INTRODUCTION:

Inosine monophosphate dehydrogenase (IMPDH) is a major target for both antitumor and immunosuppresive drug design. Over 400 publications deal with the enzyme, its mechanism, and its inhibition. We have determined a 2.9Å structure of a ternary complex between human type II IMPDH, the substrate analogue 6-Cl IMP, and the isosteric NAD analog Selenazole Adenine Dinucleotide (SAD). The ternary complex identifies the dinucleotide binding site, and reveals isozyme-specific protein-cofactor interactions which may be exploted in the design of type II-specific agents.

Inosine monophosphate dehydrogenase (IMPDH) catalyses the committed step in guanine nucleotide biosynthesis: the NAD-dependent conversion of inosine 5' monophosphate (IMP) to xanthosine 5' monophosphate. This reactions involves a B-side specific hydride transfer from the substrate inosine ring to the NAD nicotinamide moiety

IMPDH inhibitors act as both anti-tumor and immunosuppressive agents. Inhibition of IMPDH depletes guanine nucleotide pools, hampering DNA and RNA synthesis, guanine nucleotide-coupled signaling, and oncogene expression. Ultimately , IMPDH inhibition suppresses cell proliferation and induces cell differentiation.

Two isoforms of human IMPDH have been identified, having 85% sequence identity. Expression of the type II isoform is significantly up regulated in rapidly proliferating cells, including antigen-stimulated lymphocytes and neoplastic cells.The type I enzyme is constitutively expressed in normal cells. Thus, specificity of IMPDH inhibitors to the type II isoform would better target their biological impact, improving both their anti-tumor and immunosuppressive efficacies and reducing their general toxicity.

Two classes of IMPDH inhibitors have been studied extensively: analogues of the immunosuppressive agent mycophenolic acid (MPA), and analogues of the cofactor NAD. The structure of the highly homologous hamster IMPDH, in complex with MPA and substrate IMP, was recently determined (Sintchak et al, 1996, Cell, v85, 921-930). The MPA structure provides valuable information about overall enzyme structure, as well as substrate and MPA binding. However, MPA blocks NAD binding, preventing identification of any enzyme-cofactor interactions. In addition, all residues identified as directly interacting with MPA are conserved between IMPDH types I and II. This precludes identification of any explotable differences in the design of agents specific for one isoform over the other.

RESULTS:

Fig 1. The Human Type II IMPDH-SAD-6 Cl IMP complex forms a tetramer, as observed for the hamster enzyme. However, the dinucleotide binding site (SAD shown in red, below) involves residues from both the monomer which binds substrate IMP (green) and the neighboring monomer.

Fig 2: Ligand-ligand interactions: The sA-weighted Fo-Fc map in the region of the ligands(2.0s). The SAD selenazole ring occupies the putative nicotinamide site. The selenazole ring stacks with the 6-Cl IMP inosine ring (double arrow), presenting its B-side to the substrate. The carboxamide nitrogen interacts with the O2' hydroxyl of the substrate. The SAD Se is within 2.9Å of O4 of the selenazole moiety ribose, as we've observed previously in binding to alcohol dehydrogenase (Li et al. 1994, Biochemistry, 33, 23-32).

Fig 3. Adenine binding : Type I vs Type II binding. Three of the four residues interacting with the adenine ring are NOT conserved between Type I and Type II isoforms. Arrow indicates the change from Type II to Type I. These differences can be exploted in the design of type-II specific agents.

*Colby, T. D., *Vanderveen, K., *Strickler, M.D., Markham, G. D., Goldstein, B.M. Crystal Structure of Human Type II Inosine Monophosphate dehydrogenase: Implications for ligand binding and drug design. Proc. Nat. Acad. Sci. 96: 3531-3536 (1999) . REPRINT*

Goldstein, B.M. and *Colby, T.D. IMP Dehydrogenase : Structural Aspects of Inhibitor Binding. Currrent Med. Chem. 6:519-536 (1999). Medline Abstract

Goldstein, B.M. and *Colby, T.D. Conformational Constraints in NAD Analogs: Implications for Dehydrogenase Binding and Specificity. Advances in Enzyme Regulation, 40:405-426 (2000)

Lesiak, K; Watanabe, K. A., Majumdar, A., Powell, J., Seidman, M., *Vanderveen, K., Goldstein, B. M., Pankiewicz, K. W. Synthesis of Methylene Bis(phosphonate) Analogue of Mycophenolic Adenine Dinucleotide. A Glucuronidation Resistant MAD Analogue of NAD. J. Med. Chem. , 41:618-622 (1998) . REPRINT *

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