Inhibitors of inosine monophosphate dehydrogenase: probes for antiviral drug discovery

The role of inosine monophosphate dehydrogenase (IMPDH) at the metabolic branch point of de novo purine nucleotide biosynthesis makes this enzyme an attractive probe for the discovery of antiviral compounds. Introduction of unsaturation at the 2-position of IMP, the natural substrate for IMPDH, produces Michael acceptors at that position, which results in these compounds being inhibitors of IMPDH. Consistent with this mechanism-based molecular design, some of the parent nucleosides exhibited antiviral activity.     

Inhibition of inosine monophosphate dehydrogenase (IMPDH) by the antiviral compound, 2-vinylinosine monophosphate

A new enzyme-mediated synthesis of 2-vinylinosine, a compound with broad-spectrum RNA antiviral activity, is described. In order to understand the mechanism of action of this compound, we synthesized its monophosphate and investigated the behavior of that compound toward the enzyme, inosine monophosphate dehydrogenase (IMPDH), a key enzyme involved in the biosynthesis of nucleotides. 2-Vinylinosine monophosphate is a potent inhibitor of IMPDH with a K(i) of 3.98 microM (k(inact)=2.94 x 10(-2) s(-1)). The antiviral activity of 2-vinylinosine may be explained by its cellular conversion to the monophosphate through the sequential action of PNP and HGPRT and subsequent inhibition of IMPDH by the cellularly produced 2-vinylinosine 5'-monophosphate.     

INHIBITORS OF INOSINE MONOPHOSPHATE DEHYDROGENASE: PROBES FOR ANTIVIRAL DRUG DISCOVERY

The role of inosine monophosphate dehydrogenase (IMPDH) at the metabolic branch point of de novo purine nucleotide biosynthesis makes this enzyme an attractive probe for the discovery of antiviral compounds. Introduction of unsaturation at the 2-position of IMP, the natural substrate for IMPDH, produces Michael acceptors at that position, which results in these compounds being inhibitors of IMPDH. Consistent with this mechanism-based molecular design, some of the parent nucleosides exhibited antiviral activity.     

Ribavirin can be mutagenic for arenaviruses

Arenaviruses include several important human pathogens, and there are very limited options of preventive or therapeutic interventions to combat these viruses. An off-label use of the purine nucleoside analogue ribavirin (1-β-d-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is the only antiviral treatment currently available for arenavirus infections. However, the ribavirin antiviral mechanism action against arenaviruses remains unknown. Here we document that ribavirin is mutagenic for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) in cell culture. The mutagenic activity of ribavirin on LCMV was observed under single- and multiple-passage regimes and could not be accounted for by a decrease of the intracellular GTP pool promoted by ribavirin-mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). Our findings suggest that the antiviral activity of ribavirin on arenaviruses might be exerted, at least partially, by lethal mutagenesis. Implications for antiarenavirus therapy are discussed.     

A heterocyclic molecule with significant activity against dengue virus

There are no specific approved drugs or vaccines for the treatment or prevention of infectious dengue virus and there are very few compounds known that inhibit the replication of this virus. This letter describes the concise synthesis of two uracil-based multifunctional compounds. One of these compounds (1) has strong activity against dengue virus. It also exhibits low activity against a few other RNA viruses, but is highly active against yellow fever virus, a related flavivirus. It is likely that the mechanism of action of the antiviral activity of this compound is through its inhibition of the enzyme, inosine monophosphate dehydrogenase (IMPDH). Molecular modeling studies reveal that the compound can have specific hydrogen bonding interactions with a number of amino acids in the active site of IMPDH, a stacking interaction with the bound natural substrate, IMP, and the ability to interfere with the binding of NAD⁺ with IMPDH, prior to the hydration step.     

Bioprospecting the American Alligator Peptidome for antiviral peptides against Venezuelan equine encephalitis virus

The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.     

Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells.

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.     

