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WO2017035288A1 - Composition et procédé destinés à prévenir, limiter ou traiter une infection à bactéries entéropathogènes - Google Patents

Composition et procédé destinés à prévenir, limiter ou traiter une infection à bactéries entéropathogènes Download PDF

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WO2017035288A1
WO2017035288A1 PCT/US2016/048517 US2016048517W WO2017035288A1 WO 2017035288 A1 WO2017035288 A1 WO 2017035288A1 US 2016048517 W US2016048517 W US 2016048517W WO 2017035288 A1 WO2017035288 A1 WO 2017035288A1
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compound
group
alkyl
toxt
acid
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F. Jon Kull
Ronald K. Taylor
Gordon W. Gribble
Evans O. ONYANGO
Anne R. KELLEY
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Dartmouth College
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Dartmouth College
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/46—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing six-membered aromatic rings and other rings, e.g. cyclohexylphenylacetic acid
    • C07C57/50—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing six-membered aromatic rings and other rings, e.g. cyclohexylphenylacetic acid containing condensed ring systems
    • 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
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C53/00—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen
    • C07C53/132—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen containing rings
    • C07C53/136—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen containing rings containing condensed ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/30—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing six-membered aromatic rings
    • C07C57/38—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing six-membered aromatic rings polycyclic
    • C07C57/40—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing six-membered aromatic rings polycyclic containing condensed ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/30—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing six-membered aromatic rings
    • C07C57/42—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms containing six-membered aromatic rings having unsaturation outside the rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/12—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
    • C07D217/14—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals
    • C07D217/16—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/04—One of the condensed rings being a six-membered aromatic ring
    • C07C2602/10—One of the condensed rings being a six-membered aromatic ring the other ring being six-membered, e.g. tetraline
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/26—All rings being cycloaliphatic the ring system containing ten carbon atoms
    • C07C2602/28—Hydrogenated naphthalenes

Definitions

  • V. cholerae 's primary virulence factors, the toxin- coregulated pilus (TCP) and cholera toxin (CT), occurs via a transcriptional cascade involving several activator proteins, and serves as a paradigm for the regulation of bacterial virulence.
  • the VPI contains the genes responsible for the synthesis and assembly of the essential colonization factor TCP, and the CTX phage encodes the CT genes.
  • TCP and CT genes are coordinately regulated at the transcriptional level by a virulence cascade involving activator proteins encoded both within the VPI and the ancestral genome.
  • AphA and AphB initiate the expression of the cascade by a novel interaction at the tcpPH promoter.
  • AphA is a member of a new regulator family and AphB is a LysR-type activator, one of the largest transcriptional regulatory families.
  • ToxT an AraC/XylS (A/X) type regulator, then directly activates the promoters of the primary virulence factors.
  • ToxT is the paramount regulator of virulence gene expression.
  • ToxT inhibitors have been identified and shown to provide protection against intestinal colonization by V. cholerae.
  • bile Schollmacher, et al. (1999) J. Bacteriol. 181 : 1508-14
  • virstatin a small molecule 4-[N-(l,8-naphthalimide)]-n-butyric acid, has been shown to inhibit virulence regulation in V.
  • ToxT contains an almost completely buried and solvent inaccessible sixteen carbon fatty acid bound to a pocket in the N-terminal domain, which can influence its DNA binding activity.
  • virulence gene expression can be reduced between 6-8 fold with cz ' s-palmitoleic acid and 10-15 fold with oleic acid (Lowden, et al. (2010) Proc. Natl. Acad. Sci. USA 107:2860- 5).
  • This disclosure provides a compound having the structure of Formula I, or a hydrate, isomer, prodrug or pharmaceutically acceptable salt of Formula I:
  • R 1 is Ci-C 6 alkyl, Ci-C 6 alkenyl, C 4 -C 6 aryl, halo, -CF 3 , -OCH 3 , -N0 2 , -CN, - OH, - Me 2 , -COOH, or -COOCH 3 ;
  • R 2 is hydrogen, alkylidenyl, or oxo;
  • n is 0-6; and dashed lines represent bonds that are independently present or absent.
  • a pharmaceutical, nutraceutical, nutritional, medical nutrition food or functional food composition containing a compound of Formula I is also provided.
  • kits for decreasing expression of a bacterial virulence factor and preventing, mitigating, or treating an infection by a bacterium that expresses an A/X regulatory protein comprising contacting the bacterium that expresses an A/X regulatory protein with a disclosed compound, such that the expression of a virulence factor by said bacterium is decreased.
  • the bacterium is Vibrio cholerae, Escherichia coli, Shigella flexneri, Yersinia enter ocolitica, Yersinia pestis, Brucella abortus Salmonella typhi, Bacillus anthracis, Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus or Salmonella typhimurium.
  • Figure 1 shows a sequence alignment of A/X family members from V. cholerae (Vc ToxT; SEQ ID NO: 1), an E. coli ETEC strain (Ec FapR; SEQ ID NO:2), an E. coli EPEC strain (Ec PerA; SEQ ID NO:3), S typhi (St SirC; SEQ ID NO:4), S typhimurium (St HilD; SEQ ID NO:5), S flexneri (Sf VirF; SEQ ID NO:6), Y. enter ocolitica (Ye VirF; SEQ ID NO: 7), an E. coli ETEC strain (Ec Rns; SEQ ID NO: 8), Y.
  • Vc ToxT V. cholerae
  • Ec FapR E. coli ETEC strain
  • Ec FapR E. coli EPEC strain
  • Ec PerA SEQ ID NO:3
  • S typhi Styphi
  • St SirC SEQ ID NO
  • Figure 2 shows the effects of compound 1 on tcp expression.
  • V. cholerae cells were grown in LB pH 6.5 at 30°C for 18 hours ⁇ the indicated inhibitor in methanol, ⁇ - galactosidase activity of a tcp-lacZ reporter construct was determined, ⁇ -galactosidase units are shown for a wild-type strain treated with methanol (MeOH), 0.01% oleic acid (0.01% OA), 0.02% oleic acid (0.02% OA), or 0.02% compound 1 (Compl) as compared to an untreated control (ToxT) and an untreated ToxT deletion strain ( ⁇ ).
  • MeOH methanol
  • OA 0.01% oleic acid
  • 0.02% oleic acid 0.02% oleic acid
  • Compl compound 1
  • Figure 3 shows the fatty acid binding region of ToxT.
  • the carboxylate head of c/5-palmitoleate (green) interacts with Lys31 of the N-terminal domain (blue) and Lys230 of the C-terminal domain (grey).
