Monophosphate Dehydrogenase Research Articles

OS6.1 Targeting Purine Metabolism to Overcome Therapeutic Resistance in Glioblastoma

Abstract BACKGROUND A distinguishing characteristic of all cancers is uncontrolled cell division, and they require additional nucleotide bases such as purines, the building blocks of DNA and RNA, to sustain their uncontrolled growth. Purines can be synthesized from scratch by de novo pathway or salvaged by recycling surrounding nucleotides that are released by hydrolytic degradation. Even though the central nervous system (CNS), as well as CNS associated malignancies like glioblastoma (GBM), rely more heavily on the salvage pathway due to its energy efficiency, its precious role in promoting chemoresistance and GBM recurrence is yet to be elucidated. MATERIAL AND METHODS We have examined the role of purine biosynthesis in GBM by using stable isotope tracing analysis as well as utilizing a knockdown (KD) system to investigate its effect on i) DNA damage response during temozolomide (TMZ) therapy, ii) tumor engraftment and iii) therapeutic responses in vivo. RESULTS Through gene expression and protein-protein interaction analysis, we have identified ARL13B, member of ADP-ribosylation factor-like protein family, as a novel regulator of the purine biosynthesis pathway in GBM. ARL13B can physically interacting with the inosine monophosphate dehydrogenase 2 (IMPDH2), a key rate-limiting enzyme for purine biogenesis. Isotope tracer analysis under normal physiological conditions revealed that during TMZ treatment, salvage recycling activity was decreased by 50% while de novo pathway activity remains unchanged. In contrast, TMZ treatment of ARL13B knock-out cells results in a ~50% decrease in de novo pathway activity (p-value=0.004), whereas purine salvage pathway activity is upregulated ~6-fold (p-value <0.0001). ARL13B knockdown cells treated with TMZ show a robust increase in DNA double-strand breaks compared to control cells exposed to TMZ, as demonstrated by gH2X staining. Mice orthotopically engrafted with KD cells experience prolonged survival relative to mice engrafted with unmodified cells. CONCLUSION We propose that ARL13B-IMPDH2 interaction has two consequences: i) augmentation of de novo purine biosynthesis activity, and ii) inhibition of nucleotide recycling. The increasing de novo purine biosynthesis during TMZ therapy helps GBM cells reduce the recycling of nucleotides via the salvage pathway that have been modified as a result of TMZ alkylation. This, in turn, protects the cells from deleterious effects of incorporating modified nucleotides into newly-synthesized DNA while maintaining a supply of purine building blocks to support uncontrolled proliferation. Our results indicate that the interaction of ARL13B-IMPDH2 functions as a purine biosynthesis regulator that could be targeted for increasing efficacy of TMZ treatment of GBM. ​

ANKRD9 is a metabolically-controlled regulator of IMPDH2 abundance and macro-assembly

Members of a large family of Ankyrin Repeat Domain (ANKRD) proteins regulate numerous cellular processes by binding to specific protein targets and modulating their activity, stability, and other properties. The same ANKRD protein may interact with different targets and regulate distinct cellular pathways. The mechanisms responsible for switches in the ANKRDs' behavior are often unknown. We show that cells' metabolic state can markedly alter interactions of an ANKRD protein with its target and the functional outcomes of this interaction. ANKRD9 facilitates degradation of inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in GTP biosynthesis. Under basal conditions ANKRD9 is largely segregated from the cytosolic IMPDH2 in vesicle-like structures. Upon nutrient limitation, ANKRD9 loses its vesicular pattern and assembles with IMPDH2 into rodlike filaments, in which IMPDH2 is stable. Inhibition of IMPDH2 activity with ribavirin favors ANKRD9 binding to IMPDH2 rods. The formation of ANKRD9/IMPDH2 rods is reversed by guanosine, which restores ANKRD9 associations with the vesicle-like structures. The conserved Cys109Cys110 motif in ANKRD9 is required for the vesicle-to-rods transition as well as binding and regulation of IMPDH2. Oppositely to overexpression, ANKRD9 knockdown increases IMPDH2 levels and prevents formation of IMPDH2 rods upon nutrient limitation. Taken together, the results suggest that a guanosine-dependent metabolic switch determines the mode of ANKRD9 action toward IMPDH2.

The role of mycophenolate in the treatment of antineutrophil cytoplasmic antibody-associated vasculitis.

