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ANTI-MICROBIAL PROTEASE INHIBITOR

Human morbidity and mortality due to bacterial infections has become a problem of great concern due to the well-publicized dramatic increase in the frequency of infections caused by bacteria that are resistant to most, or even all, known available antibiotics. Since even modern state-of-the-art antibiotics can be ineffective for treating disease caused by multi-drug resistant "superbugs" there is an urgent need for the development of new drugs for the treatment of bacterial infections. Researchers at Dartmouth have recently identified the active site and applied a chemical method to inhibit a family of bacterial proteases involved in pathogenicity. The protease is required for infection by numerous gram-negative bacterial pathogens and has a role in the dissemination of genes such as those that encode resistance to antibiotics via a DNA-uptake system in at least several gram-negative and gram-positive bacterial species. Since the protease family is unique to bacteria, it represents a potential anti-microbial target.

A fundamental process of all living cells, including bacteria is the secretion of proteins across membranes. In recent years it has become evident that in the case of bacteria there are several systems for secreting proteins and some of these systems have unique leader peptidases associated with their cognate secreted proteins. One of these is known as the type 2 secretion system which promotes extracellular secretion of bacterial factors, most notably toxins and colonization pili that are the hallmarks of the mechanisms that promote virulence of pathogenic bacteria. A unifying component of the various type 2 secretion systems is the involvement of type 4 pilin (in the case of colonization pilus formation), or pilin-like (in the case of toxin secretion apparatus or DNA-uptake apparatus) proteins. Our laboratory has been studying the Vibrio cholerae type 4 colonization pilus TCP (toxin coregulated pilus). The pilus is the major intestinal colonization factor of V. cholerae and other highly related type 4 pili serve as the major colonization factor of up to 50 different gram-negative bacterial species. The type 4 pili are composed of a polymerized structure of type 4 pilin, designated TcpA in the case of TCP. The pilin is synthesized as a prepilin with a leader peptide that requires a type 4 leader peptidase to process the type 4 prepilin leader sequence to allow secretion of the mature protein. In the case of TcpA processing, the cognate prepilin peptidase is termed TcpJ. Pilus or peptidase mutants are unable to colonize and are avirulent in animal models and humans. In an effort to understand the requirements and mechanism by which TcpJ processes TcpA prepilin into its mature form capable of assembly into a colonization pilus, we have undertaken a genetic approach of mutating the tcpJ gene. This method led to the discovery of the mechanism of action of this protease, which is unique from any previously described in the scientific literature. Our results demonstrate that TcpJ, and likely any type 4 prepilin peptidase, works by a mechanism that represents a new subclass of proteases. The generality of this mechanism of action was then demonstrated for the type 4 prepilin peptidase, VcpD, that is required for toxin secretion. Identifying the active site residue(s) of the protease allowed for the specific modification of the residue(s) to prevent protease activity. This chemical inhibition was detected using an in vitro peptidase processing reaction that is potentially adaptable to a high-throughput screen for compounds with inhibitory activity.

This technology is claimed in the issued United States Patent No. 6,887,677 and the published United States Patent Application No. 11/071,972. We are seeking an industrial partner to further develop a protease inhibitor which would function in a therapeutic and perhaps prophylactic role to inhibit or prevent gram-negative bacterial infections which are a major cause of rapidly fatal infections such as bacterial meningitis or bacterial sepsis, and to serve to prevent DNA-uptake by gram-positive bacterial species, which is required for the evolution of multi-drug resistant strains. The development of such an antimicrobial could be analogous to the development and clinical use of viral protease inhibitors which have recently met with great success in the treatment of infections caused by human immunodeficiency virus (HIV-1). (Ref: J67)


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