  • Full structure (inset) obtained from PDB 3GBG.
  • Figures 4A-B shows that small molecule inhibitors maintain a fatty acid-like structure.
  • General structure of a compound disclosed herein ( Figure 4A) compared to the bound conformation of cis-palmitoleate ( Figure 4B).
  • Figures 5A-C show the inhibition of tcpA expression and autoagglutination activities by compounds disclosed herein.
  • Figure 5A ⁇ -galactosidase activity of tcpA- lacZ fusion construct in the presence of virstatin (vir.), ten synthesized compounds, oleic acid (OA) and palmitoleic acid (POA) at concentrations of 5 ⁇ (blue) and 0.5 ⁇ (grey), graphed as percent change.
  • ⁇ is calculated based on 100% WT. The rest are calculated based on 100% WT+DMSO.
  • Figure 5B Western blot showing TcpA expression in the corresponding lanes. Compounds were added to a final concentration of 5 ⁇ .
  • Top non-specific band is a loading control.
  • Figure 5C Autoagglutination of 0395 cultures grown in the presence of 0.5 ⁇ (top) and 0.05 ⁇ (bottom) compounds 4a, 5a, 3b, and 4b.
  • Figures 6A-C show inhibition of ToxT-DNA binding interactions by compounds disclosed herein.
  • Figure 6A ToxT EMSA in the presence of virstatin and our compounds. All lanes contain a DIG labeled, 84-bp segment of the tcpA promoter. All lanes except lane 1 contain 0.78 ⁇ ToxT. The solvent (DMSO) does not inhibit DNA binding (lane 3). An excess of the unlabeled DNA segment competes for binding (lane 4). The presence of 100 ⁇ virstatin (vir) or 100 ⁇ synthesized compounds inhibits DNA binding.
  • Figure 6B ToxT does not shift the negative control DIG CJ2.6 DNA, a mutated segment of the tcpA promoter which ToxT cannot bind.
  • Figure 6B ToxT EMS A in the presence of virstatin and four lead compounds. All lanes contain labeled DNA. Virstatin is tested at 100, 10, 1, and 0.1 ⁇ concentrations. Compounds 4a, 5a, 3b and 4b are each tested 10, 1, and 0.1 ⁇ .
  • Figures 7A-C show X-ray crystal structures of ToxT-inhibitor complexes reveal compounds displace the fatty acid and bind in the regulatory pocket.
  • Figure 7A Simulated annealing F 0 - F c omit maps of ToxT bound to compounds 3b (left) and 5a (right) contoured at 2.5 ⁇ .
  • Figure 7B Overlay of crystal structures of ToxT bound to fatty acid (PDB: 3GBG) with ToxT bound to 3b (left) and ToxT bound to 5a (right).
  • Figure 7C Overlay of crystal structures of ToxT bound to 3b (left) and ToxT bound to 5a (right) with the conformations predicted by Autodock. Autodock predictions are purple and orange, respectively.
  • Figures 8A-C show competition STD NMR of compounds 5a and 3b in the presence of virstatin.
  • Figure 8A IH-NMR spectrum of virstatin alone (top) and in the presence of 20 ⁇ ToxT (middle). The corresponding STD-difference spectrum resulting from on-resonance saturation (irradiation of ToxT) and the transfer of resonance to virstatin (bottom).
  • Figure 8B, Figure 8C IH-NMR spectrum of 100 ⁇ virstatin in the presence of 20 ⁇ ToxT ( Figures 8B and 8C top) and with the addition of compound 3b ( Figure 8B middle) or compound 5a ( Figure 8C middle).
  • Figures 9A-B show that unsaturated fatty acids do not inhibit autoagglutination.
  • Figure 9 A Autoagglutination of 0395 cultures grown in the presence of 0.5 ⁇ , 0.05 ⁇ , and 5 nM virstatin and compounds 4a, 5a, 3b, and 4b.
  • Figure 9B Cultures containing 0.5 ⁇ and 0.05 ⁇ oleic and palmitoleic acids were grown similarly; the unsaturated fatty acids were not soluble at 5 nM.
  • Figure 10 shows the fatty acid binding region of ToxT comparing cis- palmitoleate (green, from crystal structure 3GBG) and models of compounds 3a, 3b, 3c, 3d, 4a, 4b, 4c, 5a, 5b, 5c and virstatin.
  • the naphthalenes with double bonds in the carboxylate chain are dark blue, the naphthalenes with single bonds are medium blue, tetralins are orange, and virstatin is magenta. Bound conformations were predicted using Autodock.
  • Figure 11 shows an electron density map of the natural fatty acid ligand. Simulated annealing Fo - Fc omit map of ToxT bound to cis-palmitoleate contoured at 2.5 ⁇ (PDB: 3GBG).
  • Figure 12 shows the STD-AF 0 curve of virstatin as a function of ligand concentration. Data were fit to equation 3. The isotherm was constructed using the STD data from protons H e f of virstatin. The same was done for protons H a b (data not shown).
  • a class of bicyclic compounds has now been identified, which exhibits anti- virulence activity against V. cholerae.
  • the compounds of this invention were designed to bind to the pocket located in the N-terminus of ToxT (Lowden, et al. (2010) Proc. Natl. Acad. Sci. USA 107:2860-5), thereby disrupting DNA binding activity and virulence gene expression.
  • ToxT homologues are found in a wide variety bacterial pathogens
  • the compounds of this invention find use as broad spectrum anti-virulence agents in the treatment of antibiotic-resistant bacterial infections as well as in prophylactic treatment of infections, e.g., for travelers or military personnel in areas with suboptimal water and/or food quality.
  • the compounds of this invention are highly specific for pathogens, the normal bacterial flora of the gut is not affected.
  • Compounds provided herein may have the structure of Formula I, which includes hydrates, isomers, prodrugs or pharmaceutically acceptable salts of Formula I:
  • R 1 is hydrogen (H) or Ci-C 6 alkyl, Ci-C 6 cycloalkyl, alkyl Ci-C 6 alkenyl, C 4 -C 6 aryl, halo (e.g., F, CI, Br or I), -CF 3 , -OCH 3 , -N0 2 , -CN, -OH, - Me 2 , -COOH, or - COOCH 3 ;
  • n is 0-6; and dashed lines represent bonds that are independently present or absent. In certain embodiments, n is 3, 4, or 5.
  • the compound of Formula I has the structure of Formula la:
  • R 1 is selected from the group consisting of hydrogen, -OH, and Ci-C 6 alkyl, wherein the Ci-C 6 alkyl is optionally substituted with an -OH group
  • n is selected from 1, 2, 3 and 4; and a dashed line represents a bond that is either present or absent.