Mycophenolic acid, the active metabolite for mycophenolate mofetil and mycophenolic sodium, is a strong, noncompetitive, reversible inhibitor of inosine monophosphate dehydrogenase, the key enzyme in de novo synthesis of guanosine nucleotides leading to selective inhibition of lymphocyte proliferation. Mycophenolic acid has been evaluated as induction and remission maintenance agent in the treatment of antineutrophil cytoplasmic antibody-associated vasculitis (AAV). Since the course of disease of AAV usually requires long term immunosuppression, mycophenolate has been explored as a less toxic agent compared to cyclophosphamide and azathioprine. Mycophenolate is a potent immunosuppressive agent in the therapy of AAV, non-inferior to other available drugs with comparable side effect profile. Therefore, it could be a valuable alternative in cases of toxicity with life threatening side effects or intolerance to cyclophosphamide or azathioprine, in cases with high cumulative dose of cyclophosphamide, but also in cases with insufficient response. Several studies have shown a higher relapse rate following discontinuation of mycophenolate or in mycophenolate treated subjects that raises concerns about its usefulness in the treatment of AAV. This review describes the efficacy of mycophenolate in AAV as remission induction agent, as remission maintenance agent, and as therapeutic option in relapsing AAV disease, the relapse rate following discontinuation of mycophenolate, and the adverse events related to mycophenolate treatment.

FF-10501 induces caspase-8-mediated apoptotic and endoplasmic reticulum stress-mediated necrotic cell death in hematological malignant cells.

FF-10501 is a novel inhibitor of inosine monophosphate dehydrogenase (IMPDH). Clinical trials of FF-10501 for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are currently being conducted in the United States. Although it has been shown that FF-10501 induces apoptosis in hematological malignant cells, the intracellular mechanisms of this effect have not been characterized. We conducted an in vitro study to elucidate the mechanisms of FF-10501-induced cell death using 12 hematological malignant cell lines derived from myeloid and lymphoid malignancies. FF-10501 suppressed the growth of each cell line in a dose-dependent manner. However, the clinically relevant dose (40μM) of FF-10501 induced cell death in three cell lines (MOLM-13, OCI-AML3, and MOLT-3). Investigation of the cell death mechanism suggested that FF-10501 induces both apoptotic and necrotic cell death. FF-10501-induced apoptosis was mediated by caspase-8 activation followed by activation of the mitochondrial pathway in MOLM-13 and MOLT-3 cells. FF-10501 induced necrotic cell death via endoplasmic reticulum stress in OCI-AML3 cells. The present study is the first to identify intracellular pathways involved in FF-10501-induced cell death.

Effect of mycophenolate and rapamycin on renal fibrosis in lupus nephritis.

Lupus nephritis (LN) leads to chronic kidney disease (CKD) through progressive fibrosis. Mycophenolate inhibits inosine monophosphate dehydrogenase and is a standard treatment for LN. The mammalian or mechanistic target of rapamycin (mTOR) pathway is activated in LN. Rapamycin inhibits mTOR and is effective in preventing kidney transplant rejection, with the additional merits of reduced incidence of malignancies and viral infections. The effect of mycophenolate or rapamycin on kidney fibrosis in LN has not been investigated. We investigated the effects of mycophenolate and rapamycin in New Zealand Black and White first generation (NZB/W F1) murine LN and human mesangial cells (HMCs), focusing on mechanisms leading to kidney fibrosis. Treatment of mice with mycophenolate or rapamycin improved nephritis manifestations, decreased anti-double stranded (ds) DNA antibody titer and reduced immunoglobulin G (IgG) deposition in the kidney. Both mycophenolate and rapamycin, especially the latter, decreased glomerular mTOR Ser2448 phosphorylation. Renal histology in untreated mice showed mesangial proliferation and progressive glomerulosclerosis with tubular atrophy, and increased expression of transforming growth factor β1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle actin (α-SMA), fibronectin (FN) and collagen. Both mycophenolate and rapamycin ameliorated the histopathological changes. Results from in vitro experiments showed that both mycophenolate and rapamycin decreased mesangial cell proliferation and their binding with anti-dsDNA antibodies. Mycophenolate and rapamycin also down-regulated mTOR and extracellular signal-regulated kinase (ERK) phosphorylation and inhibited fibrotic responses in mesangial cells that were induced by anti-dsDNA antibodies or TGF-β1. Our findings suggest that, in addition to immunosuppression, mycophenolate and rapamycin may reduce fibrosis in LN, which has important implications in preventing CKD in patients with LN.

