Development of Novel MetAP2 inhibitors as potential therapeutics for Microsporidiosis

Abstract This application represents an approach to rational drug design for the treatment of microsporidiosis using structure activity relationships (SAR) for agents that inhibit Methionine Aminopeptidase type 2 (MetAP2). Microsporidiosis is an emerging zoonotic infection that is an opportunistic pathogen in the setting of AIDS, but is also seen in other immune compromised hosts as well as immune competent hosts. Current therapies are suboptimal. The hypothesis underlying this type of grant is that differences in the structure of the drug target between host and pathogen will permit the design of selective therapeutic agents with decreased host toxicity. The initial choice in drug design is the selection of the target among the dozens of potential targets. MetAP2 is an extremely logical therapeutic target for these pathogens. Microsporidia lack Methionine Aminopeptidase type 1 (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 humans have both MetAP1 and MetAP2. Use of Fumagillin and its derivatives, which are non-competitive inhibitors that covalently bind to and inhibit MetAP2 (but not MetAP1), has confirmed that inhibition of MetAP2 is an effective in vitro and in vivo therapeutic target for many species of microsporidia. In fact, Fumagillin has been demonstrated to have efficacy in humans infected with Enterocytozoon bieneusi; however, its use has been limited by bone marrow toxicity. Our research group has cloned, expressed and determined the crystal structure of the MetAP2 of the microsporidian Encephalitozoon cuniculi (i.e. EcMetAP2) as well as developed yeast dependent on EcMetAP2 for growth. We have identified and cloned Ent.bieneusi MetAP2 (EbMetAP2). Exploiting differences in the structure of MetAP2 between host and pathogen should permit the design of selective therapeutic competitive inhibitors of MetAP2 with decreased host toxicity. These new inhibitors will be tested in vitro and in vivo for efficacy and in an iterative process we will use this information to refine our models and improve inhibitor design. Using this Limited Rational Design (LRD) approach we will generate new libraries based on our lead compounds that will then be modified to generate new libraries to improve their selectivity and pharmacologic properties. We have already identified two lead compounds, BL6 and D63, from our initial LRD/SAR studies that have increased selectivity for microsporidian MetAP2 and efficacy in our in vitro and in vivo models of microsporidiosis. Since MetAP2 is important in other protozoa our lead compounds should also define new classes of drugs that could have broad anti-parasitic activity and prove useful in the treatment of other infections. Our assembled research group containing experts in medicinal chemistry, parasitology, bioinformatics, mass spectrometry and structural biology as well as industry consultants has the necessary complementary expertise to develop and test these compounds.