R may comprise a variety of different chemical moieties.
  • an antibacterial compound selected from the group consisting of:
  • the invention also includes analogues and mimetics of any of the compounds described above. Pharmaceutically acceptable salts and esters of the compounds are also included. Antimicrobial compositions containing any of the compounds are also included. [0012]
  • a method of identifying antibacterial agents comprises screening a library for compounds that have growth inhibitory activity against a Gram negative bacterial strain; selecting growth inhibitory compounds that exhibit at least one additional preselected parameter; identifying target of activity through high copy suppression analysis and select compounds that are suppressed by a high copy of a particular gene.
  • the bacterial strain is an E. coli strain. In a more preferred embodiment the strain is E. coli MC1061.
  • the additional parameter is selected from the group consisting of potency, solubility, purity, structure and availability.
  • the gene is one involved in lipoprotein synthesis. More preferably the gene is the lolA gene.
  • a method of determining the chemical - genetic interaction of antibacterial compounds comprises preparing an array comprising a bacterial library whose members express a high copy number of particular gene; exposing the library to increasing amounts of a test compound; identifying members of the library that display resistance to the compound and determining which gene those members express at high copy number.
  • the invention also provides for a method of treating a bacterial infection in an animal or human host.
  • the method comprises administering to one in need of treatment a compound having the general structure:
  • an antimicrobial composition comprising compound I or Il is provided.
  • FIGURE 1 illustrates a flowchart for lead discovery
  • FIGURE 2 illustrates results obtained using a high copy growth array
  • FIGURE 3 illustrates schematically the interaction between genes and antibiotics
  • FIGURE 4 shows the results of a quantitative suppression analysis of MAC0013243 by high copy lolA
  • FIGURE 5 illustrates the interaction of LoIA with MAC0013243 using NMR
  • FIGURE 6 is a series of Western blots illustrating accumulation of Lpp in the inner membrane of E. coli.
  • the invention provides a novel high throughput screening method for the identification of effector molecules based on overexpression of target genes.
  • the high throughput screening assay of the present invention involves generating or otherwise obtaining a library that over-expresses certain genes.
  • the library may be from a gram positive or a gram negative bacteria.
  • the high copy suppression assay of the present invention can also be used to screen for targets in other types of pathogens and to identify novel effector molecules.
  • the assay can be used to screen for anti-viral or anti-fungal agents and their targets.
  • the screening method can also be used to screen for targets for cancer immunotherapeutics and to identify molecules that have anti-tumor activity. This is especially useful in cases where a tumor becomes resistant to standard drug regimens.
  • the library is obtained, a large number of molecules can be screened. By comparing the activity against cells having a normal copy number of each gene and cells from the over-expressing library, it can be determined which compounds have activity. The target of those compounds can then be identified based on which gene is overexpressed. Furthermore, once a target is identified, novel therapeutics can be intelligently designed. The technology can also be applied to known effector molecules whose targets have not previously been identified.
  • the high throughput assay was used to screen for antibacterial agents, also referred to as antimicrobials or antibiotics.
  • a method for probing the genetic chemical interactions between genes and antibacterials was used to identify genes that encode antibacterials targets.
  • the invention also provides novel antibacterial agents.
  • a class of molecules that interfere with lipoprotein targeting is also provided.
  • the screening method of the present invention was used to identify a novel class of antibiotics have the general structure:
  • the core of the molecule typically comprises of a 1 ,2,3,4- tetrahydro[1 ,3,5]triazine core that may be substituted at one or more positions.
  • substituents include: -CN, -NC, -NCO, -NCS, -OH, -NO, -OCN, -SCN, -NO 2 , -F, -Cl, -Br, -I 1 - N 3 , -OR- I , -OR 2 , -N(Ri) 2 , -N(R 2 ) 2 , -N(R 1 )(R 2 ), -SRi, -SR 2 , -SOR 1 , - S(O) 2 Ri, -SOR 2 , -S(O) 2 R 2 , -00R 1 , -00R 2 -C(O)ORi, -C(O)R 1 , - C(O)OR 2 , -C(O)R 2 , -OC(O)Ri, -OC(O)R 2 , -NR 1 C(O)Ri, -NR 1 C(O)R 2
  • iii) m is an interger varying between O and 12; and iv) n is an integer varying between 0 and 12.
  • a high throughput screen was utilized to identify compounds in a small molecule screening library (-50,000 small molecules) that had growth inhibitory activity against E. coli strain MC1061 , a hyper-permeable rough lipopolysaccharide mutant (Casadaban and Cohen 1980), at a concentration of 50 ⁇ M in rich liquid media.
  • the compound library was from Maybridge pic (Cornwall, England), had an average molecular mass of 325 g/mol and was chosen for its high quality, diversity, drug likedness and re-supply rate (Brown 2003).
  • the screen was of a very high quality in terms of signal, noise and replication rate where these details have been previously published on a subset (8,640 molecules) of the screening library (Li et al. 2004).
  • Figure 1 illustrates a schematic used to identify growth inhibitory molecules for follow-up using suppression analysis.
  • the MIC of the compound was determined in rich media against the hyper-permeable screening strain (MC1061 ) and against wild-type E. coli (AG 1 ).
  • a number of actives suffer from solubility problems in aqueous formulations and so insoluble compounds were eliminated based on visual inspection following dilution into aqueous solution. Actives were confirmed for quality and identity (identical in structure to those represented in the database) using analytical liquid chromatography with mass spectrometry detection (LC/MS). Grouping of bioactive compounds by chemical class and structure was done to establish structure and activity relationships in the screening data.
  • LC/MS mass spectrometry detection
  • Figure 2 shows an example of a high copy growth array that was developed to detect suppression by mostly essential genes over expressed in E. coli.
  • 373 genes were represented in this array along with control wells that included E. coli transformed with empty expression vector and plasmid encoding efflux machinery such as acrB, tolC and marA.
  • This gene set encompassed the set of 303 putatively essential genes.
  • the clones were clustered on the plate according to function based on four broad categories, including information storage and processing, metabolism, cellular processes and poorly characterized proteins.
  • Figure 2 shows the results of a suppression study run at increasing concentrations of cycloserine, (i.e. 0, 2-, 4- and 8-fold the MIC (minimum concentration to inhibit growth)) of the antibiotic.
  • concentrations of cycloserine i.e. 0, 2-, 4- and 8-fold the MIC (minimum concentration to inhibit growth)
  • the vast majority of clones grew well on the control (no antibiotic) plate indicating that protein over expression was not generally deleterious to growth.
  • the concentration range tested provides a 'stringency' analysis.
  • 2-fold the MIC a large number of clones were capable of growth while at 4- and 8-fold only a handful of clones were able to suppress the action of cycloserine.
  • suppression was seen for clones overproducing FtsA, a cell division protein (Pages et al.
  • antibiotics such as translation inhibitors, clindamycin, tetracycline, spectinomycin and neomycin, didn't show suppression at 16-fold the MIC, registered few interactions at 8-fold and tended to interact with genes that suppress the action of multiple antibiotics. No two antibiotics showed the same chemical-genetic interactions. This data suggests that high-copy suppression analysis can provide a mechanistic fingerprint of the mechanism of action of an antibiotic.
