Enzyme Inosine Monophosphate Dehydrogenase Research Articles

Roncoroni Re-Visited: The Neuronal Intranuclear Rodlet Comes of Age.

Despite myriad technological advances in neuroscience, the nervous system harbors morphological phenomena that continue to defy explanation. First described by the classical microscopists, including Santiago Ramon y Cajal, at the end of the 19th century, the neuronal intranuclear rodlet (INR) has mystified neurohistologists and microscopists for centuries. In this review article, we will provide an overview of the discovery of the INR as well as the subsequent attempts to elucidate its nature and functional significance. We outline our own studies of this structure over the past three decades, focusing on its elusive nature, its interactions with other nuclear organelles, and on disease-related quantitative changes in Alzheimer's disease. We then describe our somewhat serendipitous discovery that these structures are filamentous aggregates of the nucleotide-synthesizing metabolic enzyme inosine monophosphate dehydrogenase. The filamentation of metabolic enzymes to form mesoscale cellular structures called "rods and rings" or "cytoophidia" (Greek for "cellular snakes") is a recently described phenomenon that remains to be systematically investigated in the nervous system. Thus, this review provides an intriguing historical juxtaposition in neuroscience, inculcating the neuronal INR, once a mere morphological curiosity, into one of the most rapidly evolving fields in contemporary cell biology.

Triple in silico targeting of IMPDH enzyme and RNA-dependent RNA polymerase of both SARS-CoV-2 and Rhizopus oryzae.

Aim: Mucormycosis has been associated with SARS-CoV-2 infections during the last year. The aim of this study was to triple-hit viral and fungal RNA-dependent RNA polymerases (RdRps) and human inosine monophosphate dehydrogenase (IMPDH). Materials & methods: Molecular docking and molecular dynamics simulationwere usedto test nucleotide inhibitors (NIs) against the RdRps of SARS-CoV-2 and Rhizopus oryzae RdRp. These same inhibitors targeted IMPDH. Results: Four NIs revealed a comparable binding affinity to the two drugs, remdesivir and sofosbuvir. Binding energies were calculated using the most abundant conformations of the RdRps after 100-ns molecular dynamics simulation. Conclusion: We suggest the triple-inhibition potential of four NIs against pathogenic RdRps and IMPDH, which is worth experimental validation.

Inosine monophosphate dehydrogenase intranuclear inclusions are markers of aging and neuronal stress in the human substantia nigra

We explored mechanisms involved in the age-dependent degeneration of human substantia nigra (SN) dopamine (DA) neurons. Owing to its important metabolic functions in post-mitotic neurons, we investigated the developmental and age-associated changes in the purine biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH). Tissue microarrays prepared from post-mortem samples of SN from 85 neurologically intact participants humans spanning the age spectrum were immunostained for IMPDH combined with other proteins. SN DA neurons contained two types of IMPDH structures: cytoplasmic IMPDH filaments and intranuclear IMPDH inclusions. The former were not age-restricted and may represent functional units involved in sustaining purine nucleotide supply in these highly metabolically active cells. The latter showed age-associated changes, including crystallization, features reminiscent of pathological inclusion bodies, and spatial associations with Marinesco bodies; structures previously associated with SN neuron dysfunction and death. We postulate dichotomous roles for these two subcellularly distinct IMPDH structures and propose a nucleus-based model for a novel mechanism of SN senescence that is independent of previously known neurodegeneration-associated proteins.

Safety and efficacy of mycophenolate in COVID-19: a nonrandomised prospective study in western India.

