Cryptosporidium parvum IMP Dehydrogenase: IDENTIFICATION OF FUNCTIONAL, STRUCTURAL, AND DYNAMIC PROPERTIES THAT CAN BE EXPLOITED FOR DRUG DESIGN

Nwakaso N Umejiego,Catherine Li,Thomas Riera,Lizbeth Hedstrom,Boris Striepen

doi:10.1074/jbc.m407121200

Abstract

The protozoan parasite Cryptosporidium parvum causes severe enteritis with substantial morbidity and mortality among AIDS patients and young children. No fully effective treatment is available. C. parvum relies on inosine 5'-monophosphate dehydrogenase (IMPDH) to produce guanine nucleotides and is highly susceptible to IMPDH inhibition. Furthermore, C. parvum obtained its IMPDH gene by lateral transfer from an epsilon-proteobacterium, suggesting that the parasite enzyme might have very different characteristics than the human counterpart. Here we describe the expression of recombinant C. parvum IMPDH in an Escherichia coli strain lacking the bacterial homolog. Expression of the parasite gene restores growth of this mutant on minimal medium, confirming that the protein has IMPDH activity. The recombinant protein was purified to homogeneity and used to probe the enzyme's mechanism, structure, and inhibition profile in a series of kinetic experiments. The mechanism of the C. parvum enzyme involves the random addition of substrates and ordered release of products with rate-limiting hydrolysis of a covalent enzyme intermediate. The pronounced resistance of C. parvum IMPDH to mycophenolic acid inhibition is in strong agreement with its bacterial origin. The values of Km for NAD and Ki for mycophenolic acid as well as the synergistic interaction between tiazofurin and ADP differ significantly from those of the human enzymes. These data suggest that the structure and dynamic properties of the NAD binding site of C. parvum IMPDH can be exploited to develop parasite-specific inhibitors.

Highlights

The protozoan parasite Cryptosporidium parvum causes severe enteritis with substantial morbidity and mortality among AIDS patients and young children
The inosine 5؅-monophosphate dehydrogenase (IMPDH) inhibitors ribavirin and mycophenolic acid (MPA) block C. parvum development in tissue culture [5, 6], and ribavirin is effective in the neonatal mouse model,2 which validates IMPDH as a target for treatment of cryptosporidiosis
Molecular Methods—The coding sequence of C. parvum IMPDH was amplified by PCR, generating artificial flanking restriction sites for BamHI and HindIII using primers 5Ј-CGTAGGATCCATGGGTACAAAAAACATAGGAAAAGG-3Ј and 5Ј-TACGAAGCTTCTATTTACTATAATTCATTACTT-3Ј and plasmid pCpIMPDHComp as template

Summary

Introduction

The protozoan parasite Cryptosporidium parvum causes severe enteritis with substantial morbidity and mortality among AIDS patients and young children. C. parvum relies on inosine 5؅-monophosphate dehydrogenase (IMPDH) to produce guanine nucleotides and is highly susceptible to IMPDH inhibition. The values of Km for NAD and Ki for mycophenolic acid as well as the synergistic interaction between tiazofurin and ADP differ significantly from those of the human enzymes These data suggest that the structure and dynamic properties of the NAD binding site of C. parvum IMPDH can be exploited to develop parasitespecific inhibitors. Effective drug treatment is urgently needed for the management of cryptosporidiosis, especially in AIDS patients. The IMPDH inhibitors ribavirin and mycophenolic acid (MPA) block C. parvum development in tissue culture [5, 6], and ribavirin is effective in the neonatal mouse model, which validates IMPDH as a target for treatment of cryptosporidiosis. IMPDH is a well developed target for antiviral and immunosuppressive therapy, and several inhibitors of human IMPDH are already in clinical use [7,8,9,10], so the design of parasite-specific IMPDH inhibitors will be greatly facilitated by the ability to “piggy-back” on existing drug development programs

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