# Patent

## Title
Methods for identifying and using MarR family polypeptide binding compounds

## Abstract

Methods for identifying MarR family inhibiting compounds are described. The methods include the use of computer aided rational based drug design programs and three dimensional structures of MarR family polypeptides.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application Ser. No. 60/388,618, entitled “Methods for Identifying and Using MarR Family Polypeptide Binding Compounds,” filed on Jun. 13, 2002; and U.S. Provisional Application Ser. No. 60/305,322, entitled “Methods for Identifying and Using MarR Family Polypeptide Binding Compounds,” filed on Jul. 13, 2001. This application is related to U.S. patent application Ser. No. 10/196,655, entitled “Crystal Structure of a MarR Family Polypeptide,” filed concurrently herewith; U.S. Provisional Application Ser. No. 60/388,622, entitled “Crystal Structure of a MarR Family Polypeptide,” filed Jun. 13, 2002; and U.S. Provisional Application Ser. No. 60/305,404, entitled “Crystal Structure of a MarR Family Polypeptide,” filed on Jul. 13, 2001. The entire contents each of the aforementioned applications are hereby incorporated herein by reference.


BACKGROUND OF THE INVENTION
The Mar phenotype in E. coli is attributed largely to the action of MarA, the expression of which is regulated by MarR (Alekshun, M. N. supra (1997)). MarA is a transcription factor that autoactivates expression of the marRAB operon and regulates the expression of a global network of more than 60 chromosomal genes (Martin, R. G. et at. J. Bact. 178, 2216–2223 (1996); Barbosa, T. M. & Levy, S. B. J. Bad. 182, 3467–3474 (2000)). Mar mutants in isolates of clinical origin have now been identified (Maneewannakul, K. & Levy, S. B. Antimicrob. Agents Chemoiher. 40, 1695–1698 (1996); Oethinger, M. et at. Antimicrob. Agents Chemother. 42, 2089–2094 (1998); Linde, H. J. et at. Antimicrob. Agents Chemother. 44, 1865–1868 (2000); Ziha-Zarifi, I., et at. Antimicrob. Agents Chemother. 43, 287–291 (1999); Koutsolioutsou, A et at. Antimicrob. Agents Chemother. 45, 38–43 (2001)). Constitutive overexpression of MarA or a MarA homolog in many, of these strains is a key contributor to the maintenance of the resistance phenotype, particularly with respect to the fluoroquinolones, and recent studies have documented the selection of Mar mutants, bearing mutations in MarR, MexR, or other homologous loci, in E. coli, Pseudomonas aeruginosa, and other organisms during antimicrobial chemotherapy (Oethinger, M supra; Linde, H. J. et at.; supra; Ziha-Zarifi, I. et al. supra; Kern, W. V., et at. Antimicrob. Agents Chemother. 44, 814–820 (2000)).
MarR is a regulator of multiple antibiotic resistance in Escherichia coli. It is the prototypic member of a family of regulatory proteins found in the Bacteria and the Archae that play important roles in the development of antibiotic resistance, a global health problem. In the absence of an appropriate stimulus, MarR negatively regulates expression of the marRAB operon (Cohen, S. P., et al. 1993. J. Bacteriol. 175: 1484–1492; Martin, R. G. and Rosner, J. L. 1995. Proc. Natl. Acad. Sci. 92: 5456–5460; Seoane, A. S. and Levy, S. B. 1995. J. Bacteriol. 177: 3414–3419, 1995). DNA footprinting experiments suggest that MarR dimerizes at two locations, sites I and II, within the mar operator (marO) (Martin and Rosner, 1995, supra). Site I is positioned among the −35 and −10 hexamers and site II spans the putative MarR ribosome binding site (reviewed in Alekshun, M. N. and Levy, S. B. 1997. Antimicrob. Agents Chemother. 10: 2067–2075).
MarR is a member of a newly recognized family of regulatory proteins (Alekshun, M. N. and Levy, S. B. 1997. Antimicrob. Agents Chemother. 10: 2067–2075. Sulavik, M. C., et al. 1995. Mol. Med. 1: 436–446) and many functional homologues have been identified in a variety of important human pathogens and have been found to regulate a variety of different processes. For example, some MarR homologues have been found to control expression of multiple antibiotic resistance operons, some regulate tissue-specific adhesive properties, some control expression of a cryptic hemolysin, some regulate protease production, and some regulate sporulation. Proteins of the MarR family control an assortment of biological functions including resistance to multiple antibiotics, organic solvents, household disinfectants, and oxidative stress agents, collectively termed the multiple antibiotic resistance (Mar) phenotype (Alekshun, M. N. & Levy, S. B. Trends Microbiol. 7, 410–413 (1999)). These proteins also regulate the synthesis of pathogenic factors in microbes that infect humans and plants (Miller, P. F. & Sulavik, M. C. Mol. Microbiol. 21, 441–448 (1996)). Insight into the three dimensional structure of MarR family proteins would be of great value in designing drugs that interact with this family of proteins and modulate MarR function, for example, antibiotic resistance and virulence.
SUMMARY OF THE INVENTION
The instant invention advances the prior art by providing the crystal structure of a MarR family polypeptide, MarR. The crystal structure of MarR provides the three-dimensional structure, as well as the shape and electronic properties of its active sites. It can be used in a comprehensive rational drug design program to develop novel chemotherapeutics targeted toward the MarR/MarA transcription system. The atomic coordinates of a MarR crystal structure cocrystallized with and without salicylate are given in FIG. 1 and FIG. 2, respectively.
In one embodiment, the invention pertains, at least in part, to methods for identifying a MarR family modulating compound. The method includes selecting a candidate MarR family modulating compound by performing rational drug design with the set of atomic coordinates in FIG. 1 or 2. The method may further include contacting the candidate MarR family modulating compound with the MarR family polypeptide, and determining the ability of the candidate MarR family modulating compound to modulate the MarR family polypeptide. The invention also pertains to compounds identified by these methods and methods of using the compounds to modulate MarR family polypeptides.
In another embodiment, the invention pertains, at least in part, to methods for identifying a MarR family modulating compound. The methods include determining the structure of a MarR family polypeptide using the structure of MarR and identifying a candidate MarR family modulating compound by performing rational drug design based on the structure. The method may further include the steps of contacting the candidate MarR family modulating compound with a MarR family peptide, and a nucleic acid molecule, and measuring the binding affinity of the MarR family polypeptide peptide with the nucleic acid molecule. The invention also pertains to compounds identified by these methods and methods of using the compounds to modulate polypeptides.
In yet another embodiment, the invention pertains, at least in part, to a method for identifying a MarR modulating compound. The method includes obtaining a set of atomic coordinates defining the three-dimensional structure of MarR and selecting a candidate MarR modulating compound by performing rational drug design with the three dimensional structure of MarR. The method may further include the steps of contacting the candidate MarR modulating compound with MarR, measuring the ability of the candidate MarR modulating compound to modulate the activity of MarR. The invention also pertains to compounds identified by these methods and methods for modulating MarR using the compounds of the invention.
In another embodiment, the invention pertains, at least in part, to a MarR modulating compound of the formula (I):

X-Y-Z  (I)

wherein X is an interacting moiety; Y is a hydrophobic moiety; and Z is a polar moiety.

The invention also pertains, at least in part, to methods for inhibiting expression of MarA, by contacting MarR with a MarR modulating compound of formula (I).
In yet another embodiment, the invention also pertains to methods for decreasing multidrug resistance in a microbe. The method includes contacting the microbe with a MarR modulating compound of formula(I).



BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
FIG. 1 shows the atomic coordinates of the MarR-salicylate co-crystal.
FIG. 2 shows the atomic coordinates of the MarR crystal without salicylate.
FIG. 3 shows the sequence alignment of MarR (SEQ ID NO. 2) with representative members of the MarR family. The representive members of the MarR family shown in FIG. 3 are MprA (SEQ ID NO. 3). MexR (SEQ ID NO. 4). Ys87 (SEQ ID NO. 5). SlvA (SEO ID NO. 6). PecS (SEO ID NO. 7). and CinR (SEQ ID NO. 8).
FIG. 4 is a ribbon representation of the salicylate containing MarR dimer with the two-fold axis near vertical. There are two salicylate molecules per monomer and each is represented by a stick model.
FIG. 5 is a representation of the N-/C-terminal domain represented by a surface (red) around the van der Waals radii of the side chain atoms only of the hydrophobic core residues. The main chain and other residues of the domain are shown in yellow for one subunit and blue for the other. Helices leading to and from the domain are shown in ribbon representation.
FIG. 6 is an electrostatic surface representation of the MarR dimer.
FIG. 7 is a Cα trace of a MarR subunit in stereo representation.
FIG. 8 is a diagram which shows interactions between the DNA-binding domains of the dimer in the region of the Arg 73–Asp 67′ salt bridges. The stereo view is coincident with the 2-fold rotation axis of the dimer. Electron density shown is a 2FO–FC map contoured at 1σ.
FIG. 9 shows a ribbon representation of the MarR dimer with the two-fold axis near vertical.
FIG. 10 is a computer model of MarR interacting with DNA.
FIG. 11 shows a sequence comparison and alignment of MarR (SEQ ID NO. 2) and SlyA (SEQ ID NO. 6).
FIG. 12 shows structurally conserved regions of SIyA (SEQ ID NO. 6) and MarR (SEQ ID NO.2) as determined by COMPOSER.
FIG. 13a represents the SCRs of MarR derived from the MarR crystal structure. The basic framework of SlyA is shown in FIG. 13b. 
FIG. 14 shows a Cα-tube representations of MarR from the crystal structure and its homology with a model of SlyA.