Design,synthesis and biological evaluation of Helicobacter pylori inosine 5′-monophosphate dehydrogenase (HpIMPDH) inhibitors. Further optimization of selectivity towards HpIMPDH over human IMPDH2

Inosine 5′-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes a crucial step in guanine nucleotide biosynthesis, thereby governing cell proliferation. In contrast to mammalian IMPDHs, microbial IMPDHs are relatively less explored as potential targets for antimicrobial drug discovery. In continuation with our previous work, here we report the discovery of moderately potent and highly selective Helicobacter pylori IMPDH (HpIMPDH) inhibitors. The present study is mainly focused around our previously identified, modestly potent and relatively nonselective (for HpIMPDH over human IMPDH2) hit molecule IX (16i). In an attempt to optimize the selectivity for the bacterial enzyme, we screened a set of 48 redesigned new chemical entities (NCEs) belonging to 5-aminoisobenzofuran-1(3H)-one series for their in vitro HpIMPDH and human IMPDH2 inhibition. A total of 12 compounds (hits) demonstrated ≥70% HpIMPDH inhibition at 10 μM concentration; none of the hits were active against hIMPDH2. Compound 24 was found to be the most potent and selective molecule (HpIMPDH IC₅₀ = 2.21 µM) in the series. The study reaffirmed the utility of 5-aminoisobenzofuran-1(3H)-one as a promising scaffold with great potential for further development of potent and selective HpIMPDH inhibitors.     

Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate,cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism

The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis. Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy. The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves. Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme. It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists. The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+). In addition, a potassium ion has been located at the dimer interface. A structural model for the kinetic mechanism is proposed.     

Cryptosporidium parvum IMP dehydrogenase: identification of functional, structural, and dynamic properties that can be exploited for drug design

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.     

Evaluation of antiviral activity of different origin compounds by flow cytometry

Against many viral diseases caused for example by HSV, EBV, CMV, HIV, RSV, HCV for which vaccines are not available, chemiotherapeutics seem to have the principal significance. High progress in development of new antiviral compounds is observed. In addition to synthetic compounds a large number of naturally occurring substances have been shown to posses antiviral activity. One of such substance is tannic acid. In this study comparison of antiviral activity of tannic acid, acyclovir (ACV) and ganciclovir (GCV) against cytomegalovirus (CMV) is presented. The MRC5 cells infected with CMV and treated with different compounds were analyzed by flow cytometry and cythopatic effect inhibition test for inhibition of virus replication and by MTT assay for cytotoxity. It has been shown that tannic acid has antiviral activity against cytomegalovirus and that expression of virus antigens measured as median fluorescence intensity (MFI) by flow cytometry can be used for evaluation of virus replication.     

Inhibitory action of polyunsaturated fatty acids on IMP dehydrogenase

We screened the inhibitor of mouse inosine 5'-monophosphate dehydrogenase (IMPDH) type II from natural compounds, and found that a fatty acid, linoleic acid (C18:2), inhibited IMPDH activity. In the C18:2 fatty acid derivatives, all trans-configuration (i.e., linoelaidic acid), ester form, alcohol form, and addition of the hydroxyl group of linoleic acid had no effect on inhibitory activity. Therefore, both parts of a carboxylic acid and an alkyl chain containing cis-type double bonds of fatty acid might be essential for inhibition. Among the various carbon atom lengths and double bonds of fatty acids examined, the strongest inhibitor was C20:2-fatty acid, eicosadienoic acid, and 50% inhibition was observed at a concentration of 16.1 microM. Eicosadienoic acid induced the inhibition of IMPDH activity and was competitive with respect to IMP (K(i)=3.1 microM). For inhibitory effect, the C20-fatty acids ranked as follows: C20:2>C20:3>C20:1> C20:4>C20:5, and C20:0 showed no inhibition. The energy-minimized three-dimensional structures of linear-chain C20-fatty acids were calculated, and it was found that a length of 20.7-22.5A and width of 4.7-7.2A in the fatty acid molecular structure was suggested to be important for IMPDH inhibition. Docking simulation of C20-fatty acids and mouse IMPDH type II, which was homology modeled from human IMPDH type II (PDB code: 1NF7), was performed, and the fatty acid could bind to Cys331, which is a amino acid residue of the active site, competitively with IMP. Based on these results, the IMPDH-inhibitory mechanism of fatty acids is discussed.     