  • R 1 is Ci-C 6 alkyl. In another particular embodiment, R 1 is methyl. In another particular embodiment, R 1 is ethyl. In another particular embodiment, R 1 is propyl. In another particular embodiment, R 1 is n- butyl. In another particular embodiment, R 1 is t-butyl. In another particular embodiment, R 1 is pentyl. In another particular embodiment, R 1 is Ci-C 6 alkyl, wherein the Ci-C 6 alkyl is substituted with an -OH group. In another particular embodiment, R 1 is -CH 2 OH. In another particular embodiment, R 1 is -CH 2 CH 2 OH. In another particular embodiment, R 1 is -CH 2 CH 2 CH 2 OH.
  • the compound of Formula la is selected from the following:
  • R 1 is Ci-C 6 alkyl, wherein the Ci-C 6 alkyl is optionally substituted with an -OH group.
  • R 1 is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl, and isomers thereof.
  • R 1 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, and neopentyl
  • R 1 is -CH 2 OH.
  • R 1 is -CH 2 CH 2 OH.
  • R 1 is -CH 2 CH 2 CH 2 OH.
  • R 1 is -CH 2 CH 2 CH 2 CH 2 OH.
  • the compound of Formula I has the structure of Formula lb:
  • R 1 is selected from the group consisting of hydrogen, hydroxyl and Ci-C 6 alkyl, wherein the Ci-C 6 alkyl is optionally substituted with an -OH group
  • X is selected from the group consisting of - H-, -CH 2 - CH 2 -, -CH 2 - H- and - H-CH 2 -
  • n is selected from 1, 2, 3 and 4
  • a dashed line represents a bond that is either present or absent.
  • R 1 is Ci-C 6 alkyl.
  • R 1 is methyl.
  • R 1 is ethyl.
  • R 1 is propyl.
  • R 1 is n- butyl.
  • R 1 is t-butyl.
  • R 1 is pentyl.
  • R 1 is Ci-C 6 alkyl, wherein the Ci-C 6 alkyl is substituted with an -OH group.
  • R 1 is -CH 2 OH.
  • R 1 is -CH 2 CH 2 OH.
  • the compound of Formula lb has the structure of Formula IbOl :
  • R 1 is Ci-C 6 alkyl, wherein the Ci-C 6 alkyl is optionally substituted with an -OH group.
  • R 1 is selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl, hexyl, and isomers thereof.
  • R 1 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, and neopentyl.
  • R 1 is -CH 2 OH.
  • R 1 is -CH 2 CH 2 OH.
  • R 1 is -CH 2 CH 2 CH 2 OH.
  • R 1 is -CH 2 CH 2 CH 2 CH 2 OH.
  • the compound of Formula I has the structure of Formula Ic:
  • R 1 is selected from the group consisting of hydrogen, hydroxyl and Ci-C 6 alkyl, wherein the Ci-C 6 alkyl is optionally substituted with an -OH group;
  • X is -CH 2 -CH 2 -;
  • n is selected from 1, 2, 3 and 4; and each dashed line independently represents a bond that is either present or absent.
  • R 1 is Ci-C 6 alkyl. In another particular embodiment, R 1 is methyl. In another particular embodiment, R 1 is ethyl. In another particular embodiment, R 1 is propyl. In another particular embodiment, R 1 is n- butyl. In another particular embodiment, R 1 is t-butyl. In another particular embodiment, R 1 is pentyl. In another particular embodiment, R 1 is Ci-C 6 alkyl, wherein the Ci-C 6 alkyl is substituted with an -OH group. In another particular embodiment, R 1 is -CH 2 OH. In another particular embodiment, R 1 is -CH 2 CH 2 OH. In another particular embodiment, R 1 is -CH 2 CH 2 CH 2 OH.
  • the invention is a compound having the structure of Formula II, which includes hydrates, isomers, prodrugs or pharmaceutically acceptable salts of Formula II:
  • R 1 is selected from the group consisting of Ci-C 6 alkyl, Ci-C 6 alkenyl, C4-C6 aryl, halo, CF 3 , OCH 3 , N0 2 , CN, OH, Me 2 , COOH, and COOCH 3 ;
  • X is -CH- or N;
  • compounds of Formula I, la, lb, Ic and II are selected from the following compounds, and pharmaceutically acceptable salts thereof:
  • compounds disclosed herein bind to the regulatory pocket of ToxT with a K d of less than about 300 ⁇ .
  • a compound comprising a carboxylate moiety capable of interacting with Lys31 of the N-terminal domain and Lys230 of the C- terminal domain of the fatty acid binding region of ToxT, and a fused bicyclic moiety.
  • the fused bicyclic moiety is selected from the group consisting of naphthalene, 1,2,3,4-tetrahydronaphthalene, 1,2,3, 4,5, 6,7,8-octahydronaphthalene, decalin, isoquinoline, and quinolone.
  • the fused bicyclic moiety is capable of interacting with Phe22 of the fatty acid binding region of ToxT.
  • the compound binds to the regulatory pocket of ToxT with a K d of less than about 300 ⁇ , as measured by a method known to those skilled in the art or described herein.
  • the compound displaces the natural (e.g., endogeneous) fatty acid ligand from the fatty acid binding region of ToxT, as measured by a method known to those skilled in the art or described herein.
  • the compound inhibits tcpA expression as determined by a ⁇ -galactosidase assay as described herein or another method known to those skilled in the art.
  • a method for preventing, mitigating, or treating an infection by a bacterium that expresses an A/X regulatory protein comprising administering to a subject in need thereof an effective amount of a disclosed compound, or a pharmaceutical composition thereof, such that the infection is prevented, mitigated, or treated.
  • the method is for preventing an infection.
  • the method is for mitigating an infection.
  • the method is for treating an infection.
  • the bacterium is selected from the group consisting of Vibrio cholerae, Escherichia coli, Shigella flexneri, Yersinia enter ocolitica, Yersinia pestis, Brucella abortus, Salmonella typhi, Bacillus anthracis, Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus, and Salmonella typhimurium.
  • a method for decreasing expression of a bacterial virulence factor comprising contacting a bacterium that expresses an A/X regulatory protein with a disclosed compound, or a pharmaceutically acceptable salt thereof, such that the expression of a virulence factor by said bacterium is decreased.
  • the bacterium is selected from the group consisting of Vibrio cholerae, Escherichia coli, Shigella flexneri, Yersinia enter ocolitica, Yersinia pestis, Brucella abortus, Salmonella typhi, Bacillus anthracis, Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus, and Salmonella typhimurium.