DETAILED DESCRIPTION OF THE INVENTION
Chemotherapeutic intervention for the treatment and prevention of disease is predicated upon the ability of small molecules (drugs) to infiltrate a biological system and to interact with the components of the biological system (e.g. proteins, RNA, DNA, membranes, etc.) in a manner that modulates their normal function. Rational drug design attempts to formulate drug design hypotheses that specify and optimize the physical contacts between the drug and target. Koshland has used a lock and key analogy to characterize drug-target interactions; a specific “key” (drug) interacts only with its respective molecular “lock” (target) (Koshland, D. E., Jr. Angew. Chem. 1994, 106, 2468–2472). This model asserts that an appropriate degree of shape and electronic complimentarily between the drug and target must occur to produce productive drug-target interactions-those that cause a desired pharmacological response. The specific location on the “lock” or target is referred to as the active or catalytic site. The three dimensional shape and electronic properties of the active site form the basis for rational drug design and provides information toward the systematic chemical modifications of potential drugs.
In one embodiment, the invention pertains to methods for identifying MarR family modulating compounds using the three-dimensional structure of a MarR family polypeptide. The method includes selecting a candidate MarR family modulating compound by performing rational drug design with the atomic coordinates of a MarR family polypeptide. The method may also include contacting the candidate MarR family modulating compound with MarR family polypeptide; and determining the ability of said candidate MarR family modulating compound to a modulate MarR family polypeptide. In one embodiment, the MarR family polypeptide is MarR. The atomic coordinates of MarR in the presence and absence of salicylate are given in FIGS. 1 and 2, respectively.
MarR Family Polypeptides and Nucleic Acid Molecules
The term “MarR family polypeptide” includes molecules related to MarR, e.g., having certain shared structural and functional features. MarR family polypeptides also include those which are structural homologs of MarR. The structural homologs include those having a crystallized form which are structurally similar to that of crystallized MarR. Generally, it is believed that there is a strong relationship between the tertiary structure of a protein and its function within the biological system. Furthermore, it is known that a protein's overall tertiary structure is related to its primary amino acid sequence. Therefore, it has been demonstrated that proteins with similar amino acid make up and sequence will possess similar overall structure and will likely share similar function. MarR family members, in addition to having similarity to MarR, may bind to DNA and regulate transcription. While some MarR family members negatively control transcription (e.g., MarR), others have positive/activator functions (e.g., SlyA, BadR, NhhD, and MexR). MarR family polypeptides comprise DNA and protein binding domains. In addition, MarR family polypeptides can interact with a variety of structurally unrelated compounds that regulate their activity.
Exemplary MarR family members are taught in the art and can be found, e.g., in Sulavik et al. (1995. Molecular Medicine. 1:436), Miller and Sulavik (1996. Molecular Microbiology. 21:441) in which alignments of MarR and related proteins are shown, or through the use of BLAST searches and other techniques known in the art. Exemplary MarR family polypeptides are also illustrated in the following chart:










MarR Family Polypeptides









Gram-negative
Gram-positive
Acid-fast





Escherichia coli
Bacillus
Mycobacterium


MarR
subtilus
tuberculosis


SlyA
YdcH
14.7 kD


EmrR (MprA)
YhbI
Rv1404


PapX
YkmA
Rv0737


PrsX
YkoM
Rv0042c


HpcR
Orf7
Yz08 (15.6 kD)


Ec17kD
YfiV
Mycobacterium



YetL
leprae


Slamonella
YdgJ
Yz08 (15.6 kD)


typhimurium
YwoH


MarR
YwaE
Archaea


SlyA
YwhA
Methanobacterium


EmrR
Hpr
thermoautotrophicum



YybA
MTH313


Pseudomonas
YxaD


aeruginosa
YsmB


MexR
YusO
Sulfolobus



YpoP
solfataricus


Erwinia
YkvE
Lrs14


chrysanthemi



PecS

Archaeoglobus




fulgidus




CinR


Rhodopseudomonas
Bacillus firmus


palustris
Orf7
Purple non-sulfur


BadR

Rhodobacter


Burkhelderia
Staphylococcus
capsulatus


pseudomallei
sciuri
PetP


OrfE
Orf145
Sinorhizobium



Orf141
meliloti



Butyrivibrio
SlyA (E293909)



