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  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
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  • A61K31/425—Thiazoles
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  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
• • A—HUMAN NECESSITIES
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  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
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• • A—HUMAN NECESSITIES
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  • A61P31/08—Antibacterial agents for leprosy
• • A—HUMAN NECESSITIES
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  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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  • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
  • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
  • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
  • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
  • C07D235/08—Radicals containing only hydrogen and carbon atoms
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  • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
  • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
  • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
  • C07D235/12—Radicals substituted by oxygen atoms
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  • C07D235/14—Radicals substituted by nitrogen atoms
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
  • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
  • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
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  • C07D267/02—Seven-membered rings
  • C07D267/08—Seven-membered rings having the hetero atoms in positions 1 and 4
  • C07D267/12—Seven-membered rings having the hetero atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems
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  • 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
  • C07D401/04—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 directly linked by a ring-member-to-ring-member bond
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  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/04—Ortho-condensed systems
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Multidrug resistance in bacteria is generally attributed to the acquisition of multiple transposons and plasmids bearing genetic determinants for different mechanisms of resistance (Gold et al. 1996. N. Engl J. Med. 335:1445).
• descriptions of intrinsic mechanisms that confer multidrug resistance have begun to emerge. The first of these was a chromosomally encoded multiple antibiotic resistance (mar) locus in Escherichia coli (George and Levy, 1983. J. Bacteriol. 155:531; George and Levy 1983 J. Bacteriol. 155:541).
• the mar locus consists of two divergently positioned transcriptional units that flank a common promoter/operator region in E. coli, Salmonella typhimurium, and other Entrobacteriacae (Alekshun and Levy.
• One operon encodes MarC, a putative integral inner membrane protein without any yet apparent function, but which appears to contribute to the Mar phenotype in some strains.
• the other operon comprises marRAB, encoding the Mar repressor (MarR), which binds marO and negatively regulates expression of marRAB (Cohen et al. 1994. J. Bacteriol 175:1484; Martin and Rosner 1995. PNAS 92:5456; Seoane and Levy. 1995 J. Bacteriol. 177:530), an activator (MarA), which controls expression of other genes on the chromosome, e.g., the mar regulon (Cohen et al.
• E. coli Exposure of E. coli to several chemicals, including tetracycline and chloramphenicol (Hachler et al 1991 J Bacteriol 173(17):5532-8; Ariza, 1994, J Bacteriol; 176(1): 143-8), sodium salicylate and its derivatives (Cohen, 1993, J Bacteriol; 175(24):7856-62) and oxidative stress agents (Seoane et al. 1995. J Bacteriol; 177(12):3414-9) induces the Mar phenotype. Some of these chemicals act directly at the level of MarR by interacting with the repressor and inactivating its function (Alekshun. 1999. J. Bacteriol.
• MarA activates the transcription of several genes that constitute the E. coli mar regulon (Alekshun, 1997, Antimicrob. Agents Chemother. 41:2067-2075; Alekshun, 1999, J. Bacteriol. 181:3303-3306).
• the increased expression of the AcrAB/TolC multidrug efflux system (Fralick, 1996, J Bacteriol. 178(19):5803-5; Okusu, 1996 J Bacteriol;178(l):306-8) and decreased synthesis of OmpF (Cohen, 1988, J Bacteriol; 170(12):5416-22) an outer membrane protein, play major roles.
• MarA is a member of the XylS/AraC family of transcriptional activators (Gallegos et al. 1993. Nucleic Acids Res. 21 :807). There are more than 100 proteins within the XylS/AraC family and a defining characteristic of this group of proteins is the presence of two helix-turn-helix (HTH) DNA binding motifs. Proteins within this family activate many different genes, some of which produce antibiotic and oxidative stress resistance or control microbial metabolism and virulence (Gallegos et al. supra). Summary of the Invention
• the instant invention identifies microbial transcription factors, e.g., transcription factors of the AraC-XylS family, as virulence factors in microbes and shows that inhibition of these factors reduces the virulence of microbial cells. Because these transcription factors control virulence, rather than essential cellular processes, the development of resistance is much less likely. Accordingly, in one aspect, the invention is directed to a method for preventing infection of a subject by a microbe comprising: administering a compound that modulates the expression or activity of a microbial transcription factor to a subject at risk of developing an infection such that infection of the subj ect is prevented.
• microbial transcription factors e.g., transcription factors of the AraC-XylS family
• the transcription factor is a member of the AraC- XylS family of transcription factors.
• the transcription factor is a member of the MarA family of transcription factors. In another embodiment, the method further comprises administering an antibiotic.
• the invention pertains to a method for preventing urinary tract infection of a subject by a microbe comprising: administering a compound that modulates the expression or activity of a microbial transcription factor to a subject at risk of developing a urinary tract infection such that infection of the subject is prevented.
• the invention pertains to a method for reducing virulence of a microbe comprising: administering a compound that modulates the expression or activity of a microbial transcription factor to a subject at risk of developing an infection with the microbe such that virulence of the microbe is reduced.
• the transcription factor is a member of the AraC- XylS family of transcription factors.
• the transcription factor is a member of the MarA family of transcription factors.
• the method further comprises administering an antibiotic.
• the invention in another aspect, pertains to a method for treating a microbial infection in a subject comprising: administering a compound that modulates the expression or activity of a transcription factor to a subject having a microbial infection such that infection of the subject is treated.
• the transcription factor is a member of the AraC- XylS family of transcription factors. In another embodiment, the transcription factor is a member of the MarA family of transcription factors.
• the invention further comprises administering an antibiotic.
• the invention pertains to a method for treating a urinary tract infection in a subject comprising: administering a compound that modulates the expression or activity of a transcription factor to a subject having a urinary tract infection such that infection of the subject is treated.
• the transcription factor is a member of the AraC- XylS family of transcription factors .
• the transcription factor is a member of the MarA family of transcription factors.
• the method further comprises administering an antibiotic.
• the invention pertains to a method for reducing virulence in a microbe comprising: administering a compound that inhibits the expression or activity of a transcription factor to a subject having a microbial infection such that virulence of the microbe is reduced.
• the transcription factor is a member of the AraC- XylS family of transcription factors.
• the transcription factor is a member of the MarA family of transcription factors.
• the method further comprises administering an antibiotic.
• the invention pertains to a method for evaluating the effectiveness of a compound that modulates the expression or activity of a microbial transcription factor at inhibiting microbial virulence comprising: infecting a non-human animal with a microbe, wherein the ability of the microbe to establish an infection in the non-human animal requires that the microbe colonize the animal; administering the compound that modulates the expression or activity of the microbial transcription factor to the non-human animal; and determining the level of infection of the non-human animal, wherein the ability of the compound to reduce the level of infection of the animal indicates that the compound is effective at inhibiting microbial virulence.
• the transcription factor is a member of the AraC- XylS family of transcription factors.
• the transcription factor is a member of the MarA family of transcription factors.
• the method further comprises administering an antibiotic.
• the level of infection of the non-human animal is determined by measuring the ability of the microbe to colonize the tissue of the non-human animal.
• the level of infection of the non-human animal is determined by enumerating the number of microbes present in the tissue of the non- human animal.
• the invention pertains to a method for identifying a compound for treating microbial infection, comprising: innoculating a non-human animal with a microbe, wherein the ability of the microbe to establish an infection in the non-human animal requires that the microbe colonize the animal; administering a compound which reduces the expression or activity of a microbial transcription factor to the animal, and determining the effect of the test compound on the ability of the microbe to colonize the animal, such that a compound for treating microbial infection is identified.
• the transcription factor is a member of the AraC- XylS family of transcription factors.
• the transcription factor is a member of the MarA family of transcription factors.
• the level of infection of the non-human animal is determined by measuring the ability of the microbe to colonize the tissue of the non-human animal.
• the level of infection of the non-human animal is determined by enumerating the number of microbes present in the tissue of the non- human animal.
• method for identifying a compound for reducing microbial virulence comprising: inoculating a non-human animal with a microbe, wherein the ability of the microbe to establish an infection in the non-human animal requires that the microbe colonize the animal; administering a compound which reduces the expression or activity of a microbial transcription factor to the animal, and determining the effect of the test compound on the ability of the microbe to colonize the animal, such that a compound for reducing microbial virulence is identified.
• the transcription factor is a member of the AraC- XylS family of transcription factors.
• the transcription factor is a member of the MarA family of transcription factors.
• the level of infection of the non-human animal is determined by measuring the ability of the microbe to colonize the tissue of the non-human animal.
• the level of infection of the non-human animal is determined by enumerating the number of microbes present in the tissue of the non- human animal.
• the invention pertains to a method for identifying transcription factors which promote microbial virulence comprising: creating a microbe in which a transcription factor to be tested is misexpressed; introducing the microbe into a non- human animal; wherein the ability of the microbe to establish an infection in the non- human animal requires that the microbe colonize the animal; and determining the ability of the microbe to colonize the animal, wherein a reduced ability of the microbe to colonize the animal as compared to a wild-type microbial cell identifies the transcription factor as a transcription factor which promotes microbial virulence.
• the transcription factor is a member of the AraC-
• the transcription factor is a member of the MarA family of transcription factors.
• the level of infection of the non-human animal is determined by measuring the ability of the microbe to colonize the tissue of the non-human animal.
• the level of infection of the non-human animal is determined by enumerating the number of microbes present in the tissue of the non- human animal.
• the invention pertains to a method for reducing the ability of a microbe to adhere to an abiotic surface comprising: contacting the abiotic surface or the microbe with a compound that modulates the activity of a transcription factor such that the ability of the microbe to adhere to the abiotic surface is reduced.
• the transcription factor is a member of the AraC- XylS family of transcription factors.
• the transcription factor is a member of the MarA family of transcription factors.
• the method further comprises contacting the abiotic surface or the microbe with a second agent that is effective at controlling the growth of the microbe.
• the abiotic surface is selected from the group consisting of: stents, catheters, and prosthetic devices.
• the invention pertains to a pharmaceutical composition comprising a compound that modulates the activity or expression of a microbial transcription factor and a pharmaceutically acceptable carrier, wherein the compound reduces microbial virulence.
• the invention pertains to a pharmaceutical composition comprising a compound that modulates the activity or expression of a microbial transcription factor and an antibiotic in a pharmaceutically acceptable carrier.
• the present invention represents arr advance over the prior art by identifying transcription factor modulating compounds, such as, but not limited to helix- turn-helix protein modulating compounds, and providing novel assays that can be used to identify compounds which modulate microbial transcription factors, such as MarA family polypeptides and AraC family polypeptides.
• Modulation of gene transcription brought about by the modulation of transcription factors, such as helix-turn-helix domain containing proteins, can control a wide variety of cellular processes. For example, in prokaryotic cells processes such as metabolism, resistance, and virulence can be controlled.
• the invention pertains to a method for reducing antibiotic resistance of a cell, e.g., a eukaryotic or prokaryotic cell, hi a preferred embodiment, the cell is a microbial cell. In one embodiment, the invention pertains to a method for reducing antibiotic resistance in a microbial cell, by contacting a cell with a transcription factor modulating compound, such that the antibiotic resistance of the cell is reduced, hi an embodiment, the transcription factor modulating compound is of the formula (I):
• the invention pertains to a method for modulating transcription.
• the method includes contacting a transcription factor with a transcription factor modulating compound, such that the transcription factor is modulated.
• the transcription factor modulating compound is of the formula (1): A-E (I) wherein A is a polar moiety; and E is a hydrophobic moiety, and pharmaceutically acceptable salts thereof.
• the invention also includes methods for identifying transcription factor modulating compounds.
• the method includes contacting a microbial cell with a test compound under conditions which allow interaction of the compound with the microbial cell and measuring the ability of the test compound to affect the cell.
• the microbial cell includes a selective marker under the direct control of a transcription factor responsive element and a transcription factor.
• the invention includes methods for identifying a transcription factor modulating compound.
• the method includes contacting a microbial cell comprising: 1) a selective marker under the control of a transcription factor responsive element and 2) a transcription factor, with a test compound under conditions which allow interaction of the compound with the microbial cell, and measuring the ability of the test compound to affect the growth (e.g., in vitro or in vivo) or survival of the microbial cell, wherein the inactivation of the transcription factor leads to a decrease in in vitro or in vivo cell survival.
• the invention also pertains to similar methods where the inactivation of the transcription factor leads to an increase in cell survival, as well as methods wherein the activation of the transcription factor leads to increased or, alternatively, decreased cell survival.
• the invention also pertains to methods for identifying a transcription factor modulating compound, by contacting a microbial cell comprising: 1) a chromosomal deletion in a guaB or purA gene, 2) heterologous guaB or purA gene under the control of its transcription factor responsive promoter, and 3) a transcription factor, with a test compound under conditions which allow interaction of the compound with the microbial cell.
• the method further includes the steps of measuring the ability of the compound to affect gene expression of the reporter or the growth or survival of the microbial cell as an indication of whether the compound modulates the activity of a transcription factor.
• the ability of the compound to modulate the activity of a transcription factor leads to an alteration in gene expression may effect cell growth or survival.
• the invention pertains to transcription factor modulating compounds
• HTH protein modulating compounds and MarA family modulating compounds identified by the methods of the invention, methods of using these compounds and pharmaceutical compositions comprising these compounds.
• the transcription factor modulating compounds of the invention include, but are not limited to, compounds of formulae (I)-(X) and Tables 4, 5, 6, 7 and 8.
• the invention also pertains to methods using computer modeling programs to identify transcription factor modulating compounds.
• the invention pertains to a method of identifying transcription factor modulating compounds.
• the method includes obtaining the structure of the transcription factor modulating compound, and using or identifying a scaffold which has an interaction energy score of - 20 or less with a portion of the transcription factor, thus identifying potential transcription factor modulating scaffolds.
• the invention also pertains, at least in part, to a kit for identifying a transcription factor modulating compound which modulates the activity of a
• the kit includes 1) a if 1 '"'" •'
• the invention also pertains, at least in part, to pharmaceutical compositions which contain an effective amount of a transcription factor modulating compound, and, optionally, a pharmaceutically acceptable carrier.
• the invention also pertains to a method of inhibiting a biofilm, by administering a composition comprising a transcription factor modulating compound such that the biofilm is inhibited.
• the invention pertains to a pharmaceutical composition
• a pharmaceutical composition comprising an effective amount of a transcription factor modulating compound, and a pharmaceutically acceptable carrier.
• the transcription factor modulating compound is of
• W is O or S
• X is O, S, or C, optionally linked to Q;
• a 1 is C-Z 4 , O, or S;
• a 2 is C-Z 5 , or N-Z 5 ;
• Z 1 , Z 2 , Z 3 , Z 4 and Z 5 are each independently hydrogen, alkoxy, hydroxy, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic, amino, or cyano;
• Z 3 is hydrogen, alkoxy, hydroxy, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic, amino, nitro, cyano, carbonyl, or thiocarbonyl;
• Q is an aromatic or heterocyclic moiety, and pharmaceutically acceptable salts thereof.
• the invention pertains to a pharmaceutical composition
• a pharmaceutical composition comprising an effective amount of a transcription factor modulating compound, and a pharmaceutically acceptable carrier.
• the compound is of the formula
• G is substituted or unsubstituted aromatic moiety, heterocyclic, alkyl, alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, or hydrogen;
• L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , and L 10 are each independently oxygen, substituted or unsubstituted nitrogen, sulfur and or substituted or unsubstituted carbon, and pharmaceutically acceptable salts thereof.
• the invention pertains to a pharmaceutical composition comprising an effective amount of a transcription factor modulating compound and a pharmaceutically acceptable carrier.
• the transcription factor modulating compound is of the formula (IV):
• Y 1 and Y 2 are each oxygen or sulfur
• J is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, cyano, nitro, amino, or halogen;
• V is substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, alkylamino, or alkylthio;
• P and K are each independently substituted or unsubstituted aryl, and pharmaceutically acceptable salts thereof.
• the invention pertains to a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a transcription factor modulating compound.
• the transcription factor modulating compound is of the formula (V): wherein
• T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently substituted or unsubstituted carbon, oxygen, substituted or unsubstituted nitrogen, or sulfur;
• M is hydrogen, alkyl, alkenyl, heterocyclic, alkynyl, or aryl, or pharmaceutically acceptable salts thereof.
• the invention pertains to a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a transcription factor modulating compound.
• the transcription factor modulating compound is of the formula
• R 1 is OH, OCOCO H, or a substituted or unsubstituted straight or branched C 1 -C 5 alkyloxy group
• R 2 is H, CO 2 (C C 5 substituted or unsubstituted, straight or branched alkyl), or a substituted or unsubstituted aryl group;
• R 4 , R 5 , R 6 , and R 7 are independently selected from the group consisting of H, (CrC 5 substituted or unsubstituted, straight or branched alkyl), CO 2 (C 1 -C 5 substituted or unsubstituted, straight or branched alkyl), CO(C C 5 substituted or unsubstituted, straight or branched alkyl), CO(substituted or unsubstituted aryl or heteroaryl), CO(C 3 -C 6 substituted or unsubstituted cycloalkyl), O(C ⁇ -C5 substituted or unsubstituted, straight or branched alkyl), C ⁇ OHX -Cs substituted or unsubstituted, straight or branched alkyl), substituted or unsubstituted amino, CO 2 H, CN, NO 2 , CONH 2 , (CO)(NHOH), and halogen.
• R 4 , R 5 , R 6 , and R 7 are all H and R 2 is unsubstituted phenyl, then R 1 is not OCH 2 CO 2 CH 2 CH 3 ; In certain aspects of fonnula Va, R 4 , R 5 , and R 7 are all H.
• R 1 of formula Va may be selected from the group consisting of OH, O(CR'R") ⁇ _ 3 H, 0(CR'R")i-3OH, O(CR'R") ⁇ - 3 CO 2 H, O(CR'R") 1 _ 3 CO 2 (CR'R") 1 _ 3 H, O(CR'R") 1 _ 3 (CO)NH 2 , O(CR'R") 1 _ 3 (CNH)NH 2 , OCOCO 2 H, O(CR'R") 1 _ 3 SO 3 H, O(CR'R") 1--3 OSO 3 H, O(CR'R") ⁇ _ 3 PO 3 H, O(CR'R") 1 _ 3 OPO 3 H, O(CR'R") 1 _ 3 N[(CR'R") 0 _ 3 H] 2 , O(CR'R") 1 _ 3 (CO)(NHOH), and O(CR'R' 3 (heteroaryl); wherein R' and R" are each independently H, a Cr-C alkyl, C
• the heteroaryl group may be a pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• R 2 of formula Va may be a substituted or unsubstituted phenyl, pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• R 6 of formula Va is H, (CR'R") ⁇ _ 3 H, (CR'R") ! _ 3 OH, (CR'R") ⁇ _ 3 NH 2 , (NOH)(CR'R") ⁇ - 3 H, CO(CR'R")o- 3 NH 2 , CO(CR'R") 1 _ 3 H, CO(CR'R") 1--3 OH, CO(CR'R")o_ 3 CF 3 , (CR'R") 0 _ 3 N[(CR'R")o_ 3 H] 2 , CO(substituted or unsubstituted heteroaryl), CO(C 3 -C 6 substituted or unsubstituted cycloalkyl), CO(substituted or unsubstituted phenyl), CO 2 (CR'R")o_ 3 H, CN, NO 2 , F, CI, Br, or I, wherein R' and R" are each independently H, a C 1 -C 3 alkyl,
• R 6 of formula Va is CO(substituted or unsubstituted heteroaryl), wherein said heteroaryl group is a pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• the invention pertains to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a transcription factor modulating compound.
• the transcription factor modulating compound may be of the formula (VI):
• G 1 , G 2 , and G 3 are each independently O, S, substituted or unsubstituted nitrogen, or substituted or unsubstituted carbon;
• E 1 , E 2 , and E 3 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, or acyl;
• E 4 is alkyl, alkenyl, alkynyl, aryl, halogen, cyano, amino, nitro, or acyl, and pharmaceutically acceptable salts thereof.
• Figure 1 is a multiple sequence alignment of AraC-XylS family polypeptides.
• Figure 2 is a multiple sequence alignment of PROSITE PS00041 and AraC family polypeptides.
• Figure 3 is a multiple sequence alignment of PROSITE PS01124 and AraC family polypeptides.
• Figure 4 is a CoMFA contour map for a representative triazinoxazepine.
• the instant invention identifies microbial transcription factors, e.g., transcription factors of the AraC-XylS family, as virulence factors in microbes and shows that inhibition of these factors reduces the virulence of microbial cells. Because these transcription factors control virulence, rather than essential cellular processes, modulation of these factors should not promote resistance.