  • the X-ray structure of ToxT revealed a 16-carbon monounsaturated fatty acid cis- palmitoleate bound in a pocket within the N-terminal domain, the homologous location as the arabinose-binding site within AraC, a related protein for which the family is named.
  • the fatty acid bridges the interface between the N-terminal dimerization domain and C-terminal DNA-binding domain of ToxT, suggesting a potential mechanism for fatty acid-mediated inhibition.
  • the long aliphatic chain abuts the hydrophobic residues within the N-terminal domain and helices 9 and 10 of the C-terminal domain, occupying the bulk of the binding pocket (Fig. 3).
  • the anionic carboxylate forms salt bridges with C-terminal and N-terminal lysine residues, presumably locking ToxT in a "closed" conformation in which it is unable to dimerize and/or bind DNA.
  • a conformational change in the ligand-binding pocket and domain interface can easily impact the nature of the DNA-binding domain or the dimerization domain.
  • the carboxylate head and hydrophobic tail responsible for inter-domain interactions is critical for inhibition, and maintaining fatty acid-like character is required for an effective V. cholerae anti-virulence drug.
  • a subset of small molecule-inhibitors have been designed with these general characteristics ( Figures 4A-B).
  • compounds disclosed herein comprise a fused ring system that should provide rigidity and lead to tighter binding.
  • the "pre-folded" bicyclic compounds disclosed herein have already overcome this entropic penalty.
  • the initial subset of compounds includes variations in both the length of the carboxylate chain and degree of saturation of the ring system.
  • Inhibitors prevent virulence factor expression and ToxT-DNA binding
  • electrophoretic mobility shift assays were performed using purified ToxT protein and a digoxigenin-labeled 84 base-pair segment of the tcpA promoter. All compounds and virstatin prevented ToxT from binding DNA (Fig. 6A). The same concentration of ToxT did not shift a similar altered probe that ToxT is unable to bind due to a mutation in the binding site at the tcpA promoter (Hulbert, R. R. & Taylor, R. K. Mechanism of ToxT-Dependent Transcriptional Activation at the Vibrio cholerae tcpA Promoter. J. Bacteriol.
  • Inhibitors bind ToxT more tightly than virstatin
  • Saturation transfer difference (STD) NMR was used to characterize the strength of ToxT-ligand binding interactions. Significant STD signal was observed for virstatin, and confirms it is binding to ToxT. As shown in Fig. 8A, saturation is transferred to the aromatic protons of virstatin upon binding the protein. The STD effect on the aliphatic protons of virstatin was not analyzed due to their spectral overlap with ToxT. Based on the STD spectra for the aromatic protons H a>b and H e>f , the K d of virstatin was determined to be 483 ⁇ 109 ⁇ and 331 ⁇ 65 ⁇ respectively (Fig. 12).
  • the relative binding affinities of compounds 3b and 5a were determined by competition STD NMR experiments. The compounds were titrated into samples containing 20 ⁇ ToxT and 100 ⁇ virstatin, causing a significant decrease in the STD signals of virstatin (Fig. 8B,C). As X-ray crystallography has confirmed the binding pocket of compounds 3b and 5a, and because the STD signal of virstatin decreased upon the addition of the competitors, it is evident that virstatin also binds in the same regulatory pocket of ToxT. Based on the calculated K d of virstatin and the decrease in the STD signal, the calculated Ki values of 3b and 5a were 10 ⁇ and 31 ⁇ , respectively. Despite a suggested tendency for virstatin to bind non-specifically at high concentrations, the relative magnitudes of these binding constants are consistent with the activity assays described above.
  • Compounds disclosed herein are inhibitors of ToxT, the master regulator of virulence for the disease cholera. These inhibitors were designed based on the conformation of the ToxT ligand found in the X-ray crystal structure, cz ' s-palmitoleic acid (Lowden, M. J. et al. Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes. Proceedings of the National Academy of Sciences 107, 2860-2865 (2010)). These "pre-folded" small molecules bind much more tightly than unsaturated fatty acids, which show no activity at the tested concentrations.
  • the compounds do not inhibit colony formation, and therefore are not bactericidal, but were shown to inhibit virulence gene production via ⁇ -galactosidase and autoagglutination assays, and ToxT-DNA binding via EMSA.
  • the compounds bind to the ToxT regulatory domain pocket as predicted by AutoDock and visualized by X-ray crystallography. Based on a calculated K d determined by STD NMR, the lead compounds have at least 10-fold stronger binding affinities than the best-known ToxT inhibitor, virstatin (Shakhnovich, E. A., Hung, D. T., Pierson, E., Lee, K. & Mekalanos, J. J.
  • Virstatin inhibits dimerization of the transcriptional activator ToxT. Proceedings of the National Academy of Sciences 104, 2372-2377 (2007); Hung, D. T., Shakhnovich, E. A., Pierson, E. & Mekalanos, J. J. Small-Molecule Inhibitor of Vibrio cholerae Virulence and Intestinal Colonization. Science 310, 670-674 (2005)).
  • the STD NMR data also indicate that virstatin binds in the same ligand-binding pocket of ToxT, which seems to accommodate only one ligand at a time.
  • compounds disclosed herein displace the fatty acid and bind in its place.
  • alkyl refers to a straight or branched chain hydrocarbon, preferably having from one to six carbon atoms ⁇ i.e., Ci-C 6 ).
  • alkyl as used herein include methyl, ethyl, propyl, isopropyl, n-butyl, isopentyl, n- pentyl, and the like, as well as substituted versions thereof.
  • an alkyl of the invention is a C 1 -C3 alkyl.
  • alkylidenyl refers to a divalent functional group derived from an alkane by removal of two hydrogen atoms from the same carbon atom, the free valencies being part of a double bond.
  • alkenyl refers to a straight or branched chain aliphatic hydrocarbon containing one or more carbon-to-carbon double bonds that may be optionally substituted, with multiple degrees of substitution included within the present invention. Examples include, but are not limited to, vinyl, allyl, and the like, as well as substituted versions thereof.
  • aryl refers to a monovalent group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a four-, five- or six-membered aromatic ring structure wherein the ring atoms are all carbon, and wherein the monovalent group is composed of carbon and hydrogen.
  • aryl groups include phenyl, methylphenyl, (dimethyl)phenyl, -ethylphenyl, propylphenyl, - C 6 H 4 CH(CH 3 ) 2 , -C 6 H 4 CH(CH 2 ) 2 , methylethylphenyl, vinylphenyl, naphthyl, and the monovalent group derived from biphenyl.