fibrisolvens



CinR



Sphingomonas



aromaticivorans



Orf158



Rhodococcus



rhodochrous



NhhD



Streptomyces



peucetius



Orf1









Preferably, a MarR family polypeptide is MarR. Other preferred MarR family polypeptides include: EmrR, Ec17kD, and MexR.
In a further embodiment, the MarR family polypeptide has a winged-helix structure, such as the three dimensional structure of MarR.
FIG. 3 shows a sequence alignment of MarR with representative MarR family polypeptides. The MarR secondary structure elements were identified in its crystal structure and are illustrated in FIG. 3 (e.g., as tubes for α-helices (α) and arrows for β-sheets (β) and the single wing region (W1)). The numbering in FIG. 3 is according to the MarR primary sequence. Furthermore, residues that are identical in all homologs are colored in red, highly conserved amino acids are colored in yellow, and moderately conserved residues are colored in blue. The MarR family polypeptides used for the alignment were from the following organisms: MarR, E. coli; MprA (EmrR), E. coli; MexR, Pseudomonas aeruginosa; YS87, Mycobacterium tuberculosis; SlyA, Salmonella typhimurium; PecS, Erwinia chrysanthemi; CinR, Butyrivibrio fibrisolvens. 
In a further embodiment, the MarR family polypeptide comprises, consists essentially of, or consists of the polypeptide sequence shown in Sequence Listing SEQ ID NO:1. Other MarR family polypeptides of interest include EmrR, YS87, PecS, CinR, SlyA, Ec17kD, MexR.
In another embodiment, the MarR family polypeptide is found, for example, in one of the following organisms Escherichia coli, Salmonella typhimurium, Salmonella enterica, Enterobacter cloacae, Enterobacter aerogenes, Erwinia chrysanthemi, Yrsinia pestis, Yersinia enterocolitica, Kluyvera cryocrescens, Edwardsiella tarda, Pseudomonas aeruginosa, Vibrio cholera, Xanthomonas axonopodis, Xanthomonas campestris, Ralstonia solanacearum, Burkholderia pseudomallei, Burkholderia cepacia, Vogesella indigofera, Mesorhizobium loti, Agrobacterium tumefaciens, Sinorhizobium meliloti, Brucella melitensis, Caulobacter crescentus, Bacillus anthracis, Bacillus subtilis, Bacillus halodurans, Listeria monocytogenes, Listeria innocua, Listeria welshimeri, Staphylococcus sciuri, Streptococcus criceti, Streptococcus pneumoniae, Clostridium perfringens, Clostridium difficile, Streptomyces coelicolor, Streptomyces avermitilis, Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium glutamicum, Thermotoga maritima, Methanosarcina acetivorans, Methanosarcina mazei, and Sulfolobus solfataricus. 
In another embodiment, the MarR family polypeptide is from an organism belonging to one of the following biological classifications: Enterobacteriaceae, Enterobacter, Yersinia, Kluyvera, Edwardsiella, Xanthomonas group, Xanthomonadales, Pseudomonaceae/Moraxellaceae group, Pseudomonadaceae, Vibrionaceae group, Burkholderia/Oxalobacter/Ralstonia group, Ralstonia group, Burkholderia group, Neisseriaceae, Vogesella, Rhizobiaceae group, Phyllobacteriaceae, Mesorhizobium, Rhizobiaceae, Sinorhizobium, Brucellaceae, Brucella, Caulobacter group, Firmicutes, Bacillus/Clostridium group, Bacilli, Bacillales, Bacillus, Bacillaceae, Bacillus cereus group, Listeria, Listeriaceae, Staphylococcaceae, Staphylococcus, Streptococcus, Lactobacillales, Streptococcaceae, Clostridium, Clostridiaceae, Clostridiales, Clostridia, Actinomycetales, Actinobacteria, Actinobacteridae, Streptomyces, Streptomycineae, Streptomycetaceae, Corynebacterineae, Mycobacterium, Mycobacteriaceae, Corynebacteriaceae, Corynebacterium, Nostocales, Nostocaceae, Nostoc, Thermotogae, Thermotogales, Thermotogaceae; Thermotoga, Methanosarcina, Euryarchaeota, Methanococci; Methanosarcinales, Methanosarcinaceae, Crenarchaeota, Thermoprotei; Sulfolobales, Sulfolobaceae, Sulfolobus, Proteobacteria, Pectobacterium, Cyanobacteria, or Archaea.
In one embodiment, the MarR family polypeptides of the invention are naturally occurring. In another embodiment, the subject crystal structures can be generated using non-naturally occurring forms of MarR family polypeptides, e.g. mutants or synthetic forms of MarR family polypeptides not found in nature.
In one embodiment, the MarR family polypeptide comprises one or more conservative mutations as compared to the wild type protein for the particular MarR family polypeptide. The term “MarR family polypeptide” also includes fragments of MarR family polypeptides which minimally retain at least a portion of the tertiary structure of the MarR family protein.
MarR family member polypeptide sequences are “structurally related” to one or more known MarR family members, preferably to MarR. This structural relatedness is shown by sequence similarity between two MarR family polypeptide sequences or between two MarR family nucleotide sequences. Sequence similarity can be shown, e.g., by optimally aligning MarR 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 or 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. When a position in one sequence is occupied by the same amino acid residue or by the same base as the corresponding position in the other sequence, then the molecules are identical at that position. If 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). The degree (percentage) of identity or similarity between sequences, therefore, can be calculated as a function of the number of identical or similar positions shared by two sequences (i.e., % homology=# of identical or similar positions/total # of positions×100). Alignment strategies are well known in the art; see, for example, Altschul et al. supra for optimal sequence alignment.
MarR family polypeptides share some amino acid sequence similarity with MarR. The nucleic acid and amino acid sequences of MarR as well as other MarR family polypeptides are available in the art. For example, the nucleic acid and amino acid sequence of MarR 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:1).
The nucleic acid and protein sequences of MarR can be used as “query sequences” to perform a search against databases (e.g., either public or private) to, for example, identify other MarR 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. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to MarR family nucleic acid molecules. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to MarR protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389–3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
MarR family members can also be identified as being structurally similiar based on their ability to specifically hybridize to the complement of nucleic acid sequences specifying MarR. Such 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 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50–65° C. Conditions for hybridizations are largely dependent on the melting temperature that is observed for half of the molecules of a substantially pure population of a double-stranded nucleic acid. The melting temperature is the temperature in °C. at which half the molecules of a given sequence are melted or single-stranded. For nucleic acids of sequence 11 to 23 bases, the melting temperature 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 melting temperature, e.g., 15° C., 20° C., 25° C. or 30° C. lower than the melting temperature. The effect of salt concentration (in M of NaCl) can also be calculated, see for example, Brown, A., “Hybridization” pp. 503–506, in The Encyclopedia of Molec. Biol., J. Kendrew, Ed., Blackwell, Oxford (1994).
Preferably, the nucleic acid sequence of a MarR 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 most preferably at least about 50%, or 60% identical or more with a MarR nucleotide sequence. Preferably, MarR family members have an amino acid sequence at least about 20%, more preferably at least about 30%, more preferably at least about 40% identical and most preferably at least about 50%, or 60% or more identical with a MarR amino acid sequence. However, it will be understood that 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). For example, the level of amino acid sequence homology between MarR and Pecs is about 31% and the level of amino acid sequence homology between MarR and PapX is about 28% when determined as described above. Accordingly, structural similarity among MarR family members can also be determined based on “three-dimensional correspondence” of amino acid residues.
The language “three-dimensional correspondence” includes residues which spatially correspond, e.g., are in the same functional position of a MarR family protein member as determined, e.g., by x-ray crystallography, but which may not correspond 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. Thus, MarR family members can be identified based on functional homology and sequence homology, e.g., as described in the art (Li et al. 2001. EMBO Journal 20:4854).
Preferred MarR family polypeptides include: MarR, EmrR, Ec17kD, MexR, PapX, SlyA, Hpr, PecS, Hpr, MprA, or (EmrR). In a more preferred embodiment, a MarR family polypeptide is selected from the group consisting of: MarR, EmrR, Ec17kD, and MexR. In a particularly preferred embodiment, a MarR family polypeptide is MarR.
In addition to sharing structural similarity, MarR family members have a MarR family polypeptide activity, i.e., they bind to DNA and regulate transcription. Some MarR family members positively regulate transcription (e.g., SlyA, BadR, NhhD, or MexR), while others negatively regulate transcription (e.g., MarR). While all MarR family members bind to DNA and regulate transcription, the different loci controlled by each family member regulate different processes in microbes. For example, MarR family polypeptides can control the expression of microbial loci involved in: regulation of antibiotic resistance [e.g., MarR (Cohen et al. 1993. J. Bacteriol. 175:1484), EmrR (Lomovskaya and Lewis. 1992. Proc. Natl. Acad. Sci. 89:8938), and Ec17kD (Sulavik et al. 1995. Mol. Med. 1:436), and MexR (Poole et al. 1996. Antimicrob. Agents. Chemother. 40:2021)], regulation of tissue-specific adhesive properties [e.g., PapX (Marklund et al., 1992. Mol. Microbiol. 6:2225)], regulation of expression of a cryptic hemolysin [e.g., SlyA (Ludwig et al. 1995 249:4740)], regulation of protease production [e.g., Hpr from B. subtilis (Perago and Hoch. 1988. J. Bacteriol. 170:2560) and PecS from Erwinia chrysanthemi (Reverchon et al., 1994. Mol. Microbiol. 11:1127)] and regulation of sporulation [e.g., Hpr (Perego and Hoch. 1988. J. Bacteriol. 170:2560)], regulation of the breakdown of plant materials [e.g., CinR (Dalymple and Swadling 1997 Microbiology)] sensing of phenolic compounds [(e.g., Sulvik et al. 1995. Mol. Med. 1:436], and repress marRAB expression when introduced into E. coli [e.g., Ec17kd (Marklund et al. 1992. Mol. Microbiol. 6:2225) and MprA (EmrR) (del Castillo et al., 1991. J. Bacteriol. 173:3924)]. The activity of MarR family polypeptides is antagonized by salicylate (Lomovskaya et al., 1995. J. Bacteriol. 177:2328; Sulavik et al. 1995. Mol. Med. 1:436).
Preferred MarR family polypeptide activities include regulation of multiple drug resistance and/or regulation of virulence.
In addition to full length MarR family polypeptide fragments MarR family polypeptide which are useful in making crystals are also within the scope of the invention. Accordingly, MarR family polypeptides for use in the instant screening assays can be full length MarR family member proteins or fragments thereof. Thus, a MarR family polypeptide can comprise, consist essentially of, or consist of an amino acid sequence derived from the full length amino acid sequence of a MarR family member. For example, in one embodiment, a polypeptide comprising a MarR family polypeptide DNA interacting domain or a polypeptide comprising a MarR family member protein interacting domain can be used.
In addition, naturally or non-naturally occurring variants of these polypeptides and nucleic acid molecules which retain the same functional activity, e.g., the ability to bind to DNA and regulate transcription. Such variants can be made, e.g., by mutation using techniques which are known in the art. Alternatively, variants can be chemically synthesized.
For example, it will be understood that the MarR family polypeptides described herein, are also meant to include equivalents thereof. For instance, mutant forms of MarR family polypeptides which are functionally equivalent, (e.g., have the ability to bind to DNA and to regulate transcription from an operon) can be made using techniques which are well known in the art. Mutations can include, e.g., at least one of a discrete point mutation which can give rise to a substitution, or by at least one deletion or insertion. For example, random mutagenesis can be used. Mutations can be made, e.g., by random mutagenesis or using cassette mutagenesis. For the former, 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. In the latter, discrete regions of a protein, corresponding either to defined structural or functional determinants (e.g., the first or second helix of a helix-turn-helix domain) are subjected to saturating or semi-random mutagenesis and these mutagenized cassettes are re-introduced into the context of the otherwise wild type allele. In one embodiment, PCR mutagenesis can be used. For example, Megaprimer PCR can be used (O. H. Landt, Gene 96:125–128).
In addition, other portions of the above described polypeptides suitable for use in the claimed assays, such as those which retain their function (e.g., the ability to bind to DNA, to regulate transcription from an operon) or those which are critical for binding to regulatory molecules (such as compounds) can be easily determined by one of ordinary skill in the art (e.g, using standard truncation or mutagenesis techniques) and used in the instant assays. Exemplary techniques are described by Gallegos et al. (1996. J. Bacteriol. 178:6427).
It shall be understood that the instant invention also pertains to isolated MarR family member polypeptides, portions thereof, and the nucleic acid molecules encoding them, including naturally occurring and mutant forms.
Preparation of MarR Family Polypeptides
Preferred MarR family polypeptides for use in screening assays are synthesized, isolated or recombinant polypeptides. In one embodiment, MarR family polypeptides can be made from nucleic acid molecules. Nucleic acid molecules encoding MarR family polypeptides can be used to produce MarR family polypeptides. For example, nucleic acid molecules encoding a MarR family polypeptide can be isolated (e.g., isolated from the sequences which naturally flank it in the genome and from cellular components) and can be used to produce a MarR family polypeptide. In one embodiment, 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 can be used to produce MarR family polypeptides. The term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Nucleic acid molecules specifying MarR family polypeptides can be placed in a vector. The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. The term “expression vector” 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). In the present specification, “plasmid” and “vector” are used interchangeably, as a plasmid is a commonly used form of vector. Moreover, the invention is intended to include other vectors which serve equivalent functions.
Exemplary expression vectors for expression of a gene encoding a MarR family polypeptide and capable of replication in a bacterium, such a bacterium from a genus selected from the group consisting of: Escherichia, Bacillus, Streptomyces, Streptococcus, 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. Pat. No. 4,745,056. Suitable vectors for a variety of organisms are described in Ausubel, F. et al., Short Protocols in Molecular Biology, Wiley, New York (1995), and for example, for Pichia, can be obtained from Invitrogen (Carlsbad, Calif.).
Useful expression control sequences, include, for example, the early and late promoters of SV40, adenovirus 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 protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-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. A useful translational enhancer sequence is described in U.S. Pat. No. 4,820,639.
It should be understood that 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 protein desired to be expressed.
“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 nucleic acid sequences with which they are operably linked. It will also be understood that a recombinant gene encoding a MarR family polypeptide 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 MarR family gene. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). For instance, 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 MarR family proteins of this invention.
Appropriate 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 MarR family polypeptides 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.
Such vectors can be introduced into cells using standard techniques, e.g., transformation or transfection. The terms “transformation” and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient or “host” cell. The term “transduction” means transfer of a nucleic acid sequence, preferably DNA, from a donor to a recipient cell, by means of infection with a virus previously grown in the donor, preferably a bacteriophage. Nucleic acids can also be introduced into microbial cells by transformation using calcium chloride or electroporation.
