The Microsporidia are emerging zoonotic pathogenic protists which are on the Bioterrorism B list as they can be transmitted by food or water. The most common human pathogenic microsporidia Enterocytozoon bieneusi is non cultivatable. Our data, and that of other groups, indicates that fumagillin functions by inhibiting microsporidian methionine aminopeptidase type 2 (MetAP2) which is an essential enzyme in these organisms. This application represents the initial steps in rationale drug design for these difficult pathogens. Exploiting differences in the structure of MetAP2 between host and pathogen should permit the design of selective therapeutic competitive agents with decreased host toxicity. MetAP2 is a logical therapeutic target as microsporidia lack MetAP1 making MetAP2 an essential enzyme. Among eukaryotes this makes them highly susceptible to MetAP2 inhibitors and limits the toxicity of these compounds in their hosts as most eukaryotes have both MetAPI and MetAP2. Use of fumagillin and its derivatives has confirmed that inhibition of MetAP2 is an effective in vitro and in vivo therapeutic target for many species of microsporidia suggesting that like Encephalitozoon cuniculi other microsporidia are dependent on MetAP2. Fumagillin has demonstrated efficacy in human infections due to Ent. bieneusi, but its use has been limited by bone marrow toxicity. We have successfully developed primers for PCR cloning and sequencing of the MetAP2 genes of the microsporidia. We plan to clone the MetAP2 gene of Ent. bieneusi using techniques we have developed from our successful cloning of MetAP2 from other human pathogenic microsporidia. We will express, purify and characterize recombinant microsporidian MetAP2s. Crystals of purified recombinant microsporidian MetAP2 will be obtained for X-ray crystallography. In addition, Saccharomyces cerevisiae will be engineered to express microsporidian MetAP2 and not yeast MetAP1 (map1) or MetAP2 (map2) in order to develop a rapid surrogate screening system for drug studies. This project will develop regents useful for the development of new therapeutic agents for microsporidiosis targeting microsporidian methionine aminopeptidase type 2. In addition, the approaches and methods developed in this R21 should also prove useful for studies on methionine aminopeptidases as therapeutic targets in other pathogenic protists. ? ? ?
|Das, Bhaskar C; Thapa, Pritam; Karki, Radha et al. (2013) Boron chemicals in diagnosis and therapeutics. Future Med Chem 5:653-76|
|Alvarado, John Jeff; Nemkal, Anjana; Sauder, J Michael et al. (2009) Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470. Mol Biochem Parasitol 168:158-67|
|Upadhya, Rajendra; Zhang, Hong Shan; Weiss, Louis M (2006) System for expression of microsporidian methionine amino peptidase type 2 (MetAP2) in the yeast Saccharomyces cerevisiae. Antimicrob Agents Chemother 50:3389-95|