  • the aryl is a phenyl group.
  • hydrate when used as a modifier to a compound means that the compound has less than one (e.g., hemihydrate), one (e.g., monohydrate), or more than one (e.g., dihydrate) water molecules associated with each compound molecule, such as in solid forms of the compound.
  • An "isomer" of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.
  • a "pharmaceutically acceptable salt” refers to a salt of a disclosed compound which is pharmaceutically acceptable, and e.g., which possesses the desired pharmacological activity.
  • Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2- hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'- methylenebis(3-hydroxy-2-ene-l -carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l- carboxylic acid, acetic acid, aliphatic mono- and di-carboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid,
  • Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases.
  • Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide.
  • Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
  • Disclosed compounds may also exist in prodrug form. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals, e.g., solubility, bioavailability, manufacturing, etc., the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates prodrugs of compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.
  • prodrugs include, for example, compounds described herein in which a hydroxy or carboxy group is bonded to any group that, when the prodrug is administered to a patient, cleaves to form a hydroxy or carboxylic acid, respectively.
  • a compound comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound.
  • Suitable esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, phosphates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-P-hydroxynaphthoate, gentisates, isethionates, di-p-toluoyl tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, ⁇ -toluenesulfonates, cyclohexylsulfamates, quinates, esters of amino acids, and the like.
  • Disclosed compounds can be prepared as described herein (see Example 1) or using any suitable methodology routinely practiced in the art, and be analyzed for their pharmacological properties by routine methodologies. For example, kinetic solubility can be measured using a direct UV absorbance method or thermodynamic solubility can be measured. In addition, stability in gastrointestinal fluids can be determined by conventional methods (Asafu-Adj aye, et al. (2007) J. Pharm. Biomed. Anal. 43 : 1854- 1859), e.g., 1 hour in simulated gastric fluid (pH 1.2, pepsin) at 37°C and/or 3 hours in simulated intestinal fluid (pH 6.8, pancreatin).
  • PAMPA Parallel Artificial Membrane Permeability Assay
  • BBB blood-brain barrier
  • a disclosed compound may be administered in a pharmaceutical composition by various routes including, but not limited to, intradermal, intramuscular, intraperitoneal (e.g., by injection), intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, transdermal, rectal, or topical administration.
  • the active compound may be coated.
  • the therapeutic compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.
  • compositions comprising a compound disclosed herein and a pharmaceutically acceptable carrier.
  • dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • Sterile injectable solutions can be prepared by incorporating the therapeutic compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the therapeutic compound into a sterile carrier which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., the therapeutic compound) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a disclosed compound can be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the therapeutic compound and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
  • the therapeutic compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the percentage of the therapeutic compound in the compositions and preparations may, of course, be varied. The amount of the therapeutic compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of a selected condition in a subject.
  • this invention also provides nutraceuticals; nutritional compositions, such as dietary supplements; medical nutrition or functional foods including a compound of Formula I.
  • nutritional compositions such as dietary supplements; medical nutrition or functional foods including a compound of Formula I.
  • Such compositions can be prepared by mixing one or more compounds of the invention with an edible nutritionally acceptable solid or liquid carriers and/or excipients, e.g., fillers, such as cellulose, lactose, sucrose, mannitol, sorbitol, and calcium phosphates; and binders, such as starch, gelatin, tragacanth, methylcellulose and/or polyvinylpyrrolidone (PVP).
  • fillers such as cellulose, lactose, sucrose, mannitol, sorbitol, and calcium phosphates
  • binders such as starch, gelatin, tragacanth, methylcellulose and/or polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrroli
  • Optional additives include lubricants and flow conditioners, e.g., silicic acid, silicon dioxide, talc, stearic acid, magnesium/calcium stearates and polyethylene glycol (PEG) diluents; disintegrating agents, e.g., starch, carboxymethyl starch, cross-linked PVP, agar, alginic acid and alginates, coloring agents, flavoring agents and melting agents.
  • the composition of the invention can optionally include conventional food additives, such as emulsifiers, stabilizers, sweeteners, preservatives, chelating agents, osmotic agents, buffers or agents for pH adjustment, acidulants, thickeners, texturizers and the like.
  • compositions of the present invention can further include antibiotics (e.g., tetracyclines), probiotics, prebiotics, anti-LPS slgA (Apter, et al. (1993) Infect. Immun. 61(12):5279-5285), as well as other monounsaturated fatty acids such as oleic acid or palmitoleic acid to facilitate the prevention, mitigation and/or treatment of a bacterial infection.
  • antibiotics e.g., tetracyclines
  • probiotics e.g., prebiotics, anti-LPS slgA (Apter, et al. (1993) Infect. Immun. 61(12):5279-5285)
  • anti-LPS slgA Apter, et al. (1993) Infect. Immun. 61(12):5279-5285
  • other monounsaturated fatty acids such as oleic acid or palmitoleic acid to facilitate the prevention, mitigation
  • Suitable product formulations include sachets, soft gel, powders, syrups, pills, capsules, tablets, liquid drops, sublinguals, patches, suppositories, liquids, injectables and the like.
  • food and beverage products containing one or more compounds of the present invention, such as solid food products, like bars (e.g., nutritional bars or cereal bars), powdered drinks, dairy products, breakfast cereals, muesli, candies, confectioneries, cookies, biscuits, crackers, chocolate, chewing-gum, desserts and the like; liquid comestibles, like soft drinks, juice, sports drinks, milk drinks, milk-shakes, yogurt drinks or soups, etc.
  • the addition of one or more compounds of the invention to animal feed is also included within the scope of this invention.
  • compositions can be provided as a component of a meal, e.g., a nutritional or dietary supplement, in the form of a health drink, a snack or a nutritionally fortified beverage, as well as a conventional pharmaceutical, e.g., a pill, a tablet or a softgel, for example.
  • a meal e.g., a nutritional or dietary supplement
  • a conventional pharmaceutical e.g., a pill, a tablet or a softgel, for example.
  • ToxT belongs to the AraC/XylS (A/X) superfamily of regulatory proteins. This family is composed of approximately 1,974 members identified in 149 bacterial genomes including Bacillus anthracis, Listeria monocytogene s ⁇ and Staphylococcus aureus (Ibarra, et al. (2008) Genetica 133 :65-76), and is known for its role in virulence gene regulation.
