Crystal structure of Tritrichomonas foetus inosine monophosphate dehydrogenase in complex with the inhibitor ribavirin monophosphate reveals a catalysis-dependent ion-binding site

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in GMP biosynthesis. The resulting intracellular pool of guanine nucleotides is of great importance to all cells for use in DNA and RNA synthesis, metabolism, and signal transduction. The enzyme binds IMP and the cofactor NAD(+) in random order, IMP is converted to XMP, NAD(+) is reduced to NADH, and finally, NADH and then XMP are released sequentially. XMP is subsequently converted into GMP by GMP synthetase. Drugs that decrease GMP synthesis by inhibiting IMPDH have been shown to have antiproliferative as well as antiviral activity. Several drugs are in use that target the substrate- or cofactor-binding site; however, due to differences between the mammalian and microbial isoforms, most drugs are far less effective against the microbial form of the enzyme than the mammalian form. The high resolution crystal structures of the protozoan parasite Tritrichomonas foetus IMPDH complexed with the inhibitor ribavirin monophosphate as well as monophosphate together with a second inhibitor, mycophenolic acid, are presented here. These structures reveal an active site cation identified previously only in the Chinese hamster IMPDH structure with covalently bound IMP. This cation was not found previously in apo IMPDH, IMPDH in complex with XMP, or covalently bound inhibitor, indicating that the cation-binding site may be catalysis-dependent. A comparison of T. foetus IMPDH with the Chinese hamster and Streptococcus pyogenes structures reveals differences in the active site loop architecture, which contributes to differences in cation binding during the catalytic sequence and the kinetic rates between bacterial, protozoan, and mammalian enzymes. Exploitation of these differences may lead to novel inhibitors, which favor the microbial form of the enzyme.     

Insulin and oleate promote translocation of inosine-5' monophosphate dehydrogenase to lipid bodies

In the present study we identify inosine-5' monophosphate dehydrogenase (IMPDH), a key enzyme in de novo guanine nucleotide biosynthesis, as a novel lipid body-associated protein. To identify new targets of insulin we performed a comprehensive 2-DE analysis of (32)P-labelled proteins isolated from 3T3-L1 adipocytes (Hill et al. J Biol Chem 2000; 275: 24313-24320). IMPDH was identified by liquid chromatography/tandem mass spectrometry as a protein which was phosphorylated in a phosphatidylinositol (PI) 3-kinase-dependent manner upon insulin treatment. Although insulin had no significant effect on IMPDH activity, we observed translocation of IMPDH to lipid bodies following insulin treatment. Induction of lipid body formation with oleic acid promoted dramatic redistribution of IMPDH to lipid bodies, which appeared to be in contact with the endoplasmic reticulum, the site of lipid body synthesis and recycling. Inhibition of PI 3-kinase blocked insulin- and oleate-induced translocation of IMPDH and reduced oleate-induced lipid accumulation. However, we found no evidence of oleate-induced IMPDH phosphorylation, suggesting phosphorylation and translocation may not be coupled events. These data support a role for IMPDH in the dynamic regulation of lipid bodies and fatty acid metabolism and regulation of its activity by subcellular redistribution in response to extracellular factors that modify lipid metabolism.     

Structure-activity relationships for inhibition of inosine monophosphate dehydrogenase and differentiation induction of K562 cells among the mycophenolic acid derivatives

Inosine monophosphate dehydrogenases (IMPDHs) are the committed step in de novo guanine nucleotide biosynthesis. There are two separate, but very closely related IMPDH isoenzymes, termed type I and type II. IMPDHs are widely believed to be major targets for cancer and transplantation therapy. Mycophenolic acid (MPA) is a potent inhibitor of IMPDHs. Previously, we found that MPA acted as a latent agonist of this nuclear hormone receptor in U2OS cells, and 6'-hydroxamic acid derivatives of MPA inhibited tubulin-specific histone deacetylase[s] (HDAC[s]) in HeLa cells. Although MPA is a promising lead compound, structure-activity relationships (SARs) for inhibition of IMPDH, and the mechanism action of MPA derivatives have not well been understood. We therefore synthesized, evaluated MPA derivatives as IMPDH inhibitor in vitro and cellular level, and explored their biological function and mechanism in cultured cells. This paper exhibits that (i) functional groups at C-5, C-7, and C-6' positions in MPA are important for inhibitory activity against IMPDH, (ii) it is difficult to improve specificity against IMPDH II by modification of 5-, 7-, and 6'-group, (iii) demethylation of 5-OMe results in increasing hydrophilicity, and lowering cell permeability, (iv) ester bonds of protective groups at C-7 and C-6' positions are hydrolyzed to give MPA in cultures, (v) the effects of a tubulin-specific HDAC[s] inhibitor on proliferation and differentiation are weaker than its inhibitory activity against IMPDH. The present work may provide insight into the development of a new class of drug lead for treating cancer and transplantation.     

