The structure of inosine 5′-monophosphate dehydrogenase and the design of novel inhibitors

Abstract

The enzyme IMPDH is a homotetramer of ∼55 kDa subunits and consists of a (β/α)₈ barrel core domain and a smaller subdomain. The active site has binding pockets for the two substrates IMP and NAD. The enzymatic reaction of oxidation of IMP to XMP proceeds through a covalent mechanism involving an active site cysteine residue.

This enzyme is a target for immunosuppressive agents because it catalyzes a key step in purine nucleotide biosynthesis which is important for the proliferation of lymphocytes.

Several X-ray structures of inhibitors bound to IMPDH have been published. The uncompetitive IMPDH inhibitor MPA is the active metabolite of the immunosuppressive agent mycophenolate mofetil (CellCept®) which is approved for the prevention of acute rejection after kidney and heart transplantation. The bicyclic ring system of MPA packs underneath the hypoxanthine ring of XMP*, thereby trapping this covalent intermediate of the enzymatic reaction. Ribavirin monophosphate, the active metabolite of the antiviral agent ribavirin, is a substrate mimic of IMP. The structure of the two inhibitors 6-Cl-IMP and SAD binding in the IMP and NAD pockets of IMPDH, respectively, gives information for the binding mode of the di-nucleotide cofactor to the enzyme.

At Vertex Pharmaceuticals a structure-based drug design program for the design of IMPDH inhibitors was initiated. Several new lead compound classes unrelated to other IMPDH inhibitors were found. Integrating structural information into an iterative drug-design process led to the design of VX-497. VX-497 is a potent uncompetitive enzyme inhibitor of IMPDH. The phenyl-oxazole moiety of the molecule packs underneath XMP*, analogous to MPA. VX-497 also makes several new interactions that are not observed in the binding of MPA. VX-497 is a potent immunosuppressive agent in vitro and in vivo. A Phase I clinical trial has been successfully concluded and the compound is currently in Phase II trials in psoriasis and hepatitis C. The rapid progress from initiation of the drug design program to a compound entering clinical trials illustrates the power of structure-based drug design to accelerate the drug discovery process.

The structural information on IMPDH has also significantly increased our knowledge about the mechanistic details of this fascinating enzyme.

Introduction

Pharmacological intervention aimed at suppressing the immune system plays an important role in the management of autoimmune diseases, the prevention of rejection after organ transplantation and the treatment of graft versus host disease. The first immunosuppressive agents used in the clinic were steroids and cytotoxic agents. Since then, a number of new agents have been introduced into clinical practice (Oliyaei et al., 1998). Perhaps most notable in this respect was the discovery and development of Cyclosporin A (reviewed in Borel and Kis, 1991). The molecular mechanism of action of this and other immunosuppressive drugs has been elucidated and is reviewed in this issue and elsewhere Herrmann et al., 2000, Liu, 1993, Matsuda and Koyasu, 2000, Schreiber, 1992, Suthanthiran et al., 1996. Despite dramatic advances in the development of immunosuppressive drugs, which have made medical procedures such as solid organ transplantation a reality, the search continues for new compounds with the ability to modulate the immune system (Morris, 1996).

The enzymes of the nucleotide biosynthesis pathway are essential for supporting cell proliferation. Typically, cells can synthesize nucleotides in two different ways. In the de novo pathway, the purine or pyrimidine ring system is assembled in a step-wise manner. In the salvage pathway, preformed nucleobases, nucleosides, and nucleotides are recycled (Kornberg and Baker, 1992).

The first unique step in the de novo biosynthesis of guanine nucleotides is catalyzed by the enzyme inosine 5′-monophosphate dehydrogenase (IMPDH, E.C. 1.1.1.205). This reaction is dependent on NAD⁺ and produces XMP, which is then aminated to GMP (catalyzed by GMP synthase, E.C. 6.3.5.2). Through the successive action of several enzymes (Fig. 1), GMP gives rise to some of the building blocks of DNA (dGTP) and RNA synthesis (GTP). GTP also plays an important role in intracellular signaling and glycoprotein biosynthesis (reviewed in Allison and Eugui, 2000). Fig. 1 also illustrates that, in addition to the de novo pathway, GMP can be generated by the salvage pathway from guanosine or guanine.

Several lines of research have converged to implicate IMPDH as an attractive target for pharmacological intervention. Interest in IMPDH as a target for antiproliferative agents has come from the observation that the enzyme is upregulated in rapidly proliferating tumor cells (Jackson et al., 1975). The work of Allison et al. (1977) has demonstrated that lymphocytes in particular are dependent on the de novo pathway of nucleotide biosynthesis, making IMPDH a promising target for immunosuppressive therapy (Allison and Eugui, 2000). Over the years several inhibitors of IMPDH enzyme have been described (see below) and it has been shown that the immediate biochemical effect of IMPDH inhibition in sensitive cell types is a decrease in intracellular guanine nucleotide levels Eugui et al., 1991, Franklin and Morris, 1994. Via this mechanism IMPDH inhibition has antimicrobial (Mizuno et al., 1974), antiparasitic Berman and Webster, 1982, Hupe et al., 1986, Wang et al., 1984, antiproliferative (Carter et al., 1969), antiviral Sidwell et al., 1972, Williams et al., 1968, and immunosuppressive (Mitsui and Suzuki, 1969) pharmacological activities.