  • A/X AraC/XylS
  • the crystal structure of ToxT identified a binding pocket enclosed by residues Y12, Y20, F22, L25, 127, K31, F33, L61, F69, L71, V81, and V83 from the N-terminal domain and residues 1226, K230, M259, V261, Y266, and M269 from the C-terminal domain.
  • the volume of this predominantly hydrophobic pocket is 780.9 A 3 as calculated by the program CASTp.
  • This pocket contains a sixteen- carbon fatty acid with a negatively charged carboxylate head group forming salt bridges with both K31 from the N-terminal domain and K230 from the C-terminal domain.
  • compositions herein can be broadly applied to treat enteric bacterial infections that cause travelers' diarrhea, salmonella, brucellosis, botulism, dysentery, and typhoid fever, diseases infecting some 4 billion people annually worldwide.
  • the present invention embraces compositions containing one or more compounds of the invention for use in methods for decreasing or inhibiting the expression of bacterial virulence genes.
  • This method is carried out by contacting a pathogenic bacterium with a composition of the present invention so that the expression of at least one virulence factor, e.g., TCP and/or CT in V. cholerae, is measurably decreased as compared to bacteria not contacted with the composition of the invention.
  • a decrease or inhibition of virulence factor expression can be measured using any conventional method for monitoring nucleic acid or protein levels in a cell, e.g., northern blot analysis, RT-PCR analysis, dot blot analysis, western blot analysis and the like.
  • the composition of the invention decreases virulence factor expression by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or as much as 100% as compared to untreated bacteria.
  • V. cholerae There are several characteristics of pathogenic V. cholerae that are important determinants of the colonization process. These include adhesins, neuraminidase, motility, chemotaxis and toxin production. If the bacteria are able to survive the gastric secretions and low pH of the stomach, they are well adapted to survival in the small intestine. V. cholerae is resistant to bile salts and can penetrate the mucus layer of the small intestine, possibly aided by secretion of neuraminidase and proteases. Specific adherence of V. cholerae to the intestinal mucosa is likely mediated by the long filamentous TCP pili which are coregulated with expression of the cholera toxin genes.
  • V. cholerae produces cholera toxin, which is composed of two A subunits and five B subunits.
  • the B subunits allow binding to a ganglioside (GMi) receptor on the intestinal epithelial cells.
  • GMi ganglioside
  • the B pentamer must bind to five corresponding GMi receptors. This binding occurs on lipid rafts, which anchor the toxin to the membrane for endocytosis of the A subunits, thereby trafficking the toxin into the cell and to the basolateral surface where it acts (Lencer (2001) Am. J. Physiol. Gastrointest. Liver Physiol. 280:G781-G786).
  • the A subunits proteolytically cleave into Al and A2 peptides.
  • the Al peptide ADP-ribosylates a GTP- binding protein, thereby preventing its inactivation.
  • the always active G protein causes adenylate cyclase to continue forming cAMP.
  • This increase in intracellular cAMP blocks absorption of sodium and chloride by microvilli and promotes the secretion of water from the intestinal crypt cells to preserve osmotic balance (Torgersen, et al. (2001) J. Cell Sci. 114:3737-3747). This water secretion causes the watery diarrhea with electrolyte concentrations isotonic to plasma.
  • the fluid loss occurs in the duodenum and upper jejunum, with the ileum less affected.
  • the colon is less sensitive to the toxin, and is therefore still able to absorb some fluid.
  • the large volume however, overwhelms the colon's absorptive capacity.
  • Escherichia coli There are several pathogenic derivatives of E. coli. Several of the most common are as follows. One is Enterohemorrhagic E. coli (EHEC), which causes a Shigella-like illness and is also known as the hamburger meat E. coli. Another is Enteropathogenic E. coli (EPEC), which causes persistent diarrhea in children. EPEC expresses a surface appendage termed the bundle forming pilus, or BFP. BFP is required for intestinal colonization by the bacterium. BFP gene expression is activated by the A/X family member PerA that meets alignment criteria described herein. A third example is Enterotoxigenic E.
  • EHEC Enterohemorrhagic E. coli
  • EPEC Enteropathogenic E. coli
  • BFP is required for intestinal colonization by the bacterium.
  • BFP gene expression is activated by the A/X family member PerA that meets alignment criteria described herein.
  • a third example is Enterotoxigenic E.
  • ETEC ETEC
  • ETEC colonization factor adhesions
  • CS1 and CS2 colonization factor adhesions
  • Rns A/X family regulator
  • cofS, IngS and CfaD regulatory proteins cofS, IngS and CfaD, which regulate the expression of virulence factors.
  • CfaD and Rns are fully interchangeable with each other (Bodero, et al. (2007) J. Bacteriol. 189: 1627-32) and recognize the same DNA binding sites.
  • Salmonella Salmonella.
  • Salmonella cause 1.4 million cases of gastroenteritis and enteric fever per year in the US and lead all other food borne pathogens as a cause of death. While there are over a thousand serotypes of Salmonella that can cause gastroenteritis, S. enteritidis (sv. Typhimurium) is the leading cause. S. enteritidis (sv. Typhimurium) infection of mice serves as a model for typhoid fever as the causative agent of this disease only infects humans. As such, this species has served as a model organism for both gastroenteritis and typhoid fever. Most of the genes that encode virulence factors are located in clusters on salmonella pathogenicity islands termed SPIs.
  • SPI-1 carries the genes for a type III secretion system (T3SS), the expression of which is critical for virulence.
  • T3SS type III secretion system
  • the master regulator of the expression of SPI-1 genes is HilA.
  • the expression of HilA itself is controlled by HilD.
  • HilD is an A/X family member that meets alignment criteria described herein.
  • Salmonella typhi (S. enterica sv. Typhi) is the leading cause of enteric fever also known as typhoid fever. Typhoid fever is estimated to affect approximately 17 million people annually, causing 600,000 deaths. S. typhi is a multi-organ organism, infecting lymphatic tissues, liver, spleen, and bloodstream. S. typhi has a gene regulatory network similar to the SPI-1 and regulation of T3SS gene expression in S. enteritidis (sv. Typhimurium). In the case of S. typhi the aligned A/X family member is designated SirC.
  • Shigella Several Shigella species are responsible for the majority of bacillary dysentery that is caused by this organism. S. dysenteriae is common in many parts of the world. S. flexneri and S. sonnei are the most common in the U.S. Most molecular analysis regarding Shigella has been performed with S. flexneri. This species requires a surface protein, IcsA, to nucleate actin and travel through and between host cells. Expression of the icsA gene is activated by VirF, which meets alignment criteria described herein.
