A novel sarR gene and sarR gene product which down regulates the expression of sarA and the resulting virulence determinants in Staphylococcus aureus is provided. Methods for modulating the expression of sarA and virulence determinants are also provided. A preferred embodiment of the present invention provides structural information relating to the gene product and enables the identification and formulation of lead compounds and reducements for treating and preventing infections by S. aureus and related bacteria.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides a new genetic locus of S. aureus and other bacteria. The gene at this locus is referred to herein as sarR. The sarR gene is involved in the regulation and expression of virulence determinants in S. aureus and other bacteria.

The present invention provides a polynucleotide sequence of the sarR gene (SEQ. ID. NO: 1) which is involved in the regulation and expression of virulence determinants in S. aureus and related bacteria. Also provided is a sarR gene product, an isolated polypeptide which is involved in regulation and expression of virulence determinants in S. aureus and related bacteria.

An object of the present invention is to provide nucleic acid sequences that regulate the expression of virulence determinants in S. aureus and related bacteria. In a preferred embodiment the nucleic acid sequence is isolated from S. aureus and in another preferred embodiment the nucleic acid sequence is a sarR gene (SEQ. ID. NO: 1).

Another object of the present invention is to provide a polypeptide which regulates the expression of virulence determinants in S. aureus and other bacteria. In a preferred embodiment the polypeptide is isolated from S. aureus and in another preferred embodiment the polypeptide is encoded by a sarR gene and correspond to the sequence set forth in SEQ ID NO: 2.

Vectors and host cells comprising nucleic acid sequences encoding these polypeptides and chemical entities that mimic or enhance the activity of such polypeptides are further objects of the invention. These agents can be used alone or in combination with antibacterial agents such as penicillin to enhance the properties of such agents.

Another object of the present invention is to provide methods for modulating regulation and expression of virulence determinants in S. aureus and related bacteria to inhibit their growth and infectivity by contacting the bacteria with an agent which interacts with sarA promoter regions.

Another object of the present invention is to provide a method of inhibiting growth and infectivity of bacteria comprising contacting the bacteria with an agent which enhances the expression of the sarR gene, or the activity of a polypeptide encoded by the sarR. gene.

Another object of the present invention is to provide a kit for identifying the presence of a sarR gene or a polypeptide encoded by a sarR gene.

A further object of the present invention is to provide nucleic acid sequences from a mutant sarR gene in S. aureus and related bacteria, and polypeptides encoded by a mutant sarR gene.

A further object of the invention is to provide a three dimensional crystal structure of the SarR protein.

A further object of the invention is to provide methods of screening for lead compounds which inhibit the expression of virulence determinants in S. aureus and related bacteria.

Another object of the present invention is to provide pharmaceutical compositions for use as anti-bacterial agents against S. aureus and other bacteria.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel gene found in S. aureus and other bacterial species. This gene is designated herein as sarR and is represented SEQ ID NO: 1. The present invention also provides for a novel gene corresponding to the sequence as essentially set forth in SEQ ID NO: 1, and analogs or homologs thereof.

The present invention also provides the sarR gene product designated herein as the SarR protein represented in SEQ ID. NO. 2, and analogs or homologs thereof. The SarR protein has a molecular weight of approximately 13.7 kDa and has a deduced basic pI of approximately 9.2. The sarR gene product is also characterized by a predominance of charged residues (34%).

The present invention also relates to newly discovered binding sites in the sarA promoter system. These binding sites enable the SarR protein to repress sarA expression by preventing sarA from initiating the expression of virulence factors

The present invention further provides a model for a SarA/SarR heterodimer which interferes with the function of the SarA homodimer. The present invention also provides alternative heterodimers which include SarA/SarR peptidomimetic combinations and other combinations resulting from lead compounds developed from structural analysis of SarR.

The present invention also provides a sarR gene product or peptidomimetic capable of directly binding to promoters of a virulence gene such as the hla promoter of the alpha hemolysin gene to provide direct inhibition of such virulence factors.

The present invention also provides a method of down; regulating sarA P1 transcription to repress the expression of sarA. This function of the sarR gene product was demonstrated by the results of mutant studies disclosed herein. The sarR mutant expressed a much higher level of the SarA protein than the parent strain. Accordingly, the present invention also provides sarR mutant strains. These strains are useful as experimental and diagnostic tools to specifically characterize the sarA expression systems.

The present invention also provides a detailed three-diminsional (3-D) crystal structure of the SarR protein. The structural data demonstrate that the protein is a member of a new family of winged helix proteins.