  • Table 2 shows the minimum inhibitor concentrations for four molecules from the lead series.
  • Pseudomonas aeruginosa (MDR B39825) 256 256 >256 128 Pseudomonas aeruginosa (MDR B7845) 32 32 32 4 Pseudomonas aeruginosa (MDR B16266) 8 16 16 8 Pseudomonas aeruginosa (MDR B7171) 8 64 128 32 Burkholderia cepacia (B 154408) 128 128 128 128 Stenotrophomonas maltophila (B22020) 256 256 256 256 256 Salmonella typhimurium (B101045) 128 256 256 256 Achromobacter Xylosidans (B76167) 256 >256 256 256 Escherichia coli (ESBL B2259) 256 >256 256 >256 >256 >256 >256 Escherichia coli (ESBL ATCC) 256 >256 256 >256 Escherichia coli (
  • Table 2 summarizes the antibacterial activity for four compounds, MAC0013243, MAC0030381 , MAC003866 and MAC0013208, in the lead series identified. All showed significant activity against Gram-negative bacteria but had no impact on Gram-positive organisms even at the highest concentrations tested. This spectrum of activity is consistent with the concept that these compounds interrupt lipoprotein targeting since this particular aspect of Gram-negative bacterial physiology is not shared with Gram- positive bacteria (Tokuda and Matsuyama 2004). It is noteworthy that MAC0013243 exhibited activity against clinical isolates of multidrug resistant P. aeruginosa, and extended spectrum beta-lactamase resistant E. coli. These organisms have high levels of resistance to conventional antibiotics and thus this activity from this lead series of compounds of a novel chemical class and exhibiting a novel mechanism is significant.
  • FIG. 4 shows the results of a quantitative analysis of the suppression of chemical lethality by MAC0013243 by pCA24N-/o/A which expresses LoIA at high copy.
  • E. coli AG 1 cells harbouring pCA24N (dark diamonds) or pCA24N-/o//A (dark circles) were exposed to MAC0013243.
  • Cells were grown overnight (LB, 30 ⁇ g/mL Chloramphenicol) and used to inoculate) growth media (200 ⁇ L of LB with 30 ⁇ g/mL Chloramphenicol and 0.1 mM IPTG ).
  • Cells were grown at 37°C in sterile 96 well plates for 16 hours and optical densities recorded at 600 nM.
  • Figure 5 shows all the peaks of MAC0013243 that interact with LoIA (Panel B).
  • the dissociation constant (K D ) for MACO013243: LoIA complex was determined to be approximately 7.5+ 3.9 ⁇ M ( Figure 5, panel D).
  • Figure 5. Interaction of MAC0013243 with LoIA by NMR.
  • A) Representation of the chemical structure of MAC0013243.
  • Panel B and C represent the 1 D STD double difference spectra and reference spectra for MAC0013243 and LoIA (100:1 ratio).
  • the peaks labeled in panel B represent the protons of the compound that interact with LoIA, where as DMSO does not interact with the protein. All the non exchangeable protons of MAC 0013243 interacting with LoIA are labeled accordingly.
  • D) The dissociation constant (K D ) for the MAC0013243:LolA complex was measured from the Water LOGSY (Dalvit et al. 2001 ) signal intensity of the C b -H resonance of MAC0013243 as a function of ligand concentration. The experiment was performed with 1024 scans, 2sec repetition delay at 295 K. The curve represents the best fit for the data obtained from the difference in intensities of the WaterLOGSY spectra recorded in presence and absence of the LoIA, respectively.
  • the results provide biochemical confirmation that MAC0013243 interacts directly with LoIA in vitro and provide further support for the conclusion that LoIA, and lipoprotein targeting, is the likely target of this and related actives.
  • E. coli lipoproteins are anchored to the periplasmic surface of the inner or outer membranes.
  • Braun's lipoprotein (Lpp) is the most abundant outer membrane (OM) lipoprotein in E coli.
  • Lipoproteins lacking an inner membrane (IM) retention signal are transported to the OM by the LoI transport system comprised of 5 proteins, LoIABCDE (Tokuda and Matsuyama 2004).
  • LoIC, LoID and LoIE form an ATP-binding cassette transporter in the IM. At the expense of ATP, this transporter releases lipoproteins into the periplasm where they are bound by the chaperone LoIA.
  • the LolA-lipoprotein complex transverses the periplasm and interacts with an OM receptor LoIB which is essential to insert the lipoproteins into OM (Matsuyama et al. 1995).
  • LoIA plays an important role of transferring the lipoproteins from LoICDE complex from the inner membrane to LoIB in the outer membrane.
  • the above findings indicate that MAC0013243 causes Lpp to accumulate in the IM in vivo, perturbing the physiological function of LoIA in E. coli.
  • the PCR amplified product was cloned into pDEST14 using the Gateway cloning and Expression Kit (Invitrogen.Canada)and the insert sequence was confirmed by DNA sequence analysis (MOBIX, McMaster University). The clone was designed to create a protein product with a C-terminal poly- histidine tag for easy purification.
  • LoIA was expressed and purified from Escherichia coli BL21 (DE3) Al cells transformed with pDEST14-/o/>4 grown at 37 0 C to an optical density (600 nm) of 0.6 in Luria-Bertani media (2L) supplemented with 100 ⁇ g/mL ampicillin. The cells were then induced with 1 mM IPTG and 0.2% w/v L-arabinose and grown for an additional 2 hours prior to harvest by centrifugation at 10,000 x g.
  • the cells were washed with a 0.85% saline solution, pelleted and re-suspended in 25 mL lysis buffer (25 mM HEPES pH 8.0, 1 mM DTT, 0.5 mg DNase, 0.5 mg RNase). All subsequent steps were performed at 4 0 C.
  • Cells were lysed by passage through a French press at 20,000 psi and clarified by centrifugation at 40,000 x g for 2 hours. Clarified lysate was purified by nickel chelate chromatography using a 1 mL HiTrap affinity column (Amersham Biosciences, Baie d'Urfe, PQ).
  • the column was washed with buffer A (2OmM Sodium phosphate buffer pH 7.4, 50OmM NaCI containing 10% glycerol and 20 mM imidazole) and eluted with a linear imidazole gradient of 20-300 mM.
  • buffer A 2OmM Sodium phosphate buffer pH 7.4, 50OmM NaCI containing 10% glycerol and 20 mM imidazole
  • Fractions of the eluant were analyzed by SDS-PAGE, and those containing pure His-tagged LoIA were pooled and concentrated using an Amicon ultracentrifugal filter device (5000 MW, Fisher Scientific) and simultaneously buffer exchanged to 2OmM Sodium phosphate buffer pH 7.4, 100 mM NaCI and 10% glycerol. Using this method about 10mg of pure LoIA was obtained.
  • Fractions rich in LoIA protein were stored in aliquots at -80 0 C. Concentration of purified protein was determined by a
  • Example 2 MAC0013243 causes accumulation of Lpp in the inner membrane of E. coli
  • Triazanes Triazanes The amine (1 eq.) was mixed well with formaldehyde (2 eq.) in dioxane. To this solution, the substituted-thiourea (1 eq.) was added. The mixture was heated until a solution was obtained and allowed to stir overnight at room temperature. The precipitate produced was isolated via filtration. While many of the triazanes thus obtained were shown to be analytically pure, additional recrystallization was required in some cases.