Bacillus anthracis Inosine 5'-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion-, Substrate-, and Product-Bound Complexes

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of Bacillus anthracis IMPDH, in a phosphate ion-bound (termed "apo") form and in complex with its substrate, inosine 5'-monophosphate (IMP), and product, xanthosine 5'-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known Cryptosporidium parvum IMPDH inhibitors was examined for the B. anthracis enzyme and compared with those of three bacterial IMPDHs from Campylobacter jejuni, Clostridium perfringens, and Vibrio cholerae. The structures contribute to the characterization of the active site and design of inhibitors that specifically target B. anthracis and other microbial IMPDH enzymes.     

Antivirals and antiviral strategies

In recent years, the demand for new antiviral strategies has increased markedly. There are many contributing factors to this increased demand, including the ever-increasing prevalence of chronic viral infections such as HIV and hepatitis B and C, and the emergence of new viruses such as the SARS coronavirus. The potential danger of haemorrhagic fever viruses and eradicated viruses such as variola virus being used as bioterrorist weapons has also increased the profile of antiviral drug discovery. Here, the virus infections for which antiviral therapy is needed and the compounds that are available, or are being developed, for the treatment of these infections are described.     

Synthesis of the inosine 5′-monophosphate dehydrogenase (IMPDH) inhibitors

Abstract

Inosine 5′-monophosphate dehydrogenase (IMPDH) is important molecular target for potential anticancer, antiviral, antibacterial and immunosuppressive agents. A lot of compounds were obtained to establish their activity toward this enzyme, and to improve therapeutic properties of IMPDH inhibitors used as the drugs. Some of the recently reported analogs exhibited promising results during in vitro and in vivo examinations in comparison to substances applied in clinic. In this review, we describe synthesis and biological activity evaluations of the newly designed IMPDH inhibitors.     

Approaches to antiviral drug development

At present, only a few drugs have been approved by the FDA for therapy of viral infections in humans. There is a great need for antiviral drugs with increased potency and decreased toxicity, as well as drugs to treat viral diseases for which no drug or vaccine is currently available. Two approaches for development of antiviral drugs are described--an empirical strategy and a rational strategy--with several examples of each. Although many compounds have potent antiviral activity in cell culture, only a small fraction of these will go on to become antiviral drugs for use in humans. At this time, only seven synthetic compounds and alpha interferon have been approved by the FDA for therapy of viral infections in humans. None of these approved drugs are without toxicities, however, and hence there is a great need for antiviral drugs with increased potency and decreased toxicity, as well as for drugs to treat viral diseases for which no drug or vaccine is currently available. Two approaches for the development of antiviral drugs--the empirical and the rational strategies--and their applications and future directions are discussed.     

A Nucleotide-Dependent Conformational Switch Controls the Polymerization of Human IMP Dehydrogenases to Modulate their Catalytic Activity

Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo GTP biosynthetic pathway and plays essential roles in cell proliferation. As a clinical target, IMPDH has been studied for decades, but it has only been within the last years that we are starting to understand the complexity of the mechanisms of its physiological regulation. Here, we report structural and functional insights into how adenine and guanine nucleotides control a conformational switch that modulates the assembly of the two human IMPDH enzymes into cytoophidia and allosterically regulates their catalytic activity. In vitro reconstituted micron-length cytoophidia-like structures show catalytic activity comparable to unassembled IMPDH but, in turn, are more resistant to GTP/GDP allosteric inhibition. Therefore, IMPDH cytoophidia formation facilitates the accumulation of high levels of guanine nucleotides when the cell requires it. Finally, we demonstrate that most of the IMPDH retinopathy-associated mutations abrogate GTP/GDP-induced allosteric inhibition and alter cytoophidia dynamics.