Antivirals and immunosuppressive agents are used with variable success in the treatment of COVID-19. Mycophenolate, an inhibitor of enzyme inosine monophosphate dehydrogenase, is an immunosuppressant used to prevent allograft rejection and other autoimmune diseases. Few laboratory studies have also reported antiviral properties of mycophenolate. The current study tried to assess the safety and efficacy of mycophenolate in patients hospitalised with COVID-19. This was a prospective non-randomised open label study with the objective to assess the effect of addition of mycophenolate to the standard of care on mortality due to COVID-19 and duration of hospital stay. The target study population was comprised of patients requiring inpatient treatment for COVID-19 during the period from Jan 15-April 15, 2021. The study was registered with Clinical Trial Registry of India (CTRI/2021/01/030477, registered on date-14/01/2021). Adult patients (n=106) requiring hospitalisation for COVID-19 received mycophenolate, 360mg, one tablet daily for one month. Mycophenolate was initiated within 48h of the diagnosis of SARS-CoV-2 infection by RT‒PCR. While patients who did not consent for mycophenolate (n=106), received only standard of care, and were considered as control group. The relevant clinical data including NEWS2 scores and high-resolution computed tomography of the thorax were collected and analysed. The mortality and hospital stay were significantly lower in the study group compared to the control group. Mycophenolate significantly reduced mortality after adjustment for other predictors (adjusted odds ratio: 0.082 with 95% CI: 0.012-0.567). Mycophenolate was an independent predictor of survival in patients hospitalised due to COVID-19. There was also no evidence of secondary bacterial infections and post-COVID complications. Mycophenolate administration is safe in COVID-19. Mycophenolate reduces mortality and duration of hospital stay in patients with COVID-19. Shri Janai Research Foundation, India.

LncRNA UCA1 Participates in De Novo Synthesis of Guanine Nucleotides in Bladder Cancer by Recruiting TWIST1 to Increase IMPDH1/2.

Metabolic dysregulation has been identified as one of the hallmarks of cancer biology. Based on metabolic heterogeneity between bladder cancer tissues and adjacent tissues, we discovered several potential driving factors for the bladder cancer occurrence and development. Metabolic genomics showed purine metabolism pathway was mainly accumulated in bladder cancer. Long noncoding RNA urothelial carcinoma-associated 1 (LncRNA UCA1) is a potential tumor biomarker for bladder cancer diagnosis and prognosis, and it increases bladder cancer cell proliferation, migration, and invasion via the glycolysis pathway. However, whether UCA1 plays a role in purine metabolism in bladder cancer is unknown. Our findings showed that UCA1 could increase the transcription activity of guanine nucleotide de novo synthesis rate limiting enzyme inosine monophosphate dehydrogenase 1 (IMPDH1) and inosine monophosphate dehydrogenase 2 (IMPDH2), triggering in guanine nucleotide metabolic reprogramming. This process was achieved by UCA1 recruiting the transcription factor TWIST1 which binds to the IMPDH1and IMPDH2 promoter region. Increased guanine nucleotide synthesis pathway products stimulate RNA polymerase-dependent production of pre-ribosomal RNA and GTPase activity in bladder cancer cells, hence increasing bladder cancer cell proliferation, migration, and invasion. We have demonstrated that UCA1 regulates IMPDH1/2-mediated guanine nucleotide production via TWIST1, providing additional evidence of metabolic reprogramming.

Targeting Some Enzymes with Repurposing Approved Pharmaceutical Drugs for Expeditious Antiviral Approaches Against Newer Strains of COVID-19.

At present, global vaccination for the SARS-CoV2 virus 2019 (COVID-19) is 95% effective. Generally, viral infections are arduous to cure due to the mutating nature of viral genomes, with the consequent quick development of resistance, posing significant fatalities or hazards. The novel corona viral strains are increasingly lethal than earlier variants, as those evolve faster than imagined. Despite the emergence of several present innovative treatment options, the vaccines, and available drugs, the latter still are the needs of the time. Therefore, repurposing the approved pharmaceutical drugs of a well-known safety profile would be ascertained to provide faster antiviral approaches for the newer strains of COVID-19. Recently, a combination of remdesivir, which has a competitively inhibitory effect on the nucleotide uptake in the virus, and the merimepodibs, an inhibitor of the enzyme inosine monophosphate dehydrogenase, which has a role in the synthesis of nucleotides of guanine bases, is in use in phase 2 clinical trials. However, new investigations suggest that using remdesivir, there is no statistically significant difference with uncertain clinical importance for moderate COVID-19 patients. Herein, an intellectual selection of approved drugs based on the safety profile is described, to target any essential enzymes that are required for the virus-receptor contact, fusion, and/or different stages of the life cycle of this virus, should help to screen drugs against newer strains of COVID-19.Graphical abstract

Guanosine triphosphate links MYC-dependent metabolic and ribosome programs in small-cell lung cancer.