  • Bacillus anthracis Bacillus anthracis is an aerobic spore-forming bacteria that causes anthrax disease. Livestock may become infected by eating or inhaling anthrax spores. Humans, especially farmers and individuals who work in slaughterhouses, may develop cutaneous anthrax through skin exposure to infected animals. Humans can also get inhalational anthrax by breathing in material contaminated with the bacteria. This bacterium also expresses an AraC family member.
  • Listeria Listeria monocytogenes is a facultative intracellular bacterium that is the causative agent of Listeriosis. It is one of the most virulent food-borne pathogens with 20 to 30 percent of clinical infections resulting in death. Listeria monocytogenes also expresses an AraC family member.
  • Staphylococcus aureus is a facultatively anaerobic, gram-positive coccus and is the most common cause of staph infections.
  • Some strains of S. aureus, which produce the exotoxin TSST-1, are the causative agents of toxic shock syndrome, whereas other strains of S. aureus also produce an enterotoxin that is the causative agent of S. aureus gastroenteritis.
  • Yersinia enterocolitica is a common pathogen of children and adults, with a strong propensity for extraintestinal complications. Gastrointestinal disorders include enterocolitis, particularly in children, and pseudoappendicitis, particularly in young adults.
  • Y. enterocolitica virulence factors include outer proteins termed Yops and YadA, which is an adhesin that is essential for colonization.
  • VirF is an A/X family member that meets alignment criteria described herein.
  • Yersinia pestis is the pathogen of human and animals that causes all three main forms of the plague including pneumonic, septicemic and bubonic plagues. Similar to VirF, the Y. pestis virulence regulon is controlled by the A/X family member LcrF (Hoe, et al. (1992) J. Bacteriol. 174:4275-86).
  • Bacillus anthracis the etiologic agent of anthrax
  • Brucella abortus which causes brucellosis
  • Clostridium botulinum the causal agent of botulism
  • bacterial infection is used to describe the process of adherence and virulence factor production by a pathogenic bacterium that expresses an A/X regulatory protein.
  • treatment or “treating” means any therapeutic intervention in a mammal, preferably a human or any other animal suffering from a enteropathogenic bacterial infection, such that symptoms and bacterial numbers are reduced or eliminated.
  • adhesion of V. cholerae to the intestinal mucosa via TCP pili, colonization will be reduced or inhibited, thereby allowing the subject to clear the bacterial infection.
  • prevention refers to prophylactic treatment, wherein clinical symptom development is delayed or inhibited, e.g., preventing infection from occurring and/or developing to a harmful state.
  • “Mitigation” or “mitigating” means arresting the development of clinical symptoms, e.g., stopping an ongoing infection to the degree that it is no longer harmful, or providing relief or regression of clinical symptoms, e.g., a decrease in fluid loss resulting from an infection.
  • Prophylactic or therapeutic treatment involves the administration of an effective amount of a compound of this invention to a subject in need thereof, thereby preventing, mitigating, or treating a bacterial infection.
  • Subjects benefiting from the method of the invention include those having a bacterial infection (e.g., exhibiting signs or symptoms) or those at risk of having a bacterial infection (e.g., a subject exposed to a contaminated food or water source).
  • the terms "effective amount” means a dosage sufficient to measurably decrease or inhibit virulence gene expression and provide prevention, mitigation and/or treatment of a bacterial infection.
  • the administered dose delays, mitigates, or reduces the signs and/or symptoms of infection in the subject by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%) relative to untreated subjects.
  • the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in preventing, mitigating or treating the disease in humans.
  • compositions of the invention to be administered are determined in the light of various relevant factors including the purpose of administration (e.g., prevention, mitigation or treatment), the age, sex and body weight of an individual subject, and/or the severity of the subject's symptoms.
  • the compositions of the invention can be administered under the supervision of a medical specialist, or may be self-administered.
  • compositions of the present invention would usually be single or multiple servings per day, e.g., once or twice daily, for acute or chronic use.
  • benefit may be derived from dosing regimens that can include consumption on a daily, weekly or monthly basis or any combination thereof.
  • Administration of compositions of the invention, e.g., treatment could continue over a period of days, weeks, months or years, until an infection has been treated.
  • the composition of the invention is consumed at least once a day on a regular basis, to prevent an infection.
  • ⁇ -galactosidase activity was quantitatively measured according to Miller (Miller, J. H. in Experiments in molecular genetics (Cold Spring Harbor Laboratory, 1972)).
  • ToxT was expressed by autoinduction from toxT-intein/CBD (chitin binding domain) fusion construct transformed in BL21-CodonPlus (DE3)-RIL E. coli, as done previously (Lowden, M. J. et al. Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes. Proceedings of the National Academy of Sciences 107, 2860-2865 (2010)). Cells were harvested by centrifugation, resuspended in medium-salt column buffer (20 mM Tris, 1 mM EDTA, 500 mM NaCl, pH 8), lysed via sonication, and clarified by centrifugation.
  • medium-salt column buffer (20 mM Tris, 1 mM EDTA, 500 mM NaCl, pH 8
  • Clarified supernatant was loaded onto a gravity flow column packed with chitin beads (New England Biolabs) equilibrated in column buffer. After elution of the supernatant, the column was washed with column buffer followed by low-salt buffer (20 mM Tris, 1 mM EDTA, 200 mM NaCl, pH 8), and equilibrated with cleavage buffer (low-salt buffer with 100 mM dithiothreitol (DTT)). The column was placed at 4 °C for 16 hours to cleave the intein with the CBD.
  • low-salt buffer 20 mM Tris, 1 mM EDTA, 200 mM NaCl, pH 8.
  • cleavage buffer low-salt buffer with 100 mM dithiothreitol (DTT)
  • ToxT- intein/CBD fusion protein that co-eluted with the cleaved ToxT was separated using a HiTrap sepharose packed fast flow cationic exchange column (GE) with the following gradient: 45% high-salt buffer (20 mM Tris, 1 mM EDTA, 1 M NaCl, pH 8) for 175 minutes at a flow rate of 0.4 ml/min.
  • GE HiTrap sepharose packed fast flow cationic exchange column
  • DIG-TCP-5 5' TGTTTCTTTCA ATGCAAGTG
  • DIG-TCP-6 5'
  • CACAAAGTCACCTACAATTG CACAAAGTCACCTACAATTG.
  • Purified ToxT protein was mixed with 0.5 ng DIG- DNA in a binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 100 mM KC1, 5 mM MgCl 2 , 1 mM DTT, 0.3 mg/ml BSA, 0.25 ⁇ g poly [d(I-C)], and 10% glycerol).