  • Pseudomonas aeruginosa (PA01) 512 256 64
  • Enterococcus faecalis >256 >256 >256
  • the envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis.
  • UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase J Biol Chem, 270(51 ): 30384-30391.

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Abstract

A novel class of antibiotics that interfere with lipoprotein targeting have been identified. A high gene copy suppression method is used to identify new compounds. Novel compositions containing the antimicrobials are disclosed as well as their uses.

Description

ANTIBACTERIAL AGENTS FIELD OF INVENTION
[0001] The present invention relates to a new class of antibiotics that are effective against Gram-negative bacteria.
BACKGROUND OF THE INVENTION
[0002] Multidrug resistant bacteria are a major problem both in hospital and community settings. Some strains of pathogens, e.g., Pseudomonas aeruginosa, Staphylococcus aureus and Mycobacterium tuberculosis, are highly resistant to most antibiotics. Remarkably, in the past forty years, only two new chemical classes of antibiotics, the oxazolidinone linezolid (Ford et al. 2001 ) and the lipopeptide daptomycin (LaPlante and Rybak 2004) classes, have reached the clinic. Furthermore, existing antibiotics are directed to a small number of targets, in particular cell wall, DNA and protein biosynthesis. It has been estimated that fewer than 30 proteins have been exploited commercially as targets for antibacterial drugs (Haselbeck et al. 2002). Multidrug resistance among bacterial pathogens exists, in large part, to the limited repertoire of antibacterial mechanisms that are known.
[0003] In recent years, infections by drug resistant Gram-negative bacteria, have proven to be particularly problematic (Rice 2006). Multidrug-resistant Gram-negative bacteria present a special challenge because they have a lower intrinsic permeability to antibacterial compounds and they also have pervasive multidrug efflux pumps that can expel antibiotics of widely different chemical classes. Due to the variety and capacity of the latter, high level resistance is especially problematic in nosocomial infections. In the community setting, resistance of Gram-negative pathogens to ampicillin and trimethoprim-sulfamethoxazole has been growing, as has the prevalence of fluoroquinolone resistance in pathogenic Escherichia coli.
[0004] Thus, there was a need for novel antimicrobial agents, particularly agents that are effective against Gram-negative pathogens. SUMMARY OF THE INVENTION
[0005] The present invention addresses the problems of the prior art. The invention is based on the discovery of a new lead series of molecules for the treatment of Gram-negative infections. An entirely new chemical class of antibiotics as demonstrated by four exemplary structurally related compounds is provided. Cell-based small molecule screening was used to search for growth-inhibitory molecules with a variety of antibacterial mechanisms. Secondary screens made use of newly available, genome-scale clone sets for the model bacterium £. coli to conduct highly parallel suppression screens and provide insights into the mechanism of action of molecules with antibacterial activity. In addition to their structural novelty, the novel antibacterials of the present invention also have an entirely novel mechanism of action, namely bacterial lipoprotein targeting. The lead series provides a basis for the establishment of synthetic molecules based on structure and activity relationships. These synthetic compounds are encompassed within the present invention.
[0006] In one aspect of the invention, an antibacterial compound that targets lipoprotein is provided.
The present invention provides compounds of the formula (I)
Figure imgf000004_0001
or a pharmaceutically acceptable salt thereof.
In a preferred embodiment, the core of the molecule consists of a 1 ,2,3,4- tetrahydro[1 ,3,5]triazine core that may be substituted at one or more positions. i) Substituents R1 and R2 are independently selected from: a) H, b) Unsubstituted or substituted, linear or branched, saturated or unsaturated C1 to C24 alkyl, cycloalkyl, heteroalkyl, aryl or heteroaryl moiety, c) An acyl group of structure R3-C=O wherein R3 is defined by i(a) or i(b) above, d) An alkoxy group of structure R4-0 wherein R4 is defined by i(a) or i(b) above, e) A sulfide group of structure R5-S wherein R5 is defined by i(a) or i(b) above, f) An amino group of structure R6-N-R7 wherein R6 and R7 are independently selected from those substituents defined in i(a) or i(b) above, g) Any combination that incorporates elements from claims i(c) to i(f) into an alkyl or cycloalkyl unit as defined in i(b), h) One or more of a substituent selected independently from the following group wherein the designated subscripted Ri and R2 functionalities are those defined in i(a) and i(b) as define above. Such substituents include:
-CN, -NC, -NCO, -NCS, -OH, -NO, -OCN, -SCN, -NO2, -F, -Cl, -Br, -I, - N3, -OR1, -OR2, -N(R1J2, -N(R2)2, -N(R1)(R2), -SR1, -SR2, -SOR1, - S(O)2R1, -SOR2, -S(O)2R2, -0OR1, -0OR2 -C(O)OR1, -C(O)R1, - C(O)OR2, -C(O)R2, -OC(O)R1, -OC(O)R2, -NR1C(O)R1, -NR1C(O)R2, - NR2C(O)R1, -NR2C(O)R2, -C(O)N(R1),, -C(O)N(R1)(R2), -C(O)N(R2)2, - C(NR1)(N(R1),), -C(NR1)(N(R2),), -C(NR1)(N(R1)(R2)), -C(NR2)(N(R1),), -C(NR2)(N(R1)(R2)), -C(NR2)(N(R2)2), -C(N(R1)(R2)(N(Ri)(R2), - N(R1)C(N(R1)(N(R1),), -N(R1)C(N(R1)XN(R1)(R2)), - N(R1)C(N(R2)XN(R1)(R2)), -N(R1)C(N(R2)(N(R1)2), - N(R2)C(N(R1)XN(R1J2, -N(R2)C(N(R2)XN(RO2, - N(R2)C(N(R1)XN(R1)(R2)), -N(R2)C(N(R2)XN(R1)(R2)), - N(R1)C(N(R1)XN(R2)Z1 -N(R2)C(N(R1)XN(R2),), -N(R1)C(N(R2)XN(R2),, ^1 =S1 =N(R1) Or =N(R2);
ii) Substituent X is independently selected from: a) A methylene (CH2) group, b) A carbonyl (C=O) group, c) A sulphur or oxygen atom or a substituted amine of type R8N, wherein R8 is defined by i(a) and i(b) described above, d) A sulfoxide (S=O) or sulfone (SO2) moiety, e) A phosphonate moiety of structure -P(=O)(OR9)2l wherein R9 is defined by i(a) or i(b) described above, f) A substituted or unsubstituted vinyl or conjugated vinyl group (CH=CH)y- where y is an integer varying between 0 and 6;
iii) m is an interger varying between 0 and 12; and iv) n is an integer varying between 0 and 12.
[0007] In another aspect of the invention, an antibacterial compound having the general structure as shown below is provided:
Figure imgf000007_0001
[0008] While Br and Cl are shown as possible substitutes, any halogen may be substituted at this position.
[0009] R may comprise a variety of different chemical moieties.