MYC stimulates both metabolism and protein synthesis, but how cells coordinate these complementary programs is unknown. Previous work reported that, in a subset of small-cell lung cancer (SCLC) cell lines, MYC activates guanosine triphosphate (GTP) synthesis and results in sensitivity to inhibitors of the GTP synthesis enzyme inosine monophosphate dehydrogenase (IMPDH). Here, we demonstrated that primary MYChi human SCLC tumors also contained abundant guanosine nucleotides. We also found that elevated MYC in SCLCs with acquired chemoresistance rendered these otherwise recalcitrant tumors dependent on IMPDH. Unexpectedly, our data indicated that IMPDH linked the metabolic and protein synthesis outputs of oncogenic MYC. Coexpression analysis placed IMPDH within the MYC-driven ribosome program, and GTP depletion prevented RNA polymerase I (Pol I) from localizing to ribosomal DNA. Furthermore, the GTPases GPN1 and GPN3 were upregulated by MYC and directed Pol I to ribosomal DNA. Constitutively GTP-bound GPN1/3 mutants mitigated the effect of GTP depletion on Pol I, protecting chemoresistant SCLC cells from IMPDH inhibition. GTP therefore functioned as a metabolic gate tethering MYC-dependent ribosome biogenesis to nucleotide sufficiency through GPN1 and GPN3. IMPDH dependence is a targetable vulnerability in chemoresistant MYChi SCLC.

Anti-rods/rings autoantibody and IMPDH filaments: an update after fifteen years of discovery.

Autoantibodies to unknown subcellular rod and ring-shaped structures were first discovered in sera from hepatitis C patients in 2005. Early studies showed a strong association between these anti-rods/rings antibodies (anti-RR) and the standard of care interferon-α plus ribavirin combination therapy (IFN/RBV), suggesting that anti-RR are drug-induced autoantibodies. In the context of hepatitis C, anti-RR have been linked with relapse from or lack of response to IFN/RBV in some patient cohorts. However, examples of anti-RR in other diseases and healthy individuals have also been reported over the years, although anti-RR remains a rare autoantibody response in general. The advent of new direct-acting antiviral drugs for chronic hepatitis C and studies of anti-RR from different parts of the world are also beginning to change the perception of anti-RR. The nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) has been identified as the major autoantigen recognized by anti-RR. Coincidentally, the assembly of IMPDH into micron-scale rod and ring-shaped structures was discovered around the same time as anti-RR. Knowledge of the fundamental biological properties and cellular functions of these structures, referred to as "IMPDH filaments" by cell biologists, has advanced in parallel to anti-RR antibodies. Recent studies have revealed that IMPDH filament assembly is a mechanism to prevent feedback inhibition of IMPDH and is therefore important for the increased nucleotide production required in hyperproliferating cells, like activated T cells. Fifteen years later, we review the history and current knowledge in both the anti-RR autoantibody and IMPDH filament fields. TAKE-HOME MESSAGE: Anti-rods/rings are recognized as an example of a drug-induced autoantibody in hepatitis C patients treated with interferon and ribavirin, although new studies suggest anti-rods/rings may be detected in other contexts and may depend on unknown environmental or genetic factors in different populations. Recent data suggest that the assembly of IMPDH into rod and ring structures, the targets of anti-rods/rings autoantibody, is a mechanism for hyperproliferating cells, like activated T cells, to maintain increased guanine nucleotide levels to support rapid cell division.