Novel adenosine-derived inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase.

We have found cyclophane-type adenosine derivatives having p-quinone amide moieties (1 and 2) as weak inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase (CpIMPDH) from the Hokkaido University Chemical Library via the luciferase-based high-throughput screening. To obtain more potent inhibitors, we synthesized four new derivatives free from cyclophane rings (3-6). The N-H derivatives 3 and 5 showed more potent activities (24.4 and 11.1 μM, respectively) in the presence of dithiothreitol (DTT), whereas the N-methyl derivative 4 indicated more potent activity (2.1 μM) without DTT. Conformational analysis of compounds 3 and 4 suggested that N-H amide 3 binds to IMP-binding site in the DTT mediated manner.

Abstract 81: ARL13B interacts with IMPDH2 to modulate purine synthesis and temozolomide resistance in glioblastoma

Abstract Glioblastoma, a universally lethal primary brain tumor, harnesses cellular plasticity to drive therapeutic adaptation. Critical factors in developing this plasticity are histone modifiers such as Polycomb Repressor Complex 2 protein EZH2. In order to examine tumor cell plasticity in depth, we conducted multiple ChIP Sequencing runs and demonstrate that EZH2 binds within an enhancer region of ARL13B during temozolomide (TMZ) therapy and induces an H3K4 mono-methylation mark. Concurrently, we observed an increase in H3K27ac at the transcription start site of ARL13B as well as a lack of H3K27 tri-methylation, EZH2’s canonical histone mark. Based on this we hypothesize that EZH2 could be non-canonically regulating ARL13B to allow for cellular plasticity and ultimately drive therapeutic adaptation. Delving further into this regulation we demonstrate that knockdown of ARL13B in patient derived xenograft cells significantly increased survival of mice in an orthotopic GBM model when compared to controls (p-value <0.0001). The Cancer Genome Atlas (TCGA) patient dataset demonstrates time to recurrence in patients with downregulated ARL13B is substantially increased as compared to ARL13B upregulated patients (log-rank p-value=0.0012). Searching for a mechanism behind this survival benefit, we preformed mass spectrometry on an ARL13B pulldown in a patient derived xenograft line during TMZ therapy and identified inosine monophosphate dehydrogenase 2 (IMPDH2), the rate-limiting enzyme in de-novo guanine nucleotide biosynthesis, as a significant binding partner of ARL13B during TMZ chemotherapy (p-value <0.0001). Probing this novel interaction further we examined the de-novo and salvage purine biosynthesis pathways using radiolabeled carbon tracing experiments. In ARL13B knockdown cells, purine salvage pathway usage is upregulated 7-fold (p-value <0.0001) while de-novo pathway usage was decreased about 50% (p-value=0.004) in a TMZ specific manner. Examination of IMPDH2 enzymatic activity using a formazin reduction assay demonstrated a decrease in activity over 8 days of TMZ exposure (p<.001). Moreover, ARL13B knockdown GBM cells treated with TMZ show a robust increase in DNA double-strand breaks compared to control cells exposed to TMZ, demonstrated by γH2X staining. Finally, a potent inhibitor of IMPDH2 (Mycophenolate Mofetil) significantly extended median survival in an orthotopic PDX mouse model only when in combination with TMZ (p<.01). Based on these data we hypothesize that EZH2 regulates a novel ARL13B and IMPDH2 interaction which when lost forces cells into salvage synthesis exclusively. This synthesis shift forces cells to uptake and incorporate purines that have been alkylated by TMZ therapy which increases DNA double strand breaks and ultimately impairs therapeutic adaptation. Citation Format: Jack Shireman, Eunus Ali, Miranda Saathoff, Cheol Park, Issam Ben-Sahra, Atique U. Ahmed. ARL13B interacts with IMPDH2 to modulate purine synthesis and temozolomide resistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 81.