Other enzymes of nucleotide biosynthesis have been investigated as targets for immunosuppressive therapy. Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) are mutated in genetic disorders which are characterized by immunodeficiency (Allison et al., 1977). Inhibitors of ADA have been described but are used only in cancer treatment. For PNP, data about the clinical usefulness of enzyme inhibition are beginning to emerge (Morris and Montgomery, 1998). The potent IMPDH inhibitor mycophenolic acid (MPA, see below) has been investigated in clinical trials in psoriasis in the late 1970s. Pharmacological activity and relative safety were demonstrated, even though the compound ultimately was not marketed (reviewed in Silverman Kitchin et al., 1997). These encouraging early clinical results combined with a number of preclinical studies clearly validate IMPDH as a promising target for immunosuppression.

Several classes of IMPDH inhibitors have been described (Pankiewicz, 1999). One class of inhibitors consists of nucleoside analogs that can be phosphorylated intracellularly to the 5′-monophosphates and which then inhibit IMPDH competitively. Examples include mizoribine (Kusumi et al., 1988), and ribavirin (Streeter et al., 1973). Examples of uncompetitive inhibitors are MPA (Franklin and Cook, 1969), first described more than 100 years ago (Gosio, 1896), and the nucleoside tiazofurin which needs to be activated to its dinucleotide to become an inhibitor (Cooney et al., 1982).

The pharmacologic effects of IMPDH inhibition have been exploited by a number of marketed drugs. An ester pro-drug of MPA, mycophenolate mofetil (CellCept®) has been developed as an immunosuppressant and was approved for the prevention of acute rejection in kidney Keown et al., 1996, Pichlmayr et al., 1995, Sollinger et al., 1995 and heart (Kobashigawa et al., 1998) transplantation in combination with steroids and cyclosporin A. Mizoribine (Bredinin®) is approved in Japan for multiple indications in transplantation and autoimmune diseases including prevention of rejection after renal transplantation, idiopathic glomerulonephritis, lupus nephritis, and rheumatoid arthritis. An aerosol formulation of the antiviral agent ribavirin called Virazole® has been approved for treatment of respiratory syncytial virus. An oral formulation (Rebetol®) recently gained approval as a combination product with interferon alpha (Rebetron™) for the treatment of both hepatitis C patients who have relapsed after interferon monotherapy (Davis et al., 1998) and naı̈ve patients McHutchison et al., 1998, Poynard et al., 1998.

The biochemistry and enzymology of IMPDH have been extensively studied. IMPDH has been described in prokaryotes and eukaryotes and the enzyme is well conserved, with 36% sequence identity between the E. coli and human proteins (Natsumeda and Carr, 1993). Many species have two genes for IMPDH, type I and type II. In humans each of the genes encodes a protein of 514 amino acids with high homology between the isoforms (84% amino acid identity, Collart and Huberman, 1988). The active enzyme is formed by a tetramer comprised of identical subunits of 55 kDa molecular weight (Carr et al., 1993).

Many studies have investigated the mechanism of the reaction catalyzed by IMPDH. Various authors have proposed an ordered bi–bi reaction mechanism for IMPDH with IMP binding before NAD⁺, and NADH being released before XMP (e.g. Holmes et al., 1974). A more recent study has demonstrated random binding of substrates while product release occurs in an ordered sequence (Wang and Hedstrom, 1997). It has been shown that IMPDH activity is dependent on monovalent cations (Xiang et al., 1996) and K⁺ is present in one of the X-ray crystal structures of the enzyme (Sintchak et al., 1996). Mass spectrometry analysis of an MPA-inhibited enzyme-product complex has identified a single cysteine residue (Cys 331 in human IMPDH) that is covalently modified during the enzymatic reaction (Link and Straub, 1996). This residue has been shown to be covalently modified by a substrate analog Antonino et al., 1994, Huete-Pérez et al., 1995. In addition, the X-ray crystal structure of a covalently bound thioimidate intermediate has been determined (discussed below, Sintchak et al., 1996). These results show that the chemistry of the reaction (Fig. 2) proceeds through nucleophilic attack by an activated Cys⁻ onto C-2 of the purine ring followed by hydride transfer to NAD⁺. After NADH release, the XMP* intermediate (the carbon atom has the oxidation state of XMP) can be trapped by uncompetitive IMPDH inhibitors like MPA (Fleming et al., 1996). Hydrolysis of the covalent bond between the active site cysteine and the purine ring system produces XMP, which is released in the last step of the enzyme reaction.

In the last 3 years our understanding of IMPDH structure and function has been significantly increased through high-resolution X-ray crystallographic studies Colby et al., 1999, Sintchak et al., 1996, Whitby et al., 1997, Zhang et al., 1999. In this review, we will first briefly present the structure of IMPDH. The focus of this discussion is the implications of the structural work for the design of inhibitors of this important target for pharmacological intervention. Therefore, we will concentrate primarily on IMPDH structures that have inhibitors bound. These are the structure of IMPDH in complex with MPA and XMP* (Sintchak et al., 1996), the complex with 6-Cl-IMP (6-chloropurine riboside 5′-monophosphate) and selenazole-4-carboxamide adenine dinucleotide (SAD) bound (Colby et al., 1999), and the structure with ribavirin 5′-monophosphate bound (Sintchak et al., 1999). The available structural information makes IMPDH a very attractive target for structure-based drug design. At Vertex Pharmaceuticals, a structure-based project for the design of IMPDH inhibitors has been initiated. Novel classes of compounds, structurally unrelated to other IMPDH inhibitors, have been designed. Results on the structural aspects of this program are presented.