• microbial transcription factors e.g., transcription factors of the AraC-XylS family
• HTH helix-turn-helix transcription factors
• AraC helix-turn-helix transcription factors
• MarA MarA
• Rob SoxS
• LysR winged helix transcription factors
• OmpR Huffman, J. L., and R. G. Brennan 2002. Curr Opin Struct Biol 12:98-106, Martinez-Hackert, E., and A. M. Stock 1997. Structure.
• the AraC-XylS family of transcription factors comprises many members. MarA, SoxS, Rma, and Rob are examples of proteins within the AraC-XylS family of transcription factors. These factors belong to a subset of the AraC-XylS family that have historically been considered to play roles in promoting resistance to multiple antibiotics and have not been considered to be virulence factors. In fact, the role of marA in virulence has been tested using a marA null mutant of Salmonella enterica serovar Typhimurium (S. typhimurium) in a mouse infection model ( Sulavik et al. 1997. J. Bacteriology 179:1857) and no such role has been found.
• this invention is based, at least in part, on the finding that the ability of microbes to cause infection in a host can be inhibited by inhibiting the expression and/or activity of microbial transcription factors.
• the instant invention validates the use of microbial transcription factors as therapeutic targets.
• the invention pertains, at least in part, to compounds which modulate transcription factors (e.g., helix-turn-helix (HTH) proteins, AraC family polypeptides, MarA family polypeptides, etc.), methods of identifying the transcription factor modulating compounds (e.g., HTH protein modulating compounds, AraC family polypeptide modulating compounds, MarA family polypeptide modulating compounds, etc.), and methods of using the compounds.
• transcription factors e.g., helix-turn-helix (HTH) proteins, AraC family polypeptides, MarA family polypeptides, etc.
• methods of identifying the transcription factor modulating compounds e.g., HTH protein modulating compounds, AraC family polypeptide modulating compounds, MarA family polypeptide modulating compounds, etc.
• transcription factor includes proteins that are involved in gene regulation in both prokaryotic and eukaryotic organisms.
• transcription factors can have a positive effect on gene expression and, thus, may be referred to as an "activator” or a “transcriptional activation factor.”
• a transcription factor can negatively effect gene expression and, thus, may be referred to as “repressors” or a “transcription repression factor.”
• Activators and repressors are generally used terms and their functions are discerned by those skilled in the art.
• the term "infectivity” or "virulence” includes the ability of a pathogenic microbe to colonize a host, a first step required in order to establish growth in a host. Infectivity or virulence is required for a microbe to be a pathogen. In addition, a virulent microbe is one which can cause a severe infection. As used herein, the term “pathogen” includes both obligate and opportunistic organisms. The ability of a microbe to resist antibiotics is also important in promoting growth in a host, however, in one embodiment, antibiotic resistance is not included in the terms "infectivity” or "virulence” as used herein.
• the instant invention pertains to methods of reducing the infectivity or virulence of a microbe without affecting (e.g., increasing or decreasing) antibiotic resistance.
• the term "infectivity or virulence” includes the ability of an organism to establish itself in a host by evading the host's barriers and immunologic defenses.
• AraC family polypeptide include an art recognized group of prokaryotic transcription factors which contains more than 100 different proteins (Gallegos et al, (1997) Micro. Mol. Biol. Rev. 61: 393; Martin and Rosner, (2001) Curr. Opin. Microbiol. 4:132).
• AraC family polypeptides include proteins defined in the PROSITE (PS) database (http://www.expasy.ch/prosite as profile PS01124.
• the AraC family polypeptides also include polypeptides described in PS0041, HTH AraC Family 1, and PS01124, and HTH AraC Family 2.
• AraC-XylS family polypeptides Multiple sequence alignments for the AraC-XylS family polypeptides, HTH AraC family 1, and HTH AraC family 2 are shown in Figures 1-3, respectively.
• the AraC family polypeptides are generally comprised of, at the level of primary sequence, by a conserved stretch of about 100 amino acids, which are believed to be responsible for the DNA binding activity of this protein (Gallegos et al, (1997) Micro. Mol. Biol. Rev. 61: 393; Martin and Rosner, (2001) Curr. Opin. Microbiol. 4: 132).
• AraC family polypeptides also may include two helix turn helix DNA binding motifs (Martin and Rosner, (2001) Curr. Opin. Microbiol.
• the invention pertains to a method for modulating an AraC family polypeptide, by contacting the AraC family polypeptide with a test compound which interacts with a portion of the polypeptide involved in DNA binding.
• the test compound interacts with a conserved aminoacid residue (capitalized) of the HTH AraC family 1 protein indicated in Figure 2.
• the term "helix-turn-helix protein,” “HTH protein,” “helix-turn-helix polypeptides,” and “HTH polypeptides,” includes proteins comprising one or more helix-turn-helix domains. Helix-turn-helix domains are known in the art and have been implicated in DNA binding (Ann Rev. of Biochem. 1984. 53:293). An example of the consensus sequence for a helix-turn domain can be found in Brunelle and Schleif (1989. J. Mol. Biol. 209:607). The domain has been illustrated by the sequence
• the helix-turn-helix domain was the first DNA-binding protein motif to be recognized. Although originally the HTH domain was identified in bacterial proteins, the HTH domain has since been found in hundreds of DNA-binding proteins from both eukaryotes and prokaryotes. It is constructed from two alpha helices connected by a short extended chain of amino acids, which constitutes the "turn.”
• a helix-turn-helix domain containing protein is a Mar A family polypeptide.
• the language "MarA family polypeptide" includes the many naturally occurring HTH proteins, such as transcription regulation proteins which have sequence similarities to MarA and which contain the MarA family signature pattern, which can also be referred to as an XylS/AraC signature pattern.
• An exemplary signature pattern which defines MarA family polypeptides is shown, e.g., on PROSITE and is represented by the sequence: [KRQ]-[LTVMA]-X(2)-[GSTALIV]- ⁇ FYWPGDN ⁇ X(2)-[LIVMSA]-X(4,9)-[L ⁇ VMF]-X(2)-[LIVMSTA]-X(2)-[GSTACIL]- X(3)-[GANQRF]-[LIVMFY]-X(4,5)-[LFY]-X(3)-[FYIVA]- ⁇ FYWHCM ⁇ -X(3)- [GSADENQKR]-X-[NSTAPKL]-[PARL], where X is any amino acid.
• MarA family polypeptides have two "helix-turn-helix” domains. This signature pattern was derived from the region that follows the first, most amino terminal, helix-turn-helix domain (HTH1) and includes the totality of the second, most carboxy terminal helix-turn-helix domain (HTH2). (See PROSITE PS00041).
• the MarA family of proteins (“MarA family polypeptides") represent one subset of AraC-XylS family polypeptides and include proteins like MarA, SoxS, Rob, Rma, AarP, PqrA, etc.
• MarA family polypeptides generally, are involved in regulating resistance to antibiotics, organic solvents, and oxidative stress agents (Alekshun and Levy, ( 991) Antimicrob. Agents. Chemother. 41: 2067).
• MarA-like proteins also generally contain two HTH motifs as exemplified by the MarA and Rob crystal structures (Kwon et al, (2000) Nat. Struct. Biol. 7: 424; Rhee et al, (1998) Proc. Natl Acad. Sci. U.S.A. 95: 10413).
• MarA family polypeptide or portion thereof comprises the first MarA family HTH domain (HTH1) (Brunelle, 1989, JMol Biol; 209(4):607-22).
• a MarA polypeptide comprises the second MarA family HTH domain (HTH2) (Caswell, 1992, Biochem J; 287:493-509.).
• HTH1 MarA family HTH domain
• HTH2 MarA family HTH domain
• a MarA polypeptide comprises both the first and second MarA family HTH domains.
• MarA family polypeptide sequences are "structurally related" to one or more known MarA family members, preferably to MarA. This relatedness can be shown by sequence or structural similarity between two MarA family polypeptide sequences or between two MarA family nucleotide sequences that specify such polypeptides. Sequence similarity can be shown, e.g., by optimally aligning MarA family member sequences using an alignment program for purposes of comparison and comparing corresponding positions. To determine the degree of similarity between sequences, they will be aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one protein for nucleic acid molecule for optimal alignment with the other protein or nucleic acid molecules). The amino acid residues or bases and corresponding amino acid positions or bases are then compared.
• amino acid residues are not identical, they may be similar.
• an amino acid residue is "similar" to another amino acid residue if the two amino acid residues are members of the same family of residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art (see, for example, Altschul et al. 1990. J. Mol. Biol.
• 215:403 including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).
• basic side chains e.g., lysine, arginine, histidine
• acidic side chains e.g., aspartic acid, glutamic acid
• uncharged polar side chains e.
• Alignment strategies are well known in the art; see, for example, Altschul et al. supra for optimal sequence alignment.
• MarA family polypeptides may share some amino acid sequence similarity with MarA.
• the nucleic acid and amino acid sequences of MarA as well as other MarA family polypeptides are available in the art.
• the nucleic acid and amino acid sequence of MarA can be found, e.g., on GeneBank (accession number M96235 or in Cohen et al. 1993. J. Bacteriol. 175:1484, or in SEQ ID NO:l and SEQ ID NO:2.
• the nucleic acid and/or amino acid sequences of MarA can be used as
• “query sequences” to perform a search against databases (e.g., either public or private) to, for example, identify other MarA family members having related sequences. Such searches can be performed, e.g., using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
• Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
• the default parameters of the respective programs e.g., XBLAST and NBLAST
• XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
• MarA family members can also be identified as being similar based on their ability to specifically hybridize to nucleic acid sequences specifying MarA.
• stringent conditions are known to those skilled in the art and can be found e.g., in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
• a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 °C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
• SSC sodium chloride/sodium citrate
• Tm melting temperature
• Tm is the temperature in °C at which half the molecules of a given sequence are melted or single-stranded.
• the Tm can be estimated in degrees C as 2(number of A+T residues) + 4(number of C+G residues).
• Hybridization or annealing of nucleic acid molecules should be conducted at a temperature lower than the Tm, e.g., 15 °C, 20°C, 25°C or 30°C lower than the Tm.
• the nucleic acid sequence of a MarA family member identified in this way is at least about 10%, 20%, more preferably at least about 30%, more preferably at least about 40% identical and preferably at least about 50%, or 60% identical to a MarA nucleotide sequence.
• the nucleic acid sequence of a MarA family member is at least about 70%, 80%, preferably at least about 90%, more preferably at least about 95% identical with a MarA nucleotide sequence.
• MarA family members have an amino acid sequence at least about 20%, preferably at least about 30%, more preferably at least about 40% identical and preferably at least about 50%, or 60% or more identical with a MarA amino acid sequence.
• the nucleic acid sequence of a MarA family member is at least about 70%, 80%, more preferably at least about 90%, or more preferably at least about 95% identical with a MarA nucleotide sequence.
• the level of sequence similarity among microbial regulators of gene transcription, even though members of the same family is not necessarily high. This is particularly true in the case of divergent genomes where the level of sequence identity may be low, e.g., less than 20% (e.g., B. burgdorferi as compared e.g., to B. subtilis). Accordingly, structural similarity among MarA family members can also be determined based on "three-dimensional correspondence" of amino acid residues.
• three-dimensional correspondence is meant to includes residues which spatially correspond, e.g., are in the same position of a MarA family polypeptide member as determined, e.g., by x-ray crystallography, but which may not conespond when aligned using a linear alignment program.
• the language “three- dimensional correspondence” also includes residues which perform the same function, e.g., bind to DNA or bind the same cofactor, as determined, e.g., by mutational analysis.
• MarA family polypeptides are shown in Table 1, Figures 1-3, and at Prosite (PS00041) and include: AarP, Ada, AdaA, AdiY, AfrR, AggR, AppY, AraC, CafR, CelD, CfaD, CsvR, D90812, EnvY, ExsA, FapR, HrpB, InF, InvF, LcrF, LumQ, MarA, MelR, MixE, MmsR, MsmR, OrfR, Orf_f375, PchR, PerA, PocR, PqrA, RafR, RamA, RhaR, RhaS, Rns, Rob, SoxS, S52856, TefD, TcpN, ThcR, TmbS, U73857, U34257, U21191, UreR, VirF, XylR, XylS, Xysl, 2, 3, 4, Ya52, Y
• D90812 CafR (29) U21191 (40) FapR (10, 11) LcrF (30) or VirF (30) AraL (41) MelR (12) ORF /375 (13, 14) Providencia stuartii Streptococcus mutans RhaR (15, 16, 17) AarP (31) MsmR (42) RhaS (18) Rob (19) Pseudomonas spp.
• Pediococcus pentosaceus U73857 (20) MmsR (32) RafR (43) XylR (21) TmbS (33) YijO (22) XylS (34) Photobacterium leiognathi Xysl,2,3,4 (35, 36) LumQ (44) Proteus vulgaris PqrA (23) Cyanobacteria Bacillus subtilis Synechocystis spp. AdaA (45)
• Salmonella typhimurium LumQ (37) YbbB (46)
• PocR (26) YzbC (49) a The smaller MarA homologs, ranging in size from 87 (U34257) to 138 (OrfR) amino acid residues, are represented in boldface. References are given in parentheses and are listed below.
• transcription factor modulating compound or transcription factor modulator
• transcription factor modulating compounds include compounds which interact with one or more transcription factors, such that the activity of the transcription factor is modulated, e.g., enhanced or inhibited.
• the term also includes both AraC family modulating compounds and MarA family modulating compounds.
• the transcription factor modulating compound is an inhibiting compound of a transcription factor, e.g., a prokaryotic transcription factor or a eukaryotic transcription activation factor.
• the transcription factor modulating compounds modulate the activity of a transcription factor as measured by assays known in the art or LANCE assays such as those described in Example 8.
• the transcription factor modulating compound inhibits a particular transcription factor by about 10%) or greater, about 40% or greater, about 50% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater, about 95% or greater, or about 100% as compared to the activity of the transcription factor with out the transcription factor modulating compound, hi another embodiment, the transcription factor modulating compound inhibits biofilm formation. In one embodiment, the transcription factor modulating compound inhibits biofilm formation as measured by assays known in the art or the Crystal Violet assay described in Example 7.
• the transcription factor of the invention inhibits the formation of a biofilm by about 25% or more, 50% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more, 99.99% or more, or by 100%, as compared to the formation of a biofilm without the transcription factor modulating compound.
• HTH protein modulating compound or “HTH protein modulator” includes compounds which interact with one or more HTH proteins such that the activity of the HTH protein is modulated, e.g., enhanced or, inhibited, hi one embodiment, the HTH protein modulating compound is a MarA family polypeptide modulating compound.
• the activity of the HTH protein is enhanced when it interacts with the HTH protein modulating compound.
• the activity of the HTH protein may be increased by greater than 10%, greater the 20%, greater than 50%, greater than 75%, greater than 80%, greater than 90%, or 100% of the activity of the HTH protein in the absence of the HTH modulating compound.
• the activity of the HTH protein is decreased upon an interaction with the HTH protein modulating compound.
• the activity of the HTH protein is decreased by about 25% or more, 50% or more, 75% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more, 99.99% or more, or by 100%, as compared to the activity of the protein of a HTH protein when not contacted with an HTH modulating compound of the invention using techniques and assays described herein. Values and ranges included and/or intermediate of the values set forth herein are also intended to be within the scope of the present invention.
• MarA family polypeptide modulating compound or “MarA family modulating compound” include compounds which interact with one or more MarA family polypeptides such that the activity of the MarA family peptide is enhanced or inhibited.
• the MarA family polypeptide modulating compound is an inhibiting compound.
• the MarA family inhibiting compound is an inhibitor of MarA, Rob, and/or SoxS.
• the MarA family polypeptide modulating compound modulates the expression of luciferase in the Luciferase Assay described in Example 9.
• the MarA family polypeptide modulating compound decreases luciferase expression by greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90% or about 100%.
• polypeptide(s) refers to a peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
• Polypeptide(s) includes both short chains, commonly referred to as peptides, oligopeptides and oligomers and longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
• Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
• Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
• Modifications include, for example, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma- carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, se
• Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
• winged helix includes dimeric transcription factors in which each monomer comprises a helix-turn-helix motif followed by one or two ⁇ -hairpin wings (Brennan. 1993. Cell. 74:773; Gajiwala and Burley. 2000. Curr. Opin. Struct. Biol. 10:110).
• the classic winged helix motif comprises two wings, three ⁇ helices, and three ⁇ strands in the sequence H1-B1-H2-T-H3-B2-W1-B3-W2 (where H is a helix, B is a ⁇ strand, T is a turn, and W is a wing), although some variation in structure has been demonstrated ( Huffman and Brennan. 2002. Current Opinion in Structural Biology. 12:98).
• looped-hinge helix included transcription factors, such as AbrB which, in the absence of DNA, have revealed a dimeric N-terminal region consisting of a four-stranded ⁇ sheet and a C-terminal DNA-binding region comprising one ⁇ helix and a "looped hinge” (see, e.g., Huffman and Brennan. 2002 Current Opinion in Structural Biology 12:98). Residues corresponding to R23 and R24 of AbrB are critical for DNA recognition and contribute to the electropositive nature of the DNA-binding region.
• Prefened polypeptides are “naturally occurring.”
• a "naturally-occurring" molecule refers to a molecule having an amino acid or a nucleotide sequence that occurs in nature (e.g. , a natural polypeptide).
• naturally or non-naturally occurring variants of the polypeptides and nucleic acid molecules which retain the same functional activity (such as, the ability to bind to target nucleic acid molecules (e.g., comprising a marbox) or to polypeptides (e.g. RNA polymerase) with a naturally occurring polypeptide are provided for.
• Such immunologic cross-reactivity can be demonstrated, e.g. , by the ability of a variant to bind to a MarA family polypeptide responsive element.
• Such variants can be made, e.g., by mutation using techniques that are known in the art. Alternatively, variants can be chemically synthesized.
• variant(s) includes nucleic acid molecules or polypeptides that differ in sequence from a reference nucleic acid molecule or polypeptide, but retain its essential properties. Changes in the nucleotide sequence of the variant may, or may not, alter the amino acid sequence of a polypeptide encoded by the reference nucleic acid molecule. Nucleotide or amino acid changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by a naturally occurring reference sequence. A typical variant of a polypeptide differs in amino acid sequence from a reference polypeptide.
• a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and/or deletions in any combination.
• a variant of a nucleic acid molecule or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally.
• Non-naturally occurring variants of nucleic acid molecules and polypeptides may be made from a reference nucleic acid molecule or polypeptide by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
• variants can be chemically synthesized.
• artificial or mutant forms of autologous polypeptides which are functionally equivalent, can be made using techniques which are well known in the art.
• Mutations can include, e.g., at least one discrete point mutation which can give rise to a substitution, or by at least one deletion or insertion.
• mutations can also be made by random mutagenesis or using cassette mutagenesis.
• the entire coding region of a molecule is mutagenized by one of several methods (chemical, PCR, doped oligonucleotide synthesis) and that collection of randomly mutated molecules is subjected to selection or screening procedures.
• discrete regions of a polypeptide, corresponding either to defined structural or functional determinants are subjected to saturating or semi-random mutagenesis and these mutagenized cassettes are re-introduced into the context of the otherwise wild type allele.
• PCR mutagenesis can be used.
• Megaprimer PCR can be used (O.H. Landt, 1990. Gene 96:125-128).
• a MarA family polypeptide excludes one or more of XylS, AraC, and MelR. In other preferred embodiments, a MarA family polypeptide is involved in antibiotic resistance. In particularly preferred embodiments, a MarA family polypeptide is selected from the group consisting of: MarA, RamA, AarP, Rob, SoxS, and PqrA.
• the language "activity of a transcription factor” includes the ability of a transcription factor to interact with DNA, e.g., to bind to a transcription factor responsive promoter, or to initiate transcription from such a promoter.
• the language expressly includes the activities of AraC family polypeptides, HTH proteins and MarA family polypeptides.
• the language "activity of a MarA family polypeptide” includes the ability of the MarA family polypeptide to interact with DNA, e.g., to bind to a MarA family polypeptide responsive promoter, or to initiate transcription from such a promoter.
• MarA functions both as a transcriptional activator (e.g., upregulating genes such as inaA, galT, micF, etc.) and as a repressor (e.g., downregulating genes such as fecA, purA, guaB, etc.) (Alekshun, 1997 , Antimicrob. Agents Chemother. 41:2067-2075; Barbosa & Levy, J. Bact. 2000, Vol. 182, p. 3467-3474; Pomposiello et al. J. Bad. 2001, Vol 183, p. 3890-3902).