“Cells,” “host cells,” “recipient cells, are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. In preferred embodiments, cells used to express MarR family polypeptides for purification, e.g., host cells, comprise a mutation which renders any endogenous MarR family polypeptide nonfunctional or causes the endogenous polypeptide to not be expressed. In other embodiments, 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.
Purification of a MarR family polypeptides, e.g., recombinantly expressed polypeptides, can be accomplished using techniques known in the art. For example, if the MarR family polypeptide is expressed in a form that is secreted from cells, the medium can be collected. Alternatively, if the MarR family polypeptide is expressed in a form that is retained by cells, the host cells can be lysed to release the MarR family polypeptide. Such spent medium or cell lysate can be used to concentrate and purify the MarR family polypeptide. For example, the medium or lysate can be passed over a column, e.g., a column to which antibodies specific for the MarR family member polypeptide have been bound. Alternatively, such antibodies can be specific for a non-MarR family member polypeptide which has been fused to the MarR family polypeptide (e.g., as a tag) to facilitate purification of the MarR family member polypeptide. Other means of purifying MarR family member polypeptides are known in the art.
Architecture of the MarR-Salicylate Co-Crystal Structure
The term “three dimensional structure” includes both pictorial representations of MarR family polypeptides (e.g., such as those shown for MarR in the Figures) as well as atomic coordinates (e.g., such as those given in FIG. 1 for MarR-salicylate cocrystal, or in FIG. 2 for MarR) and other renditions of the shape, size, or symmetry of a MarR family polypeptide of interest. In a further embodiment, the three dimensional structure of the crystallized MarR family polypeptide is determined to a resolution of 5 Å or better, 3 Å or better, 2.5 Å or better, or, advantageously, 2.3 Å or better. The three dimensional structure of MarR, a MarR family polypeptide, is described in greater detail below.
The salicylate containing MarR consists of a dimer with approximate overall dimensions of 50×55×45 Å, as shown in FIG. 4. There is one monomer in the asymmetric unit of the crystal with the dimer composed of subunits related by a crystallographic two-fold rotation. The dimeric structure is consistent with the results of earlier in vitro experiments suggesting that MarR binds the mar operator (marO) as a dimer (Martin, R. G. et al. supra (1996); Martin, R. G. & Rosner, J. L. Proc. Natl. Acad. Sci. U.S.A. 92, 5456–5460 (1995)). Another family member, MprA (EmrR) (FIG. 3) is also believed to function as a dimer (Brooun, A., et al. J. Bact. 181, 5131–5133 (1999)).
Each MarR salicylate subunit is an α/β protein with approximate dimensions of 35×25×60 Å and can be divided into two domains as shown in FIG. 4. FIG. 4 is a ribbon representation of the co-crystal structure of the MarR dimer viewed with the subunit 2-fold axis near vertical. The N- and C-terminal regions are closely juxtaposed and intertwine with the equivalent regions of the second subunit to form a domain that holds the subunits together (FIG. 5). This N-/C-terminal domain is linked to the remainder of the protein by two long antiparallel helices in each subunit. These helices lead to a globular domain that is likely to be responsible for DNA binding (see below). Although the globular DNA-binding domains of the dimer are adjacent to one another, they make minimal contact with each other and are situated to function independently. The overall organization of the N-/C-terminal domain and the two DNA-binding domains results in the formation of an approximately 6 Å wide channel through the center of the dimer (FIGS. 6 and 7). The electrostatic surface potential is consistent with the putative DNA-binding regions being strongly electropositive, as observed in other such winged-helix DNA-binding proteins (Gajiwala, K. S. & Burley, S. K. Curr. Opin. Str. Biol. 10, 110–116 (2000)).
Genetic and biochemical data have previously identified the N-terminus of MarR to be important for mediating protein-protein contacts between repressor subunits and have demonstrated that the C-terminus is important for protein function (Alekshun, M. N., et al. Mol. Microbiol. 35, 1394–404 (2000); Linde, H. J. et al. supra). The present structure shows that α-helices in the N- and C-terminal regions of each monomer fold around and interdigitate with those of the other subunit to form a well-packed hydrophobic core (FIG. 5) burying a surface area of 3,570 Å2 (the total buried surface area for the whole dimer is 3,700 Å2). The dimer is further stabilized in this region by several intermolecular hydrogen bonds, notably that between the ε-amino group of Lys 24 and the main chain carbonyl oxygen of Pro 144′ in the C-terminus of the second subunit and that between the main chain carbonyl oxygen of Glu 10 and the side chain amino group of Lys 140′.
While the DNA-binding lobe of each subunit also forms a well-packed hydrophobic core, the only interactions between these lobes of the two subunits are salt bridges formed between Asp 67 and Arg 73′ and the reciprocal pair (FIG. 8). These salt bridges stabilize the relationship between the two lobes of the dimer in the crystal form of the protein but if disrupted by other interactions, such as might occur during the binding of MarR to marO, the two lobes would be able to act independently. Relative movement of the lobes would require distortion of the helices that link them to the N-/C-terminal domain. The long linker helix region encompassing residues 103–126 (α5/α5′) appears poorly ordered in the region of Gly 116, as is the loop (residues 128–131) that connects this helix to the C-terminal helix (α6/α6′). It is possible that flexibility at these sites in MarR helps to accommodate relative shifts of the two lobes of the dimer that might occur on binding to DNA.
Architecture of the MarR Crystal Structure
The MarR without salicylate structure is a dimer and both subunits of the dimer are in the asymmetric unit. These individual subunits are joined by protein-protein interactions mediated by amino acids within both the N- and C-termini of the monomers. Like the MarR-salicylate structure, MarR without salicylate is an α/β protein. The MarR without salicylate structure is, however, conformationally different from the salicylate bound protein in that the caliper created by the dimer is more closed in the form of the protein without salicylate. Thus, the channel through the center of the dimer has been lost.
The overall architecture of the MarR without salicylate structure is comparable to that of the salicylate bound protein. The presumed DNA binding lobes or domains are linked to the remainder of the protein by two long α-helices. The positioning of the two DNA binding lobes in the MarR without salicylate structure is fixed by hydrogen bonds between the two lobes. This arrangement is believed to be mediated by interactions between Asp 67 and Arg 77′. In addition, Asp 26 is involved in hydrogen bonds with the side chains of Lys 44 and Lys 25. Together, the presumed recognition helices within the DNA binds lobes overlap by approximately one helical turn. FIG. 9 shows a ribbon representation of the MarR dimer with the two-fold axis near vertical.
The DNA Binding Domain
Previous studies have shown the region spanning amino acids 61–121 in MarR to be required for its DNA binding activity (Alekshun, M. N et al., supra, (2000)). In the crystal structure, amino acids 55–100 [β1-α3-α4-β2-W1 (wing)-β3] adopt the winged-helix fold (Clark, K. L. et al. Nature 364, 412–420 (1993)). The overall topology [H1 (α2)-S1 (β1)-H2 (α3)-H3 (α4, recognition helix)-S2 (β2)-W1-S3 (β3)] of this region is similar to other winged-helix DNA binding proteins (the terminology applied for these and subsequent structural elements is according to Gajiwala and Burley, supra (2000)) except that a third strand of sheet present in most members of the group appears to be represented in this MarR structure only by an interaction with Ile 55 (β1). The presence of this residue as the third component in the sheet interaction is similar to that observed in OmpR (Martinez-Hackert, E. & Stock, A. M. Structure 5, 109–124 (1997)), a winged helix protein, where Leu 180 interacts with the two strands of the antiparallel sheet that forms part of the “wing” in this transcription factor.
Within the winged-helix family of DNA-binding proteins, there are multiple modes of DNA binding. Members such as HNF-3γ use the recognition helix (H3) of the motif as the primary determinant for DNA-protein interactions in the major groove, and a wing region(s) (W1) to form minor groove or phosphodiester backbone nucleoprotein contacts (Clark, K. L. et al. supra (1993)). Others, such as hRFX1, use W1 to interact with the major groove and the H3 helix makes only a single minor groove contact (Gajiwala, K. S. et al. Nature 403, 916–921 (2000)). The juxtaposition of the DNA-binding lobes in the present structure does not allow for modeling of the whole dimer onto a B-DNA representation of the operator. However, since mutations in both α4 (H3) and W1 affect the DNA binding activity of MarR it is expected that amino acids from each of these regions would contribute to the DNA binding activity of the protein. For example, mutations in α4, including an R73C change, abolish MarR DNA binding activity in whole cells and in vitro(Alekshun, M. N et al., supra, (2000)). In the present crystal structure, it is the side chain of Arg 73 that is hydrogen bonded to Asp 67′ of the other subunit, an interaction that stabilizes the relative orientation of the two DNA-binding lobes. Also, an R94C mutation at the tip of W1 is inactive in a whole cell assay while a G95S “superrepressor” mutation increases the DNA binding activity of MarR 30-fold in vitro (Alekshun, M. N et al., supra, (2000); Alekshun, M. N. & Levy, S. B. J. Bact. 181, 3303–3306 (1999)). In the absence of protein-DNA co-crystal structures, the precise mechanism by which these mutations affect the DNA binding activity of the protein is uncertain.
Footprinting experiments have suggested that MarR binds as a dimer at two separate but very similar sites in marO, the protein protects  ̃21-bp of DNA on both strands at a single site, and does not bend its target (Martin, R. G. et al., supra (1996); Martin, R. G. et al. Proc. Natl. Acad Sci. U.S.A. 92, 5456–5460 (1995)). Each MarR binding site is composed of two half-sites whose organization is such that they are on different faces of the DNA double helix (Alekshun, M. N. et al. Mol. Microbiol. 35, 1394–404 (2000)), an arrangement that is very similar to the hRFX1 binding site (Gajiwala, K. S. et al. Nature 403, 916–921 (2000)). For MarR to bind as a dimer, with each winged-helix DNA binding domain contacting one half-site on B-DNA, geometric constraints suggest only a few possible modes of binding. One scenario, involving the binding of a single dimer to one MarR binding site, would require reorientation of the DNA binding lobes so that each could reach one half-site. This would be analogous to the binding of an E2F-DP heterodimer (a eukaryotic transcription factor in which each subunit also has a winged-helix DNA binding domain) to its cognate binding site (Zheng, N. et al. Genes Dev. 13, 666–74. (1999)). A second scenario would involve the binding of two dimers, on opposite faces of the double helix, to a single MarR binding site. This model would be analogous to the binding of DtxR (a bacterial protein with a winged-helix DNA binding domain) to its target, although in DtxR the half-sites are on the same face of the DNA helix (Pohl, E. et al. J. Biol. Chem. 273, 22420–22427 (1998); White, A. et al. Nature 394, 502–506 (1998)).
The term “appropriate conditions” include those conditions which result in the formation of a crystal which can by analyzed to a resolution of 5.0 Å or less. The crystals may be formed using suitable art recognized techniques, such as hanging droplet vapor diffusion. In one embodiment, the temperature of crystallization of the MarR family polypeptide is from about 1° C. to about 30° C., from about 10° C. to about 25° C., from about 15° C. to about 20° C., or abut 17° C. In a further embodiment, the conditions are selected such that crystals of said MarR family polypeptide grow within an acceptable time and reach dimensions which are suitable for structural determination, e.g., by using X-ray diffraction. In one embodiment, the acceptable time is 8 weeks or less, 6 weeks or less, 4 weeks or less, or 3 weeks or less. In an embodiment, the dimensions of the crystal are approximately 0.1 mm or greater per side, 0.2 mm or greater per said, or approximately 0.3 mm per side or greater.
In a further embodiment, the appropriate conditions include a cocrystallization agent which interacts with the protein such that the three dimensional structure of the protein can be determined.
The term “cocrystallization agent” includes substances which can be crystallized with the MarR family polypeptide such that the three dimensional structure can be determined. In an embodiment, the coocrystallization agent is a MarR family polypeptide modulator. The term “MarR family polypeptide modulator” includes compounds which interact with MarR family polypeptides, either to inhibit or enhance the activity of the MarR family polypeptides, such that they alter its activity in its non-crystallized form. In one embodiment, the MarR family polypeptide modulator is a MarR inhibitor (e.g., salicylate, plombagin, or DNP). In an embodiment, the concentration of the salicylate is about 100 mM or less, 150 mM or less, 200 mM or less, or 250 mM or less.
The crystal structure or MarR has been solved using crystals grown in the presence and in the absence of high concentrations (250 mM) of sodium salicylate. This agent, at millimolar concentrations, is known to inhibit MarR activity both in vitro and in whole cells (Alekshun, M. N. supra (1999)). It is routinely used as a model inhibitor of MarR to induce MarA expression in E. coli and S. typhimurium (Cohen, S. P. et al. J. Bact. 175, 7856–7862 (1993); Sulavik, M. C. et al. J. Bact. 179, 1857–1866 (1997)) and thus, to confer a Mar phenotype (Alekshun, M. N. supra (1999)). In one example, salicylate was included in the current crystal growth conditions to provide stable crystals. In another example, the crystal structure of MarR was determined using MarR without salicylate.
Electron density that is consistent with bound salicylate is apparent at two sites on each subunit in the present structure. These sites are on the surface of the molecule on either side of the proposed DNA-binding helix α4 (H3). In one site (SAL-A), the salicylate hydroxyl is hydrogen bonded to the hydroxyl side chain of Thr 72 in the α4 (H3) helix and the salicylate carboxylate hydrogen bonds to the guanidinium group of Arg 86. In the other site (SAL-B), the salicylate hydroxyl hydrogen bonds to the backbone carbonyl of Ala 70 and its carboxyl hydrogen bonds to Arg 77. In each of these sites, the salicylate ring sits over a hydrophobic side chain in the pocket; Pro 57 in SAL-A and Met 74 in SAL-B and other surface hydrophobes are also located laterally within 3.5 Å of the unsubstituted side of the ring. Although SAL-B is solvent exposed, SAL-A packs in the crystal with Val 96 of a symmetry mate situated 3.6 Å above the salicylate ring and adjacent to the SAL-A site of this symmetry mate. Since both SAL-A and SAL-B are close to the DNA binding helix, they may be positioned to influence DNA binding.
The crystal structure of MarR was solved by multiwavelength anomalous dispersion methods using protein containing selenomethionine. Diffraction data were collected to 2.3 Å from crystals of both seleno and native protein.
Use of the MarR Crystal Structure to Model the Structures of Other MarR Family Polypeptides
In one embodiment, the invention pertains to a method for determining the structure of a MarR family polypeptide comprising analyzing the sequence of the related polypeptide and then modeling its structure based on the structure of MarR. The invention also pertains to the use of the MarR family polypeptide structures in the methods described below, e.g., for the identification of MarR family polypeptide modulating compounds.