ANALYTICAL METHODS FOR ESTIMATION OF MYCOPHENOLIC ACID IN BULK AND IN PHARMACEUTICAL DOSAGE FORM: A REVIEW

Mycophenolic acid is an anti-metabolite immunosuppressant. It also inhibits the enzyme inosine monophosphate dehydrogenase; essential for purine synthesis. High performance liquid chromatography (HPLC) and the UV are an essential analytical tools in assessing drug product. HPLC methods should be able to isolate, detect, and enumerate the various drugs and drug associated degradants that can form on storage, or manufacturing. It should also detect and enumerate any drugs and drug-related impurities that may be introduced during synthesis. Validation is the process of establishing the performance characteristics and limits of a method and identification of the effects which may change these features and to what extent. This article discusses the current and potential uses of the drug mycophenolic acid as well as the plans and the subjects related to designing UV and HPLC method for development and validation.
 Keywords: Mycophenolic acid, Immunosuppressant, HPLC, UV, Validation

Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations.

Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas GTP promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and non-assembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.

PWE-028 A Novel Assay for The Direct Detection of Deoxythioguanosine (DTG) in DNA from Patients On Azathioprine Using Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS)

Introduction The correlation between thiopurine drug metabolite levels in red blood cells (RBC) and clinical response is currently under debate, with some studies reporting a good correlation whereas others report a poor correlation. However, without a suitable alternative assay this is the only method presently available to guide dosing of patients treated with thiopurine drugs: azathioprine (AZA), mercaptopurine (MP) or thioguanine. Biologically, this method is not perfect given that RBC lack the critical drug-metabolising enzyme inosine-monophosphate dehydrogenase, which is central to the bioconversion of MP to thioguanine nucleotides that are then incorporated as fraudulent bases into nucleic acids. To try to get a better indicator of drug response and hence clinical outcome, we have developed a sensitive liquid chromatography- tandem mass spectrometry (LC-MS/MS) assay to measure incorporated dTG in the DNA isolated from peripheral blood mononuclear cells of patients treated with thiopurines Methods DNA was isolated from blood samples collected from IBD patients on thiopurines. DNA was denatured then digested using P1 nuclease followed by treatment with alkaline phosphatase and finally diluted in MilliQ water prior to analysis. A total of 0.2 μg of DNA in 50 µL was injected for chromatographic separation followed by analysis on an API4000 triple quadrupole mass spectrometer. Standard curves and controls were validated and samples analysed to determine number of moles of dTG/105 5 moles of dA. Results From a small cohort of 20 AZA treated IBD patients (10 Crohn’s Disease and 10 Ulcerative Colitis) with dTG levels of 0.18 to 11.3 moles dTG/105 5 moles dA (approximately 5 to 340 femtomoles/μg DNA) were detected with no detectable dTGs in un-treated patients. The intra- and inter-assay variability was below 7.8% and 17.0% respectively with a detection limit of 15.5 pg (54.7 femtomoles) and was quantified in DNA samples relative to endogenous dA. Conclusion This method enables the direct detection of a cytotoxic thiopurine metabolite in an easily obtainable, stable sample and will facilitate a better understanding of the mechanisms of action of these inexpensive yet effective drugs. Disclosure of Interest None Declared

Abstract 3030: De novo engineering of chromosomal amplifications in human cells

Abstract Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to recurrent amplification events are lacking. Moreover, some common amplification events, such as that at 8p11-12 in breast cancer, harbor multiple candidate driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our general strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We chose regions recurrently amplified at high frequency in breast cancer, including 8p11-12, 17q12, and 11q13-14. After confirmation of successful gene targeting, the targeted cells are subjected to selective pressure directed at the amplifiable marker. Spontaneous DNA breakage events lead to varying degrees of co-amplification of linked loci near the targeting site. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using either the E. coli inosine monophosphate dehydrogenase enzyme IMPDH or a mutant dihydrofolate reductase gene as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR. Genome-wide array comparative genomic hybridization confirmed that amplification had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including “sawtooth” patterns, distal copy number loss suggesting a breakage-fusion-bridge mechanism, and large continuous regions of copy number gain. Amplification was confirmed by FISH. RT-PCR and immunoblotting demonstrated overexpression of selected amplified transcripts and proteins. In summary, we have used genome engineering techniques to create focal chromosomal amplification events de novo in human cell lines. These amplifications share many of the features of amplifications observed in primary human cancers. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a system to model oncogene cooperativity in amplifications harboring multiple candidate driver genes. Citation Format: Josh Lauring. De novo engineering of chromosomal amplifications in human cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3030. doi:10.1158/1538-7445.AM2015-3030