• transcription factor responsive element includes a nucleic acid sequence which can interact with a transcription factor (e.g., promoters or enhancers or operators) which are involved in initiating transcription of an operon in a microbe.
• a transcription factor e.g., promoters or enhancers or operators
• Transcription factor responsive elements responsive to various transcription factors are known in the art and additional responsive elements can be identified by one of ordinary skill in the art. For example, microarray analysis can be used to identify genes that are regulated by a transcription factor of interest. For interest, genes regulated by a transcription factor would be expressed at higher levels in wild type cells than in cells which are deleted for the transcription factor.
• genes responsive to a given transcription factor would comprise one or more target sequences responsive to the transcription factor in their promoter regions (Lyons et al. 2000.
• Exemplary responsive elements include: araBAD, araE, araFGH (responsive to AraC); melBAD (responsive to MelR); rhaSR (responsive to RhaR); rahBAD, rhaT (responsive to RhaS); Pm (responsive to XylS); fumC, inaA, micF, nfo, pai5, sodA, tolC, acrAB, fldA, fpr, mar, poxB, ribA, and zwf (responsive to MarA, SoxS, Rob); and coo, rns (responsive to Rns).
• marA family polypeptide responsive element includes a nucleic acid sequence which can interact with marA, e.g., promoters or enhancers which are involved in regulating transcription of a nucleic acid sequence in a microbe.
• MarA responsive elements comprise approximately 16 base pair marbox sequence, the sequence critical for the binding of MarA to its target, hi addition, a secondary site, the accessory marbox, upstream of the primary marbox contributes to basal and derepressed mar transcription.
• a marbox may be situated in either the forward or backward orientation. (Martin, 1999, Mol. Microbiol. 34:431-441). In the marRAB operon, the marbox is in the backward orientation and is thus located on the sense strand with respect to marRAB (Martin, 1999, Mol.
• MarA family responsive elements are promoters that are structurally or functionally related to a marA promoter, e.g., interact with MarA or a protein related to MarA.
• the marA family polypeptide responsive element is a marRAB promoter.
• marA family polypeptide responsive promoters as defined herein, e.g., the 405 -bp Thai fragment from the marO region is a marA family responsive promoter (Cohen et al. 1993. J Bact. 175:7856). h addition, MarA has been shown to bind to a 16 bp MarA binding site (referred to as the "marbox" within marO (Martin et al. 1996. J. Bacteriol. 178:2216).
• MarA also affects transcription from the acrAB; micF; mlr 1,2,3; sip; nfo; inaA; fpr; sodA; soi-17,19; zwfifumC; or rpsF promoters (Alekshun and Levy. 1997. Antimicrobial Agents and Chemother. 41:2067).
• marA family responsive promoters include: araBAD, araE, araFGH and araC, which are activated by AraC; Pm, which is activated by XylS; melAB which is activated by MelR; and oriC which is bound by Rob.
• MarA family polypeptide responsive promoter also includes portions of the above promoters which are sufficient to activate transcription upon interaction with a MarA family member protein.
• the portions of any of the MarA family polypeptide-responsive promoters which are minimally required for their activity can be easily determined by one of ordinary skill in the art, e.g., using mutagenesis. Exemplary techniques are described by Gallegos et al. (1996, J. Bacteriol. 178:6427).
• a “MarA family polypeptide responsive promoter” also includes non-naturally occurring variants of MarA family polypeptide responsive promoters which have the same function as naturally occurring MarA family promoters.
• such variants have at least 30%) or greater, 40% or greater, or 50% or greater, nucleotide sequence identity with a naturally occurring MarA family polypeptide responsive promoter.
• such variants have at least about 70% nucleotide sequence identity with a naturally occurring MarA family polypeptide responsive promoter.
• such variants have at least about 80% nucleotide sequence identity with a naturally occurring MarA family polypeptide responsive promoter.
• such variants have at least about 90% nucleotide sequence identity and preferably at least about 95% nucleotide sequence identity with a naturally occurring MarA family polypeptide responsive promoter.
• nucleic acid molecules encoding variants of MarA family polypeptide responsive promoters are capable of hybridizing under stringent conditions to nucleic acid molecules encoding naturally occurring MarA family polypeptide responsive promoters.
• the methods described herein can employ molecules identified as responding to the transcription factors of the invention, i.e., molecules in a regulon whose expression is controlled by the transcription factor.
• molecules identified as responding to the transcription factors of the invention i.e., molecules in a regulon whose expression is controlled by the transcription factor.
• compounds that modulate transcription of genes that are directly modulated by a microbial transcription factor e.g., a marA family transcription factor
• such genes can be identified as important in controlling virulence using the methods described herein.
• the term “regulon” includes two or more loci in two or more different operons whose expression is regulated by a common repressor or activator protein.
• the term “interact” includes close contact between molecules that results in a measurable effect, e.g., the binding of one molecule with another.
• a MarA family polypeptide can interact with a MarA family polypeptide responsive element and alter the level of transcription of DNA.
• compounds can interact with a MarA family polypeptide and alter the activity of a MarA family polypeptide.
• inducible promoter includes promoters that are activated to induce the synthesis of the genes they control.
• the term “constitutive promoter” includes promoters that do not require the presence of an inducer, e.g., are continuously active.
• the terms “heterologous DNA” or “heterologous nucleic acid” includes
• heterologous DNA DNA that does not occur naturally in the cell (e.g., as part of the genome) in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature or which is operatively linked to DNA to which it is not normally linked in nature (i.e., a gene that has been operatively linked to a heterologous promoter).
• Heterologous DNA is 1) not naturally occurring in a particular position (e.g. , at a particular position in the genome) or 2) is not endogenous to the cell into which it is introduced, but has been obtained from another cell.
• Heterologous DNA can be from the same species or from a different species. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which is expressed is herein encompassed by the term heterologous DNA.
• heterologous protein refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. That is, the polypeptide is expressed from a heterologous nucleic acid molecule.
• microbe includes microorganisms expressing or made to express a transcription factor, araC family polypeptide, HTH protein, or a marA family polypeptide.
• "Microbes” are of some economic importance, e.g., are environmentally important or are important as human pathogens. For example, in one embodiment microbes cause environmental problems, e.g., fouling or spoilage, or perform useful functions such as breakdown of plant matter, hi another embodiment, microbes are organisms that live in or on mammals and are medically important.
• microbes are unicellular and include bacteria, fungi, or protozoa
• microbes suitable for use in the invention are multicellular, e.g., parasites or fungi, h preferred embodiments, microbes are pathogenic for humans, animals, or plants.
• Microbes maybe used as intact cells or as sources of materials for cell-free assays.
• the microbes include prokaryotic organisms, i other embodiments, the microbes include eukaryotic organisms.
• Exemplary bacteria that comprise MarA homologs include the following:
• Yersinia enterocolitica Yersinia pestis Pseudomonas aeruginosa Enterobacter spp. Klebsiella sp. Proteus spp. Vibrio cholerae Shigella sp. Providencia stuartii Neisseria meningitidis Mycobacterium tuberculosis Mycobacterium leprae Staphylococcus aureus Streptococcus pyogenes Enterococcus faecalis Bordetella pertussis Bordetella bronchiseptica
• selective marker includes polypeptides that serve as indicators, e.g., provide a selectable or screenable trait when expressed by a cell.
• selectable marker includes both selectable markers and counterselectable markers.
• selectable marker includes markers that result in a growth advantage when a compound or molecule that fulfills the test parameter of the assay is present.
• counterselectable marker includes markers that result in a growth disadvantage unless a compound or molecule is present which disrupts a condition giving rise to expression of the counterselectable marker.
• exemplary selective markers include cytotoxic gene products, gene products that confer antibiotic resistance, gene products that are essential for growth, gene products that confer a selective growth disadvantage when expressed in the presence of a particular metabolic substrate (e.g., the expression of the URA3 gene confers a growth disadvantage in the presence of 5- fluoroorotic acid).
• reporter gene includes any gene which encodes an easily detectable product which is operably linked to a regulatory sequence, e.g., to a transcription factor responsive promoter.
• operably linked it is meant that under appropriate conditions an RNA polymerase may bind to the promoter of the regulatory region and proceed to transcribe the nucleotide sequence such that the reporter gene is transcribed.
• a reporter gene consists of the transcription factor responsive promoter linked in frame to the reporter gene. In certain embodiments, however, it maybe desirable to include other sequences, e.g, transcriptional regulatory sequences, in the reporter gene construct.
• modulation of the activity of the promoter may be effected by altering the RNA polymerase binding to the promoter region, or, alternatively, by interfering with initiation of transcription or elongation of the mRNA.
• sequences which are herein collectively referred to as transcriptional regulatory elements or sequences may also be included in the reporter gene construct, hi addition, the construct may include sequences of nucleotides that alter translation of the resulting mRNA, thereby altering the amount of reporter gene product.
• reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1 : 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); PhoA, alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem.
• CAT chloramphenicol acetyl transferase
• isolated or recombinant includes nucleic acid molecules which have been, e.g., (1) amplified in vitro by, for example, polymerase chain reaction (PCR); (2) recombinantly produced by cloning, or (3) purified, as by cleavage and gel separation; or (4) synthesized by, for example, chemical synthesis.
• PCR polymerase chain reaction
• Such a nucleic acid molecule is isolated from the sequences which naturally flank it in the genome and from cellular components.
• a substantially purified or recombinant transcription factor can be obtained from cells which have been engineered to express an isolated or recombinant nucleic acid molecule which encodes a transcription factor.
• a bacterial cell can be transformed with a plasmid which encodes a transcription factor.
• the transcription factor can then be purified from the bacterial cells and used, for example, in the cell-free assays described herein or known in the art.
• antibiotic includes antimicrobial agents isolated from natural sources or chemically synthesized.
• antibiotic refers to antimicrobial agents for use in human therapy.
• Preferred antibiotics include: tetracycline, fluoroquinolones, chloramphenicol, penicillins, cephalosporins, puromycin, nalidixic acid, and rifampin.
• test compound includes any reagent or test agent which is employed in the assays of the invention and assayed for its ability to influence the activity of a transcription factor, e.g., an AraC family polypeptide, an HTH protein, or a MarA family polypeptide, e.g., by binding to the polypeptide or to a molecule with which it interacts. More than one compound, e.g., a plurality of compounds, can be tested at the same time for their ability to modulate the activity of a transcription factor, e.g., an AraC family polypeptide, an HTH protein, or a MarA family polypeptide, activity in a screening assay.
• the test compound is a MarA family modulating compound.
• Test compounds that can be tested in the subject assays include antibiotic and non-antibiotic compounds.
• test compounds include candidate detergent or disinfectant compounds.
• Exemplary test compounds which can be screened for activity include, but are not limited to, peptides, non-peptidic compounds, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides), and natural product extract libraries.
• non-peptidic test compound includes compounds that are comprised, at least in part, of molecular structures different from naturally-occurring L- amino acid residues linked by natural peptide bonds.
• non-peptidic test compounds also include compounds composed, in whole or in part, of peptidomimetic structures, such as D-amino acids, non-naturally-occurring L-amino acids, modified peptide backbones and the like, as well as compounds that are composed, in whole or in part, of molecular structures unrelated to naturally-occurring L-amino acid residues linked by natural peptide bonds.
• Non-peptidic test compounds also are intended to include natural products.
• small molecules can be used as test compounds.
• the term "small molecule” is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight, hi one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. 1998. Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds, hi a further embodiment, a small molecule is not biosynthetic.
• the term "antagonist” includes transcription factor modulating compounds (e.g., AraC family polypeptide modulating compounds, HTH protein modulating compounds, MarA family polypeptide modulating compounds, etc.) which inhibit the activity of a transcription factor by binding to and inactivating the transcription factor (e.g., an AraC family modulating compound, an MarA family polypeptide modulating compound, etc.), by binding to a nucleic acid target with which the transcription factor interacts (e.g., for MarA, a marbox), by disrupting a signal transduction pathway responsible for activation of a particular regulon (e.g.
• transcription factor modulating compounds e.g., AraC family polypeptide modulating compounds, HTH protein modulating compounds, MarA family polypeptide modulating compounds, etc.
• a nucleic acid target with which the transcription factor interacts e.g., for MarA, a marbox
• a signal transduction pathway responsible for activation of a particular regulon e.g.
• Antagonists may include, for example, naturally or chemically synthesized compounds such as small cell permeable organic molecules, nucleic acid interchelators, peptides, etc.
• agonist includes transcription factor modulating compounds (e.g., AraC family polypeptide modulating compounds, HTH protein modulating compounds, MarA family polypeptide modulating compounds, etc.) which promote the activity of a transcription factor by binding to and activating the transcription factor (e.g., an AraC family modulating compound, an MarA family polypeptide modulating compound, etc.), by binding to a nucleic acid target with which the transcription factor interacts (e.g., for MarA, a marbox), by facilitating a signal transduction pathway responsible for activation of a particular regulon (e.g., for Mar, the inactivation of MarR or activation of MarA synthesis), and/or by facilitating a critical protein-protein interaction (e.g., MarA-RNA polymerase interactions that are required for MarA to function as a transcription factor.)
• Agonists may include, for example, naturally or chemically synthesized compounds such as small cell permeable organic molecules, nucleic acid interchelators, peptides,
• Helix-turn-helix domains are known in the art and have been implicated in DNA binding (Ann Rev. of Biochem. 1984. 53:293).
• An example of the consensus sequence for a helix-turn domain can be found in Brunelle and Schleif (1989, J. Mol.
• the domain has been illustrated by the sequence XXXPhoAlaXXPhoGlyPhoXXXXPhoXXPhoXX, where X is any amino acid and Pho is a hydrophobic amino acid.
• the crystal structure of MarA has been determined and the first (most amino terminal) HTH domain of MarA has been identified as comprising from about amino acid 31 to about amino acid 52 and the second HTH domain of MarA has been identified as comprising from about amino acid 79 to about amino acid 102 (Rhee et al. 1998. Proc. Natl Acad. Sci. USA. 95:10413).
• helix-turn-helix domains in other MarA family members as well as other HTH proteins can easily be found by one of skill in the art.
• an alignment program e.g., the ProDom program or other programs known in the art
• a portion of the MarA amino acid sequence e.g., comprising one or both HTH domains of MarA (such as from about amino acid 30 to about amino acid 107 of MarA) to produce an alignment.
• the amino acid sequences corresponding to the HTH domains of MarA can be identified in other MarA family member proteins.
• An exemplary consensus sequence for the first helix-turn-helix domain of a MarA family polypeptide can be illustrated as XXXXAXXXXXSXXXLXXXFX, where X is any amino acid.
• An exemplary consensus sequence for the second helix-turn-helix domain of a MarA family polypeptide is illustrated as XXIXXIAXXXGFXSXXXFXXX[F Y], where X is any amino acid.
• a MarA family polypeptide first helix-turn-helix domain comprises the consensus sequence E/D-X-V/L-A-D/E-X-A/S-G-X-S-X3-L-Q-X2-F- K/R/E-X2-T/I.
• a MarA family polypeptide second helix-turn-helix domain comprises the consensus sequence I-X-D-I-A-X3-G-F-X-S-X2-F-X3-F-X4.
• a MarA family member HTH domain is a MarA HTH domain.
• the first and second helix-turn-helix domains of MarA are, respectively, EKVSERSGYSKWHLQRMFKKET and TLYLAERYGFESQQTLTRTFKJ YF.
• Other exemplary MarA family helix-turn-helix domains include: about amino acid 210 to about amino acid 229 and about amino acid 259 to about amino acid 278 of MelR; about amino acid 196 to about amino acid 215 and about amino acid 245 to about amino acid 264 of AraC; and about amino acid 230 to about amino acid 249 (or 233-253) and about amino acid 281 to about amino acid 301 (or 282-302) of XylS (see e.g., Brunelle et al. 1989. J. Mol. Biol. 209:607; Niland et al 1996. J. Mol. Biol. 264:667; Gallegos et al. 1997. Microbiology and Molecular Biology Reviews. 61:393).
• MarA family polypeptide helix-turn-helix domains are derived from or are homologous to the helix-turn-helix domains found in the MarA family polypeptides as described supra.
• a MarA family polypeptide excludes one or more of XylS, AraC, and MelR.
• a MarA family polypeptide is selected from the group consisting of: MarA, RamA, AarP, Rob, SoxS, and PqrA. Both of the helix-turn-helix domains present in MarA family polypeptides are in the carboxy terminal end of the protein. Proteins or portions thereof comprising either or both of these domains can be used in the instant methods.
• a polypeptide which is used in screening for compounds comprises the helix-turn-helix domain most proximal to the carboxy terminus (HTH2) of the MarA family polypeptide from which it is derived.
• such a polypeptide comprises the helix-turn-helix domain most proximal to the amino terminus (HTH1) of the MarA family polypeptide from which it is derived
• other polypeptide sequences may also be present, e.g., sequences that might facilitate immobilizing the domain on a support, or, alternatively, might facilitate the purification of the domain.
• such a polypeptide consists essentially of the helix- tum-helix domain most proximal to the carboxy terminus of the MarA family polypeptide from which it is derived. In other prefened embodiments, such a polypeptide consists essentially of the helix-turn-helix domain most proximal to the amino terminus of the MarA family polypeptide from which it is derived.
• such a polypeptide consists of the helix-tum-helix domain most proximal to the carboxy terminus of the AraC family polypeptide or MarA family polypeptide from which it is derived.
• such a polypeptide consists of the helix-tum-helix domain most proximal to the amino terminus of the AraC family polypeptide or MarA family polypeptide from which it is derived.
• MarA family polypeptide or AraC family polypeptide helix-tum-helix domains can be made using techniques which are known in the art.
• the nucleic acid and amino acid sequences of transcription factors, such as MarA family polypeptides are available, for example, from GenBank. Using this information, the helix-turn-helix consensus motif and mutational analysis provided herein, one of ordinary skill in the art can identify MarA family or AraC family polypeptide helix-tum-helix domains.
• nucleic acid molecules encoding transcription factors or portions thereof (e.g., HTH protein helix-tum-helix domains, AraC family helix-tum- helix domains, MarA family helix-turn-helix domains or mutant forms thereof).
• isolated or recombinant is meant a nucleic acid molecule which has been (1) amplified in vitro by, for example, polymerase chain reaction (PCR); (2) recombinantly produced by cloning, or (3) purified, as by cleavage and gel separation; or (4) synthesized by, for example, chemical synthesis.
• PCR polymerase chain reaction
• nucleic acid molecule is isolated from the sequences which naturally flank it in the genome and from cellular components.
• the isolated or recombinant nucleic acid molecules encoding transcription factors e.g., HTH protein helix-tum-helix domains, AraC family helix- tum-helix domains, MarA family helix-tum-helix domains or mutant forms thereof
• transcription factors e.g., HTH protein helix-tum-helix domains, AraC family helix- tum-helix domains, MarA family helix-tum-helix domains or mutant forms thereof
• HTH protein helix-tum-helix domains e.g., MarA family helix-tum-helix domains or mutant forms thereof.
• Such polypeptides can be purified from cells which have been engineered to express an isolated or recombinant nucleic acid molecule which encodes a HTH protein helix-tum-helix domain (e.g., MarA family helix-turn-helix domain or mutant forms thereof).
• a bacterial cell can be transformed with a plasmid which encodes a MarA family helix-tum-helix domain.
• the MarA family helix- tum-helix protein can then be purified from the bacterial cells and used, for example, in the cell-free assays described herein.
• HTH protein helix-tum-helix domain e.g., MarA family helix-tum-helix domain
• Purification of a HTH protein helix-tum-helix domain can be accomplished using techniques known in the art. For example, column chromatography could be used, or antibodies specific for the domain or for a polypeptide fused to the domain can be employed, for example on a column or in a panning assay.
• cells used to express HTH protein helix-tum- helix domains e.g., MarA family helix-turn-helix domains or mutant forms thereof
• host cells comprise a mutation which renders any endogenous HTH proteins nonfunctional or causes the endogenous protein to not be expressed.
• mutations may also be made in MarR or related genes of the host cell, such that repressor proteins which bind to the same promoter as a MarA family polypeptide are not expressed by the host cell.
• a mutant form of a HTH protein helix-turn helix domain e.g., a non-naturally occurring form of a MarA family helix-tum-helix domain which has altered activity, e.g., does not retain wild type MarA family polypeptide helix-tum-helix domain activity, or which has reduced activity or which is more active when compared to a wild-type MarA family polypeptide helix-tum-helix domain.