The present invention provides methods for treating microbial pathogenesis by the administration of SarR analogs in the form of small molecule compounds which alter the functioning of a microbial sarA expression. Reducing or eliminating the expression of sarA by such analogs can greatly alter the course and effects of a bacterial infection. This treatment approach is distinct from most prior bactericidal or bacteristatic antimicrobial treatments which attempted to either kill the microbial cells, or directly prevent them from reproducing. The antimicrobial action of the compounds of these standard antimicrobial treatments is exerted both in vivo, in an infection, and in vitro, in a culture, unless some specific compensating factor(s) is provided which allows survival or growth in the presence of the antimicrobial agent. In contrast, this invention is directed at the regulation of sarA expression which is involved in the pathogenesis process, but is not necessarily essential for microbial survival or growth in vitro. By selectively targeting the expression of virulence factors and not directly killing the bacterium there is less pressure to develop antimicrobial resistance. Because this type of therapy is less likely to alter the local flora, the incidence of superinfection (e.g. fungal or other bacterial infections such as pseudomonas or enterococcus) is reduced.

According to one aspect of the present invention, an analog library is provided to produce a very large number of potential molecules for regulating the sarA expression system, and in general the greater:the number of analogs in the library, the greater the likelihood that at least one member of the library will effectively regulate the sarA expression system. Designed libraries following a particular template structure and limiting amino acid variation at particular positions are much preferred, since a single library can encompass all the designed analogs and the included sequences will be known and presented in roughly equal numbers. By contrast, random substitution at only six positions in an amino acid sequence provides over 60 million analogs, which is a library size that begins to present practical limitations even when utilizing screening techniques as powerful as phage display. Libraries larger than this would pose problems in handling, e.g., fermentation vessels would need to be of extraordinary size, and more importantly, the likelihood of having all of the planned polypeptide sequence variations represented in the prepared library would decrease sharply. It is therefore preferred to create a designed or biased library, in which the amino acid positions designated for variation are considered so as to maximize the effect of substitution on the sarA regulation characteristics of the analog, and the amino acid residues allowed or planned for use in substitutions are limited.

The use of replicable genetic packages, such as the bacteriophages, is one method of generating novel polypeptide entities that regulate sarA expression. This method generally consists of introducing a novel, exogenous DNA segments into the genome of a bacteriophage (or other amplifiable genetic package) so that the polypeptide encoded by the non-native DNA appears on the surface of the phage. When the inserted DNA contains sequence diversity, then each recipient phage displays one variant of the template (parental) amino acid sequence encoded by the DNA, and the phage population (library) displays a vast number of different but related amino acid sequences.

Such techniques make it possible not only to screen a large number of potential binding molecules but make it practical to repeat the binding/elution cycles and to build secondary, biased libraries for screening analog-displaying packages that meet initial criteria.

It is well-known to those normally skilled in the art that it is possible to replace peptides with peptidomimetics. Peptidomimetics are generally preferable as therapeutic agents to peptides owing to their enhanced bioavailability and relative lack of attack from proteolytic enzymes. Techniques of molecular modeling may be used to design a peptidomimetics which mimic the structure of the SarR peptide disclosed herein. Accordingly, the present invention also provides peptidomimetics and other lead compounds which can be identified based on the data obtained from structural analysis of the SarR protein disclosed herein. A potential SarR analog is examined through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK. This procedure can include computer fitting of potential SarR analogs. Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of an analog to a potential binding site. Generally the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force) the more potent the potential drug will be since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential drug the more likely that the drug will not interfere with other properties of the sarA expression system. This will minimize potential side-effects due to unwanted interactions with other proteins.

Initially a potential SarR analog could be obtained by screening a random peptide library produced by a recombinant bacteriophage, for example, or a chemical library. A analog ligand selected in this manner could be then be systematically modified by computer modeling programs until one or more promising potential ligands are identified.

Such computer modeling allows the selection of a finite number of rational chemical modifications, as opposed to the countless number of essentially random chemical modifications that could be made, and of which any one might lead to a useful drug. Thus through the use of the three-dimensional structure disclosed herein and computer modeling, a large number of compounds is rapidly screened and a few likely candidates can be determined without the laborious synthesis of untold numbers of compounds.

Once a potential SarR analog is identified it can be either selected from a library of chemicals commercially available from most large chemical companies including Merck, GlaxoWelcome, Bristol Meyers Squib, Monsanto/Searle, Eli Lilly, Novartis and Pharmacia UpJohn, or alternatively the potential ligand is synthesized de novo. As mentioned above, the de novo synthesis of one or even a relatively small group of specific compounds is reasonable in the art of drug design.

In a further aspect, this invention provides methods of treating a bacterial infection by administering a pharmaceutical composition comprising a SarR analog, peptide, or peptidomimitic to inhibit the expression of sarA. The present invention also provides a method for prophylactic treatment of a mammal, in particular a human, in order to prevent a bacterial infection. Such treatment comprises administering a pharmaceutical preparation comprising a SarR analog, peptide, or peptidomimetic to the mammal. Preferably such treatment would be used when the patient is at risk of contracting or developing a bacterial infection. Such a prophylactic treatment method may have particular benefit, for example, for treating patients prior to surgical operations.