Regulation of the interaction of inosine monophosphate dehydrogenase with mycophenolic Acid by GTP

Inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in the de novo synthesis of guanine nucleotides, is a major therapeutic target. A prototypic uncompetitive inhibitor of IMPDH, mycophenolic acid (MPA), is the active form of mycophenolate mofeteil (CellCept), a widely used immunosuppressive drug. We have found that MPA interacts with intracellular IMPDH in vivo to alter its mobility on SDS-polyacrylamide gels. MPA also induces a striking conformational change in IMPDH protein in intact cells, resulting in the formation of annular aggregates of protein with concomitant inhibition of IMPDH activity. These aggregates are not associated with any known intracellular organelles and are reversible by incubating cells with guanosine, which repletes intracellular GTP, or with GTPgammaS. GTP also restores IMPDH activity. Treatment of highly purified IMPDH with MPA also results in the formation of large aggregates of protein, a process that is both prevented and reversed by the addition of GTP. Finally, GTP binds to IMPDH at physiologic concentrations, induces the formation of linear arrays of tetrameric protein, and prevents the aggregation of protein induced by MPA. We conclude that intracellular GTP acts as an antagonist to MPA by directly binding to IMPDH and reversing the conformational changes in the protein.     

Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum

Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCl). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.     

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.     

HPLC/tandem ion trap mass detector methods for determination of inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT)

The efficiency of Mycophenolate mofetil (MMF) and Azathioprine (AZA) as immunosuppressive agents depends on the activity of 2 enzymes, inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT) respectively. We present preliminary evaluation of nonradioactive methods that apply HPLC with ion-trap mass detection to measure the activities of IMPDH in peripheral blood mononuclear cells (PBMC) and TPMT in the erythrocytes (RBC). We found IMPDH activity of 0.9 +/- 0.2 nmol/hour/10(6) PBMC and TPMT activity of 19.9 +/- 4.7 nmol/hour/ml RBC in healthy subjects. These methods, following its further validation, could be useful for monitoring the activity in a clinical and experimental setting.     

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.     

Cofactor-type inhibitors of inosine monophosphate dehydrogenase via modular approach: targeting the pyrophosphate binding sub-domain

Cofactor-type inhibitors of inosine monophosphate dehydrogenase (IMPDH) that target the nicotinamide adenine dinucleotide (NAD) binding domain of the enzyme are modular in nature. They interact with the three sub-sites of the cofactor binding domain; the nicotinamide monophosphate (NMN) binding sub-site (N sub-site), the adenosine monophosphate (AMP) binding sub-site (A sub-site), and the pyrophosphate binding sub-site (P sub-site or P-groove). Mycophenolic acid (MPA) shows high affinity to the N sub-site of human IMPDH mimicking NMN binding. We found that the attachment of adenosine to the MPA through variety of linkers afforded numerous mycophenolic adenine dinucleotide (MAD) analogues that inhibit the two isoforms of the human enzyme in low nanomolar to low micromolar range. An analogue 4, in which 2-ethyladenosine is attached to the mycophenolic alcohol moiety through the difluoromethylenebis(phosphonate) linker, was found to be a potent inhibitor of hIMPDH1 (K(i)=5 nM), and one of the most potent, sub-micromolar inhibitor of leukemia K562 cells proliferation (IC(50)=0.45 μM). Compound 4 was as potent as Gleevec (IC(50)=0.56 μM) heralded as a 'magic bullet' against chronic myelogenous leukemia (CML). MAD analogues 7 and 8 containing an extended ethylenebis(phosphonate) linkage showed low nanomolar inhibition of IMPDH and low micromolar inhibition of K562 cells proliferation. Some novel MAD analogues described herein containing linkers of different length and geometry were found to inhibit IMPDH with K(i)'s lower than 100 nM. Thus, such linkers can be used for connection of other molecular fragments with high affinity to the N- and A-sub-site of IMPDH.     