Plasma and intracellular pharmacokinetic–pharmacodynamic analysis of mycophenolic acid in de novo kidney transplant patients

ObjectivesLittle is known about the correlation between the inosine-monophosphate dehydrogenase (IMPDH) activity and mycophenolic acid (MPA) concentrations in peripheral-blood-mononuclear cells (PBMCs), where the drug is acting. The aim of this study was to analyze the relationship between plasma or PBMC MPA levels, as pharmacokinetic (PK) markers, and the intracellular IMPDH enzyme activity, as a pharmacodynamic (PD) biomarker, in kidney transplantation. Design and methodsForty de novo renal transplant patients were enrolled in this prospective study. The sampling was performed on the day before transplantation and at T0, T1.5 and T3.5 following the morning dose, on days 2, 4 and 10 post-transplantation. All subjects were treated with a fixed MMF dose (500mg twice-a-day). IMPDH activities were determined by HPLC, and MPA plasma or PBMC concentrations were obtained by LC–MSMS. ResultsImportant inter-patient variability was observed both for the PK and PD biomakers. Pre-dose IMPDH activity, surprisingly, increased during the 10days post-transplantation. As expected, a significant inverse relationship was found between IMPDH activities and MPA concentrations in both plasma and PBMCs. A significant correlation was found between plasma and PBMC MPA values. Maximum IMPDH inhibition was found mostly at T1.5, before returning to its pre-dose levels at T3.5. IMPDH inhibition at T1.5 better correlated with plasma MPA AUC0–3.5 (p=0.027) than with PBMC AUC0–3.5 (p=0.323). Mean MPA plasma concentrations paralleled the enzyme inhibition profiles and decreased strongly at T3.5, whereas the decreasing slope of MPA concentrations in PBMCs appeared slower. ConclusionsThese findings suggest that PBMC MPA concentrations do not provide any better correlation with the IMPDH activity than plasma MPA values, most likely due to the correlation between plasma and PBMC MPA levels and to the important interpatient variability both in MPA levels and enzyme activities.

Cloning, characterization and validation of inosine 5′-monophosphate dehydrogenase of Babesia gibsoni as molecular drug target

The inosine monophosphate dehydrogenase (IMPDH) enzyme has been characterized and validated as a molecular drug target in other apicomplexans but not in the genus Babesia. Subsequently, we cloned and expressed a Babesia gibsoni IMPDH (BgIMPDH) cDNA in Escherichia coli. We also determined the inhibitory effect of mycophenolic acid (MPA) on recombinant BgIMPDH (rBgIMPDH) activity and the Babesia-growths in vitro. The translated BgIMPDH peptide contained thirteen amino acid residues responsible for substrate and cofactor binding in its catalytic domain with Gly374 in BgIMPDH being replaced by Ser388 in mammalian IMPDH. The native BgIMPDH enzyme in the parasite was approximately 54-kDa a mass similar to His-tag rBgIMPDH protein. The Km values of the rBgIMPDH were 8.18±0.878 (mean±standard error of the mean) μM and 360.80±43.41μM for IMP and NAD+, respectively. MPA inhibited the rBgIMPDH activity yielding a Ki value of 20.93±1.83μM with respect to NAD+. For Babesia growths, the IC50s were 0.95±0.21 and 2.88±0.49μM for B. gibsoni and B. bovis, respectively. Therefore, our results suggest that MPA may inhibit the replication of Babesia parasites by targeting IMPDH enzyme of the purine pathway.