• mutant forms can be made using techniques which are well known in the art. For example, random mutagenesis can be used. Using random mutagenesis one can mutagenize an entire molecule or one can proceed by cassette mutagenesis. In the former instance, the entire coding region of a molecule is mutagenized by one of several methods (chemical, PCR, doped oligonucleotide synthesis) and that collection of randomly mutated molecules is subjected to selection or screemng procedures.
• random mutagenesis can be used. Using random mutagenesis one can mutagenize an entire molecule or one can proceed by cassette mutagenesis. In the former instance, the entire coding region of a molecule is mutagenized by one of several methods (chemical, PCR, doped oligonucleotide synthesis) and that collection of randomly mutated molecules is subjected to selection or screemng procedures.
• PCR mutagenesis is used.
• Example 2 describes the use of Megaprimer PCR (O.H. Landt, Gene 96:125-128) used to introduce an N7zel restriction site into the centers of both the helix A (position 1989) and helix B (position 2016) regions of the marA gene.
• such mutant helix-turn-helix domains comprise one or more mutations in the helix-turn-helix domain most proximal to the carboxy terminus (HTH2) of the MarA family polypeptide molecule.
• the mutation comprises an insertion into helix A and helix B of the helix-turn-helix domain most proximal to the carboxy terminus of the MarA family polypeptide.
• such mutant helix-turn-helix domains comprise one or more mutations in the helix-turn-helix domain most proximal to the amino terminus (HTH1) of the MarA family polypeptide molecule.
• the mutation comprises an insertion into helix A and helix B of the helix-tum-helix domain most proximal to the amino terminus of the MarA family polypeptide.
• the mutation is selected from the group consisting of: an insertion at an amino acid conesponding to about position 33 of MarA and an insertion at an amino acid position conesponding to about position 42 of MarA.
• Conesponding amino acids can be determined, e.g., using an alignment of the helix-tum-helix domains.
• Such mutant forms of MarA family helix-turn-helix motifs are useful as controls to verify the specificity of antiinfective compounds for a MarA family helix- tum-helix domain or as controls for the identification of genetic loci which affect resistance to antiinfectives.
• the mutant MarA family helix-turn-helix domains described in the appended Examples demonstrate that insertional inactivation of MarA at either helix A or helix B in the first HTH domain abolished the multidrug resistance phenotype in both E. coli and M. smegmatis.
• the isolated or recombinant nucleic acid molecules encoding transcription factors can then, for example, be utilized in binding assays, can be expressed in a cell, or can be expressed on the surface of phage, as discussed further below.
• transcription factors e.g., HTH protein helix-tum-helix domains, AraC family helix- tum-helix domains, MarA family helix-turn-helix domains or mutant forms thereof
• it will be desirable to obtain a substantially purified or recombinant HTH protein helix-tum-helix domains e.g., MarA family helix-turn-helix domains or mutant forms thereof.
• Such polypeptides can be purified from cells which have been engineered to express an isolated or recombinant nucleic acid molecule which encodes a HTH protein helix-tum-helix domain (e.g., MarA family helix-turn-helix domain or mutant forms thereof).
• a bacterial cell can be transformed with a plasmid which encodes a MarA family helix-turn-hehx domain.
• the MarA family helix- tum-helix protein can then be purified from the bacterial cells and used, for example, in the cell-free assays described herein.
• HTH protein helix-tum-helix domain e.g., MarA family helix-turn-helix domain
• column chromatography could be used, or antibodies specific for the domain or for a polypeptide fused to the domain can be employed, for example on a column or in a panning assay.
• cells used to express HTH protein helix-tum- helix domains e.g., MarA family helix-tum-helix domains or mutant forms thereof
• host cells comprise a mutation which renders any endogenous HTH proteins nonfunctional or causes the endogenous protein to not be expressed
• mutations may also be made in MarR or related genes of the host cell, such that repressor proteins which bind to the same promoter as a MarA family polypeptide are not expressed by the host cell.
• a mutant form of a HTH protein helix-turn helix domain e.g., a non-naturally occurring form of a MarA family helix-turn-helix domain which has altered activity, e.g. , does not retain wild type MarA family polypeptide helix-tum-helix domain activity, or which has reduced activity or which is more active when compared to a wild-type MarA family polypeptide helix-tum-helix domain.
• mutant forms can be made using techniques which are well known in the art. For example, random mutagenesis can be used. Using random mutagenesis one can mutagenize an entire molecule or one can proceed by cassette mutagenesis. In the former instance, the entire coding region of a molecule is mutagenized by one of several methods (chemical, PCR, doped oligonucleotide synthesis) and that collection of
• Nucleic acids encoding transcription factors can be expressed in cells using vectors.
• transcription factors such as AraC family polypeptides, HTH proteins, e.g., MarA family polypeptides or selectable markers (or portions thereof that retain an activity of the full-length polypeptide, e.g., are capable of binding to a transcription factor responsive element or retain their indicator function)
• vectors Almost any conventional delivery vector can be used.
• Such vectors are widely available commercially and it is within the knowledge and discretion of one of ordinary skill in the art to choose a vector which is appropriate for use with a given microbial cell.
• the sequences encoding these domains can be introduced into a cell on a self-replicating vector or may be introduced into the chromosome of a microbe using homologous recombination or by an insertion element such as a transposon.
• nucleic acids can be introduced into microbial cells using standard techniques, for example, by transformation using calcium chloride or electroporation. Such techniques for the introduction of DNA into microbes are well known in the art.
• a nucleic acid molecule which has been amplified in vitro by, for example, polymerase chain reaction (PCR); recombinantly produced by cloning, or) purified, as by cleavage and gel separation; or synthesized by, for example, chemical synthesis can be used to produce MarA family polypeptides (George, A. M. & Levy, S. B. (1983)J. Bacteriol. 155, 541-548; Cohen, S. P. et al. (1993) J Infect. Dis. 168,
• PCR polymerase chain reaction
• Host cells can be genetically engineered to incorporate nucleic acid molecules of the invention.
• nucleic acid molecules specifying transcription factors can be placed in a vector.
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
• expression vector or "expression system” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a promoter).
• plasmid and “vector” are used interchangeably, as a plasmid is a commonly used form of vector.
• vectors which serve equivalent functions.
• expression systems can be used to produce the polypeptides of the invention.
• vectors include, among others, chromosomal, episomal and vims- derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and
• vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
• telomeres can be introduced into a cell on a self- replicating vector or maybe introduced into the chromosome of a microbe using homologous recombination or by an insertion element such as a transposon.
• the expression system constructs may contain control regions that regulate expression.
• Transcriptional regulatory sequence is a generic term to refer to DNA sequences, such as initiation signals, enhancers, operators, and promoters, which induce or control transcription of polypeptide coding sequences with which they are operably linked.
• a recombinant gene encoding a transcription factor gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring transcription factor gene.
• transcriptional regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
• any of a wide variety of expression control sequences, that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the polypeptide.
• any system or vector suitable to maintain, propagate or express nucleic acid molecules and/or to express a polypeptide in a host may be used for expression in this regard.
• the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning, A Laboratory Manual, (supra).
• Exemplary expression vectors for expression of a gene encoding a polypeptide and capable of replication in a bacterium e.g., a gram positive, gram negative, or in a cell of a simple eukaryotic fungus such as a Saccharomyces or, Pichia, or in a cell of a eukaryotic organism such as an insect, a bird, a mammal, or a plant, are known in the art.
• Such vectors may carry functional replication-specifying sequences (replicons) both for a host for expression, for example a Streptomyces, and for a host, for example, E. coli, for genetic manipulations and vector construction. See, e.g., U.S.
• Useful expression control sequences include, for example, the early and late promoters of SV40, adenovims or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat polypeptide, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
• the early and late promoters of SV40, adenovims or cytomegalovirus immediate early promoter the lac system, the trp system, the T
• an inducible promoter will be employed to express a polypeptide of the invention.
• trp induced by tryptophan
• tac induced by lactose
• tet induced by tetracycline
• GAL1 induced by galactose
• a constitutive promoter can be used to express a polypeptide of the invention.
• the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of polypeptide desired to be expressed.
• appropriate hosts include bacterial cells, such as gram positive, gram negative cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoplera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.
• cells used to express heterologous polypeptides of the invention comprise a mutation which renders one or more endogenous transcription factors, such as a AraC family polypeptide or a MarA family polypeptide, nonfunctional or causes one or more endogenous polypeptide to not be expressed.
• Endogenous transcription factors such as a AraC family polypeptide or a MarA family polypeptide
• Manipulation of the genetic background in this manner allows for screening for compounds that modulate specific transcription factors, such as MarA family members or AraC family members, or more than one transcription factors.
• mutations may also be made in other related genes of the host cell, such that there will be no interference from the endogenous host loci, h yet another embodiment, a mutation maybe made in a chromosomal gene to create a heterotroph.
• transformation can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology, (1986) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold
• polypeptides e.g., recombinanfly expressed polypeptides
• purification of polypeptides can be accomplished using techniques known in the art. For example, if the polypeptide is expressed in a form that is secreted from cells, the medium can be collected. Alternatively, if the polypeptide is expressed in a form that is retained by cells, the host cells can be lysed to release the polypeptide.
• Such spent medium or cell lysate can be used to concentrate and purify the polypeptide.
• the medium or lysate can be passed over a column, e.g., a column to which antibodies specific for the polypeptide have been bound.
• such antibodies can be specific for a second polypeptide which has been fused to the first polypeptide (e.g., as a tag) to facilitate purification of the first polypeptide.
• Other means of purifying polypeptides are known in the art.
• Transcription factor agonists and antagonists can be assayed in a variety of ways.
• the invention provides for methods for identifying a compound which modulates an transcription factor, e.g., by measuring the ability of the compound to interact with an transcription factor nucleic acid molecule or an transcription factor polypeptide or the ability of a compound to modulate the activity or expression of an transcription factor polypeptide.
• the ability of a compound to modulate the binding of an transcription factor polypeptide or transcription factor nucleic acid molecule to a molecule to which they normally bind e.g., a nucleic acid or protein molecule can be tested.
• a transcription factor and its cognate DNA sequence can be present in a cell free system, e.g., a cell lysate and the effect of the compound on that interaction can be measured using techniques known in the art.
• the assay system is a cell-based system.
• Compounds identified using the subject methods are useful, e.g., to interfere with the ability of a microbe to grow in a host and/or in reducing microbial virulence and, thereby, and in reducing the ability of the microbe to cause infection in a host.
• the ability of the test compound to modulate the expression and/or activity of a transcription factor can be determined in a variety of ways. Exemplary methods which can be used in the instant assays are known in the art and are described,
• the invention provides for methods of identifying a test compound which modulates the expression and/or activity of a transcription factor, (e.g., an HTH protein, a MarA family polypeptide, an AraC family polypeptide, etc.) by contacting a cell expressing a transcription factor (or portion thereof) with a test compound under conditions which allow interaction of the test compound with the cell.
• a transcription factor e.g., an HTH protein, a MarA family polypeptide, an AraC family polypeptide, etc.
• the expression of a selectable marker that confers a selective growth disadvantage or lethality is placed under the direct control of a MarA. responsive element in a cell expressing marA.
• marA is plasmid encoded, i one embodiment, the genetic background of the host organism is manipulated, e.g., to delete one or more chromosomal marA genes or marA homolog genes. h one embodiment, expression of marA is controlled by a highly regulated and inducible promoter. For example, in one embodiment, a promoter selected from the group consisting of trp, tac, or tet in bacterial cells or GAL1 in yeast cells can be used. In another embodiment, expression of marA is constitutive.
• a selective marker is a cytotoxic gene product (e.g., ccdB).
• a selective marker is a gene that confers antibiotic resistance (e.g., kan, cat, or bla).
• a selective marker is an essential gene (e.g., purA or guaB in a purine or guanine heterotroph).
• a selective marker is a gene that confers a selective growth disadvantage in the presence of a particular metabolic substrate (e.g., the expression of URA3 in the presence of 5-fluoroorotic acid [5-FOA] in yeast).
• compounds that modulate transcription factors e.g.,
• HTH proteins, AraC family polypeptides, or MarA family polypeptides are identified using a one-hybrid screening assay.
• one-hybrid screen includes assays that detect the disruption of protein-nucleic acid interactions.
• a transcription factor e.g., an HTH protein, a AraC family polypeptide, or a MarA family polypeptide
• a particular target e.g., DNA containing, for MarA, a marbox
• compounds of the invention are identified using a two-hybrid screening assay.
• the term "two-hybrid screen” as used herein includes assays that detect the disruption of protein-protein interactions. Such two hybrid assays can be used to interfere with crucial piOtein-transcription factor interactions (e.g., HTH protein interactions, AraC family polypeptide interactions, MarA family polypeptide interactions). One example would be to prevent RNA polymerase- MarA family polypeptide contacts, that are necessary for the MarA family polypeptide to function as a transcription factor (either positive acting or negative acting).
• compounds of the invention are identified using a three-hybrid screening assay.
• the term "three-hybrid screen” as used herein includes assays that will detect the disruption of a signal transduction pathway(s) required for the activation of a particular regulon of interest.
• the three-hybrid screen is used to detect disruption of a signal transduction pathway(s) required for the activation of the Mar regulon, i.e., synthesis of MarA. (Li and Park. J. Bact. 181 :4824) .
• the assay can be used to identify compounds that may be responsible for activating transcription factor expression, e.g., Mar induction by antibiotics may proceed in this manner.
• the expression of a selective marker is put under the direct control of an activatable MarA responsive activatable promoter (e.g., inaA, galT, micF).
• an activatable MarA responsive activatable promoter e.g., inaA, galT, micF.
• the expression of the selective marker would be silent.
• the gene would be silent and the cells would survive.
• Synthesis of MarA from an inducible plasmid in a suitable host would result in the activation of the MarA responsive activatable promoter and expression of the selective marker.
• ccdB the gene would be expressed and result in cell death.
• Compounds that inhibit MarA would be identified as those that permit cell survival under conditions of MarA expression.
• a selectable marker is put under the direct control of a repressible MarA responsive promoter (e.g., fecA).
• a repressible MarA responsive promoter e.g., fecA
• the expression of the selectable marker would be silent.
• ccdB this would be the case of ccdB.
• a purine or guanine heterotroph can be constructed by the inactivation of the chromosomal guaB or purA genes in E. coli.
• the guaB or purA gene would then be cloned into a suitable vector, under the control of its natural promoter. This constmct would then be transformed into the heterotrophic host.
• the heterotroph will not grow if MarA expression is constitutive and if cells are grown on media lacking purines or guanine. This can be attributed to MarA mediated repression of guaB or purA synthesis.
• Candidate inhibiting compounds of MarA can be identified as compounds that restored growth, i.e., relieved MarA mediated repression of guaB and purA expression, hi another embodiment, genes that are required for growth in vivo, for example in an animal model of infection. hi prefened embodiments, controls may be included to ensure that any compounds which are identified using the subject assays do not merely appear to modulate the activity of a transcription factor, because they inhibit protein synthesis.
• a compound appears to inhibit the synthesis of a protein being translated from RNA which is transcribed upon activation of a MarA family responsive element
• a control e.g., a protein which is being translated from RNA which is not transcribed upon activation of a MarA family responsive element
• the amount of the MarA family polypeptide being made and compared to the amount of an endogenous protein being made.
• the microbe could be transformed with another plasmid comprising a promoter which is not a MarA family responsive promoter and a protein operably linked to that promoter.
• the expression of the control protein could be used to normalize the amount of protein produced in the presence and absence of compound.
• microbes are suitable for testing in the instant assays. As such, they may be used as intact cells or as sources of material, e.g., nucleic acid molecules or polypeptides as described herein.
• microbes for use in the claimed methods are bacteria, either Gram negative or Gram positive bacteria. More specifically, any bacteria that are shown to become resistant to antibiotics , e.g., to display a Mar phenotype are prefened for use in the claimed methods, or that are infectious or potentially infectious.
• microbes suitable for testing include, but are not limited to, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia,
• microbes suitable for testing are bacteria from the family Enter obacteriaceae.
• the compound is effective against a bacteria of a genus selected from the group consisting of: Escherichia, Proteus, Salmonella, Klebsiella, Providencia, Enterobacter, Burkholderia, Pseudomonas, Aeromonas, Haemophilus, Yersinia, Neisseria, and Mycobacteria.
• the microbes to be tested are Gram positive bacteria and are from a genus selected from the group consisting of: LactobacUlus, Azorhizobium, Streptomyces, Pediococcus, Photobacterium, Bacillus, Enterococcus, Staphylococcus, Clostridium, and Streptococcus.
• the microbes to be tested are fungi. In a prefened embodiment the fungus is from the genus Mucor or Candida, e.g., Mucor racmeosus or Candida albicans.
• the microbes to be tested are protozoa.
• the microbe is a malaria or cryptosporidium parasite.
• transcription factor modulating compounds and Test Compounds can be derived from a variety of different sources and can be known or can be novel.
• libraries of compounds are tested in the instant methods to identify transcriptional activation factor modulating compounds, e.g., HTH protein modulating compounds, AraC family polypeptide modulating compounds, MarA family polypeptide modulating compounds, etc.
• known compounds are tested in the instant methods to identify transcription factor modulating compounds (such as, for example, HTH protein modulating compounds, AraC family polypeptide modulating compounds, MarA family polypeptide modulating compounds, etc.).
• transcription factor modulating compounds such as, for example, HTH protein modulating compounds, AraC family polypeptide modulating compounds, MarA family polypeptide modulating compounds, etc.
• compounds among the list of compounds generally regarded as safe (GRAS) by the Environmental Protection Agency are tested in the instant methods.
• the transcription factors which are modulated by the modulating compounds are of prokaryotic microbes.
• a recent trend in medicinal chemistry includes the production of mixtures of compounds, refened to as libraries. While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin et al. 1992. J. Am. Chem. Soc. 114:10987; DeWitt et al. 1993. Proc. Natl. Acad. Sci. USA 90:6909) peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678) oligocarbamates (Cho et al. 1993. Science. 261:1303), and hydantoins (DeWitt et al. supra).
• the compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the One-bead one-compound' library method, and synthetic library methods using affinity chromatography selection.
• biological libraries include biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the One-bead one-compound' library method, and synthetic library methods using affinity chromatography selection.
• the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. Anticancer Drug Des. 1997. 12:145).
• test compound is a peptide or
• the compounds are small, organic non-peptidic compounds.
• combinatorial polypeptides can be produced from a cDNA library.
• the compounds can be nucleic acid molecules.
• nucleic acid molecules for testing are small oligonucleotides.
• Such oligonucleotides can be randomly generated libraries of oligonucleotides or can be specifically designed to reduce the activity of a transcription factor, e.g., a HTH protein, a MarA family polypeptide, or an AraC family polypeptide.
• these oligonucleotides are sense or antisense oligonucleotides.
• oligonucleotides for testing are sense to the binding site of a particular transcription factor, e.g., a MarA family polypeptide helix-tum-helix domain.
• computer programs can be used to identify individual compounds or classes of compounds with an increased likelihood of modulating a transcription factor activity, e.g., an HTH protein, a AraC family polypeptide, or a MarA family polypeptide activity.
• a transcription factor activity e.g., an HTH protein, a AraC family polypeptide, or a MarA family polypeptide activity.
• Such programs can screen for compounds with the proper molecular and chemical complementarities with a chosen transcription factor. In this manner, the efficiency of screening for transcription factor modulating compounds in the assays described above can be enhanced.
• the invention also pertains to the use of molecular design techniques to design transcription factor modulating compounds, e.g., HTH protein modulating compounds, AraC family modulating compounds, MarA family modulating compounds,
• MarA modulating compounds which are capable of binding or interacting with one or more transcription factors (e.g., of a prokaryotic or eukaryotic organism).
• the invention pertains to both the transcription factor modulating compounds identified by the methods as well as the modeling methods, and compositions comprising the compounds identified by the methods.
• the invention pertains to a method of identifying transcription factor modulating compounds.
• the method includes obtaining the structure of a transcription factor of interest, and using GLIDE to identify a scaffold which has an interaction energy score of -20 or less (e.g., -40 or less, e.g., -60 or less) with a portion of the transcription factor.
• the invention pertains, at least in part, to a computational screening of small molecule databases for chemical entities or compounds that can bind in whole, or in part, to a transcription factor, such as a HTH protein, an AraC family polypeptide, a MarA family polypeptide, e.g., MarA.