Dynamic compartmentalization of purine nucleotide metabolic enzymes at leading edge in highly motile renal cell carcinoma

Compartmentalization is vital for biological systems at multiple levels, including biochemical reactions in metabolism. Organelle-based compartments such as mitochondria and peroxisomes sequester the responsible enzymes and increase the efficiency of metabolism while simultaneously protecting the cell from dangerous intermediates, such as radical oxygen species. Recent studies show intracellular nucleotides, such as ATP and GTP, are heterogeneously distributed in cells with high concentrations at the lamellipodial and filopodial projections, or leading edge. However, the intracellular distribution of purine nucleotide enzymes remains unclear. Here, we report the enhanced localization of GTP-biosynthetic enzymes, including inosine monophosphate dehydrogenase (IMPDH isotype 1 and 2), GMP synthase (GMPS), guanylate kinase (GUK1) and nucleoside diphosphate kinase-A (NDPK-A) at the leading edge in renal cell carcinoma cells. They show significant co-localization at the membrane subdomain, and their co-localization pattern at the membrane is distinct from that of the cell body. While other purine nucleotide biosynthetic enzymes also show significant localization at the leading edge, their co-localization pattern with IMPDH is divergent. In contrast, a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), predominantly localized in the cytoplasm. Mechanistically, we found that plasma membrane localization of IMPDH isozymes requires active actin polymerization. Our results demonstrate the formation of a discrete metabolic compartment for localized purine biosynthesis at the leading edge, which may promote localized nucleotide metabolism for cell migration and metastasis in cancers.

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

Expression of inosine monophosphate dehydrogenase 1 in breast cancer and clinical significance

Objective To study the expression level of inosine monophosphate dehydrogenase 1 (IMPDH1) in the breast cancer, analyze the correlation between IMPDH1 and clinicopathological characteristics of breast cancer, and explore possible the mechanisms of the involvement of IMPDH1 in the occurrence and development of breast cancer. Methods Seventy-two cases of breast carcinoma and para-carcinoma tissues were collected. Western blotting was used to detect the protein expression of IMPDH1 in breast carcinoma and adjacent tissues. Immunohistochemistry was applied to detect the expression of IMPDH1, and the correlation between IMPDH1 and clinicopathological characteristics of breast cancer was analyzed. Results The positive rate of IMPDH1 expression in breast cancer (79.17%) was significantly higher than in the adjacent tissues (15.28%) (P 0.05; χ2=2.027, P>0.05; χ2=0.397, P>0.05), but to lymph node metastasis, proliferation cell nuclear antigen (Ki-67) and TNM staging (χ2=8.530, P<0.01; χ2=4.308, P<0.05; χ2=4.721, P<0.05). Western blotting showed that the expression of IMPDH1 in breast cancer was significantly higher than in the normal tissue (P<0.01). Conclusion The expression of IMPDH1 in breast cancer is higher than in normal tissues, and is correlated with lymph node metastasis, Ki-67 and TNM staging, suggesting its roles in the breast cancer. Key words: Breast carcinoma; Inosine monophosphate dehydrogenase 1; Clinicopathological characteristics

Characterizing the pharmacokinetics and pharmacodynamics of immunosuppressant medicines and patient outcomes in elderly renal transplant patients.

This review examines what is currently known about the pharmacokinetics and pharmacodynamics of commonly prescribed immunosuppressant medicines, tacrolimus, cyclosporine, mycophenolate and prednisolone, in elderly renal transplant recipients, and reported patient outcomes in this cohort. Renal transplantation is increasing rapidly in the elderly, however, currently, long-term patient outcomes are relatively poor compared to younger adults. Some studies have suggested that elderly recipients may have higher dose-adjusted exposure and/or lower clearance of the calcineurin inhibitors tacrolimus and cyclosporine; with one study reporting up to 50% reduction in tacrolimus exposure in the elderly. Elderly transplant recipients do not appear to have higher dosage-adjusted exposure to mycophenolic acid (MPA). The effects of ageing on the pharmacokinetics of prednisolone are unknown. Only one study has examined how aging effects drug target enzymes, reporting no difference in baseline inosine 5'-monophosphate dehydrogenase (IMPDH) activity and MPA-induced IMPDH activity in elderly compared to younger adult renal transplant recipients. In elderly transplant recipients, immunosenescence likely lowers the risk of acute rejection, but increases the risk of drug-related adverse effects. Currently, the three main causes of death in elderly renal transplant recipients are cardiovascular disease, infection and malignancy. One study has showed that renal transplant recipients aged over 65 years are seven times more likely to die with a functioning graft compared with young adults (aged 18-29 years). This suggests that an optimal balance between immunosuppressant medicine efficacy and toxicity is not achieved in elderly recipients, and further studies are needed to foster long-term graft and patient survival. Lower maintenance immunosuppressant targets in elderly recipients may decrease patient susceptibility to drug side effects, however, further studies are required and appropriate targets need to be established.

