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.

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.

Protein translocation is essential for bacterial survival and the most important translocation mechanism in bacteria is the secretion (Sec) pathway. Thus targeting Sec pathway is a promising strategy for developing novel antibacterial therapeutics. We report the design, syntheses, mechanistic studies and structure-activity relationship studies using HQSAR and 3-D QSAR Topomer CoMFA analyses of 4-oxo-5-cyano thiouracil derivatives. In summary, introduction of polar group such as –N3 and linker groups such as –CH2-O- enhanced the potency as well as logP and logS several fold. We also report the discovery, optimization and structure-activity relationship study of 1,2,4-triazole containing pyrimidines as novel, highly potent antimicrobial agents. A number of inhibitors have been found to inhibit microbial growth at high nanomolar concentrations.

Protein translocation is essential for bacterial survival and the most important translocation mechanism in bacteria is the secretion (Sec) pathway. Thus targeting Sec pathway is a promising strategy for developing novel antibacterial therapeutics. We report the design, syntheses, mechanistic studies and structure-activity relationship studies using HQSAR and 3-D QSAR Topomer CoMFA analyses of 4-oxo-5-cyano thiouracil derivatives. In summary, introduction of polar group such as –N3 and linker groups such as –CH2-O- enhanced the potency as well as logP and logS several fold. We also report the discovery, optimization and structure-activity relationship study of 1,2,4-triazole containing pyrimidines as novel, highly potent antimicrobial agents. A number of inhibitors have been found to inhibit microbial growth at high nanomolar concentrations.

SecA ATPase is a critical member of the Sec system, which is important in the translocation of membrane and secreted polypeptides/proteins in bacteria. Small molecule inhibitors can be very useful research tools as well as leads for future antimicrobial agent development. Based on previous virtual screening work, we optimized the structures of two hit compounds and obtained SecA ATPase inhibitors with IC50 in the single digit micromolar range. These represent the first low micromolar inhibitors of bacterial SecA and will be very useful for mechanistic studies. Post synthetic modification is an important and efficient way of DNA functionalization especially in DNA aptamer selection. In this research, the feasibility of norbornene (Neo) modified thymidine triphosphate incorporation was described. Besides, substituted tetrazines have been found to undergo facile inversed electron demand Diels-Alder reactions with "tunable" reaction rates. This finding paves the way to utilize tetrazine conjugation reactions for not only DNA but also other labeling work.