Date of Award

Fall 12-14-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Phang C. Tai

Second Advisor

Zehava Eichenbaum

Third Advisor

Parjit Kaur

Fourth Advisor

Chung-Dar Lu

Abstract

Due to the emergence and dissemination of multidrug resistance, bacterial pathogens have been causing a serious public health problem in recent years. To address the existing drug resistant problem, there is an urgent need to find new antimicrobials, especially those against drug-resistant bacteria. SecA is the central component of Sec-dependent secretion pathway, which is responsible for the secretion of many essential proteins as well as many toxins and virulence factors. Two SecA homologues are indentified in some important Gram-positive pathogens. SecA1 is involved in general secretion pathway and essential for viability, whereas SecA2 contribute to secretion of specific virulence factors. The high conservation among a wide range of bacteria and no human counterpart make SecA homologues attractive targets for exploring novel antimicrobials. We hypothesize that inhibition of these SecA homologues could reduce virulence, inhibit bacteria growth, and kill bacteria. SecA1 and SecA2 from four different species were cloned, purified, and characterized. All these SecA homologues show ATPase activities, thus screening ATPase inhibitors might help to develop new antimicrobials. In this study, three structurally different classes of SecA inhibitors were developed and optimized: 1) Rose Bengal (RB) and RB analogs derived from systematical dissection RB and Structure-Activity relationship (SAR) study; 2) pyrimidine analogs derived from virtual screening based on the ATP binding pocket of EcSecA and SAR study; and 3) bistriazole analogs derived from random screening and SAR study. Several potent SecA inhibitors show promising enzymatic inhibition against SecA homologues as well as bacteriostatic and bactericidal effects. Two major efflux pumps of S. aureus, NorA and MepA, have little negative effect on the antimicrobial activities of SecA inhibitors, suggesting that targeting SecA could by-pass efflux pumps. Moreover, these inhibitors impair the secretion of important toxins of S. aureus and B. anthracis, indicating the inhibition of in vivo SecA function could reduce virulence. Target identification assays confirm that these inhibitors could directly bind to SecA homologues, and specifically identify SecA from whole cell lysate of E. coli and S. aureus, suggesting that these inhibitors are really targeting on SecA. These studies validate that SecA is a good target for development antimicrobials.

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