[0010] In another aspect of the invention, an antibacterial compound selected from the group consisting of:
Figure imgf000007_0002
[001 1] The invention also includes analogues and mimetics of any of the compounds described above. Pharmaceutically acceptable salts and esters of the compounds are also included. Antimicrobial compositions containing any of the compounds are also included. [0012] In a further aspect of the invention, a method of identifying antibacterial agents is provided. The method comprises screening a library for compounds that have growth inhibitory activity against a Gram negative bacterial strain; selecting growth inhibitory compounds that exhibit at least one additional preselected parameter; identifying target of activity through high copy suppression analysis and select compounds that are suppressed by a high copy of a particular gene.
[0013] In one preferred embodiment, the bacterial strain is an E. coli strain. In a more preferred embodiment the strain is E. coli MC1061.
[0014] In another preferred embodiment, the additional parameter is selected from the group consisting of potency, solubility, purity, structure and availability.
[0015] In yet another preferred embodiment, the gene is one involved in lipoprotein synthesis. More preferably the gene is the lolA gene.
[0016] In another aspect of the invention, a method of determining the chemical - genetic interaction of antibacterial compounds is provided. The method comprises preparing an array comprising a bacterial library whose members express a high copy number of particular gene; exposing the library to increasing amounts of a test compound; identifying members of the library that display resistance to the compound and determining which gene those members express at high copy number.
[0017] The invention also provides for a method of treating a bacterial infection in an animal or human host. The method comprises administering to one in need of treatment a compound having the general structure:
Figure imgf000008_0001
(II) [0018] In yet another aspect of the invention an antimicrobial composition comprising compound I or Il is provided.
[0019] This summary of the invention does not necessarily describe all features of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
[0021] FIGURE 1 illustrates a flowchart for lead discovery;
[0022] FIGURE 2 illustrates results obtained using a high copy growth array;
[0023] FIGURE 3 illustrates schematically the interaction between genes and antibiotics;
[0024] FIGURE 4 shows the results of a quantitative suppression analysis of MAC0013243 by high copy lolA;
[0025] FIGURE 5 illustrates the interaction of LoIA with MAC0013243 using NMR; and
[0026] FIGURE 6 is a series of Western blots illustrating accumulation of Lpp in the inner membrane of E. coli.
DETAILED DESCRIPTION
[0027] The invention provides a novel high throughput screening method for the identification of effector molecules based on overexpression of target genes.
[0028] The high throughput screening assay of the present invention involves generating or otherwise obtaining a library that over-expresses certain genes. Depending on the activity that is desired, the library may be from a gram positive or a gram negative bacteria. In addition to its usefulness for screening for novel anti-bacterials and antibacterial targets, the high copy suppression assay of the present invention can also be used to screen for targets in other types of pathogens and to identify novel effector molecules. For example, the assay can be used to screen for anti-viral or anti-fungal agents and their targets. The screening method can also be used to screen for targets for cancer immunotherapeutics and to identify molecules that have anti-tumor activity. This is especially useful in cases where a tumor becomes resistant to standard drug regimens.
[0029] Once the library is obtained, a large number of molecules can be screened. By comparing the activity against cells having a normal copy number of each gene and cells from the over-expressing library, it can be determined which compounds have activity. The target of those compounds can then be identified based on which gene is overexpressed. Furthermore, once a target is identified, novel therapeutics can be intelligently designed. The technology can also be applied to known effector molecules whose targets have not previously been identified.
[0030] The following description is of a preferred embodiment.
[0031] The high throughput assay was used to screen for antibacterial agents, also referred to as antimicrobials or antibiotics. A method for probing the genetic chemical interactions between genes and antibacterials was used to identify genes that encode antibacterials targets. The invention also provides novel antibacterial agents. A class of molecules that interfere with lipoprotein targeting is also provided.
[0032] The screening method of the present invention was used to identify a novel class of antibiotics have the general structure:
Figure imgf000010_0001
The core of the molecule typically comprises of a 1 ,2,3,4- tetrahydro[1 ,3,5]triazine core that may be substituted at one or more positions. i) Substituents R1 and R2 are independently selected from: a) H, b) Unsubstituted or substituted, linear or branched, saturated or unsaturated C1 to C24 alkyl, cycloalkyl, heteroalkyl, aryl or heteroaryl moiety, c) An acyl group of structure R3-C=O wherein R3 is defined by i(a) or i(b) above, d) An alkoxy group of structure R4-0 wherein R4 is defined by i(a) or i(b) above, e) A sulfide group of structure R5-S wherein R5 is defined by i(a) or i(b) above, f) An amino group of structure R6-N-R7 wherein R6 and R7 are independently selected from those substituents defined in i(a) or i(b) above, g) Any combination that incorporates elements from claims i(c) to i(f) into an alkyl or cycloalkyl unit as defined in i(b), h) One or more of a substituent selected independently from the following group wherein the designated subscripted Ri and R2 functionalities are those defined in i(a) and i(b) as define above. Such substituents include: -CN, -NC, -NCO, -NCS, -OH, -NO, -OCN, -SCN, -NO2, -F, -Cl, -Br, -I1 - N3, -OR-I, -OR2, -N(Ri)2, -N(R2)2, -N(R1)(R2), -SRi, -SR2, -SOR1, - S(O)2Ri, -SOR2, -S(O)2R2, -00R1, -00R2 -C(O)ORi, -C(O)R1, - C(O)OR2, -C(O)R2, -OC(O)Ri, -OC(O)R2, -NR1C(O)Ri, -NR1C(O)R2, - NR2C(O)R1, -NR2C(O)R2, -C(O)N(R1J2, -C(O)N(Ri)(R2), -C(O)N(R2J2, - C(NRi)(N(Ri)2), -C(NR1)(N(R2),), -C(NRi)(N(Ri)(R2)), -C(NR2)(N(RO2), -C(NR2)(N(R1)(R2)), -C(NR2)(N(R2),), -C(N(R1)(R2)(N(R1)(R2), - N(R1)C(N(Ri)(N(R1),), -N(Ri)C(N(Ri))(N(R1)(R2)), - N(R1)C(N(R2)XN(RiXR2)), -N(Ri)C(N(R2)(N(Ri)2), - N(R2)C(N(R1)XN(Ri)2, -N(R2)C(N(R2)XN(R1),, - N(R2)C(N(R1)XN(R1)(R2)), -N(R2)C(N(R2)XN(R1)(R2)), - N(R1)C(N(R1)XN(R2),, -N(R2)C(N(Ri))(N(R2)2), -N(R1)C(N(R2)XN(R2),, ^1 =S1 =N(R1) Or =N(R2);
ii) Substituent X is independently selected from: a) A methylene (CH2) group, b) A carbonyl (C=O) group, c) A sulphur or oxygen atom or a substituted amine of type R8N, wherein R8 is defined by i(a) and i(b) described above, d) A sulfoxide (S=O) or sulfone (SO2) moiety, e) A phosphonate moiety of structure -P(=O)(OR9)2, wherein R9 is defined by i(a) or i(b) described above, f) A substituted or unsubstituted vinyl or conjugated vinyl group - (CH=CH)y- where y is an integer varying between O and 6;
iii) m is an interger varying between O and 12; and iv) n is an integer varying between 0 and 12.