The immunosuppressive agent mizoribine monophosphate forms a transition state analogue complex with inosine monophosphate dehydrogenase

Mizoribine monophosphate (MZP) is the active metabolite of the immunosuppressive agent mizoribine and a potent inhibitor of IMP dehydrogenase (IMPDH). This enzyme catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD via a covalent intermediate at Cys319 (E-XMP). Surprisingly, mutational analysis indicates that MZP is a transition state analogue although its structure does not resemble that of the expected transition state. Here we report the X-ray crystal structure of the E.MZP complex at 2.0 A resolution that reveals a transition state-like structure and solves the mechanistic puzzle of the IMPDH reaction. The protein assumes a new conformation where a flap folds into the NAD site and MZP, Cys319, and a water molecule are arranged in a geometry resembling the transition state. The water appears to be activated by interactions with a conserved Arg418-Tyr419 dyad. Mutagenesis experiments confirm that this new closed conformation is required for the hydrolysis of E-XMP, but not for the reduction of NAD. The closed conformation provides a structural explanation for the differences in drug selectivity and catalytic efficiency of IMPDH isozymes.     

Inhibition of inosine monophosphate dehydrogenase reduces adipogenesis and diet-induced obesity

We previously described a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in lipid accumulation. Here we present data which demonstrate that IMPDH activity is required for differentiation of preadipocytes into mature, lipid-laden adipocytes and maintenance of adipose tissue mass. In 3T3-L1 preadipocytes inhibition of IMPDH with mycophenolic acid (MPA) reduced intracellular GTP levels by 60% (p < 0.05) and blocked adipogenesis (p < 0.05). Co-treatment with guanosine, a substrate in the salvage pathway of nucleotide biosynthesis, restored GTP levels and adipogenesis demonstrating the specificity of these effects. Treatment of diet-induced obese mice with mycophenolate mofetil (MMF), the prodrug of MPA, for 28 days did not affect food intake or lean body mass but reduced body fat content (by 36%, p = 0.002) and adipocyte size (p = 0.03) and number. These data suggest that inhibition of IMPDH may represent a novel strategy to reduce adipose tissue mass.     

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.     

PKB/Akt interacts with inosine-5' monophosphate dehydrogenase through its pleckstrin homology domain

The pleckstrin homology (PH) domain of the protooncogenic serine/threonine protein kinase PKB/Akt can bind phosphoinositides. A yeast-based two-hybrid system was employed which identified inosine-5' monophosphate dehydrogenase (IMPDH) type II as specifically interacting with PKB/Akts PH domain. IMPDH catalyzes the rate-limiting step of de novo guanosine-triphosphate (GTP) biosynthesis. Using purified fusion proteins, PKB/Akts PH domain and IMPDH associated in vitro and this association moderately activated IMPDH. Purified PKB/Akt also associated with IMPDH in vitro. We could specifically pull-down PKB/Akt or IMPDH from mammalian cell lysates using glutathione-S-transferase (GST)-IMPDH or GST-PH domain fusion proteins, respectively. Additionally, PKB/Akt and IMPDH could be co-immunoprecipitated from COS cell lysates and active PKB/Akt could phosphorylate IMPDH in vitro. These results implicate PKB/Akt in the regulation of GTP biosynthesis through its interaction with IMPDH, which is involved in providing the GTP pool used by signal transducing G-proteins.     

Identification of novel Mt-Guab2 inhibitor series active against M. tuberculosis

Tuberculosis (TB) remains a leading cause of mortality worldwide. With the emergence of multidrug resistant TB, extensively drug resistant TB and HIV-associated TB it is imperative that new drug targets be identified. The potential of Mycobacterium tuberculosis inosine monophosphate dehydrogenase (IMPDH) as a novel drug target was explored in the present study. IMPDH exclusively catalyzes the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) in the presence of the cofactor nicotinamide adenine dinucleotide (NAD(+)). Although the enzyme is a dehydrogenase, the enzyme does not catalyze the reverse reaction i.e. the conversion of XMP to IMP. Unlike other bacteria, M. tuberculosis harbors three IMPDH-like genes, designated as Mt-guaB1, Mt-guaB2 and Mt-guaB3 respectively. Of the three putative IMPDH's, we previously confirmed that Mt-GuaB2 was the only functional ortholog by characterizing the enzyme kinetically. Using an in silico approach based on designed scaffolds, a series of novel classes of inhibitors was identified. The inhibitors possess good activity against M. tuberculosis with MIC values in the range of 0.4 to 11.4 μg mL(-1). Among the identified ligands, two inhibitors have nanomolar K(i)s against the Mt-GuaB2 enzyme.     