• a transcription factor such as a HTH protein, an AraC family polypeptide, a MarA family polypeptide, e.g., MarA.
• the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity or by estimated interaction energy (Meng, E. C et al, 1992, J. Coma. Chem., 13:505-524).
• Such a procedure allows for the screening of a very large library of potential transcription factor modulating compounds for the proper molecular and chemical complementarities with a selected protein or class or proteins.
• Transcription factor modulating compounds identified through computational screening can later be passed through the in vivo assays described herein as further screens.
• a MarA inhibiting compound identified through computational screening could be tested for its ability to promote cell survival in a cell system containing a counterselectable marker under the control a MarA activated promoter. The promotion of cell survival in the foregoing assay would be indicative of a compound that inhibits MarA's activity as a transcriptional activator.
• Other suitable assays are described in the Examples and through the specification.
• the crystal structures of both MarA (PDB ID code 1BL0) and its homolog Rob (PDB ID code 1DY5) are available in the Protein Data Bank (http ://www.rcsb.org/pdb . These structures were used to identify sites on the proteins that could be targeted by small molecule chemical inhibiting compounds.
• a total of at least eight potential small molecule binding sites on MarA (Table 2) and four sites on Rob (Table 3) were identified as potential small molecule binding sites.
• the invention pertains, at least in part, to MarA modulating compounds which interact with any one of the following sites of MarA (based on the sequence given in SEQ ID NO. 2).
• the GLIDE docking method was then used to fit combinatorial chemistry scaffolds into these sites and an interaction energy was calculated for each.
• Eight scaffolds were predicted to bind to site 1, encompassing amino acids tryptophan 42 to lysine 50, with an interaction energy score of-60 or less. These scaffolds are shown below:
• Three scaffolds were identified for site 2 of MarA (e.g., residues histidine 54 to serine 62).
• scaffold includes the compounds identified by the computer modeling program. These compounds may or may not be themselves transcription factor modulating compounds.
• An ordinarily skilled artisan will be able to analyze a scaffold obtained from the computer modeling program and modify the scaffold such that the resulting compounds have enhanced chemical properties over the initial scaffold compound, e.g., are more stable for administration, less toxic, have enhanced affinity for a particular transcription factor, etc.
• the invention pertains not only to the scaffolds identified, but also the transcription factor modulating compounds which are developed using the scaffolds.
• Table 3 lists portions of Rob which were identified as possible interaction sites for a modulating compound.
• the invention pertains, at least in part, to any compounds modeled to bind to these regions of Rob.
• the numbering conesponds to that given in SEQ TD NO. 4.
• scaffolds were identified as small molecules that may interact with site 2 of Rob (residues 43-52), a MarA family polypeptide.
• the design of compounds that bind to, modulate, or inhibit franscription factors generally involves consideration of two factors.
• the compound must be capable of physically and structurally associating with a particular transcription factor.
• Non- covalent molecular interactions important in the association of a transcription factor with a modulating compound include hydrogen bonding, van der Waals and hydrophobic interactions.
• the modulating compound must be able to assume a conformation that allows it to associate with the selected transcription factor. Although certain portions of the inhibiting compound will not directly participate in this association with the transcription factor, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency.
• Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site, e.g., active site or accessory binding site of a particular transcription factor such as MarA, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with the particular transcription factor.
• the potential modulating effect of a chemical compound on a selected transcription factor is analyzed prior to its actual synthesis and testing by the use of computer modeling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association between it and the selected transcription factor, synthesis and testing of the compound is avoided. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized
• a transcription factor modulating compound or other binding compound e.g., an HTH protein modulating compound, an AraC family polypeptide modulating compound, or a MarA family inhibiting compound, e.g., a MarA inhibiting compound
• an HTH protein modulating compound e.g., an AraC family polypeptide modulating compound, or a MarA family inhibiting compound, e.g., a MarA inhibiting compound
• a transcription factor modulating compound or other binding compound may be computationally evaluated and designed by screening and selecting chemical entities or fragments for their ability to associate with the individual small molecule binding sites or other areas of a transcription factor.
• One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a selected transcription factor and more particularly with the individual small molecule binding sites of the particular transcription activation factor. This process may begin by visually inspecting the structure of the transcription factor on a computer screen based on the atomic coordinates of the transcription factor crystals. Selected chemical entities may then be positioned in a variety of orientations, or docked, within an individual binding site of the transcription factor. Docking may be performed using software such as Quanta and Sybyl, followed by energy minimization with standard molecular mechanics forcefields or dynamics with programs such as CHARMM (Brooks, B. R. et al., 1983, J. Comp. Chem., 4:187-217) or AMBER (Weiner, S. J. et al., 1984, J Am. Chem. Soc, 106:765- 784).
• CHARMM Brooks, B. R. et al., 1983, J. Comp. Chem., 4:187-217
• Specialized computer programs may also assist in the process of selecting molecules that bind to a selected transcription factor, (e.g., an HTH protein, an AraC family polypeptide, or a MarA family polypeptide, e.g., MarA).
• the programs include, but are not limited to: 1. GRID (Goodford, P. J., 1985, "A Computational Procedure for
• MCSS (Miranker, A. and M. Karplus, 1991, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins: Structure, Function and Genetics, 11:29-34). MCSS is available from Molecular Simulations, Burlington, Mass. >
• MACCS-3D (Martin, Y. C, 1992, J. Med. Chem., 35:2145-2154) is a 3D database system available from MDL Information Systems, San Leandro, Calif.
• DOCK Macromolecule-Ligand Interactions" J. Mol. Biol, 161:269-288.
• DOCK is available from University of California, San Francisco, Calif. DOCK is based on a description of the negative image of a space-filling representation of the molecule (i.e. the selected transcription factor) that should be filled by the inhibiting compound. DOCK includes a force-field for energy evaluation, limited conformational flexibility and consideration of hydrophobicity in the energy evaluation.
• MCDLNG Monte Carlo De Novo Ligand Generator
• Affinity is a suite of programs for automatically docking a ligand (i.e. a transcription factor modulating compound) to a receptor (i.e. a transcription factor). Commands in Affinity automatically find the best binding structures of the ligand to the receptor based on the energy of the ligand/receptor complex. As described below, Affinity allows for the simulation of flexible-flexible docking.
• 53 - Affinity consists of two commands, GridDocking and fixedDocking, under the new pulldown Affinity in the Docking module of the Insight ⁇ program.
• Both commands use the same, Monte Carlo type procedure to dock a guest molecule (i.e. HTH protein modulating compound) to a host (i.e., a transcription factor).
• a host i.e., a transcription factor.
• the "bulk" of the receptor i.e. transcription factor
• the commands differ, however, in their treatment of nonbond interactions.
• GridDocking interactions between bulk and movable atoms are approximated by the very accurate and efficient molecular mechanical/grid (MM/Grid) method developed by Luty et al. 1995. J. Comp. Chem. 16:454, while interactions among movable atoms are treated exactly.
• GridDocking also includes the solvation method of Stouten et al. 1993. Molecular Simulation 10:97.
• the fixedDocking command computes nonbond interactions using methods in the Discover program (cutoff methods and the cell multipole method) and it does not include any solvation terms.
• Affinity does not, generally, require any intervention from the user during the docking. It automatically moves the ligand (i.e. modulating compound), evaluates energies, and checks if the stracture is acceptable. Moreover, the ligand and the binding site of the receptor (i.e. the selected transcription modulator) are flexible during the search.
• Aided Mol Design 8:51 FLOG (Miller et al. 1994. J. Comp. Aided Molec. Design 8:153), and PRO_LIGAND (Clark et al 1995. J. Comp. Aided Molec. Design 9:13), to name a few.
• Affinity differs from these methods in several aspects. First, it uses full molecular mechanics in searching for and evaluating docked structures. In contrast descriptor-based methods use empirical rales which usually take into account only hydrogen bonding, hydrophobic interactions, and steric effects. This simplified description of ligand/receptor interaction is insufficient in some cases. For example, Meng et al 1992. J. Compt. Chem.
• the ligand itself is flexible in Affinity which permits different conformations of a ligand (i.e. transcription factor modulating compound) to be docked to a receptor (i.e. transcription factor).
• a ligand i.e. transcription factor modulating compound
• a receptor i.e. transcription factor
• FLOG is also able to treat flexible ligand by including different conformations for each structure in the database (Miller et al. 1995. J. Comp. Aided Molec. Design. 8:153). Most other methods are limited to rigid ligands.
• There are also a few energy based docking methods Kuntz et al. 1994. Ace.
• Affinity uses a Monte Carlo procedure in docking ligands, but there are important distinctions over the prior art methods.
• the Monte Carlo procedure in Affinity can be used in conjunction either with energy minimization (to mimic the Monte Carlo minimization method of Li & Scheraga. 1987. Proc. Natl. Acad. Sci. U.S.A. 84:6611) or with molecular dynamics (to mimic the hybrid Monte Carlo method, Clamp et al. 1994. J. Comput. Chem. 15:838, or the smart Monte Carlo method, Senderowitz et al. 1995. J. Am. Chem. Soc. 117:8211).
• This flexibility allows Affinity to be applied to a variety of docking problems.
• finity employs energy differences obtained from molecular mechanics, while the methods discussed above use empirical rules or descriptors. Therefore, Affinity is more
• Affinity allows the binding site of the receptor to relax, while the methods discussed above fix the entire receptor.
• Affinity employs two new nonbond techniques which are both accurate and efficient to make docking practical.
• One is the Grid/MM method of Luty et al which represents the bulk of the receptor by grids (Luty et al. 1995. J. Comp. Chem. 16:454). This method is 10-20 times faster than the no-cutoff method with almost no loss in accuracy. It also incorporates the solvation method of Stouten et al. (1993. Molecular Simulation 10:97). The other is the cell multipole method. This method is about 50% slower than the Grid/MM method, but it does not require grid setup. Thus, a typical docking calculation takes about 1-3 hours of CPU time on an Indigo R4400 workstation.
• suitable chemical fragments Once suitable chemical fragments have been selected, they can be assembled into a single compound or inhibiting compound. Assembly may be proceed by visual inspection of the relationship of the fragments to each other on a three-dimensional image display on a computer screen in relation to the structure coordinates of a particular transcription factor, e.g., MarA. This may be followed by manual model building using software such as Quanta or Sybyl.
• a particular transcription factor e.g., MarA.
• 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif. This area is reviewed in Martin, Y. C, 1992, “3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154).
• CAVEAT Bartlett, P. A. et al, 1989, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", h Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182-196). CAVEAT is available from the University of California, Berkeley, Calif. CAVEAT suggests inhibiting compounds to MarA based on desired bond vectors.
• HOOK available from Molecular Simulations, Burlington, Mass.). HOOK proposes docking sites by using multiple copies of functional groups in simultaneous searches.
• transcription factor modulating compounds may be designed as a whole or "de novo" using either an empty active site or optionally including some ⁇ ortion(s) of a known inhibiting compound(s). These methods include:
• LUDI (Boh , H.-J., "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inl ibiting compounds", J. ComR. Aid. Molec. Design, 6, pp. 61-78 (1992)).
• LUDI is available from Biosym Technologies, San Diego, Calif.
• LUDI is a program based on fragments rather than on descriptors.
• LUDI proposes somewhat larger fragments to match with the interaction sites of a macromolecule and scores its hits based on geometric criteria taken from the Cambridge Structural Database (CSD), the Protein Data Bank (PDB) and on criteria based on binding data.
• LUDI is a library based method for docking fragments onto a binding site.
• Fragments are aligned with 4 directional interaction sites (lipophilic-aliphatic, lipophilic-aromatic, hydrogen donor, and hydrogen acceptor) and scored for their degree of overlap. Fragments are then connected (i.e. a linker of the proper length is attached to each terminal atom in the fragments). Note that conformational flexibility can be accounted for only by including multiple conformations of a particular fragment in the library.
• LEGEND (Nishibata, Y. and A. Itai, Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations, Burlington, Mass.
• CoMFA Conformational Molecular Field Analysis
• CoMFA defines 3- dimensional molecular shape descriptors to represent properties such as hydrophobic regions, sterics, and electrostatics. Compounds from a database are then overlaid on the 3D pharmacophore model and rated for their degree of overlap. Small molecule databased that be searched include: ACD (over 1,000,000 compounds), Maybridge (about 500,000 compounds), NCI (about 500,000 compounds), and CCSD. In measuring the goodness of the fit, molecules can either be fit to the 3D molecular shape descriptors or to the active conformation of a known inhibiting compound.
• FlexX ( ⁇ 1993-2002 GMD German National Research Center for Information Technology; Rarey, M. et alJ. Mol. Biol, 261:407-489) is a fast, flexible docking method that uses an incremental construction algorithm to place ligands into
• Ligands e.g., transcription factor modulating compounds
• Ligands that are capable of "fitting" into the active site are then scored according to any number of available scoring schemes to determine the quality of the complimentarity between the active site and ligand.
• Other molecular modeling techniques may also be employed in accordance with this invention. See, e.g., Cohen, N. C. et al., "Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990). See also, Navia, M. A. and M. A. Murcko, "The Use of Structural Information in Drug Design", Cunent Opinions in Structural Biology, 2, pp.
• Candidate transcription factor modulating compounds can be evaluated for their modulating, e.g., inhibitory, activity using conventional techniques which may involve determimng the location and binding proximity of a given moiety, the occupied space of a bound inhibiting compound, the deformation energy of binding of a given compound and electrostatic interaction energies.
• conventional techniques useful in the above evaluations include, but are not limited to, quantum mechanics, molecular dynamics, Monte Carlo sampling, systematic searches and distance geometry methods (Marshall, G. R., 1987, Ann. Ref. Pharmacol. Toxicol, 27:193).
• Examples of computer programs for such uses include, but are not limited to, Gaussian 92, revision E2 (Gaussian, Inc.
• Transcription factor modulating compounds may interact with the selected transcription factor in more than one conformation that is similar in overall binding energy.
• the deformation energy of binding may be taken to be the difference between the energy of the free compound and the average energy of the conformations observed when the inhibiting compound binds to the enzyme.
• a compound designed or selected as interacting with a selected transcription factor e.g., a MarA family polypeptide, e.g., MarA, maybe further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme.
• a selected transcription factor e.g., a MarA family polypeptide, e.g., MarA
• Such non-complementary e.g., electrostatic
• the modulating compound and the enzyme when the modulating compound is bound to the selected transcription factor preferably make a neutral or favorable contribution to the enthalpy of binding.
• substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Initial substitutions are preferable conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Substitutions known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency of fit to the selected transcription factor by the same computer methods described above.
• Computer programs can be used to identify unoccupied (aqueous) space between the van der Waals surface of a compound and the surface defined by residues in the binding site. These gaps in atom-atom contact represent volume that could be occupied by new functional groups on a modified version of the lead compound. More efficient use of the unoccupied space in the binding site could lead to a stronger binding compound if the overall energy of such a change is favorable.
• a region of the binding pocket which has unoccupied volume large enough to accommodate the volume of a group equal to or larger than a covalently bonded carbon atom can be identified as a promising position for functional group substitution. Functional group substitution at this region can constitute substituting something other than a carbon atom, such as oxygen.
• Similarity Screening Once a transcription factor modulating compound has been selected or designed, computational methods to assess its overall likeness or similarity to other molecules can be used to search for additional compounds with similar biochemical behavior, hi such a way, for instance, HTS derived hits can be tested to assure that they are bona fide ligands against a particular active site, and to eliminate the possibility that a particular hit is an artifact of the screening process. There are cunently several methods and approaches to determine a particular compound's similarity to members of a virtual database of compounds. One example is the OPTISIM methodology that is distributed in the Tripos package, SYBYL ( ⁇ 1991-2002 Tripos, Inc., St. Louis, MO).
• OPTISIM exploits the fact that each 3-dimensional representation of a molecule can be broken down into a set of 2-dimensional fragments and encoded into a pre-defined binary string. The result is that each compound within a particular set is represented by a unique numerical code or fingerprint that is amenable to mathematical manipulations such as sorting and comparison. OPTISIM is automated to calculate and report the percent difference in the fingerprints of the respective compounds for instance according to the using a formalism known as the Tanimoto coefficient. For instance, a compound that is similar in stracture to another will share a high coefficient. Large virtual databases of commercially available compounds or of hypothetical compounds can be quickly screened to identify compounds with high Tanimoto coefficient.
• CoMFA molecular conelation
• a particularly useful feature in CoMFA is that the individual contribution for each grid-point is known; the importance of the grid points upon the overall conelation can be visualized graphically in what is refened to as a CoMFA field. When this field is combined with the original compound alignment, it becomes a powerful tool to rationalize the activities of the individual compounds from whence the model was derived, and to predict how chemical modification of a reference compound would be effected.
• a QSAR model was developed for a set of 92 benzodiazepines using the method described above.
• a representative CoMFA field is shown in Figure 4; the region delineated by wire mesh (adjacent to the referenced triazinoxazepine) is the region where chemical modification characterized by increasing steric bulk would lead to favorable effects in transcription factor modulation.
• the invention pertains, per se, to not only the methods for identifying the transcription factor modulating compounds, but to the compounds identified by the methods of the invention as well as methods for using the identified compounds.
• the invention pertains to methods for modulating a transcription factor, e.g., an HTH protein, an AraC family polypeptide, or a MarA family polypeptide.
• the method includes contacting the transcription factor, e.g., a MarA family polypeptide, with a transcription factor modulutating compound of the formula
• A is a polar moiety
• E is a hydrophobic moiety
• the transcription factor modulating compound e.g., a MarA family modulating compound, may comprise one or more polar moieties and/or one or more hydrophobic moieties.
• the invention pertains to methods for reducing antibiotic resistance of a microbial cell.
• the method includes contacting the cell with a transcription factor modulating compound, e.g., a MarA family modulating compound, such that the antibiotic resistance of the cell is reduced.
• a transcription factor modulating compound e.g., a MarA family modulating compound
• the invention pertains to inhibiting transcription, comprising contacting a transcription factor with a transcription factor modulating compound, such that transcription is inhibited, h a further embodiment, the
• the transcription factor modulating compound is a compound of anyone of formulae (I)-(X).
• antibiotic resistance includes resistance of a microbial cell to a antibiotic compound, especially an antibiotic compound which had been previously used to treat similar microbial organisms successfully.
• polar moiety includes moieties with at least one heterocycle. It also includes moieties such as, but not limited to, hydroxyl, halogens, thioethers, carboxylic acids, metals (e.g. alkali, alkaline, Au, Hg, Ag, Mn, Co, Cu, Zn, etc.), nitro, amino, alkoxy, and other moieties which allow the compound to perform its intended function.
• polar moiety includes moieties which allow the transcription factor modulating compound to perform its intended function, e.g., modulate a transcription factor, e.g., an AraC family polypeptide or a MarA family polypeptide.
• a heterocyclic polar moiety may comprise one or more rings, one or more of which maybe aromatic. In an embodiment, one or more rings of the polar moiety are fused.
• the heterocyclic polar moiety may also be bicyclic.
• the heterocyclic polar moiety may comprise one or more nitrogen, sulfur, or oxygen atoms.
• heterocycles include benzodioxazole, benzofuran, benzoimidazole, benzoxazole, benzothiazole, benzothiophene, chromenone, deazapurine, furan, imidazole, imidazopyridine, indole, indolizine, isooxazole, isothiaozole, isoquinoline, methylenedioxyphenyl, napthridine, oxazole, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pynole, pyrollidine, quinoline, tetrazole, thiazole, thiophene, triazole, and triazoletetrazole.
• the polar moiety may be substituted when chemically feasible.
• the polar moiety may be substituted with one or more substituents such as alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino,
• substituents also include nitro, alkoxy, aryl, amidyl, ester, thioester, alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, etc.), araalkyl (e.g., substituted or unsubstituted benzyl), hydroxy, halogen (e.g., fluorine, bromine, chlorine, iodine, etc.).
• alkyl e.g., methyl, ethyl, propyl, butyl, pentyl, etc.
• araalkyl e.g., substituted or unsubstituted benzyl
• hydroxy e.g., fluorine, bromine, chlorine, iodine, etc.
• hydrophobic moiety includes moieties such as which allow the transcription factor modulating compound (e.g., an HTH protein modulating compound, an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, etc.) to perform its intended function, e.g., modulate a transcription factor.
• examples of hydrophobic moieties include, for example, hydrogen, alkyl, alkenyl, alkynyl, and aryl moieties.