Synthesis and In Vitro Enzymatic Studies of New 3-Aryldiazenyl Indoles as Promising Helicobacter pylori IMPDH Inhibitors.

Helicobacter pylori infection is one of the primary causes of peptic ulcer followed by gastric cancer in the world population. Due to increased occurrences of multi-drug resistance to the currently available antibiotics, there is an urgent need for a new class of drugs against H. pylori. Inosine 5'-monophosphate dehydrogenase (IMPDH), a metabolic enzyme plays a significant role in cell proliferation and cell growth. It catalyses guanine nucleotide synthesis. IMPDH enzyme has been exploited as a target for antiviral, anticancer and immunosuppressive drugs. Recently, bacterial IMPDH has been studied as a potential target for treating bacterial infections. Differences in the structural and kinetic parameters of the eukaryotic and prokaryotic IMPDH make it possible to target bacterial enzyme selectively. In the current work, we have synthesised and studied the effect of substituted 3-aryldiazenyl indoles on Helicobacter pylori IMPDH (HpIMPDH) activity. The synthesised molecules were examined for their inhibitory potential against recombinant HpIMPDH. In this study, compounds 1 and 2 were found to be the most potent inhibitors amongst the database with IC50 of 0.8 ± 0.02µM and 1 ± 0.03 µM, respectively. When compared to the most potent known HpIMPDH inhibitor molecule C91, 1 was only four-fold less potent and can be a good lead for further development of selective and potent inhibitors of HpIMPDH.

Synthesis and Structure–Activity relationship of 1-(5-isoquinolinesulfonyl)piperazine analogues as inhibitors of Mycobacterium tuberculosis IMPDH

Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5′-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure–activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.

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.

Acute rejection in kidney transplantation and the evaluation of associated polymorphisms (SNPs): the importance of sample size.

Background Acute rejection (AR) is one of the most frequent complications after kidney transplantation (KT). Scientific evidence reports that some single-nucleotide polymorphisms (SNPs) located in genes involved in the immune response and in the pharmacokinetics and pharmacodynamics of immunosuppressive drugs are associated with rejection in renal transplant patients. The aim of this study was to evaluate some SNPs located in six genes: interleukin-10 (IL-10), tumor necrosis factor (TNF), adenosine triphosphate-binding cassette sub-family B member 1 (ABCB1), uridine diphosphate glucuronosyltransferase family 1 member A9 (UGT1A9), inosine monophosphate dehydrogenase 1 (IMPDH1) and IMPDH2. Methods We enrolled cases with at least one AR after KT and two groups of controls: patients without any AR after KT and healthy blood donors. Genetic analysis on DNA was performed. The heterozygosity (HET) was determined and the Hardy-Weinberg equilibrium (HWE) test was performed for each SNP. The sample size was calculated using the QUANTO program and the genetic associations were calculated using the SAS program (SAS Institute Inc., Cary, NC, USA). Results In our previous preliminary study (sample size was not reached for cases), the results showed that patients with the C allele in the SNP rs1045642 and the A allele in the SNP rs2032582 of the ABCB1 gene had more frequent AR. In contrast, with the achievement of sample size, the trend of the previous data was not confirmed. Conclusions Our study highlights a fundamental aspect of scientific research that is generally presumed, i.e. the sample size of groups enrolled for a scientific study. We believe that our study will make a significant contribution to the scientific community in the discussion of the importance of the analysis and the achievement of sample size to evaluate the associations between SNPs and the studied event.

Investigation of IMPDH2 overexpression in renal cell carcinoma (RCC) &amp; its association with oncologic outcomes.