[0033] A high throughput screen was utilized to identify compounds in a small molecule screening library (-50,000 small molecules) that had growth inhibitory activity against E. coli strain MC1061 , a hyper-permeable rough lipopolysaccharide mutant (Casadaban and Cohen 1980), at a concentration of 50 μM in rich liquid media. The compound library was from Maybridge pic (Cornwall, England), had an average molecular mass of 325 g/mol and was chosen for its high quality, diversity, drug likedness and re-supply rate (Brown 2003). The screen was of a very high quality in terms of signal, noise and replication rate where these details have been previously published on a subset (8,640 molecules) of the screening library (Li et al. 2004).
[0034] Figure 1 illustrates a schematic used to identify growth inhibitory molecules for follow-up using suppression analysis. To follow up on novel actives, the MIC of the compound was determined in rich media against the hyper-permeable screening strain (MC1061 ) and against wild-type E. coli (AG 1 ). A number of actives suffer from solubility problems in aqueous formulations and so insoluble compounds were eliminated based on visual inspection following dilution into aqueous solution. Actives were confirmed for quality and identity (identical in structure to those represented in the database) using analytical liquid chromatography with mass spectrometry detection (LC/MS). Grouping of bioactive compounds by chemical class and structure was done to establish structure and activity relationships in the screening data.
[0035] Several factors, including potency, solubility, purity, identity and diversity, were used to select a set of priority actives i.e. compounds with antibacterial activity. While metrics like purity, identity and solubility are largely clear decision points, potency and structural diversity are measures that lead to more intuitive selection of priority actives. Potency data against the wild-type strain, in particular, figured prominently in selecting priority actives since there was a paucity of molecules active against this strain.
- i i - [0036] One of the most significant hurdles to the discovery of useful lead molecules is in connecting the cellular phenotype of a small molecule and its molecular target(s).
[0037] In one method of the present invention, genetic suppression through high protein copy was used to identify the target of new leads.
[0038] In the present invention, a strategy of screening for multi-copy suppressors that takes advantage of an ordered, high expression clone set of all the essential genes from E. coli was utilized.
[0039] The analysis was largely restricted to the essential genes since the targets of growth inhibitory molecules will most frequently be a component of the essential gene set. It is clearly apparent, however, that the methods of the invention can be applied to any gene. Figure 2 shows an example of a high copy growth array that was developed to detect suppression by mostly essential genes over expressed in E. coli. In this exemplary array, 373 genes were represented in this array along with control wells that included E. coli transformed with empty expression vector and plasmid encoding efflux machinery such as acrB, tolC and marA. This gene set encompassed the set of 303 putatively essential genes. The clones were clustered on the plate according to function based on four broad categories, including information storage and processing, metabolism, cellular processes and poorly characterized proteins.
[0040] Figure 2 shows the results of a suppression study run at increasing concentrations of cycloserine, (i.e. 0, 2-, 4- and 8-fold the MIC (minimum concentration to inhibit growth)) of the antibiotic. The vast majority of clones grew well on the control (no antibiotic) plate indicating that protein over expression was not generally deleterious to growth. The concentration range tested provides a 'stringency' analysis. At 2-fold the MIC, a large number of clones were capable of growth while at 4- and 8-fold only a handful of clones were able to suppress the action of cycloserine. At 8-fold, suppression was seen for clones overproducing FtsA, a cell division protein (Pages et al. 1975), YadR, an uncharacterized protein, and DdI, a known target of cycloserine, D-ala-D-ala ligase of peptidoglycan biosynthesis (Neuhaus and Lynch 1964). At 4-fold, three more clones registered as suppressors, namely, rpsR encoding a protein in the small subunit of the ribosome (Isono and Kitakawa 1978), yjeE encoding an essential ATPase (Allali-Hassani et al. 2004) and envA encoding the deacetylase of lipid A biosynthesis (Young et al. 1995). At 16-fold the MIC (not shown), the recognized target DdI showed a suppressor phenotype in addition to FtsA, YadR and EnvA.
[0041] Referring now to Figure 3, the chemical-genetic interactions of 14 antibiotics are shown. The data are presented using the visualization tool Osprey (Breitkreutz et al. 2003) because of the network behaviour of the interactions. Lines connect the gene capable of suppression at high copy and the antibiotic whose action is suppressed. Chemical-genetic interactions with the recognized targets tended to register at high stringency (i.e., 16-fold the MIC) in the cases where these targets are encoded by a single gene: cycloserine and ddl (ddlB), fosfomycin and murA, trimethoprim and folA. In contrast, antibiotics such as translation inhibitors, clindamycin, tetracycline, spectinomycin and neomycin, didn't show suppression at 16-fold the MIC, registered few interactions at 8-fold and tended to interact with genes that suppress the action of multiple antibiotics. No two antibiotics showed the same chemical-genetic interactions. This data suggests that high-copy suppression analysis can provide a mechanistic fingerprint of the mechanism of action of an antibiotic.
[0042] The chemical-genetic interactions observed for known antibiotics fall largely into two classes, general and specific. Cycloserine is an illustrative example. The promiscuous suppression behavior of yjeE and rpsR suggests that the chemical-genetic interaction of these two genes with cylcoserine is a non-specific one. There are also specific chemical-genetic interactions. In the case of cycloserine, ddl (ddlB), ftsA and envA are proximal on the E. coli chromosome at a gene cluster that has been defined by its crucial roles in cell division. The work with known antibiotics indicates that the high-copy suppression method of the present invention is a useful methodology to identify new chemical matter with antibacterial properties and particularly those that have a novel mechanism of action.
[0043] In an analysis of the priority actives generated in one study, four structurally-related molecules were identified as being suppressed by LoIA at high copy. The results are shown in Table 1.
Table 1. Priority actives discovered in this study that are suppressed by LoIA at high-copy comprise a lead series of common structure.
Structure
Figure imgf000016_0001
Common structure
Figure imgf000016_0002
[0044] Suppression by pCA24N-/o//4 of the molecules identified in Table 1 was found to vary from between 4- to 16-fold the MIC for these compounds with LoIA being the only high-stringency suppressor identified. The high-copy suppressor LoIA is of particular significance since this locus had not been identified as a suppressor of any of the known antibiotics. LoIA is encoded by an essential gene in E. coli and is a component of lipoprotein targeting machinery of Escherichia coli and other Gram-negative bacteria (Tokuda and Matsuyama 2004). None of the lipoprotein targeting machinery are known targets of existing antibiotics. The four molecules encompass a common structure (Table 1 ).
[0045] Table 2 shows the minimum inhibitor concentrations for four molecules from the lead series.
Table 2. Minimum inhibitor concentrations (MIC) for the priority actives suppressed by LoIA.