Novel 1,2,4-triazole and imidazole derivatives of L-ascorbic and imino-ascorbic acid: synthesis, anti-HCV and antitumor activity evaluations

Several novel 1,2,4-triazole and imidazole L-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7 and 8) derivatives were prepared and evaluated for their inhibitory activity against hepatitis C virus (HCV) replication and human tumour cell proliferation. Compounds 6 and 9 exerted the most pronounced cytostatic effects in all tumour cell lines tested, and were highly selective for human T-cell acute lymphoblastic leukaemia cells (CEM/0) with IC(50)s of 10 ± 4 and 7.3 ± 0.1 μM, respectively. Unlike compound 9, compound 6 showed no toxicity in human diploid fibroblasts. One of the possible mechanisms of action of compound 6 accounting for observed cytostatic activity towards haematological malignancies might be inhibition of inosine monophosphate dehydrogenase (IMPDH) activity, a key enzyme of de novo purine nucleotide biosynthesis providing the cells with precursors for DNA and RNA synthesis indispensable for cell growth and division, which has emerged as an important target for antileukemic therapy. In addition, this compound proved to be the most potent inhibitor of the hepatitis C virus replication as well. However, observed antiviral effect was most likely associated with the effect that the compound exerted on the host cell rather than with selective effect on the replication of the virus itself. In conclusion, results of this study put forward compound 6 as a potential novel antitumor agent (IMPDH inhibitor) for treating leukaemia. Its significant biological activity and low toxicity in human diploid fibroblasts encourage further development of this compound as a lead.     

Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase

A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.     

Properties of adenosine monophosphate deaminase of Candida albicans.

Adenosine monophosphate deaminase (AMPD; EC 3.5.4.6) catalyses the hydrolysis of adenosine monophosphate (AMP) to commensurate amounts of inosine monophosphate (IMP) and ammonia. The production of AMP deaminase in Candida albicans was measured in Lee's medium grown cultures. The highest AMPD activity was observed at 24 h of growth. The enzyme had an optimum pH and temperature at 6-7 and 28 degrees C, respectively. This enzyme was inhibited under iron-limited growth conditions as well as by protease inhibitors. The AMPD of C. albicans showed a moderate increase in activity when cultures were grown in the presence of the divalent cations Mg2+, Ca2+, and Zn2+. Moreover, ADP, ATP, adenine, adenosine, deoxyribose and hypoxanthine increased the enzyme activity. Cultures grown in trypticase soy broth exhibited maximum AMPD activity compared with those grown in Sabouraud dextrose broth or Lee's medium.     

Inhibitors of inosine monophosphate dehydrogenase: SARs about the N-[3-Methoxy-4-(5-oxazolyl)phenyl moiety

The first reported structure-activity relationships (SARs) about the N-[3-methoxy-4-(5-oxazolyl)phenyl moiety for a series of recently disclosed inosine monophosphate dehydrogenase (IMPDH) inhibitors are described. The syntheses and in vitro inhibitory values for IMPDH II, and T-cell proliferation (for select analogues) are given.     

Enzymatic dephosphorylation of retinyl monophosphate by rat liver

Rat liver has been shown to contain an enzyme that catalyzes the dephosphorylation of retinyl monophosphate. This activity was extracted with 0.1 M Tris buffer (pH 7.5). Maximum reaction rate was observed at a pH range of 7.0-7.5. It did not require metal ions for activity and was sensitive to fluoride ion. The retinyl monophosphate phosphatase activity was proportional to time and protein and substrate concentration. Triton X-100 (range of 0.05-0.10%) increased the activity 100%, whereas other detergents (Tween 80, cholate, and deoxycholate) did not activate the enzyme. A number of phosphorylated compounds tested as inhibitors of retinyl monophosphatase activity, such as glucose 6-phosphate (20 mM), glycerophosphate (20 mM), phosphatidic acid (8 mM), and dolichyl phosphate (3 mM), did not compete with retinyl monophosphate as substrate. However, at 20 mM concentration, ATP, ADP, 5'-AMP, and pyrophosphate were inhibitors of the enzyme. It is not possible at present to give further details about the specificity of the phosphatase activity. The enzyme described could play a regulatory role in retinol-mediated glycosylations, by altering the endogenous level of retinyl monophosphate.     

Molecular cloning and characterization of a cDNA encoding soybean nodule IMP dehydrogenase

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo purine biosynthesis and is a postulated key enzyme in nitrogen assimilation in ureide-exporting nodules. A 2016 bp cDNA for IMPDH, designated as IMPDH, was cloned from a soybean nodule cDNA library. IMPDH encodes a polypeptide of 502 amino acids with a predicted molecular weight of 53000 and a pI of 5.54. The deduced IMPDH is 70.5% identical to that in Arabidopsis, with a 100% homology in the putative active-site region. Expressing the cloned cDNA in Escherichia coli mutant strain KLC381 (DeltaguaB) restored IMPDH activity, permitting bacterial growth on minimal medium. Southern blot analysis suggested a single copy of IMPDH gene in the soybean genome. Northern blot analysis showed that the expression of IMPDH gene is apparently nodule-specific.