An insight to the dynamics of conserved water-mediated salt bridge interaction and interdomain recognition in hIMPDH isoforms

Inosine monophosphate dehydrogenase (IMPDH) is involved in de novo biosynthesis pathway of guanosine nucleotide. Type II isoform of this enzyme is selectively upregulated in lymphocytes and chronic myelogenous leukemia (CML) cells, and is an excellent target for antileukemic agent. The molecular dynamics simulation results (15 ns) of three unliganded 1B3O, 1JCN, and 1JR1 structures have clearly revealed that IN, IC (N- and C-terminal of catalytic domains) and C1, C2 (cystathionine-beta-synthase-1 and 2) domains of IMPDH enzyme have been stabilized by six conserved water (center) mediated salt bridge interactions. These conserved water molecules could be involved in interdomain or intradomain recognition, intradomain coupling, and charge transfer processes. The binding propensity of cystathionine-beta-synthase domain to catalytic domain (through conserved water-mediated salt bridges) has provided a new insight to the biochemistry of IMPDH. Stereospecific interaction of IN with C2 domain through conserved water molecule (K109–WII 1–D215/D216) is observed to be unique in the simulated structure of hIMPDH-II. The geometrical/structural consequences and topological feature around the WII 1 water center may be utilized for isoform specific inhibitor design for CML cancer. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:1

Metabolic Enzyme IMPDH Is Also a Transcription Factor Regulated by Cellular State

Cells need to coordinate gene expression and metabolic state. Inosine monophosphate dehydrogenase (IMPDH) controls the guanine nucleotide pool and, thereby, cell proliferation. We found that Drosophila IMPDH is also a DNA-binding transcriptional repressor. IMPDH attenuates expression of histone genes and E2f, a key driver of cell proliferation. Nuclear IMPDH accumulates during the G2 phase of the cell cycle or following replicative or oxidative stress. Thus, IMPDH can couple the expression of histones and E2F to cellular state. Genome-wide profiling and in vitro binding assays established that IMPDH binds sequence specifically to single-stranded, CT-rich DNA elements. Surprisingly, this DNA-binding function is conserved in E. coli IMPDH. The catalytic function of IMPDH is not required for DNA binding. Yet substitutions that correspond to human retinitis pigmentosa mutations disrupt IMPDH binding to CT-rich, single-stranded DNA elements. By doubling as nucleotide biosynthetic enzyme or transcription factor, IMPDH can either enable or restrict cell proliferation.

Role of Salt Bridge Dynamics in Inter Domain Recognition of Human IMPDH Isoforms: An Insight to Inhibitor Topology for Isoform-II

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in the biosynthesis pathway of guanosine nucleotide. Type II isoform of the enzyme is selectively upregulated in neoplastic fast replicating lymphocytes and CML cancer cells. The hIMPDH-II is an excellent target for antileukemic agent. The detailed investigation during MD-Simulation (15 ns) of three different unliganded structures (1B3O, 1JCN and 1JR1) have clearly explored the salt bridge mediated stabilization of inter or intra domain (catalytic domains IN, with res. Id. 28–111 and 233–504, whereas two CBS domains C1, C2 are 112–171 and 172–232) in IMPDH enzyme which are mostly inaccessible in their X-rays structures. The salt bridge interaction in IN—C1 inter-domain of hIMPDH-I, IN—C2 of IMPDH-II and C1—IC of nhIMPDH-II are discriminative features among the isoforms. The IN—C2 recognition in hIMPDH-II (1B3O) is missing in type-I isoform (1JCN). The salt bridge interaction D232—K238 at the surface of protein and the involvement of three conserved water molecules or the hydrophilic centers (WA232 OD1, WB232 OD2 and W238 NZ) to those acidic and basic residues seem to be unique in hIMPDH-II. The hydrophilic susceptibility, geometrical and electronic consequences of this salt bridge interaction could be useful to design the topology of specific inhibitor for hIMPDH-II which may not be effective for hIMPDH-I. Possibly, the aliphatic ligand containing carboxyl, amide or hydrophilic groups with flexible structure may be implicated for hIMPDH-II inhibitor design using the conserved water mimic drug design protocol.