• the hydrophobic moieties may be unsubstituted or substituted, if chemically feasible (e.g., not hydrogen). In an embodiment, the hydrophobic moiety is substituted or unsubstituted phenyl.
• substituents include alkyl, alkenyl, alkynyl, alkoxy, halogen, amino, thiol, hydroxy, nitro, aryl, and heteroaryl.
• the substituents can be substituted or unsubstituted.
• the phenyl hydrophobic moiety is/r ⁇ ra-substituted, e.g., alkyl (methyl, ethyl, propyl, butyl, pentyl, etc.), halogen (e.g., fluorine, bromine, chlorine, iodine, etc.), hydroxy, substituted.
• the hydrophobic moiety is heterocyclic.
• heterocyclic hydrophobic moieties include imidazopyridine, quinolinyl, pyridinyl, etc.
• the transcription factor modulating compound e.g., MarA family polypeptide modulating compound, AraC family polypeptide modulating compound, etc.
• the transcription factor modulating compound is of the
• W is NH, O or S
• X is O, S, or C, optionally linked to Q;
• a 1 is C-Z 1 , O, or S;
• a 2 is C-Z 2 , O, or S;
• a 3 is C-Z 3 , O, or S;
• a 4 is C-Z 4 , 0, or S
• a 5 is C-Z 5 , orN-Z 5 ;
• Z 1 , Z 2 , Z 3 , and Z 4 are each independently selected from the group consisting of hydrogen, alkoxy, hydroxy, halogen, and alkyl;
• Z 5 is hydrogen, alkoxy, hydroxy, halogen, alkyl, or carbonyl
• Q is hydrogen, alkyl, alkenyl, alkynyl, halogen, hydroxy, aryl, and pharmaceutically acceptable salts thereof.
• the transcription factor modulating compound e.g., the MarA family polypeptide modulating compound, AraC family polypeptide modulating compound, etc.
• W is O or S
• X is O, S, or C, optionally linked to Q;
• a 1 is C-Z 4 , O, or S;
• a 2 is C-Z 5 , orN-Z 5 ;
• Z 1 , Z 2 , Z 3 , Z 4 and Z 5 are each independently hydrogen, alkoxy, hydroxy, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic, amino, or cyano;
• Z 3 is hydrogen, alkoxy, hydroxy, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic, amino, nitro, cyano, carbonyl, or thiocarbonyl;
• Q is an aromatic or heterocyclic moiety, and pharmaceutically acceptable salts thereof.
• W may be oxygen and X may be oxygen.
• a 1 and A 2 may be C-Z 4 and C-Z 5 , respectively.
• Z 4 and Z 5 include hydrogen and hydroxy.
• Z 1 and Z 2 include hydrogen and hydroxy.
• Other examples of Z 2 also include halogen, e.g., fluorine, chlorine, bromine, and iodine.
• Z 3 include, for example, hydrogen, alkoxy and hydroxy.
• Q include substituted and unsubstituted phenyl. The phenyl may be/? ra-substituted.
• substituents include hydroxyl, halogen (e.g., fluorine, bromine, chlorine, iodine, etc.), amino, alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, etc.), nitro, cyano, etc.
• the transcription factor modulating compound is a MarA modulating compound, and in a further embodiment, a MarA inhibiting compound.
• the transcription factor modulating compound e.g., an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, etc.
• 64 G is a substituted or unsubstituted aromatic moiety, heterocyclic, alkyl, alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, or hydrogen;
• L 1 , L 2 , L 3 , L 4 , L 9 and L 10 are each independently oxygen, sulfur, substituted or unsubstituted nitrogen, and substituted or unsubstituted carbon;
• L 5 and L 6 are each independently hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, acyl, heterocyclic, amino, nitro, hydroxy, cyano, alkoxy, or aryl, and L 5 and L 6 may optionally be linked with a chain of one to six atoms to form a fused ring, and pharmaceutically acceptable salts thereof.
• the transcription factor modulating compound e.g., an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, etc.
• the transcription factor modulating compound is of the formula (IX):
• G is substituted or unsubstituted aromatic moiety, heterocyclic, alkyl, alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, or hydrogen;
• L 1 , L 2 , L 3 , and L 4 are each independently oxygen, sulfur, substituted or unsubstituted nitrogen, and substituted or unsubstituted carbon;
• R , L and L are each independently hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, acyl, heterocyclic, amino, nitro, hydroxy, cyano, alkoxy, or aryl, and L 5 and L 6 may optionally be linked with a chain of one to six atoms to form a fused ring, and pharmaceutically acceptable salts thereof.
• the transcription factor modulating compound e.g., an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, etc.
• 65 G is substituted or unsubstituted aromatic moiety, heterocyclic, alkyl, alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, or hydrogen;
• L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , and L 10 are each independently oxygen, substituted or unsubstituted nitrogen, sulfur and or substituted or unsubstituted carbon, and pharmaceutically acceptable salts thereof.
• L 9 is N-R 9 , wherein R 9 is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, acyl, or aryl.
• L 10 is oxygen.
• R 9 is hydrogen.
• G is substituted or unsubstituted phenyl or heteroaryl.
• G is cycloalkenyl, e.g., cyclohexenyl.
• L 1 , L 2 , L 3 , and L 4 are each substituted or unsubstituted carbon and L 5 , L 6 , and L 8 are each nitrogen.
• L 7 may be substituted carbon, e.g., substituted with a thioether moiety
• L 9 and L 10 are each nitrogen.
• the invention pertains to compounds of formula (TTT), wherein L 9 is nitrogen, L 10 is oxygen, L J -L 8 are each C-H, the dotted line represents a double bond and where G is not hydrogen or methyl.
• the transcription factor modulating compound e.g., an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, etc.
• the transcription factor modulating compound is of the formula (X):
• Y 1 and Y 2 are each oxygen, sulfur, or substituted or unsubstituted carbon; J 1 , J 2 , J 3 , and J 4 are each oxygen, nitrogen, or optionally substituted carbon, and pharmaceutically acceptable salts thereof.
• the transcription factor modulating compound e.g., an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, etc.
• the transcription factor modulating compound is of the formula (IV):
• - 66 Y and Y are each oxygen or sulfur
• J is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, cyano, nitro, amino, or halogen;
• V is substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, alkylamino, or alkylthio;
• P and K are each independently substituted or unsubstituted aryl, and pharmaceutically acceptable salts thereof.
• Y and Y are each oxygen, V is alkoxy and J is lower alkyl.
• P is substituted or unsubstituted phenyl.
• K maybe substituted or unsubstituted heteroaryl.
• the transcription factor modulating compound e.g., an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, etc.
• V the formula (V):
• T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently substituted or unsubstituted carbon, oxygen, substituted or unsubstituted nitrogen, or sulfur; M is hydrogen, alkyl, alkenyl, alkynyl, heterocyclic or aryl, or pharmaceutically acceptable salts thereof.
• T 5 is N-W or C-W, wherein W is alkyl, alkenyl, alkynyl, aryl, heterocyclic, acyl, hydroxy, alkoxy, alkthio, amino, nitro, halogen, or hydrogen.
• T 6 is N.
• M is substituted or unsubstituted aryl.
• W may be substituted or unsubstituted alkyl.
• T 1 , T 2 , T 3 and T 4 are each substituted or unsubstituted carbon.
• at least one of T 1 , T 2 , T 3 , and T 4 is nitrogen, and the remaining T moieties are substituted or unsubstituted carbon.
• the transcription factor modulating compound e.g., an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, etc.
• Va is of the formula (Va):
• R 1 is OH, OCOCO 2 H, or a substituted or unsubstituted straight or branched -C 5 alkyloxy group
• R is H, CO 2 (C ⁇ -C 5 substituted or unsubstituted, straight or branched alkyl), or a substituted or unsubstituted aryl group;
• R 4 , R 5 , R 6 , and R 7 are independently selected from the group consisting of H, (Q-C 5 substituted or unsubstituted, straight or branched alkyl), CO 2 (C C 5 substituted or unsubstituted, straight or branched alkyl), CO(C C 5 substituted or unsubstituted, straight or branched alkyl), CO(substituted or unsubstituted aryl or heteroaryl), CO(C 3 -C 6 substituted or unsubstituted cycloalkyl), O(C C 5 substituted or unsubstituted, straight or branched alkyl), C(NOH)(C ⁇ -C 5 substituted or unsubstituted, straight or branched alkyl), substituted or unsubstituted amino, CO 2 H, CN, NO 2 , CONH 2 , (CO)(NHOH), and halogen.
• R 4 , R 5 , R 6 , and R 7 are all H and R 2 is unsubstituted phenyl, then R 1 is not OCH 2 CO 2 CH 2 CH 3 ; In certain aspects of formula Va, R 4 , R 5 , and R 7 are all H.
• R 1 of formula Va maybe selected from the group consisting of OH, O(CR'R") ⁇ - 3 H, O(CR'R") ⁇ _ 3 OH, 0(CR'R")i_ 3 CO 2 H, O(CR'R") 1 _ 3 CO 2 (CR'R") 1 _ 3 H, O(CR'R") 1 _ 3 (CO)NH 2 , O(CR'R") ⁇ _ 3 (CNH)NH 2 , OCOCO 2 H, O(CR'R")!_ 3 SO 3 H, O(CR'R") ⁇ _3 ⁇ S ⁇ 3 H, O(CR'R") 1 _ 3 PO 3 H, O(CR'R")i- 3 OPO3H, O(CR'R") ⁇ -3N[(CR , R") ⁇ -3H]2, O(CR'R") ⁇ _ 3 (CO)(NHOH), and 0(CR'R") ⁇ - 3 (heteroaryl);
• R' and R" are each independently H, a C1-C3 alkyl, C 2 -C 3 alkenyl, or C 2 -C 3 alkynyl group.
• Each R' and R" is preferably H or CH 3 .
• R 1 of formula Va is the heteroaryl group may be a pynolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• R 2 of formula Va maybe a substituted or unsubstituted phenyl, pynolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• R 6 of formula Va is H, (CR'R") ⁇ - 3 H, (CR'R") ⁇ - 3 OH, (CR'R") ⁇ -3NH 2 , (NOH)(CR'R") 1 _ 3 H, CO(CR'R") 0 - 3 NH 2 , CO(CR'R") 1 _ 3 H, CO(CR'R") ⁇ _ 3 OH, CO(CR'R")o_ 3 CF 3 , (CR'R") 0 - 3 N[(CR'R")o_ 3 H] 2 , CO(substituted or unsubstituted heteroaryl), CO(C 3 -C 6 substituted or unsubstituted cycloalkyl), O(CR'R") ! _ 3 H,
• R' and R" is independently H or CH 3 .
• R 6 of formula Va is CO(substituted or unsubstituted heteroaryl), wherein said heteroaryl group is a pynolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• R 4 , R 5 , and R 7 are each hydrogen; R 6 is NO 2 , and R 1 is hydroxyl.
• R 2 is substituted aryl, e.g., substituted phenyl, substituted furanyl, or substituted benzoimidazole.
• R 2 when R is substituted phenyl, R 2 is substituted with an optionally substituted arylcarbonylamino group, an amino group, a dialkyl amino group, or a carboxylate group. The aryl carbonylamino group may be substituted with dialkyl amino, alkyl, or halogens.
• R 2 when R 2 is a substituted furanyl group, R 2 is substituted with an aryl group, e.g., phenyl.
• R 2 when R 2 is an optionally substituted benzoimidazole, it is substituted with an alkyl group.
• the transcription factor modulating compound e.g., an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, etc.
• the transcription factor modulating compound is of the formula (VI):
• G 1 , G 2 , and G 3 are each independently O, S, substituted or unsubstituted nitrogen, or substituted or unsubstituted carbon;
• E 1 , E 2 , and E 3 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, or acyl;
• E 4 is alkyl, alkenyl, alkynyl, aryl, halogen, cyano, amino, nitro, or acyl, and pharmaceutically acceptable salts thereof.
• G 1 , G 2 and G 3 are each oxygen.
• transcription factor modulating compounds are shown in Table 3.
• the invention pertains to each of these compounds, methods (both therapeutic and otherwise) using each of the compounds, and compositions comprising at least one of the compounds of Table 4, Table 5, Table 6, Table 7, Table 8 or of formulae (I), (TT), (m), (W), (y), (v ⁇ ), (v ⁇ ), ( ⁇ ), ( ⁇ x) or(X).
• the invention also pertains to each of the following compounds:
• 6-Nitro-2-phenyl-l-(3-trifluoromethyl-benzyloxy)-lH-benzoimidazole 6-Nitro-2-phenyl-l-(3-trifluoromethyl-benzyloxy)-lH-benzoimidazole; (6-Nitro-2-phenyl-benzoimidazol- 1 -yloxy)-acetic acid; 1 -Benzyloxy-6-nitro-2-phenyl- 1 H-benzoimidazole; l-(4-Methyl-benzyloxy)-6-nitro-2-phenyl-lH-benzoimidazole;
• the transcription factor modulating compound is not apigenin.
• alkyl includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
• straight-chain alkyl groups e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,
• alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.
• a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C ! -C 6 for straight chain, C 3 - for branched chain), and more preferably 4 or fewer.
• prefened cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring stracture.
• Q-Ce includes alkyl groups containing 1 to 6 carbon atoms.
• alkyl includes both "unsubstituted alkyls" and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
• substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sul
• Cycloalkyls can be further substituted, e.g., with the substituents described above.
• An "alkylaryr or an “arylalkyi” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
• the term “alkyl” also includes the side chains of natural and unnatural amino acids.
• aryl includes groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, phenyl, pynole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
• aryl includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine.
• aryl groups having heteroatoms in the ring stracture may also be refened to as "aryl heterocycles", “heterocycles,” “heteroaryls” or “heteroaromatics”.
• the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminoacarbonyl, arylalkyi aminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
• aryl also includes multicyclic aryl groups such as porphrins, phthalocyanines, etc.
• alkenyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond.
• alkenyl includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups.
• alkenyl includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonen
• alkenyl further includes alkenyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.
• a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain).
• cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
• C 2 -C 6 includes alkenyl groups containing 2 to 6 carbon atoms.
• alkenyl includes both "unsubstituted alkenyls" and “substituted alkenyls”, the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
• substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
• alkynyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond.
• alkynyl includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups.
• alkynyl further includes alkynyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon
• a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain).
• C 2 -C 6 includes alkynyl groups containing 2 to 6 carbon atoms.
• alkynyl includes both "unsubstituted alkynyls" and
• substituted alkynyls refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
• substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino,
• lower alkyl as used herein means an alkyl group, as defined above, but having from one to five carbon atoms in its backbone structure.
• Lower alkenyl and “lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.
• acyl includes compounds and moieties which contain the acyl radical (CH 3 CO-) or a carbonyl group.
• substituted acyl includes acyl groups where one or more of the hydrogen atoms are replaced by for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, ary
• acylamino includes moieties wherein an acyl moiety is bonded to an amino group.
• the term includes alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.
• aroyl includes compounds and moieties with an aryl or heteroaromatic moiety bound to a carbonyl group. Examples of aroyl groups include phenylcarboxy, naphthyl carboxy, etc.
• alkoxyalkyl examples include alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.
• alkoxy includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom.
• alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups.
• substituted alkoxy groups include halogenated alkoxy groups.
• the alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate
• amine or "amino” includes compounds where a nitrogen atom is covalently bonded to at least one carbon or heteroatom.
• alkyl amino includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group.
• dialkyl amino includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups.
• arylamino and “diarylamino” include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively.
• alkylarylamino alkylaminoaryl or “arylaminoalkyl” refers to an amino group which is bound to at least one alkyl group and at least one aryl group.
• alkaminoalkyl refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.
• amide or “aminocarboxy” includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group.
• alkaminocarboxy groups which include alkyl, alkenyl, or alkynyl groups bound to an amino group bound to a carboxy group. It includes arylaminocarboxy groups which include aryl or heteroaryl moieties bound to an amino
• alkylaminocarboxy alkenylaminocarboxy
• alkynylaminocarboxy alkynylaminocarboxy
• arylaminocarboxy include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties, respectively, are bound to a nitrogen atom which is in turn bound to the carbon of a carbonyl group.
• carbonyl or “carboxy” includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom.
• moieties which contain a carbonyl include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.
• thiocarbonyl or “thiocarboxy” includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom.
• ether includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms.
• alkoxyalkyl which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.
• esters includes compounds and moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group.
• ester includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.
• alkyl, alkenyl, or alkynyl groups are as defined above.
• thioether includes compounds and moieties which contain a sulfur atom bonded to two different carbon or hetero atoms.
• examples of thioethers include, but are not limited to alkthioalkyls, alkthioalkenyls, and alkthioalkynyls.
• alkthioalkyls include compounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bonded to an alkyl group.
• alkthioalkenyls and alkthioalkynyls refer to compounds or moieties wherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkynyl group.
• hydroxy or “hydroxyl” includes groups with an -OH or -O " .
• halogen includes fluorine, bromine, chlorine, iodine, etc.
• perhalogenated generally refers to a moiety wherein all hydrogens are replaced by halogen atoms.
• polycyclyl or “polycyclic radical” refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
• Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
• alkylaminoacarboiiyl arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, arylalkyi carbonyl, alkenylcarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
• heteroatom includes atoms of any element other than carbon or hydrogen. Prefened heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
• the structure of some of the compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof.
• Bonds represented by " " in a structural formula mean that the bond may be either a single or a double bond.
• compositions which include a therapeutically- effective amount or dose of a transcription factor modulating compound and/or a compound identified in any of the instant assays and one or more carriers (e.g., pharmaceutically acceptable additives and/or diluents).
• the pharmaceutical compositions of the invention may comprise any compound described in this application as a transcription factor modulating compound, an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, a MarA family inhibiting compound, a MarA inhibiting compound, compounds of formulae (I), (TT), (TTT), (TV), (V), (VI), (VU), (VTTT), (TK), (X), Table 4, Table 5, Table 6, Table 7, Table 8, scaffold, etc.
• a composition can also include a second antimicrobial agent, e.g., an antibiotic.
• the invention pertains to pharmaceutical compositions comprising an effective amount of a transcription factor modulating compound (e.g., a MarA family polypeptide modulating compound or an AraC family polypeptide modulating compound), and a pharmaceutically acceptable carrier.
• a transcription factor modulating compound e.g., a MarA family polypeptide modulating compound or an AraC family polypeptide modulating compound
• a pharmaceutically acceptable carrier e.g., a transcription factor modulating compound, e.g., a MarA family polypeptide modulating compound or an AraC family polypeptide modulating compound
• the transcription factor modulating compound is of the formula (U):
• W is O or S
• X is O, S, or C, optionally linked to Q;
• a 1 is C-Z 4 , O, or S;
• a 2 is C-Z 5 , or N-Z 5 ;
• Z 1 , Z 2 , Z 3 , Z 4 and Z 5 are each independently hydrogen, alkoxy, hydroxy, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic, amino, or cyano;
• Z 3 is hydrogen, alkoxy, hydroxy, halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic, amino, nitro, cyano, carbonyl, or thiocarbonyl;
• Q is an aromatic or heterocyclic moiety, and pharmaceutically acceptable salts thereof.
• compositions of the invention include an effective amount of a transcription factor modulating compound of the formula (Ifl):
• G is substituted or unsubstituted aromatic moiety, heterocyclic, alkyl, alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, or hydrogen;
• L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , and L 10 are each independently oxygen, substituted or unsubstituted nitrogen, sulfur and or substituted or unsubstituted carbon, and pharmaceutically acceptable salts thereof.
• compositions of the invention include a pharmaceutically acceptable carrier (optional) and an effective amount of a transcription factor modulating compound of the formula (TV):
• Y 1 and Y 2 are each oxygen or sulfur
• J is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl, cyano, nitro, amino, or halogen;
• V is substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, alkylamino, or alkylthio;
• P and K are each independently substituted or unsubstituted aryl, and pharmaceutically acceptable salts thereof.