MlC1 (μg/mL)
13208 30381 3866 13243
Lab strains
Escherichia coli (MG1655) 8 8 16 16 Escherichia coli (MC1061 ) 8 8 16 32 Escherichia coli (ATCC 25922) 8 8 256 16 Pseudomonas aeruginosa (PA01 ) 128 128 >256 64 Burkhoderia cepacia 128 128 >256 128 Salmonella typhimurium 128 128 >256 128 Bacillus subtilis 168 >256 >256 >256 >256 Micrococcus luteus >256 >256 >256 >256 Staphylococcus aureus (ATCC) >256 >256 >256 >256 Enterococcus faecalis (ATCC) >256 >256 >256 >256 Streptococcus pyogenes >256 >256 >256 >256 Mycobacterium smegmatis >256 >256 >256 >256
Clinical isolates
Pseudomonas aeruginosa (MDR B39825) 256 256 >256 128 Pseudomonas aeruginosa (MDR B7845) 32 32 32 4 Pseudomonas aeruginosa (MDR B16266) 8 16 16 8 Pseudomonas aeruginosa (MDR B7171) 8 64 128 32 Burkholderia cepacia (B 154408) 128 128 128 128 Stenotrophomonas maltophila (B22020) 256 256 256 256 Salmonella typhimurium (B101045) 128 256 256 256 Achromobacter Xylosidans (B76167) 256 >256 256 256 Escherichia coli (ESBL B2259) 256 >256 256 >256 Klebsiella pneumoniae (ESBL ATCC) 256 >256 256 >256 Escherichia coli (B70640 ESBL) 256 >256 256 >256 Klebsiella pneumoniae (B75296 ESBL) 256 256 >256 >256 Acinetobacter baumanii 64 256 64 256
1 MICs were determined according to NCCLS methods. (National Committee for Clinical Laboratory Standards. 2000. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically— Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.)
[0046] Table 2 summarizes the antibacterial activity for four compounds, MAC0013243, MAC0030381 , MAC003866 and MAC0013208, in the lead series identified. All showed significant activity against Gram-negative bacteria but had no impact on Gram-positive organisms even at the highest concentrations tested. This spectrum of activity is consistent with the concept that these compounds interrupt lipoprotein targeting since this particular aspect of Gram-negative bacterial physiology is not shared with Gram- positive bacteria (Tokuda and Matsuyama 2004). It is noteworthy that MAC0013243 exhibited activity against clinical isolates of multidrug resistant P. aeruginosa, and extended spectrum beta-lactamase resistant E. coli. These organisms have high levels of resistance to conventional antibiotics and thus this activity from this lead series of compounds of a novel chemical class and exhibiting a novel mechanism is significant.
[0047] Figure 4 shows the results of a quantitative analysis of the suppression of chemical lethality by MAC0013243 by pCA24N-/o/A which expresses LoIA at high copy. E. coli AG 1 cells harbouring pCA24N (dark diamonds) or pCA24N-/o//A (dark circles) were exposed to MAC0013243. Cells were grown overnight (LB, 30 μg/mL Chloramphenicol) and used to inoculate) growth media (200μL of LB with 30 μg/mL Chloramphenicol and 0.1 mM IPTG ). Cells were grown at 37°C in sterile 96 well plates for 16 hours and optical densities recorded at 600 nM. The compound shows an MIC in this experiment against wild type E. coli of about 6 μg/mL while that for the overexpressing strain is approximately 16-fold higher. These results are consistent with the identification of LoIA as a high stringency suppressor for this lead series of molecules and suggest that LoIA may be the target of these molecules. [0048] In order to further test the hypothesis that MAC0013243 targets LoIA the ability of the compound to bind to pure, recombinant LoIA in vitro was determined by NMR spectroscopy.
[0049] Figure 5 shows all the peaks of MAC0013243 that interact with LoIA (Panel B). The dissociation constant (KD) for MACO013243: LoIA complex was determined to be approximately 7.5+ 3.9 μM (Figure 5, panel D). Figure 5. Interaction of MAC0013243 with LoIA by NMR. A) Representation of the chemical structure of MAC0013243. Panel B and C represent the 1 D STD double difference spectra and reference spectra for MAC0013243 and LoIA (100:1 ratio). B) The STD double difference spectra of LoIA and MAC0013243 was acquired with Bruker AV 700 spectrometer equipped with a TCI cryo-probe in 10OmM phosphate buffer pH 6.8 in 100% D2O at 295K. The double difference spectrum was obtained from the difference of STD spectra of the ligand alone and ligand + protein respectively. C) 1 D reference spectrum of MAC0013243 in presence of LoIA, DMSO signal is present as impurity in the compound. The spectra were recorded with 1024 scans, 100ms repetition delay. The residual protein signal was removed by applying a 30 ms Tip filter. The peaks labeled in panel B represent the protons of the compound that interact with LoIA, where as DMSO does not interact with the protein. All the non exchangeable protons of MAC 0013243 interacting with LoIA are labeled accordingly. D) The dissociation constant (KD) for the MAC0013243:LolA complex was measured from the Water LOGSY (Dalvit et al. 2001 ) signal intensity of the Cb-H resonance of MAC0013243 as a function of ligand concentration. The experiment was performed with 1024 scans, 2sec repetition delay at 295 K. The curve represents the best fit for the data obtained from the difference in intensities of the WaterLOGSY spectra recorded in presence and absence of the LoIA, respectively. The results provide biochemical confirmation that MAC0013243 interacts directly with LoIA in vitro and provide further support for the conclusion that LoIA, and lipoprotein targeting, is the likely target of this and related actives.
[0050] E. coli lipoproteins are anchored to the periplasmic surface of the inner or outer membranes. Braun's lipoprotein (Lpp) is the most abundant outer membrane (OM) lipoprotein in E coli. Lipoproteins lacking an inner membrane (IM) retention signal are transported to the OM by the LoI transport system comprised of 5 proteins, LoIABCDE (Tokuda and Matsuyama 2004). LoIC, LoID and LoIE form an ATP-binding cassette transporter in the IM. At the expense of ATP, this transporter releases lipoproteins into the periplasm where they are bound by the chaperone LoIA. The LolA-lipoprotein complex transverses the periplasm and interacts with an OM receptor LoIB which is essential to insert the lipoproteins into OM (Matsuyama et al. 1995).
[0051] The fate of newly formed Lpp was monitored in the inner and outer membranes of E. coli by immunoprecipitation with α Lpp antibody and analyzed by SDS PAGE and autoradiography. Panel C in figure 6 shows the accumulation of Lpp in the inner membrane of E. coli only when cells are treated with MAC0013243. On the other hand untreated cells contained Lpp only in the OM fractions.
[0052] LoIA plays an important role of transferring the lipoproteins from LoICDE complex from the inner membrane to LoIB in the outer membrane. The above findings indicate that MAC0013243 causes Lpp to accumulate in the IM in vivo, perturbing the physiological function of LoIA in E. coli.
[0053] The above disclosure generally describes the present invention. It is believed that one of ordinary skill in the art can, using the preceding description, make and use the compositions and practice the methods of the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely to illustrate preferred embodiments of the present invention and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Other generic configurations will be apparent to one skilled in the art. All reference documents referred to herein are hereby incorporated by reference. EXAMPLES
[0054] Although specific terms have been used in these examples, such terms are intended in a descriptive sense and not for purposes of limitation. Methods of microbiology, molecular biology and chemistry referred to but not explicitly described in the disclosure and these examples are reported in the scientific literature and are well known to those skilled in the art.