Given the sequence-structure relationship described, the MarR crystal structure (described below) in the presence or absence of a MarR family polypeptide modulating compound can be used as a template to generate a computational three-dimensional model of any of the other members of the MarR protein family. In another embodiment, both crystals can be compared and the resulting information (including information regarding the binding site of the MarR family modulator) can be used. The resulting structure(s) can be subjected to the entire complement of computational approaches discussed and demonstrated above. Computer software packages such as COMPOSER (SYBYL. Tripos, Inc. 1699 Hanley Rd. St. Louis, Mo. 63144; Sutcliffe, M. J. et al. Protein Eng. 1987, 1, 385–392), MODELLER (Accelrys, Inc. 9685 Scranton Road San Diego, Calif. 92121–3752 U.S.A.; Sali, A. B. J. Mol. Biol. 1993, 234, 779–815) are widely utilized. The process of generating a structure as described is known as homology modeling or comparative molecular modeling. Generically, the process includes overall protein sequence alignment, determination of structurally conserved regions (SCR's), transposition of the template structure onto the undetermined sequence, loop building and refinement. As an example of how the MarR structure can be used for this purpose, a three-dimensional model of SlyA was generated as described in the appended examples.
Design of MarR Family Modulating Compounds Using Rational Drug Design Techniques
The term “MarR family modulating compound” includes small molecules and other chemical entities which are capable of modulating, e.g., increasing or decreasing or otherwise altering the activity of a MarR family polypeptide or its down stream products, e.g., a MarR modulating compound may modulate the binding of MarR to DNA (e.g., the marO operon) or otherwise alter the expression of MarA. In one embodiment, the MarR family modulating compound is a MarR activator that enhances the binding of MarR to DNA (e.g., the marO operon), such that MarA expression is reduced.
The term “MarR family modulating compound candidate” includes compounds which are being screened or otherwise tested (e.g., computationally or in the laboratory) to determine whether or not they modulate MarR or a MarR family polypeptide.
The term “rational drug design” includes both computer aided and non-computer techniques where a protein is analyzed for active sites, and then modulating compound candidates are designed to interact with the particular spatial and electrochemical requirements of the particular site.
The term “active site” includes regions of a protein where a MarR family modulating compound physically interacts with a MarR family polypeptide. Any portion of the surface of a MarR family polypeptide can be considered an active site region or locus. In one embodiment, the portion of the MarR family protein immediately adjacent to the binding site of a MarR family modulating compound (e.g., a salicylate moiety) is referred to as the active site for the MarR family polypeptide. Other active sites include the DNA binding regions and regions necessary for interactions with other biological components, e.g., DNA or protein.
The term “interacts” includes interactions between the MarR family polypeptide and the MarR family modulating compound which result in modulation of a MarR family associated activity, e.g., expression of MarA when the MarR family polypeptide is MarR. The term also includes interactions which are determined by the shape and electronic complementarity between the MarR family polypeptide and the MarR family modulating compound. The term “interact” includes detectable interactions between molecules. The term interact is also meant to include “binding” interactions between molecules. Exemplary interactions include protein-protein and protein-nucleic acid interactions.
Specific knowledge of the three-dimensional shape and electronic properties of the MarR family polypeptide's active site provides information on how a MarR family modulating compound candidate may be modified to optimize interactions with a MarR family polypeptide. Several computer programs may be used to graphically depict the shape and electronic properties of the active site. These include, but are not restricted to CoMFA, (See Podlogar, B. L.; et al. Drug Des. Discov. 2000, 17, 4–12. and references therein), GRID (See GRID: Molecular Discovery Ltd., 4 Chandos St., London, W1A 3AQ, Goodford, P. J. et al. J. Med. Chem. 1985, 28, 849–857), and LIG BUILDER (See LIGBUILDER: Wang, R. et al. J. Mol. Model. 2000, 6, 498–516).
In these approaches, the active site is postulated and then placed within a three-dimensional lattice of evenly distributed grid points. A small molecular fragment or atom is placed on each lattice point, and a mathematical evaluation is made to determine the electronic and spatial properties at that point. After each lattice point within the active site is thus defined, the spatial and electronic “values” are contoured to generate maps or graphical representations that indicate the locations within the active site that are capable of accommodating additional “atomic bulk” and whether the atomic bulk should be charge positive, negative or neutral. It is the general theory that “filling” the active site with appropriate “atomic bulk” will optimize the drug-target interaction, thereby producing the maximal pharmacological response.
For example, the program LigBuilder was used to characterize one of the MarR active sites (SAL-A) in terms of its spatial and electronic properties. The results from this program represent a collection of colored crosses that depict an “inverse cast” of the MarR active site. Each cross represents a point where a mathematical determination was made. The shape of the inverse cast is dependent upon the van der Waals radii of the target's atoms constituting the active site as defined by the crystal structure of MarR. The colors indicate where the active site prefers positive or negative charge complemetarity. For example, arginine # 86 of MarR is positively charged at physiologic pH. Consequently, atoms or atom fragments that are negatively charged would produce the optimal complimentarily about that point, which is correctly depicted by the LigBuilder program.
Once the active site has been graphically defined, the spatial and electronic representations of a MarR family modulating compound candidate can be fit or docked within the target active site. Specific modifications of an initial candidate can be made electronically, and then tested to determine whether the complementarity between the active site and the modulating compound candidate has been increased.
To demonstrate the use of the crystal structure for docking, the coordinates of the salicylate (a MarR modulator, which, in one embodiment, can be cocrystalized with MarR) were artificially removed from the MarR active site. Using this newly created empty active site as input, the program FLEXX is able to predict the proper binding orientation of salicylate with MarR (FLEXX Module, in SYBYL. Tripos, Inc. 1699 Hanley Rd. St. Louis, Mo. 63144. Rarey, M. et al. J. Mol. Bio. 1996, 261, 470–489). The result of this docking experiment is shown in green, which can be compared to the original salicylate orientation as determined crystallographically. As shown, the dominant molecular interactions between the cocrystalized salicylate molecule and the active site residues are predicted by the docking algorithm, e.g. the carboxylate and hydroxyl groups. The greatest variation between the computationally predicted docking and that determined by experiment occurs at the 4- and 5-positions of the aromatic ring. These correspond to the regions of the cocrystalized salicylate molecule with the largest crystallographic b-factors, and indicate that the carboxylate and hydroxyl groups of the salicylate moiety create the primary interactions within the active site. These two major interactions create a hinge point where the aromatic ring pivots within the active site.
As used in a drug discovery program, small modifications of the salicylate molecule can be made computationally, and then subjected to the identical FLEXX docking as demonstrated above for salicylate. The score of the modified salicylate can be compared to the original to ascertain the modification's benefit to overall target site complimentarily. This is a time consuming process, and is typically utilized only as part of the lead optimization process in an active drug discovery program. A variant of this approach is the automated application to large virtual libraries of potential drug candidates, known as automated ligand docking (Muegge, I.; Rarey, M. Small Molecule Docking and Scoring. Reviews in Computational Chemistry; Wiley: New York, 2000). This approach is typically employed as a part of the lead identification or screening process of a drug discovery program, since a large number of modulating compound candidates can quickly be assessed for active site complimentarily. Programs available include, but are not restricted to DOCK (DOCK Suite of Programs: Reagents of the University of California: DesJarlais et al. J. Med. Chem. 1988, 31, 722–729), AUTO_DOCK (AUTO_DOCK: Olson, A. J., SCRIPPS, La Jolla, Calif. Goodsell, D. S., et al. J. Mol. Recognit. 1996, 9), GLIDE, and FLEXX (FLEXX Module, SYBYL. Tripos, Inc.; Rarey, M. et al. J. Mol. Bio. 1996, 261, 470–489). Each requires an electronic representation of a library of potential drug candidates. Early versions of these approaches treated the drug candidate as a rigid body, wherein conformational flexibility was neglected. Algorithmic improvements and increases in computational speed now allow the flexibility of a potential candidate to be included. Based on the relative values of these scores, a virtual library of structures can be quickly screened for members that would produce the best interaction within the active site. As such, large libraries, originating from commercial vendors, from proprietary template enumeration or other sources, can be culled to eliminate compounds that are not promising (data reduction) and/or prioritized to highlight compounds warranting further consideration. Compounds with better overall docking scores will be placed higher on the list.
Each of the techniques described above are included as rational drug design methods. Other rational drug design techniques include de novo drug design which utilizes the structure of the protein to generate molecules to dock within the active site. In this approach, a “seed” atom, or seed-molecule with pre-defined attachment points is placed within the active site. Programs are available to systematically “grow” chemical modifications at the attachment points resulting in novel molecules. Through an iterative process of growing and assessing the complimentarily of the new structures, productive attachments can be saved, while unproductive attachments are discarded. Subsequent redefinition of the seed based on productive attachments can produce large number of drug candidates for the specified target. This is an unbiased approach since the resulting compound is not taken from a pre-existing virtual library, and is often used to generate compounds that would otherwise not be considered based on current proprietary knowledge or chemist's intuition. For example, this approach was applied to one of the MarR active sites using the program LigBuilder to produce a list of novel potential drug candidates. The compounds generated by LigBuilder are merely representative of one class of compounds which may be useful as MarR family protein modulating compounds. The invention also pertains to other compounds which may interact with other portions and thus have little or no structural similarity to these compounds.
Rational drug design also may involve the identification of pharmacophoric elements. In drug design, important functional groups are referred to as pharmacophoric elements and are useful for productive drug-target interaction. For example, for MarR salicylate site A (SAL-A), certain interactions between the salicylate moiety and the MarR active site may be attributable to the two main functional groups of the salicylate moiety, namely the carboxylate and the hydroxyl groups. At this site, the carboxylate creates a charge-charge interaction with arginine #86, and the hydroxyl group interacts strongly with threonine #72 by virtue of hydrogen bonding. Furthermore, the absence of either of these elements may diminish the degree of complemintarity. The collection of pharmacophoric elements and their mutual spatial disposition within the active site defines the pharmacophore of the active site (See, e.g., WO 97/27219). In one embodiment, a MarR family modulating compound of the invention interacts with an amino acid corresponding (e.g., linearly or three dimensionally) to arginine at position #86 of SEQ ID NO:1 and/or threonine at position #72 SEQ ID NO:1.
For MarR, the carboxylate and hydroxyl groups of an inhibitor are separated by a distance of about 1.5 Å. As such, any compound with a similar functional groups thus positioned will possess the pharmacophore for MarR. Such information can be deduced from a known collection of compounds that demonstrate interaction with MarR. However, the crystal structure of MarR and its active site can be used to define a series of testable pharmacophore hypothesis. Programs, such as CoMFA, GRID and LigBuilder are instrumental in defining these hypotheses in a manner similar to that detailed by Clackson. In one embodiment, a MarR family modulating compound of the invention comprises a carboxylate and a hydroxyl group separated by a distance of 1.5 Angstroms.
In one embodiment, a known drug candidate is co-crystallized in the active site (e.g., salicylate, plumbagin, or DNP for MarR), since the exact coordinates of the pharmacophore can be determined. In another embodiment, the MarR family member is crystalized without a cocrystalizing agent. In another embodiment, the crystal structures of the MarR family member in the presence and absence of the co-crystallizing agent are compared to determine the effect of binding of the cocrystalizing agent. Thus, with or without a co-crystallizing compound, the pharmacophore can be used as a search query to identify structures from virtual libraries of commercial (known) or hypothetical structures. Programs including, but not restricted to UNITY (UNITY Module, in SYBYL. Tripos, Inc. 1699 Hanley Rd. St. Louis, Mo. 63144), CATALYST (CATALYST, Accelrys, Inc. 9685 Scranton Road San Diego, Calif. 92121–3752 U.S.A. Sprague, P. W. Comput.-Assisted Lead Find Optim., [Eur. Symp. Quant. Struct.-Act. Relat.] 1997, 225–240) may be used for this purpose (Greer, J. et al. J. Med. Chem. 1994, 37, 1035–1054; WO 99/45389). The pharmacophore elements can also be used as the seeds for de novo design. LigBuilder was applied to the active site of MarR using the carboxylate and hydroxyl groups as “seed” groups to approximate the pharmacophore hypothesis. Common among these structures are the actual elements of the pharmacophore as expected, but in nearly all of the structures examined, another hydrogen bond acceptor was present, indicating the possibility of yet another pharmacophoric element in the pharmacophore.
In another embodiment, the invention pertains to a method for identifying a MarR family modulating compound using the three-dimensional structure of a MarR family polypeptide. The method includes selecting a candidate MarR family modulating compound by performing rational drug design with the set of atomic coordinates in FIG. 1 and/or FIG. 2 using computer aided techniques, as described herein. In one embodiment, the method also includes contacting the candidate MarR family modulating compound with a MarR family peptide, and a nucleic acid molecule, and then measuring the binding affinity of the MarR family polypeptide peptide with the nucleic acid molecule, such that MarR family modulating compounds are identified. In one embodiment, the nucleic acid molecule is a nucleic acid molecule to which a particular MarR family member is known to bind. For example, for MarR, the nucleic acid used for the binding acid may be, for example, marO.
In a further embodiment, the MarR family modulating compound is a MarR activator that acts, e.g., to inhibit the expression of MarA.
The invention also pertains to a method of identifying a MarR family member modulating compound. The method includes obtaining a set of atomic coordinates defining the three-dimensional structure of MarR or a MarR family polypeptide; selecting a candidate MarR family modulating compound by performing rational drug design with said three dimensional structure of the MarR family polypeptide; contacting said candidate MarR family modulating compound with MarR family polypeptide; and measuring the ability of the candidate MarR family modulating compound to modulate the activity of the MarR family polypeptide, thus identifying a MarR modulating compound.
In one embodiment, the rational drug design is aided by a computer program described supra. In one embodiment, the MarR family polypeptide is MarR and has the polypeptide sequence given in SEQ ID NO. 1 and has the atomic coordinates given in FIG. 1, when cocrystallized with salicylate or FIG. 2, when crystallized without.
In another embodiment, the invention pertains to compounds generated by the methods of the invention, described above. For example, the invention pertains to the MarR family modulating compounds and MarR modulating compounds generated by the rational drug design techniques described above. Examples of MarR modulating compounds include those of the formula(I):

