INHIBITORS OF FLUROROQUINOLONE EFFLUX IN GRAM + PATHOGENS

The goal of this project is to determine the feasibility of developing inhibitors of the Staphylococcus aureus multidrug-efflux transporter NorA. NorA provides S. aureus with intrinsic resistance to fluoroquinolone antibiotics by reducing their concentration inside the bacterial cell. Inhibition of NorA has been demonstrated to dramatically potentiate the antibacterial action of fluoroquinolones and prevent the emergence of antibiotic-resistant variants of S. aureus. One NorA inhibitor 1 reserpine, has already been identified by the authors. Unfortunately, the use of reserpine is limited by its neurotoxicity. Preliminary studies have demonstrated that reserpine is not unique in its ability to inhibit NorA; screening of 2,500 organic compounds has yielded a number of structurally diverse molecules also capable of suppressing NorA activity. During the first, exploratory phase of the project, 7,500 more compounds will be screened for their ability to inhibit NorA. To eliminate toxic compounds, the identified inhibitors will be tested for their toxicity to a panel of human cell lines. The compounds to which NorA can become insensitive through mutations will also be eliminated. The remaining inhibitors of highest potency will be used in Phase Il of the project as lead compounds, on the basis of which we expect to develop a clinically useful NorA inhibitor. PROVIDE COMMERCIAL APPLICATIONS This project is to develop nontoxic inhibitors of the S. aureus multidrug transporter NorA, the protein involved in resistance of these bacteria to fluoroquinolone antibiotics. The envisioned commercial product is a combination of such an inhibitor with fluoroquinolone. Given that staphylococcal infections are widespread and rapidly become resistant to antibiotic therapy, the market for this product is likely to be comparable to the market for major antibiotics. $ = TOTAL AWARD AMTS & NOT LIMITED TO PORTION OF PROJECT RELATED TO SUBJECT OF SEARCH SUBPROJECT $ = TOTAL AWARD AMOUNT DIVIDED BY NUMBER OF SUBPROJECTS SOURCE: CRISP FORMAT F FY 97 LAST UPDATE 04-07-98 1QUERY 1536 ID SEARCH 06/01/98 PAGE 330 --PROJECT NUMBER......1 R43 GM55462-01 INVESTIGATOR NAME/ADDRESS FY 97 HODJAT, HOSSEIN K IRG/INTRAMURAL UNIT..ZRG3 OMNI CHROM, INC AWARD AMOUNT......... $100,000 10-B HENSHAW ST WOBURN, MA 01801 PERFORMING ORGANIZATION: OMNI CHROM, INC. TITLE DISPLACEMENT PURIFICATIONS OF ODNAS WITH NOVEL COLUMNS ABSTRACT: A novel ion-exchange stationary phase will be developed for displacement chromatography of synthetic phosphorothioate oligonucleotide. To date, this class of compounds are purified using ion-exchange and reversed phase-chromatography with non-linear elution techniques. These methods suffer from low recovery, non-predictable selectivity and very un- economical use of the stationary phases. The proposed research will focus on the optimization of the surface chemistry with the use of minimal amount of oligonucleotides. The base material will be made of non-porous polystyrene divinylbenzene with high chemical and physical stability. The surface of the polystyrene support will be coated first with a covalent, non-charged, hydroxyl rich, hydrophilic layer. The hydroxyl groups are then reacted with amine functionalities and, secondary interactions are controlled by derivatizing the resulting amines with activated glycols. The optimized surface will separate phosphorothioate oligonucleotides based exclusively on their length even at overloaded conditions. The separation power of displacement chromatography on the new columns will be demonstrated by micropreparative purification of crude synthetic phosphorothioate oligonucleotides. The proposed research will provide new test methods for analyzing chromatographic surfaces for displacement chromatography as well.