Example 1. NMR spectroscopy to probe the interaction of MAC0013243 with LoIA
[0055] In order to produce pure recombinant protein, a strain of E. coli that overexpressed recombinant LoIA was created. The gene encoding LoIA from E. coli was amplified from genomic DNA using VENT DNA polymerase (NEB biosciences). Primers were (5 -
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCATC ATGAAAAAAATTGCCATCACC-3') and (51- GGGGACCACTTTGTACAAGAAAGCTGGGTC
TTAGTGGTGGTGGTGGTGGTGCTTACGTTGATCATCTACCGTGACGCC- 3'). The PCR amplified product was cloned into pDEST14 using the Gateway cloning and Expression Kit (Invitrogen.Canada)and the insert sequence was confirmed by DNA sequence analysis (MOBIX, McMaster University). The clone was designed to create a protein product with a C-terminal poly- histidine tag for easy purification. LoIA was expressed and purified from Escherichia coli BL21 (DE3) Al cells transformed with pDEST14-/o/>4 grown at 370C to an optical density (600 nm) of 0.6 in Luria-Bertani media (2L) supplemented with 100 μg/mL ampicillin. The cells were then induced with 1 mM IPTG and 0.2% w/v L-arabinose and grown for an additional 2 hours prior to harvest by centrifugation at 10,000 x g. The cells were washed with a 0.85% saline solution, pelleted and re-suspended in 25 mL lysis buffer (25 mM HEPES pH 8.0, 1 mM DTT, 0.5 mg DNase, 0.5 mg RNase). All subsequent steps were performed at 40C. Cells were lysed by passage through a French press at 20,000 psi and clarified by centrifugation at 40,000 x g for 2 hours. Clarified lysate was purified by nickel chelate chromatography using a 1 mL HiTrap affinity column (Amersham Biosciences, Baie d'Urfe, PQ). The column was washed with buffer A (2OmM Sodium phosphate buffer pH 7.4, 50OmM NaCI containing 10% glycerol and 20 mM imidazole) and eluted with a linear imidazole gradient of 20-300 mM. Fractions of the eluant were analyzed by SDS-PAGE, and those containing pure His-tagged LoIA were pooled and concentrated using an Amicon ultracentrifugal filter device (5000 MW, Fisher Scientific) and simultaneously buffer exchanged to 2OmM Sodium phosphate buffer pH 7.4, 100 mM NaCI and 10% glycerol. Using this method about 10mg of pure LoIA was obtained. Fractions rich in LoIA protein were stored in aliquots at -800C. Concentration of purified protein was determined by a method described by Gill and von Hippel (Gill and von Hippel 1989).
Example 2. MAC0013243 causes accumulation of Lpp in the inner membrane of E. coli
[0056] E. coli cells were grown to log phase (OD6oo =0.3) in M9 minimal media then treated with MAC0013243 for 0, 5 and 20 minutes. All the three samples were pulse labelled with S35 Methionine and chased with cold Methionine for 2 minutes. Total membranes were isolated and fractionated on sucrose gradients to isolate inner and outer membrane fractions. Membrane fractions were resolved on 12% and 15% SDS polyacrylamide gels respectively and analyzed by western blot analysis using polyclonal α ToIC (panel A) identifying the OM fractions or α YidC antibodies (panel B) to identify the IM fractions. To detect the newly formed lpp, fractions were immunoprecipitated with an α Lpp antibody and analyzed by SDS PAGE and autoradiography. Panel C shows all of the Lpp in the OM when cells are not treated with MAC0013243. Treatment with MAC0013243 for 5 and 20 minutes causes accumulation of Lpp in the inner membrane fractions.
Example 3. Synthetic chemical exploration and optimization of the lead series
[0057] The general synthetic scheme for the preparation of all molecules was:
Figure imgf000023_0001
[0058] The general synthetic procedure was as follows.
Procedure for Preparation of Substituted-thioureas. The substituted alkyl or aryl halide or tosylate (1 eq.) and thiourea (1 eq.) were dissolved in absolute ethanol and refluxed for 2 hours. The solution was then removed from heat and allowed to stir overnight at room temperature. The precipitate was collected via filtration and recrystallized from concentrated HCI and H2O (1 :1 ).
General Procedures for Preparation of Triazanes. The amine (1 eq.) was mixed well with formaldehyde (2 eq.) in dioxane. To this solution, the substituted-thiourea (1 eq.) was added. The mixture was heated until a solution was obtained and allowed to stir overnight at room temperature. The precipitate produced was isolated via filtration. While many of the triazanes thus obtained were shown to be analytically pure, additional recrystallization was required in some cases.
[0059] 6-(2,4-dichloro-benzylsulfanyl)-3-[2-(3,4-dimethoxy-phenyl)-ethyl]- 1 ,2,3,4-tetrahydro-1 ,3,5-triazine (Compound 14):
1H NMR (200.13 MHz, DMSO-d6) δ
2.60-2.67 (m, 4H, -CH2-CH2-), 3.72 (s, 3H, -OCH3), 3.74 (s, 3H, -OCH3), 4.39 (s, 4H, N-CH2-N), 4.65 (s, 2H, benzyl-
Figure imgf000023_0002
H), 6.69-6.73 (m, 1 H, aryl-H), 6.84- 6.89 (m, 2H, aryl-H), 7.37-7.38 (m, 1 H, aryl-H), 7.65-7.69 (m, 2H, aryl-H), 10.57 (br s, 2H, N-H). 13C NMR (50.32 MHz, DMSO-d6) δ 31.9, 33.2, 51.9, 55.5, 61.5, 1 1 .9, 1 12.6, 120.4, 127.7, 129.3, 131.2, 132.6, 133.7, 134.2, 147.0, 148.5, 160.0. ES-HRMS: Calculated for C20H24N3O2SCI2 [M]+: 440.0966. Found: 440.0978.
[0060] [4-(4-chloro-benzylsulfanyl)-3,6-dihydro-2H-1 ,3,5-triazin-1 -yl]-acetic acid ethyl ester (Compound 28):
1H NMR (200.13 MHz, DMSO-d6) δ 1.22 (t,
3H, -CH3), 3.17 (s, 2H, CO-CH2-N), 4.10 (m,
Figure imgf000024_0001
2H, -CH2-CH3), 4.37 (s, 4H, N-CH2-N), 4.65 (s, 2H, benzyl-H), 7.35 (dd, 4H, aryl-H). 13C NMR (50.32 MHz, DMSO-d6) δ 14.0, 33.5, 50.8, 60.5, 61.5, 128.7, 130.9, 132.6, 134.9, 160.2, 169.1. ES-HRMS: Calculated for C14H19N3O2SCI [M]+: 328.0887. Found: 328.0893.
[0061] 6-(4-chloro-benzylsulfanyl)-3-propyl-1 ,2,3,4-tetrahydro-1 ,3,5-triazine (Compound 5)
1H NMR (200.13 MHz, DMSO-d6) δ. 0.90 (t, 3H, CH3), 1.44 (2H, m, CH3-CH2), 2.46 (2H, t,
Figure imgf000024_0002
CH2-CH2), 4.33 (s, 4H, N-CH2-N), 4.59 (s, 2H, benzyl-H), 7.24-7.35 (dd, 4H, aryl-H). 13C NMR (50.32 MHz, DMSO-d6) δ. 1 1.9, 20.9, 36.9, 51.6, 66.9, 60.8, 128.8, 130.8, 132.7, 134.1 , 157.5; ES- HRMS: Calculated for C13H18CIN3S [M]+: 283.0910. Found: 283.0909.
