The Immunosuppressive Agent Mizoribine Monophosphate Forms a Transition State Analogue Complex with Inosine Monophosphate Dehydrogenase

Gan, Lu; Seyedsayamdost, Mohammad R.; Shuto, Satoshi; Matsuda, Akira; Petsko, Gregory A.; Hedstrom, Lizbeth

doi:10.1021/bi0271401

Cited by 60 publications

(106 citation statements)

References 34 publications

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“…MZP induces the closed flap conformation in T. foetus IMPDH as well as movement in the loop that contains the catalytic Cys (41). MZP probably induces a similar conformation in human IMPDH (a similar conformation is induced by the related inhibitor ribavirin monophosphate) (42).…”

Section: Discussionmentioning

confidence: 99%

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Cryptosporidium parvum IMP Dehydrogenase

Umejiego¹,

Li²,

Riera

et al. 2004

Journal of Biological Chemistry

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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 K m for NAD and K i 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.

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Section: Discussionmentioning

confidence: 99%

“…Drug selectivity is also determined by dynamic conformational changes within the dinucleotide site (31,41). MPA binds to the vacant NADH site of the E-XMP* intermediate and blocks the movement of the flap required for the hydrolysis reaction (Fig.…”

Section: Discussionmentioning

confidence: 99%

Cryptosporidium parvum IMP Dehydrogenase

Umejiego¹,

Li²,

Riera

et al. 2004

Journal of Biological Chemistry

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“…1). IMPDH undergoes a large conformational change in mid-catalytic cycle that converts the enzyme from a dehydrogenase to a hydrolase (15). In the first half of the reaction, substrates bind, and hydride transfer occurs to form the covalent E⅐XMP* intermediate.…”

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confidence: 99%

The Functional Basis of Mycophenolic Acid Resistance in Candida albicans IMP Dehydrogenase

Köhler

Gong

Bentink

et al. 2005

Journal of Biological Chemistry

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Candida albicans is an important fungal pathogen of immunocompromised patients. In cell culture, C. albicans is sensitive to mycophenolic acid (MPA) and mizoribine, both natural product inhibitors of IMP dehydrogenase (IMPDH). These drugs have opposing interactions with the enzyme. MPA prevents formation of the closed enzyme conformation by binding to the same site as a mobile flap. In contrast, mizoribine monophosphate, the active metabolite of mizoribine, induces the closed conformation. Here, we report the characterization of IMPDH from wild-type and MPA-resistant strains of C. albicans. The wild-type enzyme displays significant differences from human IMPDHs, suggesting that selective inhibitors that could be novel antifungal agents may be developed. IMPDH from the MPA-resistant strain contains a single substitution (A251T) that is far from the MPA-binding site. The A251T variant was 4-fold less sensitive to MPA as expected. This substitution did not affect the k cat value, but did decrease the K m values for both substrates, so the mutant enzyme is more catalytically efficient as measured by the value of k cat / K m . These simple criteria suggest that the A251T variant would be the evolutionarily superior enzyme. However, the A251T substitution caused the enzyme to be 40-fold more sensitive to mizoribine monophosphate. This result suggests that A251T stabilizes the closed conformation, and this hypothesis is supported by further inhibitor analysis. Likewise, the MPA-resistant strain was more sensitive to mizoribine in cell culture. These observations illustrate the evolutionary challenge posed by the gauntlet of chemical warfare at the microbial level.

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“…Recently, details have been reported other immunosuppressive activities, including inhibition of B-cell function and antibody production (11,25). Our of the mechanism underlying inhibition of IMPDH activity by MZ and mycophenolic acid (MPA), the results thus indicate that MZ could be one candidate for islet xenotransplantation in combination with other active form of MMF (6) immunosuppressive drug for islet xenotransplantation. …”

Section: Histological Examination Histologically Marked Infiltrationmentioning

confidence: 99%

Mizoribine as Sole Immunosuppressive Agent in Islet Xenotransplantation Models: A Candidate Immunosuppressant Causing no Adverse Effects on Islets

et al. 2012

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Mizoribine (MZ) inhibits the differentiation and proliferation of helper T and B cells after antigen recognition by suppressing the purine biosynthesis pathway and nucleic acid synthesis. MZ has been used in kidney transplantation, but distinct data are unavailable for islet transplantation. The present study investigated the efficacy of MZ for islet xenotransplantation. Immunosuppressive effects of MZ were determined by mixed lymphocyte reaction (MLR) assay in vitro. Toxicities for Wistar rat islets were determined by adenosine triphosphate (ATP) contents of islets during 3-day culture and stimulation index in response to glucose after culture. Immunosuppressive effects in vivo were tested in a Wistar-to-B6 islet xenotransplantation model. MZ was administered continuously for 28 days subcutaneously or intramuscularly. MZ inhibited MLR response by approximately 50% at 0.1 µg/ml. ATP contents decreased with MZ >100 µg/ml, while stimulation index was maintained. Continuous infusion of MZ at 10 mg/kg maintained blood concentrations at 0.13-0.19 µg/ml, while intramuscular injection of MZ at 100 mg/kg/day (peak 520 µg/ml at 1 h postinjection) resulted in below measurable levels (<0.03 µg/ml) within 24 h. Graft survival was significantly prolonged following continuous infusion of 10 mg/kg/day compared to controls (31.0 ± 9.5 vs. 13.2 ± 5.2 days; p = 0.002). Furthermore, animals with intramuscular injection at doses of 3.2, 10, or 100 mg/kg/day showed significantly longer graft survival (20.0 ± 7.5, 22.0 ± 7.31, and 24.5 ± 8.1 days, respectively; p < 0.05 each). Histological examination showed significant suppression of lymphocyte infiltration by MZ administration. MZ showed immunosuppressive effects in an experimental islet xenotransplantation model without adverse effects on endocrine function of islet grafts.

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The Immunosuppressive Agent Mizoribine Monophosphate Forms a Transition State Analogue Complex with Inosine Monophosphate Dehydrogenase

Cited by 60 publications

References 34 publications

Cryptosporidium parvum IMP Dehydrogenase

Cryptosporidium parvum IMP Dehydrogenase

The Functional Basis of Mycophenolic Acid Resistance in Candida albicans IMP Dehydrogenase

Mizoribine as Sole Immunosuppressive Agent in Islet Xenotransplantation Models: A Candidate Immunosuppressant Causing no Adverse Effects on Islets

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