594 Background: Approximately 40% of locoregional RCC patients relapse after nephrectomy leading to lower 5 year survival rates of 53% (stage III) &amp; 8% (metastatic). No biomarkers of prognostic significance are in clinical use at this time. It is important to identify biomarkers that can better inform clinicians the nature of disease &amp; personalize treatment. It is even better if the biomarkers are targetable with drugs. Inosine 5”-mono-phosphate dehydrogenase type II (IMPDH2), a rate limiting enzyme in the de novo guanine nucleotide biosynthesis is upregulated in many tumor types. Work by Sasaki laboratory showed increased GTP synthesis by IMPDH2 upregulation is important for cell growth, metastasis &amp; cell maintenance in Gliobastoma Multiforme cell lines. Hypothesis: We are investigating the feasibility of using IMPDH2 expression in RCC as a diagnostic &amp; prognostic biomarker. Methods: 45 cases of clear cell RCC (all stages) were identified by chart review. Slides reviewed to identify blocks with carcinoma, normal tissue &amp; interface. Tissue microarray (TMA) was constructed using the fully automated TMA Master. TMA sectioned and stained with H &amp;E &amp; IMPDH2 antibody. Staining interpretation: TMA stained with IMPDH2 were reviewed based on IRS scoring system (PP x SI) - percentage positive(PP) cells (Negative-0, ≤ 10% -1, ≥ 11%- ≤ 50%- 2, ≥ 51%-≤ 80% -3, and ≥ 81%-4) &amp; staining intensity (SI) (Negative-0, Weak-1, Moderate-2, and Strong-3). The scoring pathologist was blinded to the clinical data &amp; outcomes of patients. Results: Preliminary results show IMPDH2 is overexpressed in RCC and tumor-normal interface compared to normal kidney. Further analyses are ongoing if IMPDH2 overexpression correlates with pertinent clinical &amp; pathological variables including TNM stage, histologic grade (Fuhrman Grade) &amp; oncologic outcomes including overall survival (OS) &amp; risk of relapse. Conclusions: In the era of Sunitinib being approved as an adjuvant therapy (but not widely used due to lack of OS data), the above data will be important to verify if IMPDH2 can be used as a clinically useful test &amp; may give insight to future personalized &amp; targeted treatment strategies (IMPDH2 inhibitors like mycophenolate) for RCC.

Oxanosine Monophosphate Is a Covalent Inhibitor of Inosine 5'-Monophosphate Dehydrogenase.

Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In contrast, the effects of RNS damaged metabolites are less appreciated. 5-Amino-3-β-(d-ribofuranosyl)-3 H-imidazo-[4,5- d][1,3]oxazine-7-one (oxanosine) and its nucleotides are products of guanosine nitrosation. Here we demonstrate that oxanosine monophosphate (OxMP) is a potent reversible competitive inhibitor of IMPDH. The value of Ki varies from 50 to 340 nM among IMPDHs from five different organisms. UV spectroscopy and X-ray crystallography indicate that OxMP forms a ring-opened covalent adduct with the active site Cys (E-OxMP*). Unlike the covalent intermediate of the normal catalytic reaction, E-OxMP* does not hydrolyze, but instead recyclizes to OxMP. IMPDH inhibitors block proliferation and can induce apoptosis, so the inhibition of IMPDH by OxMP presents another potential mechanism for RNS toxicity.

First-in-class allosteric inhibitors of bacterial IMPDHs

Inosine-5‘-monophosphate dehydrogenase (IMPDH) is an essential enzyme in many bacterial pathogens and is considered as a potential drug target for the development of new antibacterial agents. Our recent work has revealed the crucial role of one of the two structural domains (i.e. Bateman domain) in the regulation of the quaternary structure and enzymatic activity of bacterial IMPDHs. Thus, we have screened chemical libraries to search for compounds targeting the Bateman domain and identified first in-class allosteric inhibitors of a bacterial IMPDH. These inhibitors were shown to counteract the activation by the natural positive effector, MgATP, and to block the enzyme in its apo conformation (low affinity for IMP). Our structural studies demonstrate the versatility of the Bateman domain to accommodate totally unrelated chemical scaffolds and pave the way for the development of allosteric inhibitors, an avenue little explored until now.

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.

Identification of selective inhibitors of Helicobacter pylori IMPDH as a targeted therapy for the infection

Helicobacter pylori (H. pylori), the major cause of several gastric disorders has been recognied as a type I carcinogen. By virtue of resistance developed by H. pylori strains, currently used antibiotic based treatments rather demonstrate high failure rates. Hence, there is an emerging need for identification of new targets to treat H. pylori infection. Inosine-5′-monophosphate dehydrogenase (IMPDH) has been studied as a potential target to treat H. pylori infection. Here, a detailed enzyme kinetic study of recombinant expressed H. pylori inosine-5′-monophosphate dehydrogenase (HpIMPDH) is presented. A new in-house synthesized indole-based scaffold is identified as an inhibitor for HpIMPDH. These indole-based compounds showed non-competitive inhibition against IMP and NAD+ whereas the benzimidazole compounds were found be uncompetitive inhibitors. The new indole scaffold ensures specificity due to its high selectivity for bacterial IMPDH over human IMPDH II. Our work aims to overcome the drawback of existing inhibitors by introducing new indole scaffold for targeting bacterial IMPDH.