[0062] MIC determinations against our panel of test organisms are described in Table 3 for the three examples shown. The compounds conform to a general formula indicated below. Table 3. Examples of synthetic modification to priority actives identified as leads.
MIC1 (μg/mL)
Compound 14 Compound 28 Compound 5
Lab strains
Escherichia coli (MG1655) 8 256 8
Pseudomonas aeruginosa (PA01) 512 256 64
Burkhoderia cepacia 64 64 128
Salmonella typhimurium 8 64 128
Bacillus subtilis 168 >256 >256 >256
Micrococcus luteus >256 >256 >256
Staphylococcus aureus (ATCC) >256 >256 >256
Enterococcus faecalis (ATCC) >256 >256 >256
Streptococcus pyogenes >256 >256 >256
Mycobacterium smegmatis >256 >256 >256
1 MICs were determined according to NCCLS methods. (National Committee for Clinical Laboratory Standards. 2000. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically— Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.)
[0063] All citations are hereby incorporated by reference.
[0064] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
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Claims

WHAT IS CLAIMED IS:
1. A lipoprotein targeting pathway specific antibacterial compound.
2. A compound according to claim 1 having the formula (I)
Figure imgf000029_0001
or a pharmaceutically acceptable salt thereof,
wherein the compound comprises a 1 ,2,3,4-tetrahydro[1 ,3,5]triazine core.
3. A compound according to claim 2 wherein:
(i) R1 and R2 are independently selected from the group consisting of:
a) H, b) Unsubstituted or substituted, linear or branched, saturated or unsaturated C1 to C24 alkyl, cycloalkyl, heteroalkyl, aryl or heteroaryl moiety, c) An acyl group of structure R3-C=O wherein R3 is H or unsubstituted or substituted, linear or branched, saturated or unsaturated C1 to C24 alkyl, cycloalkyl, heteroalkyl, aryl or heteroaryl moiety, d) An alkoxy group of structure R4-0 wherein R4 is H or unsubstituted or substituted, linear or branched, saturated or unsaturated C1 to C24 alkyl, cycloalkyl, heteroalkyl, aryl or heteroaryl moiety, e) A sulfide group of structure R5-S wherein R5 is H or unsubstituted or substituted, linear or branched, saturated or unsaturated C1 to C24 alkyl, cycloalkyl, heteroalkyl, aryl or heteroaryl moiety, f) An amino group of structure R6-N-R7 wherein R6 and R7 are independently selected from those substituents defined in H or unsubstituted or substituted, linear or branched, saturated or unsaturated C1 to C24 alkyl, cycloalkyl, heteroalkyl, aryl or heteroaryl moiety, g) Any combination that incorporates elements from 3(i)(c) to 3(i)(f) into an alkyl or cycloalkyl unit as defined in 3(i)(b), h) One or more of a substituent selected independently from the following group wherein R1 and R2 are defined in 3(i)(a) and 3(i)(b) as above, such substituents including:
-CN, -NC, -NCO, -NCS, -OH1 -NO, -OCN, -SCN, -NO2, -F, -Cl, -Br, -I1 - N3, -OR1, -OR2, -N(Ri)2, -N(R2)Z, -N(R1)(R2), -SR1, -SR2, -SOR1, - S(O)2R1, -SOR2, -S(O)2R2, -0OR1, -0OR2 -C(O)OR1, -C(O)R1, - C(O)OR2, -C(O)R2, -OC(O)R1, -OC(O)R2, -NR1C(O)R1, -NR1C(O)R2, - NR2C(O)R1, -NR2C(O)R2, -C(O)N(R1),, -C(O)N(R1)(R2), -C(O)N(Rz)2. - C(NR1)(N(R1J2), -C(NR1)(N(R2),), -C(NR1)(N(R1)(R2)), -C(NRzXN(R1)Z), -C(NR2)(N(R1)(R2)), -C(NR2)(N(R2),), -C(N(R1)(R2)(N(R1)(R2), - N(R1)C(N(R1)(N(R1),), -N(R1)C(N(R1)XN(R1)(R2)), - N(R1)C(N(R2)XN(R1)(R2)), -N(R1)C(N(R2)(N(RO2), - N(R2)C(N(R1)XN(R1J2, -N(R2)C(N(R2)XN(R1),, - N(R2)C(N(R1)XN(R1)(R2)), -N(R2)C(N(R2)XN(R1)(R2)), - N(R1)C(N(R1)XN(Rz)2, -N(R2)C(N(Ri)XN(R2)2), -N(Ri)C(N(R2)XN(R2)Z, O1 =S1 =N(R1) Or =N(R2);
ii) Substituent X is independently selected from: a) A methylene (CH2) group, b) A carbonyl (C=O) group, c) A sulphur or oxygen atom or a substituted amine of type R8N, wherein R8 is defined by 1 ia) and l ib) described above, d) A sulfoxide (S=O) or sulfone (SO2) moiety, e) A phosphonate moiety of structure -P(=O)(OR9)2, wherein R9 is defined by 3(i)(a) or 3(i)(b) described above, f) A substituted or unsubstituted vinyl or conjugated vinyl group - (CH=CH)y- where y is an integer varying between O and 6; iii) m is an integer varying between O and 12; and iv) n is an integer varying between 0 and 12.
4. An antibacterial compound according to claim 1 having the general structure as shown below:
Figure imgf000031_0001
5. A pharmaceutically acceptable salt or ester of the compound as defined in claim 4.
6. An antibacterial compound having a structure selected from the group below:
Figure imgf000032_0001
Compound 14
Compound 28
Figure imgf000032_0002
Compound 5
Figure imgf000032_0003
, and analogues, mimetics, pharmaceutically acceptable salts and esters thereof.
7. A method of identifying antibacterial agents, said method comprising screening a bacterial library of clones over-expressing a gene for compounds that have growth inhibitory activity against a Gram negative bacterial strain; selecting growth inhibitory compounds that exhibit at least one additional pre-selected parameter; identifying target of activity through high copy suppression analysis and selecting compounds that are suppressed by a high copy of a particular gene.
8. The method of claim 7 wherein the method comprises screening for at least one additional preselected parameter.
9. A method according to claim 7 wherein the bacterial library comprises an E. co// strain.
10. A method according to claim 9 wherein the strain is E. coli MC1061.
1 1 .A method according to claim 8 wherein the additional parameter is selected from the group consisting of potency, solubility, purity, structure and availability.
12.A method according to claim 7 wherein the gene is a lipoprotein encoding gene.
13. A method according to claim 12 wherein the gene is the lolA gene.
14.A method of determining the chemical - genetic interaction of antibacterial compounds, said method comprising preparing an array from a bacterial library wherein each member of the library expresses a high copy number of particular gene; exposing the library to increasing amounts of a test compound; identifying members of the library that display resistance to the compound and determining which gene those members express at high copy number.
15.A method of treating a bacterial infection in an animal, said method comprising administering to one in need of treatment a compound having the general structure:
Figure imgf000034_0001
16. Use of a product encoded by a gene selected from the group consisting of ftsA, yadR, rpsR, yjeE and envA as an antimicrobial agent.
17.An antimicrobial composition comprising a compound of formula (I) and a pharmaceutically acceptable excipient.
18.An antimicrobial composition comprising an antibacterial compound as defined in claim 6.
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