  • Compounds in DMSO were added to a final concentration of 0.1-100 ⁇ , using the same volume of DMSO as a control.
  • ToxT was co-crystallized with the synthesized compounds in hanging drops containing 50% protein buffer (20 mM Tris, 1 mM EDTA, -300 mM NaCl, pH 7.5) and 50% reservoir solution (0.1 M MES pH 6.5 and 15% (w/v) PEG 400).
  • the compound was added to 1.47 mg/ml ToxT at a 20: 1 molar excess, and the complex was incubated at 30 °C for 15 minutes before setting up drops.
  • the cryoprotectant for ToxT crystals contained 0.1 M MES pH 6.5, 18% (w/v) PEG 400, and either 30% 1,4-butanediol or glycerol.
  • Sample preparation Virstatin was solubilized in DMSO-d 6 and compounds 5a and 3b in DMSO. Pure ToxT in protein buffer (20 mM Tris, 1 mM EDTA, -300 mM NaCl, pH 8) was used at a final concentration of 20 ⁇ . All samples contained 50 ⁇ TSP as an internal standard and 5% D 2 0. All NMR experiments were carried out on a Bruker Avance 600 MHz or 700 MHz spectrometer equipped with a TCI cryogenic probe. Samples were stored at 4 °C prior to acquisition.
  • K of virstatin For on-resonance spectra, the protein was saturated at 500 Hz or 583.33 Hz for the 600 MHz and 700 MHz NMR spectrometers, respectively, for 1-4 seconds utilizing a train of 50 ms Gaussian pulses. For off- resonance spectra, the sample was irradiated at -2000 Hz. The total relaxation delay was 7 seconds. Data acquisition consisted of 128 scans and 32,768 points. The STD-effect was calculated by measuring the intensity of the virstatin aromatic proton peaks in the on- and off-resonance spectra:
  • the initial slopes STD-AF 0 (the initial growth rates of the STD amplification factors, which corresponds to ASTD at zero tsAi-) are obtained from:
  • the K D of virstatin was calculated using Michaelis-Menten kinetics.
  • Virstatin was used as the STD indicator at a concentration of 100 ⁇ .
  • Compound 1 was tested in a reporter assay to determine its effect on inhibiting the activity of ToxT. This analysis indicated that compound 1 effectively inhibited the activity of ToxT as determined in a tcp reporter assay ( Figure 2).
  • this disclosure also includes derivatives of compound 1.
  • additional derivatives of compounds 1-3 can be prepared. For example, by varying the anhydride (or a functionalized dicarboxylic acid), the carbon chain linking the naphthalene ring to the carboxylate head group can be lengthened or shortened. Thus, reaction of l-bromo-8- methylnaphthalene with glutaric anhydride will afford a compound similar to compounds 1-3 having three carbons between the ketone carbonyl and the carboxylate group.
  • the acylation reaction can be carried out with any suitable dicarboxylic acid, e.g., malonic, succinic, glutaric, adipic, pimelic or suberic acid/anhydride, to achieve a compound having between 1 and 6 carbons between the ketone carbonyl and the carboxylate group.
  • any suitable dicarboxylic acid e.g., malonic, succinic, glutaric, adipic, pimelic or suberic acid/anhydride
  • the methyl group attached to the naphthalene ring can be substituted with any other alkyl or polar group, either by de novo synthesis from a suitable 2- alkylfuran and 3-bromobenzyne (see synthesis of l-bromo-8-methylnaphthalene in Scheme 1) or by modification of the methyl group in the final product. For example, appropriate oxidation will convert this methyl group to a carboxylate or to a hydroxyl group.
  • the synthesis of l-bromo-8-methylnaphthalene automatically gives the isomeric l-bromo-5-methylnaphthalene, which provides a set of control compounds isomeric to compounds 1-3.
  • the aromatic carbonyl ketone group can be readily reduced with sodium borohydride in trifluoroacetic acid (Gribble, et al. (1978) Synthesis 763) or other two-step reduction procedures known in the art.
  • the resulting compounds can be further reduced by catalytic hydrogenation to a derivative of compound 3.
  • the reduction conditions e.g., Birch reduction
  • compounds having one reduced ring i.e., a tetralin analogue [1,2,3,4-tetrahydronaphthalene]
  • the ketone carbonyl in Compound 1 can be converted to an alkene, so as to "stiffen" the carbon chain.
  • the naphthalene ring is replace with 4-methylindole, which could be deemed an isostere to 8-methylnaphthalene.
  • Introduction of carboxylate chains onto the indole C-3 position is facile as this position is extremely susceptible to electrophilic substitution.
  • the indole double bond is readily reduced, providing additional flexibility for polarity of the basic two-ring structure. This is shown in Compound 4.
  • the 8-methylnaphthalene unit can be replaced with the isostere 5- methylquinoline.
  • the C-4 position of quinoline like C-3 in indole, is very easily substituted and will provide a ring structure for conversion to derivatives of compound 1 and, by reduction, to derivatives of compound 3. This is shown in compound 5, and, for isoquinoline, compound 6.
  • the “anchor” for the palladium-catalyzed C-C coupling was l -bromo-8-methylnaphthalene (1), whose synthesis has been previously optimized (Onyango, E. O., Kelley, A. R., Qian, D. C. & Gribble, G. W. Syntheses of l-Bromo-8-methylnaphthalene and l-Bromo-5- methylnaphthalene. J. Org. Chem. 80, 5970-5972 (2015)).
  • R H, Me, or Et
  • n 0-3
  • the crude mixture an orange oil, was purified via silica gel chromatography (20: 1 to 2: 1 hexanes/ethyl acetate) to afford a mixture of hex-5-enoic acid, 3d, and 3d' *. Further purification by trituration first with hot hexanes (to remove the hexenoic acid) and second by EtOAc (to remove 3d') left the pure product 3d as a white solid (65.5 mg, 57%).

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Abstract

La présente invention concerne des composés bicycliques permettant de réduire l'expression de facteurs de virulence chez les bactéries de manière à prévenir, limiter ou traiter une infection bactérienne.
PCT/US2016/048517 2015-08-27 2016-08-25 Composition et procédé destinés à prévenir, limiter ou traiter une infection à bactéries entéropathogènes Ceased WO2017035288A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109928867A (zh) * 2017-12-15 2019-06-25 江苏暨明医药科技有限公司 3,5-二羟基戊苯的合成方法
CN109928867B (zh) * 2017-12-15 2022-04-12 江苏暨明医药科技有限公司 3,5-二羟基戊苯的合成方法

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