• compositions of the invention include a pharmaceutically acceptable carrier (optional) and an effective amount of a transcription factor modulating compound of the formula (V):
• T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently substituted or unsubstituted carbon, oxygen, substituted or unsubstituted nitrogen, or sulfur;
• compositions of the invention include a pharmaceutically acceptable carrier (optional) and an effective amount of a transcription factor modulating compound of the formula (Va):
• R 1 is OH, OCOCO H, or a substituted or unsubstituted straight or branched C C 5 alkyloxy group
• R 2 is H, CO 2 (Ci-C 5 substituted or unsubstituted, straight or branched alkyl), or a substituted or unsubstituted aryl group;
• R 4 , R 5 , R 6 , and R 7 are independently selected from the group consisting of H, (C 1 -C 5 substituted or unsubstituted, straight or branched alkyl), CO 2 (C ⁇ -C 5 substituted or unsubstituted, straight or branched alkyl), CO(C ⁇ -C 5 substituted or unsubstituted, straight or branched alkyl), CO(substituted or unsubstituted aryl or heteroaryl), CO(C 3 -C 6 substituted or unsubstituted cycloalkyl), O ⁇ -Cs substituted or unsubstituted, straight or branched alkyl), C(NOH)(CrC 5 substituted or unsubstituted, straight or branched alkyl), substituted or unsubstituted amino, CO 2 H, CN, NO 2 , CONH 2 , (CO)(NHOH), and halogen.
• R 1 is not O(CHCH 3 )(CO 2 )CH 2 CH 3 or OCH 2 CO 2 H.
• R 6 is H or NO
• R 1 is not a phenyl-substituted alkyloxy group.
• R , R , R , and R are all H and R is para- methoxyphenyl, then R 1 is not OH.
• R 1 is not OCH 2 CO 2 CH 2 CH 3 ; hi certain aspects of formula Va, R 4 , R 5 , and R 7 are all H.
• R 1 of formula Va maybe selected from the group consisting of OH, O CR'R' sH, O(CR'R") !
• the heteroaryl group may be a pynolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• R 2 of formula Va may be a substituted or unsubstituted phenyl, pynolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• R 6 of formula Va is H, (CR'R") ⁇ _ 3 H, (CR'R") ⁇ _ 3 OH, (CR'R") ⁇ -3NH 2 , (NOH)(CR'R") ⁇ _ 3 H, CO(CR'R")o_ 3 NH 2 , CO(CR'R") 1 _ 3 H, CO(CR'R") 1 _ 3 OH, CO(CR'R'V 3 CF 3 , (CR'R")o- 3 N[(CR'R")o-3H] 2 , CO(substituted or unsubstituted heteroaryl), CO(C 3 -C 6 substituted or unsubstituted cycloalkyl), 0(CR'R") ⁇ -3H,
• R' and R" is independently H or CH 3 .
• R 6 of formula Va is CO(substituted or unsubstituted heteroaryl), wherein said heteroaryl group is a pynolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• said heteroaryl group is a pynolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.
• R 1 is OH, OCOCO 2 H, or a substituted straight or branched C 1 -C 5 alkyloxy group, provided that R 1 is not a 2-amino- substituted ethoxy group or a substituted or unsubstituted benzyloxy group; R is H, CO 2 (C !
• R 4 , R 5 , R 6 , and R 7 are independently selected from the group consisting of H, (Ci-C 5 substituted or unsubstituted, straight or branched alkyl), CO 2 (C C5 substituted or unsubstituted, straight or branched alkyl), CO(CrC 5 substituted or unsubstituted, straight or branched alkyl), CO(substituted or unsubstituted aryl or heteroaryl), CO(C 3 - C 6 substituted or unsubstituted cycloalkyl), O(C C5 substituted or unsubstituted, straight or branched alkyl), C(NOH)(Ci-C 5 substituted or unsubstituted or unsubstit
• R 1 is not OH; and provided that when R 4 , R 5 , R 6 , and R 7 are all H and R 2 is unsubstituted phenyl, or when R 4 , R 5 , and R 7 are all H, R 6 is CI, and R 2 is ⁇ ra-methyl-phenyl, then R 1 is not OCH 2 CO 2 CH 2 CH 3 .
• compositions of the invention include a pharmaceutically acceptable carrier (optional) and an effective amount of a transcription factor modulating compound of the formula (VI):
• G , G , and G are each independently O, S, substituted or unsubstituted nitrogen, or substituted or unsubstituted carbon;
• E 1 , E 2 , and E 3 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, or acyl;
• E 4 is alkyl, alkenyl, alkynyl, aryl, halogen, cyano, amino, nitro, or acyl, and pharmaceutically acceptable salts thereof.
• compositions of the invention comprise an effective amount of a transcription factor modulating compound listed below or found in Table 4, Table 5, Table 6, Table 7, Table 8: 2-(4-isopropylphenyl)-4H-chromen-4-one; 2-(3,4-Dihydroxy-phenyl)-3,5,7-trihydroxy-chromen-4-one N-isopropyl-2- [(4-methyl-5-quinolin-6-yl-4H- 1 ,2,4-triazol-3 - yl)thio] acetamide;
• Cobalt 5,10,15 ,20-Tetra-pyridin-3 -yl-po ⁇ hyrine; Zinc 5, 10, 15,20-Tetra- ⁇ yridin-4-yl-porphyrine; 2-(4-hydroxyphenyl)-4H-chromen-4-one, and pharmaceutically acceptable salts thereof.
• the method for preventing a bacterial associated state in a subject comprising administering to the subject an effective amount of a transcription factor modulating compound, such that the bacterial associated state is prevented.
• a transcription factor modulating compound such that the bacterial associated state is prevented.
• subject includes plants and animals (e.g., vertebrates, amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep, rodents, rabbits, squinels, bears, primates (e.g., chimpanzees, gorillas, and humans) which are capable of suffering from a bacterial associated disorder.
• the term “subject” also comprises immunocompromised subjects, who may be at a higher risk for infection.
• preventing the administration of an effective amount of the transcription factor modulating compound to prevent a bacterial associated state from occurring.
• bacterial associated state includes states characterized by the presence of bacteria which can be prevented by administering the transcription factor modulating compounds of the invention.
• the term includes biofilm associated states and other infections or the undesirable presence of a bacteria on or in a subject.
• the pharmaceutical compositions can be formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, aqueous or non-aqueous solutions or suspensions, tablets, boluses, powders, granules, pastes; (2) parental admimsfration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary,
• aerosol for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
• pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the antiinfective agents or compounds of the invention from one organ, or portion of the body, to another organ, or portion of the body without affecting its biological effect.
• Each carrier should be “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject.
• materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum, such
• compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microbes maybe ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microbes maybe ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions
• a drag in order to prolong the effect of a drag, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drag then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drag in an oil vehicle.
• compositions of the present invention maybe administered to epithelial surfaces of the body orally, parenterally, topically, rectally, nasally, intravaginally, intracisternaliy. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal or vaginal suppositories.
• parenteral administration and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transfracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
• systemic administration means the administration of a sucrose octasulfate and/or an antibacterial, drug or other material other than directly into the central nervous system, such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
• compositions of the invention can be topically administered to any epithelial surface.
• An "epithelial surface” is defined as an area of tissue that covers external surfaces of a body, or which lines hollow structures including, but not limited to, cutaneous and mucosal surfaces.
• Such epithelial surfaces include oral, pharyngeal, esophageal, pulmonary, ocular, aural, nasal, buccal, lingual, vaginal, cervical, genitourinary, alimentary, and anorectal surfaces.
• Compositions can be formulated in a variety of conventional forms employed for topical admimsfration.
• semi-solid and liquid dosage forms such as liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions, slurries, powders, sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms, douches, drops, troches, chewing gums, lozenges, mouthwashes, rinses.
• semi-solid and liquid dosage forms such as liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions, slurries, powders, sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms, douches, drops, troches, chewing gums, lozenges, mouthwashes, rinses.
• Conventionally used carriers for topical applications include pectin, gelatin and derivatives thereof, polylactic acid or polyglycolic acid polymers or copolymers thereof, cellulose derivatives such as methyl cellulose, carboxymethyl cellulose, or oxidized cellulose, guar gum, acacia gum, karaya gum, tragacanth gum, bentonite, agar, carbomer, bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum, hectorite, ispaghula husk, polyvinypynolidone, silica and derivatives thereof, xanthan gum, kaolin, talc, starch and derivatives thereof, paraffin, water, vegetable and animal oils, polyethylene, polyethylene oxide, polyethylene glycol, polypropylene glycol,
• glycerol ethanol, propanol, propylene glycol (glycols, alcohols), fixed oils, sodium, potassium, aluminum, magnesium or calcium salts (such as chloride, carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptate or tarfrate).
• compositions can be particularly useful, for example, for treatment or prevention of an unwanted cell, e.g., vaginal Neisseria gonorrhoeae, or infections of the oral cavity, including cold sores, infections of eye, the skin, or the lower intestinal tract.
• Standard composition strategies for topical agents can be applied to the antiinfective compounds or a pharmaceutically acceptable salt thereof in order to enhance the persistence and residence time of the drug, and to improve the prophylactic efficacy achieved.
• a rectal suppository for topical application to be used in the lower intestinal tract or vaginally, a rectal suppository, a suitable enema, a gel, an ointment, a solution, a suspension or an insert can be used.
• Topical transdermal patches may also be used.
• Transdermal patches have the added advantage of providing controlled delivery of the compositions of the invention to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium.
• compositions of the invention can be administered in the form of suppositories for rectal or vaginal administration.
• suppositories for rectal or vaginal administration.
• These can be prepared by mixing the agent with a suitable non-irritating carrier which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum or vagina to release the drag.
• suitable non-irritating carrier which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum or vagina to release the drag.
• suitable non-irritating carrier which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum or vagina to release the drag.
• suitable non-irritating carrier which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum or vagina to release the drag.
• Such materials include cocoa butter, beeswax, polyethylene glycols, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at
• compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, films, or spray compositions containing such carriers as are known in the art to be appropriate.
• the carrier employed in the sucrose octasulfate /contraceptive agent should be compatible with vaginal administration and/or coating of contraceptive devices.
• Combinations can be in solid, semi-solid and liquid dosage forms, such as diaphragm, jelly, douches, foams, films, ointments, creams, balms, gels, salves, pastes, slurries, vaginal suppositories, sexual lubricants, and coatings for devices, such as condoms, contraceptive sponges, cervical caps and diaphragms.
• solid, semi-solid and liquid dosage forms such as diaphragm, jelly, douches, foams, films, ointments, creams, balms, gels, salves, pastes, slurries, vaginal suppositories, sexual lubricants, and coatings for devices, such as condoms, contraceptive sponges, cervical caps and diaphragms.
• the pharmaceutical compositions can be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
• the compositions can be formulated in an ointment such as petrolatum.
• Exemplary ophthalmic compositions include eye ointments, powders, solutions and the like.
• Powders and sprays can contain, in addition to sucrose octasulfate and/or antibiotic or contraceptive agent(s), carriers such as lactose, talc, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
• Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
• an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers.
• the carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.
• Aerosols generally are prepared from isotonic solutions.
• compositions of the invention can also be orally administered in any orally-acceptable dosage form including, but not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of sucrose octasulfate and/or antibiotic or contraceptive agent(s) as an active ingredient.
• capsules such as gelatin and glycerin, or sucrose and acacia
• a compound may also be administered as a bolus, electuary or paste.
• carriers which are commonly used include lactose and com starch.
• Lubricating agents such as magnesium stearate, are also typically added.
• useful diluents include lactose and dried com starch.
• the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
• Tablets, and other solid dosage forms such as dragees, capsules, pills and granules, maybe scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the phamiaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyhnethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
• compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
• These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tefrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
• the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
• adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
• Suspensions in addition to the antiinfective agent(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• Sterile injectable forms of the compositions of this invention can be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
• a nontoxic parenterally-acceptable diluent or solvent for example as a solution in 1,3-butanediol.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution, h addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono-or di-glycerides.
• Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
• oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
• These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.
• the antiinfective agent or a pharmaceutically acceptable salt thereof will represent some percentage of the total dose in other dosage forms in a material forming a combination product, including liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions slurries, soaps, shampoos, detergents, powders, sprays, lipsticks, foams, pastes, toothpastes, ointments, salves, balms, douches, drops, troches, lozenges, mouthwashes, rinses and others.
• Creams and gels for example, are typically limited by the physical chemical properties of the delivery medium to concentrations less than 20% (e.g., 200 mg/gm).
• the pharmaceutical composition of the invention can comprise sucrose octasulfate in an amount of 0.001-99%, typically 0.01-75%, more typically 0.1-20%, especially 1-10% by weight of the total preparation.
• a prefened concentration thereof in the preparation is 0.5-50%, especially 0.5-25%, such as 1-10%. It can be suitably applied 1-10 times a day, depending on the type and severity of the condition to be treated or prevented.
• the pharmaceutical composition of the invention can be applied prior to potential infection.
• the timing of application prior to potential infection can be optimized to maximize the prophylactic effectiveness of the compound.
• the timing of application will vary depending on the mode of administration, the epithelial surface to which it is applied, the surface area, doses, the stability and effectiveness of composition under the pH of the epithelial surface, the frequency of application, e.g., single application or multiple applications.
• One skilled in the art will be able to determine the most appropriate time interval required to maximize prophylactic effectiveness of the compound.
• the practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, genetics, microbiology, recombinant D ⁇ A, and immunology, which are within the skill of the art.
• the invention pertains to a method for dispersing or preventing the formation of a biofilm on a surface or in an area, by administering an effective amount of a transcription factor modulating compound, e.g., a HTH protein modulating compound, an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, or a MarA inhibiting compound.
• a transcription factor modulating compound e.g., a HTH protein modulating compound, an AraC family polypeptide modulating compound, a MarA family polypeptide modulating compound, or a MarA inhibiting compound.
• biofilm includes biological films that develop and persist at interfaces in aqueous and other environments. Biofilms are composed of microorganisms embedded in an organic gelatinous structure composed of one or more matrix polymers which are secreted by the resident microorganisms.
• biofilm also includes bacteria that are attached to a surface in sufficient numbers to be detected or communities of microorganisms attached to a surface (Costerton, J. W., et al. (1987) Ann. Rev. Microbiol. 41 :435-464; Shapiro, J. A. (1988) Sci Am. 256:82-89; O'Toole, G. et al. (2000) Annu Rev Microbiol. 54:49-79).
• the invention pertains to methods of treating biofilm associated states in a subject, by administering to said subject an effective amount of a transcription factor modulating compound, e.g., a MarA family inhibiting compound, such that the biofilm associated state is treated.
• a transcription factor modulating compound e.g., a MarA family inhibiting compound
• biofilm associated states includes disorders which are characterized by the presence or potential presence of a bacterial biofilm. Many medically important pathogens form biofilms and biofilm formation is often one component of the infectious process (Costerton, J. W. et al. (1999) Science 284:1318-1322). Examples of biofilm associated states include, but are not limited to, middle ear infections, cystic fibrosis, osteomyelitis, acne, dental cavities, and prostatitis. Biofilm associated states also include infection of the subject by one or more bacteria, e.g., Pseudomonas aeruginosa.
• biofilm formation bacteria within biofilms are generally less susceptible to a range of different antibiotics relative to their planktonic counterparts.
• the invention also pertains to methods for preventing the formation of biofilms on surfaces or in areas, by contacting the area with an effective amount of a transcription factor modulating compound, e.g., a MarA family inhibiting compound, etc.
• a transcription factor modulating compound e.g., a MarA family inhibiting compound, etc.
• AraC family inhibiting compounds and MarA family inhibiting compounds, of the present invention are useful in a variety of environments including industrial, clinical, the household, and personal care.
• the compositions of the invention may comprise one or more compounds of the invention as an active ingredient acting alone, additively, or synergistically against the target organism.
• the MarA family inhibiting compounds and modulating compounds of the invention may be formulated in a composition suitable for use in environments including industry, pharmaceutics, household, and personal care, hi an embodiment, the compounds of the invention are soluble in water.
• the modulating compounds may be applied or delivered with an acceptable carrier system.
• the composition may be applied or delivered with a suitable carrier system such that the active ingredient (e.g., transcription factor modulating compound of the invention such as a MarA family modulating compound, e.g., a MarA family polypeptide inhibiting compound) maybe dispersed or dissolved in a stable manner so that the active ingredient, when it is administered directly or indirectly, is present in a form in which it is available in a advantageous way.
• the active ingredient e.g., transcription factor modulating compound of the invention such as a MarA family modulating compound, e.g., a MarA family polypeptide inhibiting compound
• compositions of the invention may be preblended or each component may be added separately to the same environment according to a predetermined dosage for the purpose of achieving the desired concentration level of the treatment components and so long as the components eventually come into intimate admixture with each other.
• present invention may be administered or delivered on a continuous or intermittent basis.
• a franscription factor modulating compound when present in a composition will generally be present in an amount from about 0.000001% to about 100%, more preferably from about 0.001% to about 50%, and most preferably from about 0.01% to about 25%.
• compositions of the present invention comprising a carrier
• the composition comprises, for example, from about 1% to about 99%>, preferably from about 50% to about 99%, and most preferably from about 75% to about 99% by weight of at least one carrier.
• the franscription factor modulating compound, e.g., the MarA family polypeptide inhibiting compound, of the invention may be formulated with any suitable carrier and prepared for delivery in forms, such as, solutions, microemulsions, suspensions or aerosols. Generation of the aerosol or any other means of delivery of the present invention may be accomplished by any of the methods known in the art. For example, in the case of aerosol delivery, the compound is supplied in a finely divided form along with any suitable carrier with a propellant.
• Liquefied propellants are typically gases at ambient conditions and are condensed under pressure.
• the propellant may be any acceptable and known in the art including propane and butane, or other lower alkanes, such as those of up to 5 carbons.
• the composition is held within a container with an appropriate propellant and valve, and maintained at elevated pressure until released by action of the valve.
• compositions of the invention maybe prepared in a conventional form suitable for, but not limited to topical or local application such as an ointment, paste, gel, spray and liquid, by including stabilizers, peneverss and the carrier or diluent with the compound according to a known technique in the art.
• These preparations may be prepared in a conventional form suitable for enteral, parenteral, topical or inhalational applications.
• the present invention may be used in compositions suitable for household use.
• compounds of the present invention are also useful as active antimicrobial ingredients in household products such as cleansers, detergents, disinfectants, dishwashing liquids, soaps and detergents.
• the transcription factor modulating compound of the present invention may be delivered in an amount and form effective for the prevention, removal or termination of microbes.
• the compositions of the invention for household use comprise, for example, at least one franscription factor modulating compound of the invention and at least one suitable carrier.
• the composition may comprise from about 0.00001% to about 50%, preferably from about 0.0001% to about 25%, most preferably
• the transcription factor modulating compound of the present invention may also be used in hygiene compositions for personal care.
• compounds of the invention can be used as an active ingredient in personal care products such as facial cleansers, astringents, body wash, shampoos, conditioners, cosmetics and other hygiene products.
• the hygiene composition may comprise any carrier or vehicle known in the art to obtain the desired form (such as solid, liquid, semisolid or aerosol) as long as the effects of the compound of the present invention are not impaired.
• Methods of preparation of hygiene compositions are not described herein in detail, but are known in the art. For its discussion of such methods, The CTFA Cosmetic Ingredient Handbook. Second Edition, 1992, and pages 5-484 of A Formulary of Cosmetic Preparations (Vol. 2, Chapters 7-16) are incorporated herein by reference.
• the hygiene composition for use in personal care comprise generally at least one modulating compound of the present application and at least one suitable carrier.
• the composition may comprise from about 0.00001% to about 50%, preferably from about 0.0001% to about 25%, more preferably from about 0.0005% to about 10% by weight of the franscription factor modulating compound of the invention based on the weight percentage of the total composition.
• the transcription factor modulating compound of the present invention may be used in industry. In the industrial setting, the presence of microbes can be problematic, as microbes are often responsible for industrial contamination and biofouling.
• Compositions of the invention for industrial applications may comprise an effective amount of the compound of the present invention in a composition for industrial use with at least one acceptable carrier or vehicle known in the art to be useful in the treatment of such systems.
• Such carriers or vehicles may include diluents, deflocculating agents, peneverss, spreading agents, surfactants, suspending agents, wetting agents, stabilizing agents, compatibility agents, sticking agents, waxes, oils, co- solvents, coupling agents, foams, antifoaming agents, natural or synthetic polymers, elastomers and synergists.
• Methods of preparation, delivery systems and carriers for such compositions are not described here in detail, but are known in the art. For its discussion of such methods, U.S. Patent No. 5,939,086 is herein incorporated by reference.
• the prefened amount of the composition to be used may vary according to the active ingredient(s) and situation in which the composition is being applied.
• the transcription factor modulating compounds e.g., MarA family polypeptide inhibiting compounds, and compositions of the present invention maybe useful in nonaqueous environments.