X-Y-Z  (I)

wherein

    
    
        X is an interacting moiety;
        Y is a hydrophobic moiety; and
        Z is a polar moiety.
    
    


The term “interacting moiety” includes moieties which are capable of interacting with a MarR family member. Preferably, such interacting moieties interact with Thr 72 of SEQ ID. 1 or an amino acid molecule that corresponds to Thr 72 in a MarR family polypeptide. In a further embodiment, the interacting moiety is capable of interacting by hydrogen bonding. Examples of interacting moieties include, but are not limited to, hydroxyl, thiol, sulfanyl, sulfonyl, amino, carbonyl, alkyl, and acyl moieties. The term “interacting moiety” includes moieties which allow the MarR modulating compound to perform its intended function, e.g., modulate MarR family member activity. In a further embodiment, the interacting moiety is hydroxy, thiol, or amino.
The term “hydrophobic moiety” includes moieties which are capable of interacting with the MarR family polypeptide such that the compound is capable of performing its intended function, e.g., modulate MarR. In certain embodiments, the hydrophobic moiety may be substituted with substituents capable of hydrogen bonding such as, but not limited to, hydroxy, thiol, carbonyl, amino, carboxylate, or thiol. Examples of hydrophobic moieties include, but are not limited to, substituted and unsubstituted alkyl, alkenyl, alkynyl, and aryl moieties.
In certain embodiments, the hydrophobic moiety is aryl. The aryl moiety may be cyclic, bicyclic or tricyclic. Preferably, the hydrophobic moiety is selected such that it is capable of interacting with MarR, such that its activity is modulated. In a further embodiment, the MarR family modulating compound is selected such that it is capable of interacting with hydrophobic or neutral amino acid residues, such as, but not limited to, Pro 57 or Met 74 or an amino acid residue corresponding to these amino acids of SEQ ID NO:1.
The term “polar moiety” includes moieties which are capable of interacting with MarR family polypeptide such that the activity of the MarR family polypeptide is modulated. In one embodiment, the polar moiety interacts with Arg 86 or Arg 77 or an amino acid residue corresponding to these amino acids of SEQ ID NO:1. In one embodiment, polar moiety is negatively charged. Examples of polar moieties include carboxylate and isoteres thereof. Other examples include, but are not limited to, phosphate, phosphite, sulfate, sulfite, nitrate, nitrite, nitro, hydroxy, oxalate, and perchlororate.
In one embodiment, the MarR family modulating compound is a MarR inhibitor. In another embodiment, the polar moiety and the interacting moiety are separated by a distance of about 1.5 Angstroms.
In a further embodiment, the MarR modulating compound is of the formula:




wherein Y is a substituted or unsubstituted cyclic or bicyclic moiety, and pharmaceutically acceptable salts and esters thereof. In a further embodiment, X is hydroxyl. In another further embodiment, Y is monocyclic or bicyclic, optionally substituted with a hydrophilic substituent. Examples of MarR modulating compounds include those listed below.