Inhibition of Junin virus RNA synthesis by an antiviral acridone derivative

There are no specific approved drugs for the treatment of agents of viral hemorrhagic fevers (HF) and antiviral therapies against these viruses are urgently needed. The present study characterizes the potent and selective antiviral activity against the HF causing arenavirus Junin virus (JUNV) of the compound 10-allyl-6-chloro-4-methoxy-9(10H)-acridone, designated 3f. The effectiveness of 3f to inhibit JUNV multiplication was not importantly affected by the initial multiplicity of infection, with similar effective concentration 50% (EC50) values in virus yield inhibition assays performed in Vero cells in the range of 0.2–40 plaque forming units (PFU)/cell. Mechanistic studies demonstrated that 3f did not affect the initial steps of adsorption and internalization. The subsequent process of viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR in compound-treated cells relative to non-treated cells. The addition of exogenous guanosine rescued the infectivity and RNA synthesis of JUNV in 3f-treated cells in a dose-dependent manner, but the reversal was partial, suggesting that the reduction of the GTP pool contributed to the antiviral activity of 3f, but it was not the main operative mechanism. The comparison of 3f with two other viral RNA inhibitors, ribavirin and mycophenolic acid, showed that ribavirin did not act against JUNV through the cellular enzyme inosine monophosphate dehydrogenase (IMPDH) inhibition whereas the anti-JUNV activity of mycophenolic acid was mainly targeted at this enzyme.

Depletion of GTP pool is not the predominant mechanism by which ribavirin exerts its antiviral effect on Lassa virus

Ribavirin (1-β- d-ribofuranosyl-1,2,4-triazole-3-carboxamide) is the standard treatment for Lassa fever, though its mode of action is unknown. One possibility is depletion of the intracellular GTP pool via inhibition of the cellular enzyme inosine monophosphate dehydrogenase (IMPDH). This study compared the anti-arenaviral effect of ribavirin with that of two other IMPDH inhibitors, mycophenolic acid (MPA) and 5-ethynyl-1-β- d-ribofuranosylimidazole-4-carboxamide (EICAR). All three compounds were able to inhibit Lassa virus replication by ⩾2 log units in cell culture. Restoring the intracellular GTP pool by exogenous addition of guanosine reversed the inhibitory effects of MPA and EICAR, while ribavirin remained fully active. Analogous experiments performed with Zaire Ebola virus showed that IMPDH inhibitors are also active against this virus, although to a lesser extent than against Lassa virus. In conclusion, the experiments with MPA and EICAR indicate that replication of Lassa and Ebola virus is sensitive to depletion of the GTP pool mediated via inhibition of IMPDH. However, this is not the predominant mechanism by which ribavirin exerts its in-vitro antiviral effect on Lassa virus.

Inosine Monophosphate Dehydrogenase (IMPDH) Activity as a Pharmacodynamic Biomarker of Mycophenolic Acid Effects in Pediatric Kidney Transplant Recipients

Monitoring inosine monophosphate dehydrogenase (IMPDH) activity as a biomarker of mycophenolic acid (MPA)-induced immunosuppression may serve as a novel approach in pharmacokinetics (PK)/pharmacodynamics (PD)-guided therapy. The authors prospectively studied MPA pharmacokinetics and IMPDH inhibition in 28 pediatric de novo kidney transplant recipients. Pretransplant IMPDH activity and full PK/PD profiles were obtained at 3 different occasions: 1 to 3 days, 4 to 9 days, and approximately 6 months after transplant. Large intra- and interpatient variability was noted in MPA pharmacokinetics and exposure and IMPDH inhibition. MPA exposure (AUC(0-12 h)) was low early posttransplant and increased over time and stabilized at months 3 to 6. Mean pretransplant IMPDH activity (6.4 ± 4.6 nmol/h/mg protein) was lower than previously reported in adults. In most of the patients, IMPDH enzyme activity decreased with increasing MPA plasma concentration, with maximum inhibition coinciding with maximum MPA concentration. The overall relationship between MPA concentration and IMPDH activity was described by a direct inhibitory E(max) model (EC(50) = 0.97 mg/L). This study suggests the importance of early PK/PD monitoring to improve drug exposure. Because IMPDH inhibition is well correlated to MPA concentration, pretransplant IMPDH activity may serve as an early marker to guide the initial level of MPA exposure required in a pediatric population.