• Such nonaqueous environments may include, but
• - 97 - are not limited to, tenestrial environments, dry surfaces or semi-dry surfaces in which the compound or composition is applied in a manner and amount suitable for the situation.
• the transcription factor modulating compounds e.g., MarA family polypeptide modulating compounds, e.g., MarA inhibiting compounds, of the present invention may be used to form contact-killing coatings or layers on a variety of substrates including personal care products (such as toothbrushes, contact lens cases and dental equipment), healthcare products, household products, food preparation surfaces and packaging, and laboratory and scientific equipment.
• substrates include medical devices such as catheters, urological devices, blood collection and transfer devices, tracheotomy devices, intraocular lenses, wound dressings, sutures, surgical staples, membranes, shunts, gloves, tissue patches, prosthetic devices (e.g., heart valves) and wound drainage tubes.
• other substrates include textile products such as carpets and fabrics, paints and joint cement. A further use is as an antimicrobial soil fumigant.
• the transcription factor modulating compounds of the invention may also be incorporated into polymers, such as polysaccharides (cellulose, cellulose derivatives, starch, pectins, alginate, chitin, guar, canageenan), glycol polymers, polyesters, polyurethanes, polyacrylates, polyacrylonitrile, polyamides (e.g., nylons), polyolefins, polystyrenes, vinyl polymers, polypropylene, silks or biopolymers.
• the modulating compounds may be conjugated to any polymeric material such as those with the following specified functionality: 1) carboxy acid, 2) amino group, 3) hydroxyl group and/or 4) haloalkyl group.
• composition for treatment of nonaqueous environments maybe comprise at least one transcription factor modulating compound of the present application and at least one suitable carrier.
• the composition comprises from about 0.001% to about 75%, advantageously from about 0.01% to about 50%, and preferably from about 0.1% to about 25% by weight of a transcription factor modulating compound of the invention based on the weight percentage of the total composition.
• the transcription factor modulating compounds and compositions of the invention may also be useful in aqueous environments.
• “Aqueous environments” include any type of system containing water, including, but not limited to, natural bodies of water such as lakes or ponds; artificial, recreational bodies of water such as swimming pools and hot tubs; and drinking reservoirs such as wells.
• the compositions of the present invention may be useful in treating microbial growth in these aqueous environments and maybe applied, for example, at or near the surface of water.
• compositions of the invention for treatment of aqueous environments may comprise at least one transcription factor modulating compound of the present invention and at least one suitable carrier.
• the composition comprises from about 0.001%) to about 50%, advantageously from about 0.003% to about 15%, preferably from about 0.01% to about 5% by weight of the compound of the invention based on the weight percentage of the total composition.
• the present invention also provides a process for the production of an antibiofouling composition for industrial use.
• Such process comprises bringing at least one of any industrially acceptable carrier known in the art into intimate admixture with a transcription factor modulating compound of the present invention.
• the carrier may be any suitable carrier discussed above or known in the art.
• the suitable antibiofouling compositions may be in any acceptable form for delivery of the composition to a site potentially having, or having at least one living microbe.
• the antibiofouling compositions may be delivered with at least one suitably selected carrier as hereinbefore discussed using standard formulations.
• the mode of delivery may be such as to have a binding inhibiting effective amount of the antibiofouling composition at a site potentially having, or having at least one living microbe.
• the antibiofouling compositions of the present invention are useful in treating microbial growth that contributes to biofouling, such as scum or slime formation, in these aqueous environments. Examples of industrial processes in which these compounds might be effective include cooling water systems, reverse osmosis membranes, pulp and paper systems, air washer systems and the food processing industry.
• the antibiofouling composition may be delivered in an amount and form effective for the prevention, removal or termination of microbes.
• the antibiofouling composition of the present invention generally comprise at least one compound of the invention.
• the composition may comprise from about 0.001% to about 50%, more preferably from about 0.003% to about 15%, most preferably from about 0.01% to about 5% by weight of the compound of the invention based on the weight percentage of the total composition.
• the amount of antibiofouling composition may be delivered in an amount of about 1 mg/1 to about 1000 mg/1, advantageously from about 2 mg/1 to about 500 mg/1, and preferably from about 20 mg/1 to about 140 mg/1.
• Antibiofouling compositions for water treatment generally comprise franscription factor modulating compounds of the invention in amounts from about 0.001% to about 50% by weight of the total composition.
• Other components in the antibiofouling compositions may include, for example, 2-bromo- 2-nitropropane-l,3-diol (BNPD), ⁇ -nifrostyrene (BNS), dodecylguanidine hydrochloride, 2,2-dibromo-3-nitrilopropionamide (DBNPA), glutaraldehyde, isothiazolin, methylene
• compositions of the invention include biodispersants (about 0.1% to about 15% by weight of the total composition), water, glycols (about 20-30%) or Pluronic (at approximately 7% by weight of the total composition).
• concentration of antibiofouling composition for continuous or semi- continuous use is about 5 to about 70 mg/1.
• Antibiofouling compositions for industrial water treatment may comprise compounds of the invention in amounts from about 0.001% to about 50% based on the weight of the total composition.
• the amount of compound of the invention in antibiofouling compositions for aqueous water treatment may be adjusted depending on the particular environment. Shock dose ranges are generally about 20 to about 140 mg/1; the concentration for semi-continuous use is about 0.5X of these concentrations.
• the invention also pertains, at least in part, to a method of regulating biofilm development.
• the method includes administering a composition which contains a transcription factor modulating compound of the invention.
• the composition can also include other components which enhance the ability of the composition to degrade biofilms.
• compositions can be formulated as a cleaning product, e.g., a household or an industrial cleaner to remove, prevent, inhibit, or modulate biofilm development.
• biofilm is adversely affected by the administration of the compound of the invention, e.g., biofilm development is diminished.
• compositions may include compounds such as disinfectants, soaps, detergents, as well as other surfactants.
• surfactants include, for example, sodium dodecyl sulfate; quaternary ammonium compounds; alkyl pyridinium iodides; TWEEN 80, TWEEN 85, TRITON X-100; BRIJ 56; biological surfactants; rhamnolipid, surfactin, visconsin, and sulfonates.
• composition of the invention may be applied in known areas and surfaces where disinfection is required, including but not limited to drains, shower curtains, grout, toilets and flooring. A particular application is on hospital surfaces and medical instruments.
• the disinfectant of the invention may be useful as a disinfectant for bacteria such as, but not limited to, Pseudomonadaceae, Azatobacteraceae, Rhizabiaceae, Mthylococcaceae, Halobaderiaceae, Acetobaderaceae, Legionellaceae, Neisseriaceae, and other genera.
• the invention also pertains to a method for cleaning and disinfecting contact lenses.
• the method includes contacting the contact lenses with a solution of at least one compound of the invention in an acceptable carrier.
• the invention also pertains to the solution comprising the compound, packaged with directions for using the solution to clean contact lenses.
• the invention also includes a method of treating medical indwelling devices.
• the method includes contacting at least one compound of the invention with a medical indwelling device, such as to prevent or substantially inhibit the formation of a biofilm.
• medical indwelling devices include catheters, orthopedic devices and implants.
• a dentifrice or mouthwash containing the compounds of the invention may be formulated by adding the compounds of the invention to dentifrice and mouthwash formulations, e.g. , as set forth in Remington 's Pharmaceutical Sciences, 1 Sth Ed., Mack Publishing Co., 1990, Chapter 109 (incorporated herein by reference in its entirety).
• the dentifrice may be formulated as a gel, paste, powder or slurry.
• the dentifrice may include binders, abrasives, flavoring agents, foaming agents and humectants. Mouthwash formulations are known in the art, and the compounds of the invention may be advantageously added to them.
• the invention pertains to each of the transcription factor modulating compounds described herein in Tables 4, 5, 6, 7 and 8, and in Formulae (I)-(X).
• the transcriptional modulating compounds described in this application can be synthesized by art recognized techniques or using the methods described herein.
• alkyl or substituted alkyl halides were used instead of n-butyliodide following the similar method.
• the amide thus prepared, was then stined overnight with methyl triflate in dichloromethane, diluted with ether, and the precipitate was filtered, washed, and dried to obtain an imidatonium triflate salt.
• This salt was dissolved in dichloromethane, cooled to 0 °C, and added slowly, with stirring, to a cold (0 °C) solution of sodium methoxide in dry methanol over a period of ca. 30-60 min. The solvent was evaporated to dryness and the residue was extracted in n-hexane. Upon evaporation of hexane, the white solid was obtained, which was dissolved in dry methanol and treated with glacial acetic acid. After 10 minutes of stirring, the excess acid was neutralized with potassium carbonate (solid), and the solvent removed under vacuum. The residue was extracted in ether, washed with water, and dried over
• the crade material was purified by preparative HPLC.
• the same material can also be prepared by the previous method, (Suzuki coupling conditions) starting from 4-iodo-3-aminothioanisole, and purified by preparative HPLC.
• the hydrochloride salt of the diazepine was dried under vacuum to afford a light yellow solid, hi order to obtain a free base of the diazepine, the above hydrochloride salt was suspended in methanol, and treated with 10% aq. NaOH solution. After stirring at room temperature for ca. 10 min, the precipitate was filtered, washed with water, and dried under vacuum.
• Example 2 In this example, the expression of a selective marker (e.g., ccdB) is put under the direct control of a promoter activated by MarA (e.g. , inaA, galT, or micF). In the absence of MarA, the expression of the selective marker is silent and cells survive. Synthesis of MarA from an inducible plasmid in a bacterial or yeast cell leads to the activation of the inaA promoter, expression of ccdB, and subsequently results in cell death. Compounds that inhibit MarA are those that permit cell survival under conditions of MarA expression. The results of this assay are given in Table 4. hi Table 4, * indicates good inhibition of MarA and ** indicates very good inhibition of MarA.
• a selective marker e.g., ccdB
• Example 3 hi this example, the expression of luciferase is put under the direct control of a promoter activated by MarA (e.g., inaA, galT, or micF) in a cell constitutively expressing MarA. In the absence of MarA, cells luminesce. Upon modulating of MarA activity, the expression of the reporter is altered.
• MarA e.g., inaA, galT, or micF
• a selective marker e.g., ccdB
• MarA e.g., fecA, purA, guaB
• constitutive MarA synthesis e.g., using a constitutive mar (mar c ) mutant
• the expression of ccdB is silent.
• the synthesis of ccdB results in cell death.
• Example 5 hi this example, the expression of a selective marker (e.g., URA3) is put under the direct control of a promoter repressed by MarA (e.g., fecA, purA, guaB). Under conditions of constitutive MarA synthesis (e.g., using a constitutive mar (mar c ) mutant), the expression of URA3 is silent. Following inactivation of MarA, and in the presence of 5-FOA the synthesis of URA3 results in cell death.
• a selective marker e.g., URA3
• MarA e.g., fecA, purA, guaB
• Example 6 hi this example, a purine or guanine heterotroph is constructed by inactivation of either of the chromosomal guaB or purA genes in E. coli. The guaB or purA gene is then placed into a suitable vector under the control of its natural promoter and transformed into the heterofrophic host.
• the biofilm assay screens test compounds for their ability to inhibit bacteria from forming a biofilm.
• M9 M9
• casamino acids a test compound that was dissolved in lOmg/mL DMSO stock solution.
• Inoculum was started the day of the experiment by adding a colony or glycerol stock stab to 2mL M9. The tube was placed in the 37 °C shaker incubator for approximately 4-6 hours. This inoculum was refened to as the "Starter inoculum.” The inoculum was then removed from the shaker incubator and diluted to 1 X 10 6 cells/mL in M9.
• test compounds were screened at 20 ⁇ g/mL. 2.5 ⁇ L of the test compound were taken from a plate containing lOmg/mL stock and added to 200 ⁇ L of M9 and mixed. 25 ⁇ L of the diluted test compound was added to 50 ⁇ L of M9 in the assay plate. The resulting concentration of the test compound was 40 ⁇ g/mL
• the crystal violet was then removed and the plates were washed several times with tap water. 150 ⁇ L of ethanol were then added to each well, after mixing. The
• Example 8 LANCE Screening Assay for Inhibitors of MarA, SoxS, or Rob DNA- binding
• This example describes a method for the identification of test compounds that inhibit the interactions of purified transcription factor such as MarA, SoxS and/or Rob with a target DNA sequence in an in vitro system. Such molecules will hopefully be able to inhibit this interaction in vivo, leading to inhibition of transcriptional regulation by these factors and ultimately in inhibition of the drug resistance and/or virulence phenotypes associated with MarA, SoxS and Rob.
• the N-term-biotinylated double-stranded DNA has a sequence of CCG ATT TAG CAA AAC GTG GCA TCG GTC (SEQ ID NO. 5).
• the antibody used was the LANCE Eu-labeled anti-6xHis Antibody (Eu-oHis) (Perkin Elmer cat # ADOl 10) which had at least 6 Europium molecules per antibody.
• Streptavidin conjugated to SureLightTM-Allo ⁇ hycocyanin (S A-APC) was obtained from Perkin Ehner (cat #
• the Assay buffer contained 20mM Hepes pH 7.6, ImM EDTA, lOmM (NH 4 ) 2 SO 4 , and 30mM KC1, and 0.2% Tween-20.
• the plates or vials of the compounds to be tested were thawed. These stocks were at a concentration of lOmg/ml in DMSO. The solutions were allowed to thaw completely, and the plates were briefly shaken on the Titermix to redissolve any precipitated compound. Thawed aliquots of MarA, SoxS and Rob protein from the stock stored at -80°C and 1M stock of dithiothreitol stored at -20°C were then placed on ice. Dilutions at 1 : 100 of the compounds were made into a fresh 96-well polystyrene plate. The dilutions were prepared with 100% DMSO to give a final concentration of 100 ⁇ g/ml solutions. The dilutions were vortexed on a Titermix.
• Fresh DTT was added to 25-50 mL of assay buffer to produce a ImM final concentration.
• 90 ⁇ l of assay buffer was added to each of the 10 ⁇ l protein aliquots, and the solution was mixed by pipetting.
• These proteins were diluted to give the required amount of each of the diluted proteins, resulting in 20 ⁇ l of diluted protein per well, hi preparing the solutions, 20% excess was made to allow enough for control wells.
• 1000-2000 fmoles of each protein was required per well.
• the diluted protein solutions were the placed on ice.
• the DNA solution was prepared, with enough for at least 20% more wells than were tested. 15 ⁇ l (0.4 fmoles) was added per well. Then the DNA was diluted in assay buffer, and vortexed briefly to mix. The plate sealer was removed, and 15 ⁇ l of DNA solution was added to all of the wells, the plates were then resealed, and returned to the Titermix for a further 30 minutes.
• the antibody solution was prepared. 0.4 fmoles of SA- APC and 0.125 fmoles of Eu-oHis were added per well in a total volume of lO ⁇ l. Amounts were prepared sufficient for at least 20% excess. The plate sealer was the removed and 10 ⁇ l of antibody solution was added to every well. The plates were subsequently resealed, placed on the Titermix, and covered with aluminum foil.
• the protocol used was identical to that outlined above, except that only 10 compounds were assayed per plate.
• the testing concentrations started at 10 ⁇ g/ml and were diluted two-fold from 10 to 0.078 ⁇ g/ml.
• Percent inhibition was calculated as shown above. Percent inhibition was then plotted vs. log (cone. Inhibitor) using Graph pad Prism software. The ICso concentration was determined as the concentration that gives 50% inhibition.
• Example 9 Luciferase Assay The luciferase assay is used to determine if any of the compounds tested reduce the luminescent signal. This indicates that the test compounds affect regulation of micF, which in turn is regulated by Mar.
• Bacteria were E.coli AGl 12.
• the test compounds were prepared in a lOmg/mL DMSO stock solution.
• Inoculum (or “Starter Inoculum”) was started the night before the day of the experiment by adding either a colony or stab of a glycerol stock to 2mL of LB Broth. The Starter Inoculum was then placed in a 37 °C shaker incubator and allowed to grow overnight.
• Starter Inoculum was removed from the shaker and added to fresh LB Broth.
• 6mL of LB broth was prepared, with 5-1 O ⁇ L of Starter Inoculum being added per mL of added LB to form the "Test Inoculum".
• 4 plates of test compounds were assayed.
• 6mL of LB Broth was used for each plate, or 24 mL of LB, followed by the addition of 5 ⁇ L/mL of Starter Inoculum, or 120 ⁇ L of Starter Inoculum to form the Test Inoculum.
• Test moculum was placed in a 37 degree Celsius shaker and incubated for about 4 hours.
• Inoculum was monitored for bacterial growth by taking OD readings at 535 nm on a spectrophotometer. The Test inoculum should be removed when the OD reaches between 0.6 and 1.5.
• Positive and negative controls were created by adding 2uL DMSO to 198uL LB Broth. At least 4 of each control were generated. Typically, there were 8 of each. 50uL of diluted DMSO was added to 50uL LB Broth in the assay plates.
• the compounds were screened at 25ug/mL. Two identical plates of each compound were set up: 1 clear plate for growth (or “Clear Plate”), 1 white plate for luminescence (or “White Plate”). Next, 2 ⁇ L of each compound was taken from the daughter plate (containing lOmg/mL stock) and added to 198 ⁇ L of LB Broth. The sample was then mixed. Next, 25 ⁇ L of the diluted test compound was added to 25 ⁇ L of LB Broth in all of the assay plates. The concentration of the compound at this stage was 50 ⁇ g/mL.
• the Clear Plates were placed in the plate reader and read at OD 535 . This was the "Initial” growth read. The plates were then incubated plates for 5 hours at 37 degrees Celsius. After 5 hours, the plates were removed from the incubator. The Clear Plates were placed in the plate reader and read at OD5 3 5. This was the "Final" growth read.
• ND indicates that a particular test compound did not appear to decrease the lumninesce of in this particular assay. * indicates that the luminescence was decreased somewhat and ** indicates that the luminescence was decreased a substantial amount. The results from this assay are also shown in Table 5.
• 6-Nitro-benzoimidazol-l-ol hydrochloride (4) A mixture of l-hydroxy-6- nitro-lH-benzoimidazole-2-carboxylic acid ethyl ester (3) (5g, 20 mmol ) and concentrated HCl (100 mL) was refluxed for 3 hours. After cooling the mixture to room temperature, the resulting solid was collected by filtration. Yield 1.9 g (44 %) of the HCl salt.
• R ! NO 2 , F, CI, Br, NH 2 , NHAc, COMe, COPh, CF 3 , COOH, OMe, CN, CONH 2 , t Bu, COOR, etc.
• R substituted or unsubstituted phenyl, substituted or unsubstituted heterocycle
• Ri NO 2 , F, CI, Br, NH 2 , NHAc, COMe, COPh, CF 3 , COOH, OMe, CN, CONH 2 , 'Bu, COOR, etc.
• R 2 substituted or unsubstituted phenyl, substituted or unsubstituted heterocycle
• Ri NO 2 , F, CI, Br, NH 2 , NHAc, COMe, COPh, CF 3 , COOH, OMe, CN,
• R 2 substituted or unsubstituted phenyl, substituted or unsubstituted heterocycle
• the product thus obtained is usually pure, but when needed, it could be recrystallized from DMF/ether or methanol/ether or dichloromethane/hexane solvent systems.
• the quenched reaction mixture was concentrated to a volume where the product started to crash out.
• the final product was purified via chromatography in such cases or in cases that the crade material is impure.
• the final product was characterized using HPLC, MS, ! H NMR spectroscopy, and in some representative cases, using CHN analyses and C NMR spectroscopy as well.
• test compounds were diluted in DMSO to the required concenfration and added to the appropriate wells.
• Protein (SoxS) was added to the wells in EMSA buffer at a concentration that was determined to cause a 50% shift of the DNA.
• the plates were then covered, mixed and shaked for 30 minutes at room temperature to allow for compound-protein binding.
• the probe alone well showed a single DNA species (unbound) of an apparent low molecular weight.
• Controls containing protein showed approximately 50% of the DNA at a shifted or bound position (apparent higher mwt) and 50% at the same position as the probe alone (free DNA).
• Samples containing test compounds showed ratios of bands between these two controls.
• a compound that completely inhibited protein-DNA binding appeared to be similar to that of the probe alone.
• Table 6 shows the results of this assay. Compounds which showed superior inihibition of DNA binding are indicated by "*** 5 " compounds which showed very good or good inhibition of DNA binding are indicated by "**" or "*” respectively. Compounds which showed some inhibition of DNA binding are indicated by "-.”

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