The compounds described herein can be synthesized by methods known in the art. An ordinarily skilled artisan will be able to consult the chemical literature and will be able to synthesize the compounds described herein.
The term “alkenyl” includes unsaturated aliphatic groups, including straight-chain alkenyl groups, branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups, alkenyl substituted cycloalkyl or cycloalkenyl groups, and cycloalkenyl substituted alkyl or alkenyl groups. The term alkenyl further includes alkenyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. In preferred embodiments, a straight chain or branched chain alkenyl group has 10 or fewer carbon atoms in its backbone (e.g., C1–C10 for straight chain, C3–C10 for branched chain), and more preferably 6 or fewer. Likewise, preferred cycloalkenyl groups have from 4–7 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure, e.g., cyclopentene or cyclohexene.
The term “alkyl” includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. In preferred embodiments, a straight chain or branched chain alkyl has 10 or fewer carbon atoms in its backbone (e.g., C1–C10 for straight chain, C3–C10 for branched chain), and more preferably 6 or fewer. Likewise, preferred cycloalkyls have from 4–7 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
Moreover, the term 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. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, 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, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, e.g., with the substituents described above. An “alkylaryl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
The term “aryl” includes aryl groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl 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, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, 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, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
The terms “alkenyl” and “alkynyl” include unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively. Examples of substituents of alkynyl groups include, for example alkyl, alkenyl (e.g., cycloalkenyl, e.g., cyclohenxenyl), and aryl groups.
Unless the number of carbons is otherwise specified, “lower alkyl” includes an alkyl group, as defined above, but having from one to three carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths.
The terms “alkoxyalkyl”, “polyaminoalkyl” and “thioalkoxyalkyl” 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.
The terms “polycyclyl” or “polycyclic radical” refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, 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, 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, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term “heteroatom” includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
The term “alkylsulfinyl” include groups which have one or more sulfinyl (SO) linkages, typically 1 to about 5 or 6 sulfinyl linkages. Advantageous alkylsulfinyl groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.
The term “alkylsulfonyl” includes groups which have one or more sulfonyl (SO2) linkages, typically 1 to about 5 or 6 sulfonyl linkages. Advantageous alkylsulfonyl groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.
The term “alkanoyl” includes groups having 1 to about 4 or 5 carbonyl groups. The term “aroyl” includes aryl groups, such as phenyl and other carbocyclic aryls, which have carbonyl substituents. The term “alkaroyl” includes aryl groups with alkylcarbonyl substituents, e.g., phenylacetyl.
The invention also includes a method for inhibiting expression of MarA. The method includes contacting MarR with a MarR inhibiting compound. In an embodiment, the MarR inhibiting compound is of the formula(I):

X-Y-Z  (I)

wherein X is an interacting moiety, Y is a hydrophobic moiety; and Z is a polar moiety, and acceptable salts thereof. In an embodiment, the MarR inhibiting compound inhibits the binding of MarR to DNA (e.g., the marO operon).

Biological systems generally function through carefully choreographed interactions of their respective components. The operative mechanisms for many disease states implicate protein-protein interactions as key. For transcription factors, such as MarR, protein-DNA and protein-RNA interactions control the regulation events for the biological system. The drug design approaches discussed above are targeted in part to disrupt the interaction between MarR and the mar operon. Knowledge of the three dimensional structure of the MarR-marO complex can provide clues as to the key interactions (pharmacophore) made between them. A computer model of an interaction between MarR and DNA is shown in FIG. 10.
The invention also pertains to a method for decreasing multidrug resistance in a microbe, e.g., E. coli. The invention includes contacting E. coli with a MarR inhibiting compound, such that said multidrug resistance in E. coli is decreased. In an embodiment, the MarR inhibiting compound is of the formula(I):

X-Y-Z  (I)

wherein X is an interacting moiety; Y is a hydrophobic moiety; and Z is a polar moiety, and acceptable salts thereof.

The invention also pertains to methods for modulating activity of a MarR family polypeptide. The method includes contacting a MarR family polypeptide with a MarR family modulating compound identified by any method described herein (e.g., the computer modeling techniques, etc.). The invention also pertains to any compound discovered using techniques described herein.
The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.
EXEMPLIFICATION OF THE INVENTION
Example 1: Crystallization of MarR with Salicylate
Protein Production and Purification
Native and selenomethionine (Se-Met) containing MarR was prepared from E. coli BL21(DE3) (Novagen) bearing pMarR-WT, a wild type MarR expression vector that has been previously described (Alekshun, M. N. & Levy, S. B. J. Bact. 181, 4669–4672 (1999)). Native MarR was produced in whole cells according to previous methods (Alekshun, M. N. & Levy, S. B. J. Bact. 181, 4669–4672 (1999)). Se-Met MarR was produced by diluting an overnight culture of E. coli BL21(DE3)+pMarR-WT 1:1000 in M9 medium supplemented with 2 mM MgSO4, 0.2% glucose, 0.1 mM CaCl2, 0.00005% thiamine, 0.04 mg ml−1 each of the following amino acids phenylalanine, leucine, isoleucine, valine, serine, threonine, tyrosine, histidine, lysine, aspartic acid, glutamic acid, tryptophan, and tryptophan, and kanamycin (Miller, J. H. In Experiments in Molecular Genetics. (Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.; 1972). This culture was grown at 37° C. to an OD600≈0.6 and 100 mg each of amino acids threonine, lysine-hydrochloride, phenylalanine, 50 mg each of amino acids leucine, isoleucine, and valine (single letter abbreviations), and 60 mg L-(+)-selenomethionine (Sigma) were then added. The culture was grown for 15 min at 37° C.; IPTG was subsequently added to a final concentration of 1 mM and protein production was allowed to proceed for 14.5 hr at 37° C. Cell pellets were collected and processed as previously described (Alekshun, M. N. & Levy, S. B. J. Bact. 181, 4669–4672 (1999)).
Frozen cell pellets containing native or Se-Met MarR were resuspended in 100 mM sodium phosphate buffer (pH 7.4) containing a bacterial protease inhibitor cocktail (Sigma) and sonicated on ice. All buffers contained 2 mM DTT when Se-Met MarR was prepared. Insoluble matter was removed by centrifugation at 4° C. at 30,000×g for 40 min. The supernatant was passed over prepacked 5 ml SP-sepharose HiTrap columns (Amersham Pharmacia Biotech) previously equilibrated with 10 mM sodium phosphate buffer (pH 7.4). The column was washed with 50 ml of 10 mM sodium phosphate buffer (pH 7.4) and the pure proteins were eluted with a linear gradient (0–0.5 M) of NaCl in 10 mM sodium phosphate buffer (pH 7.4). Protein containing fractions were dialyzed vs. 10 mM HEPES (pH 7.4), 200 mM NaCl, and 1 mM DTT, or 2 mM DTT in the case of Se-Met MarR, and the protein in these samples was judged to be greater than 99% pure via SDS-PAGE and electrospray ionization mass spectrophotometry. The latter also demonstrated that more than 95% of the three methionine residues in Se-Met MarR were substituted with selenomethionine.
Crystallization:
MarR crystals were originally grown in 18% PEG MME 5000, 200 mM ammonium sulfate, 100 mM citrate buffer (pH 5.6) but showed anisotropic disorder in the diffraction data that made them unsuitable for structure determination. To stabilize the protein, the citrate was substituted by the known inhibitor salicylate. Crystals of the MarR-salicylate complex were grown at 17° C. by hanging droplet vapor diffusion. 6 μl of a 11.4 mg ml−1 protein solution in 200 mM NaCl, 20 mM HEPES (pH 7.4), and 10 mM DTT were added to 2 μl of reservoir buffer (18% PEG MME 5000, 50 mM ammonium sulfate, 250 mM sodium salicylate, 10 mM DTT, and 15% glycerol, pH 5.5), and 0.8 μl 15% heptanetriol. The droplets were equilibrated with 1 ml of reservoir buffer. Crystals grew within 1 week reaching dimensions of approximately 0.3 mm per side.
X-ray Data Collection, Structure Determination, and Refinement:
Diffraction data were collected at the Brookhaven National Synchrotron Light Source, beamline X8C. Crystals were flash frozen in mother liquor at the beam line before data collection. All data were processed and reduced using DENZO and SCALEPACK (Otwinowski, Z. In CCP4 Proceedings. 56–62 (Daresbury Laboratory, Warrington, UK, 1993). The space group of the MarR-salicylate co-crystals was determined to be I4122 with one molecule in the asymmetric unit and with unit cell dimensions of a=b=62.0 Å, c=132.9 Å, α=β=γ=90° for both the native and the selenoprotein. Data were collected on the selenoprotein crystals at three wavelengths to enable MAD phasing. Phases were determined from the MAD data using the program SOLVE (Terwilliger, T. C. & Berendzen, J. Acta Crystallogr. D. 55, 849–861 (1999)). This showed two selenium sites per asymmetric unit, with the third selenomethionine, at the N-terminus, apparently disordered. Maps were solvent-flattened using the program DM and the model was built into density using the program O (Collaborative Computational Project, Number 4. Acta Crystallogr. D. 50, 760–763 (1994); Jones, T. A. et al. Acta Crystallogr. A 47, 110–119 (1991)). Model and refinement parameters for salicylate were obtained from the Hetero Compound Information Center (Kleywegt, G. J. & Jones, T. A. Acta Crystallogr. D. 54, 1119–1131 (1998)). Model refinement was performed using CNS and cycles of rebuilding and refinement continued to give the final model (Brunger, A. T. et al. Acta Crystallogr. D. 54, 905–921 (1998)). Model quality was assessed by sa-omit, Fo-Fc, maps generated over the whole molecule omitting no more than 7% of the structure at a time. The model extends from residue 6 to the C-terminus at residue 144. In common with several other transcription factors (e.g. TetR, (1A6I), ArgR (1B4B) and TreR (1BYK)), MarR shows relatively high thermal mobility throughout the structure, as reflected by the B-factors. Certain regions appear to be particularly mobile, including the extended structure at the N-terminus, the tip of the “wing” (residues 91–94), parts of the a5 helix, especially around Gly 116 and the connecting loop (128–131) between the a5 and the C-terminal α6 helix. Consistent with the high B-factors, the molecule shows few well-ordered solvent molecules. PROCHECK reports overall g-factors of 0.25 (dihedrals) and 0.55 (main chain covalent forces) and shows that 91% of the residues fall within the most favored region of the Ramachandran plot, with only residue Ala 53 in a disallowed region. This residue is located at the start of the loop connecting the α2 and α3 helices.
The coordinates of the MarR-salicylate cocrystal are shown in FIG. 1. Data collection, phasing and refinement statistics for the MarR-sal cocrystal structure is shown in Table 1.