Pharmaceutical compositions according to the present invention may comprise peptides and peptidomimetics of the present invention in association with a pharmaceutically acceptable carrier or excipient, adapted for use in human or veterinary medicine. The compositions may contain from 0.001-99% of the active material. Such compositions may be presented for use in conventional manner in admixture with one or more physiologically acceptable carriers of excipients. The pharmaceutical compositions according to the invention may also contain other active ingredients such as antimicrobial agents, or preservatives. The compositions may optionally further contain one or more other therapeutic agents which may, if desired, be a chemotherapeutic antiviral agent.

Pharmaceutically acceptable salts of the peptides of this invention may be formed conventionally by reaction with an appropriate acid. The additional salts so formed from addition by acid may be identified by hydrochloric, hydrobromic, phosphoric, acetic, fumaric, salicylic, citric, lactic, mandelic, tartaric, oxalic, methanesulfonic, and the like.

Thus, the peptides and peptidomimetics according to the present invention may be formulated for oral, buccal, parenteral, topical or rectal administration. In particular, these peptides and peptidomimetics may be formulated for injection or for infusion and may be presented in unit dose form in ampoules or in multidose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

The present invention further provides a process for preparing a pharmaceutical composition which comprises bringing a peptide or peptidomimetic of the invention into association with a pharmaceutically acceptable excipient or carrier.

The dosage of the peptide or peptidomimetic used in the treatment will vary, depending on the seriousness of the disorder, the weight of the patient, the relative efficacy of the peptide and the judgment of the treating physician. Unit dosages may be administered more than once a day, e g., two or three times a day. Such therapy may extend for several weeks, in an intermittent or uninterrupted manner, until the patient's symptoms are eliminated.

The present invention also provides pharmaceutical compositions which comprise a pharmaceutically effective amount of the peptides of this invention, or pharmaceutically acceptable salts thereof, and, preferably, a pharmaceutically acceptable carrier or adjuvant. Therapeutic methods of this invention comprise the step of treating patients in a pharmaceutically acceptable manner with those peptides or compositions. Such compositions may be in the form of tablets, capsules, caplets, powders, granules, lozenges, suppositories, reconstitutable powders, or liquid preparations, such as oral or sterile parenteral solutions or suspensions.

In order to obtain consistency of administration, it is preferred that a composition of the invention is in the form of a unit dose. The unit dose presentation forms for oral administration may be tablets and capsules and may contain conventional expedients. For example binding agents, such as acacia, gelatin, sorbitol, or polyvinylpyrrolidone; fillers, such as lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants such as magnesium stearate; disintegrants, such as starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate.

The solid oral compositions may be prepared by conventional methods of blending, filling, tabletting, or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are, of course, conventional in the art. The tablets may be coated according to methods well-known in normal pharmaceutical practice, in particular with an enteric coating.

Oral liquid preparations may be in the form of emulsions, syrups, or elixirs, or may be presented as a dry product-for reconstitution with water or other suitable vehicle before use. Such liquid preparations may or may not contain conventional additives. For example suspending agents, such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats; emulsifying agents, such as sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters selected from the group consisting of glycerine, propylene glycol, ethylene glycol, and ethyl alcohol; preservatives, for instance methyl para-hydroxybenzoate, ethyl para-hydroxybenzoate, n-propyl parahydroxybenzoate, or n-butyl parahydroxybenzoate or sorbic acid; and, if desired, conventional flavoring or coloring agents.

For parenteral administration, fluid unit dosage forms may be prepared by utilizing the peptide and a sterile vehicle, and, depending on the concentration employed, may be either suspended or dissolved in the vehicle. In preparing solutions, the peptides of this invention may be dissolved in water, whereas opiates used heretofore showed only marginal solubility in aqueous media or physiological fluids. Once in solution, the peptide may be injected and filter sterilized before filling a suitable vial or ampoule and subsequently sealing the carrier or storage package. Adjuvants, such as a local anaesthetic, a preservative or a buffering agent, may be dissolved in the vehicle prior to use. Stability of the pharmaceutical composition may be enhanced by freezing the composition after filling the vial and removing the water under vacuum, e.g., freeze drying the composition. Parenteral suspensions may be prepared in substantially the same manner, except that the peptide should be suspended in the vehicle rather than being dissolved. A surfactant or wetting solution may be advantageously included in the composition to facilitate uniform distribution of the peptide.
 

Claim 1 of 5 Claims

1. An isolated nucleic acid molecule which regulates the expression of virulence determinants in gram positive bacteria, wherein said isolated nucleic acid molecule comprises the sequence set forth in SEQ ID NO:1.

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