The antibiotic discovery project combines chemistry and biology to identify new compounds of utility in treating or preventing disease caused by Gram-negative bacteria. The project develops high throughput screens, performs chemical synthesis as part of hit to lead chemistry, measures the biological effect of compounds with purified enzymes and in whole bacteria, and studies compound efficacy in small animal models of disease. Currently three separate programs are active: (1) Gram-negative virulence proteins secretion inhibition by thiazolidinones (2) Diverse strategies including chemical synthesis of analogs and high throughput screen development to discover compounds which inhibit the three steps of the second messenger di-c-GMP metabolism and (3) Testing of a quinolone that has no intrinsic antibiotic activity but promotes the activity of the cationic antibiotics polymyxins and aminoglycosides. In our first specific aim we propose to (a) advance this thiazolidinone chemotype to define a lead compound for therapeutic development by using a combination of SAR studies and analysis of target compounds in in vitro benchmark studies and animal models;(b) characterize the structural mechanisms by which thiazolidinones inhibit bacterial secretion systems implementing a combination of genetic and biochemical approaches;and to (c) identify additional broad spectrum inhibitors of bacterial secretion systems by means of high-throughput screens and corresponding secondary assays In our second specific aim (a) compounds that are potential functional inhibitors of c-di-GMP will be tested in biochemical assays for enzyme inhibition and inhibition of receptor binding of di-c-GMP (b) inhibition of c-di-GMP metabolism and inhibition of a variety of c-di-GMP regulated properties will be determined using functional assays for the effect of compounds on live bacteria and (c) new compounds will be developed that inhibit the components of di-c-GMP metabolism by performance of high throughput and virtual screens. In our third specific aim we propose to (a) investigate compounds that that increase sensitivity to cationic antibiotics for the ability to inhibit bacterial infections using animal models;(b) identify additional chemotypes that increase sensitivity to antibiotics by repeating our high-throughput screen with Burkholderia thailandensis, a model non-BSL3 organism with constitutive resistance to polymyxin and a variety of other antibiotics;and (c) identify the mechanism of action by which relevant compounds increase sensitivity to polymyxin and aminoglycosides by using a genetic approach to isolate resistant mutants.
Gram-negative bacterial resistance to current antibiotics is increasing and can be engineered. The treatment of agents of potential bio-warfare and emerging pathogens could require the development of broad spectrum counter measures that are unique to this class of organisms and this proposal addresses this problem.
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