TABLE 1





Data set
Native
Se-met edge
e-met peak
e-met remote



















Wavelength (Å)
1.072
0.9795
0.9793
0.9500


Resolution range
50–2.3
50–2.3
50–2.3
50–2.3


(Å)


Measured
56,495
84,173
96,582
87,365


reflections


Unique
6,069
5,534
5,564
5,472


reflections


Completeness (%)
99.5(100)
91.3(99.8)
91.7(99.8)
90.4(99.7)


overall


(final shell)


<I/σI>
21.1(12.0)
12.2(7.2)
12.0(7.0)
12.9(7.9)


(final shell)


Rmerge (%)
6.0(20.0)
6.4(29.7)
5.7(30.3)
4.9(25.5)


(final shell)


Rano (%)

4.9
5.0
3.5


Overall FOM
0.59/0.71


(centric/acentric)


Resolution
50–2.3


Rfree
28.7%


Rcryst
24.7%


Atoms/AU


Protein
1078


Salicylate
20


Water
18


Average B


(Å2)


main chain
49.7


side chain
59.2


salicylate
42.7


water
50.0


R.m.s. deviation


Bonds (Å)
0.009


Angles (°)
1.3









Example 2: Crystallization of MarR
MarR was produced and purified as described in Example 1.
Crystallization:
Crystals of MarR were grown by hanging droplet vapor diffusion. 3 μl of a 10 mg ml−1 2:1 (mol:mol) DNA-protein solution in 200 mM NaCl, 20 mM HEPES, pH 7.4, 20 mM TRIS-HCl, pH 8.0, and 2 mM MgCl2 was added to 1 μl of reservoir buffer (23% PEG MME 5000, 100 mM sodium citrate, 200 mM ammonium sulfate, 10 mM DTT, 10% glycerol, 5% Isopropanol, pH 5.6), and 0.4 μl 15% heptanetriol. The droplets were equilibrated with 0.5 ml of reservoir buffer.
X-ray Data Collection, Structure Determination, and Refinement:
Diffraction data were collected at the Brookhaven National Synchrotron Light Source, beamline X8C. Crystals were flash frozen in mother liquor at the beam line before data collection. All data were processed and reduced using DENZO and SCALEPACK (Otwinowski, Z. In CCP4 Proceedings. 56–62 (Daresbury Laboratory, Warrington, UK, 1993).
The coordinates of the MarR crystal without salicylate are shown in FIG. 2. Data collection, phasing, and refinement statistics for the MarR co-crystal structure is shown in Table 2.










TABLE 2









Space group
C222



Unit cell (Å)
a = 65.8, b = 137.7, c = 96.4



Resolution
50–2.7



Rfree
26.7%



Rcryst
23.2%



Atoms/AU



Protein
2093



Water
14



Average B (Å2)



main chain
40.0



side chain
48.0



Water
32.6



R.m.s. deviation



Bonds (Å)
0.009



Angles (°)
1.3










Example 3: Use of the Crystal Structure of MarR to Model Other MarR Family Polypeptides
The amino acid sequences of MarR and SlyA are shown in FIG. 11. This alignment is generated automatically using the subroutines in COMPOSER, however it can be generated by a variety of other programs. FIG. 12 shows the results of the COMPOSER program in identifying the structurally conserved regions (SCRs).
The amino acids colored magenta are the regions of MarR and SlyA where the amino acid sequences are predicted to exhibit the same tertiary structure. These predictions are based on a knowledge base of information derived from the compilation of known crystal structures. Specifically, statistical correlations are made for protein tertiary structure with the respective amino acid sequences, and it was found that the correlations could be used in a predictive manner.
In the comparative molecular modeling process, the three-dimensional coordinates of the MarR backbone in the SCRs were directly transposed to create a general framework for SlyA as seen in FIGS. 13a and 13b. FIG. 13a is the Cα-trace of MarR with the SCRs highlighted as orange tubes. The SCRs were “extracted” in their same mutual orientation to produce the basic framework of SlyA, which is shown in FIG. 13b. The process at this point generally includes only the backbone chain coordinates; the sidechains are added computationally to the SCR's on the left to create the SlyA protein. This model can, in all respects, be subjected to the identical regimen of computational protocols as the bona fide MarR crystal structure (Podlogar, B. L. et al. J. Med. Chem. 1997, 40, 3453–3455).
The regions in yellow (FIG. 12) are the “loops” that connect the SCRs. Loop regions, in general, exhibit the greatest variation among members in the same family. As such, no logical template for their construction is available. Again, use is made of the vast knowledge contained in the database of determined protein structures to construct the loop regions. FIG. 14 shows the fully constructed SlyA structure (purple) in comparison to the template protein, MarR.
Example 4: Use of the Computer Modeling to Characterize the MarR Active Site
For example, the program LigBuilder was used to characterize the MarR active site in terms of its spatial and electronic properties. The results represent a collection of colored crosses that depict an “inverse cast” of the MarR active site. Each cross represents a point where a mathematical determination was made. The shape of the inverse cast is dependent upon the van der Waals radii of the target's atoms constituting the active site as defined by the crystal structure of MarR. The colors indicate where the active site prefers positive or negative charge complemetarity. For example, arginine # 86 of MarR is positively charged at physiologic pH. Consequently, atoms or atom fragments that are negatively charged would produce the optimal complimentarily about that point, which is correctly depicted by the LigBuilder program.
Once the active site has been graphically defined, the spatial and electronic representations of a MarR modulating compound candidate can be fit or docked within the target active site. Specific modifications of an initial candidate can be made electronically, and then tested to determine whether the complementarity between the active site and the modulating compound candidate has been increased. To demonstrate the use of the crystal structure for docking, the coordinates of the salicylate were artificially removed from the MarR active site. Using this newly created empty active site as input, the program FLEXX is able to predict the proper binding orientation of salicylate with MarR (FLEXX Module, in SYBYL. Tripos, Inc. 1699 Hanley Rd. St. Louis, Mo. 63144. Rarey, M. et al. J. Mol. Bio. 1996, 261, 470–489). The result of this docking experiment can be compared to the original salicylate orientation as determined crystallographically. The dominant molecular interactions between the salicylate and the active site residues may be predicted by the docking algorithm, e.g. the carboxylate and hydroxyl groups.
Example 5: Use of Rational Drug Design to Identify MarR Modulating Compounds
One method of rational drug design techniques includes de novo drug design which utilizes the structure of the protein to generate molecules to dock within the active site. In this approach, a “seed” atom, or seed-molecule with pre-defined attachment points is placed within the active site. Programs are available to systematically “grow” chemical modifications at the attachment points resulting in novel molecules. Through an iterative process of growing and assessing the complimentarily of the new structures, productive attachments can be saved, while unproductive attachments are discarded. Subsequent redefinition of the seed based on productive attachments can produce large number of drug candidates for the specified target. This is an unbiased approach since the result is not taken from a pre-existing virtual library, and is often used to generate compounds that would otherwise not be considered based on current proprietary knowledge or chemist's intuition.
This approach was applied to the MarR protein using the program LigBuilder to produce a list of novel potential drug candidates.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.
All patents, patent applications, and literature references cited herein are hereby expressly incorporated by reference. The entire contents of Alekshun et al. “The Crystal Structure of MarR a Regulator of Multiple Antibiotic Resistance at 2.3 Å resolution,” Nature Structural Biology 8(8) is hereby incorporated herein by reference.

## Claims

1. A MarR modulating compound of the formula:




wherein
X is an interacting moiety;
wherein the interacting moiety is selected from the group consisting of hydroxyl, thiol, sulfanyl, sulfonyl, amino, carbonyl, alkyl, and acyl moieties
Y is a substituted or unsubstituted indole and pharmaceutically acceptable salts and esters thereof; and
wherein X and a carboxylate group of said formula are separated by a distance of about 1.5 Å.

2. The MarR modulating compound of claim 1, wherein said MarR modulating compound is

3. The MarR modulating compound of claim 1, wherein said compound is:

4. The MarR modulating compound of claim 1, wherein said interacting moiety is capable of hydrogen bonding.

5. The MarR modulating compound of claim 4, wherein said interacting moiety interacts with an amino acid, wherein the amino acid is Asn at position 72 of SEQ ID. 1.

6. The MarR modulating compound of claim 4, wherein said interacting moiety interacts with an amino acid, wherein the amino acid is Asn at position 70 of SEQ lID No. 1.

7. The MarR modulating compound of claim 1, wherein said MarR modulating compound is a MarR inhibitor.