Microsporidiosis:A Therapeutic Target

DESCRIPTION (Provided by the applicant): "Microsporidia" is a nontaxonomic term used to refer to a group of obligate, intracellular, parasites belonging to the phylum Microspora consisting of 144 genera and over 1200 species. These parasites are ubiquitous in nature and infections have been described in all classes of vertebrates as well as in most invertebrates. They are important agricultural parasites in commercially significant insects, fish, laboratory rodents, rabbits, fur-bearing animals and primates. Microsporidia are emerging pathogens in humans (especially in immunosuppressed hosts) as well as in animals. Data suggest that the antibiotic fumagillin and its derivatives are potentially useful agents for the treatment of microsporidiosis. We have demonstrated that TNP-470, a fumagillin derivative developed as an anti-angiogenesis drug, is active both in vitro and in vivo against the microsporidia. TNP-470 and ovalicin have been demonstrated to bind irreversibly to a common bifunctional protein identified by mass spectrometry as methionine aminopeptidase type 2 (MetAP2). These drugs do not bind or inhibit the activity of methionine aminopeptidase type 1 (MetAP1). We believe that fumagillin and its analogs function by inhibiting microsporidian methionine aminopeptidase type 2 (MetAP2) and that MetAP2 is a critical enzyme in the microsporidia. Homology PCR was used to isolate a MetAP2 homologue from Encephalitozoon hellem. We plan to finish cloning the Enc. hellem methionine aminopeptidase type 2 (EhMetAP2) and subsequently clone and express MetAP2 from Vittaforma corneae. The interaction of fumagillin with EhMetAP2 will be confirmed. Using information obtained on microsporidian MetAP2 genes, the MetAP2 gene of Enterocytozoon bieneusi will then be cloned using a homology PCR approach. In order to evaluate the inhibition of microsporidian MetAP2 as a target for therapy we plan to express enzymatically active recombinant microsporidian MetAp2, characterize the properties of recombinant and native microsporidian MetAP2 and develop an in vitro assay for inhibition of microsporidian MetAP2. In addition we plan to investigate fumagillin and ovalicin analogs as anti-microsporidian agents in vitro and in vivo based on data obtained on microsporidian MetAp2. This study should yield important information on microsporidian MetAP2, provide an in vitro rapid assay for agents with anti-microsporidian MetAp2 activity and provide data on new fumagillin analogs as agents for the treatment of experimental microsporidiosis.