Monophosphate Dehydrogenase Research Articles

Enzyme polymerization in nucleotide biosynthesis

Polymerization of metabolic enzymes into micron‐scale assemblies is an emerging phenomenon. However the role assembly plays in regulating metabolic flux differs between enzymes and in many cases is unknown. Furthermore, the signaling pathways that regulate assembly are often not well understood. Our work focuses on enzymes mediating pyrimidine and purine biosynthetic pathways that undergo regulated assembly. To address the biological function of these assemblies we have developed point mutants of CTP synthase and inosine monophosphate dehydrogenase (IMPDH), that prevent their assembly without eliminating their catalytic activity. We will present new data using these novel tools in mice and Drosophila to elucidate the role of polymerization of these enzymes in native contexts in which they assemble. Our findings demonstrate a critical role for assembly of CTPS and IMPDH in supporting the growth and proliferation of cells with elevated nucleotide demands. Thus, we propose that assembly of these nucleotide biosynthetic enzymes has been harnessed in metazoans as a homeostatic mechanism to ensure that metabolic biosynthetic flux meets demand.Support or Funding InformationNIH GM083025

The expression and prognostic role of IMPDH2 in ovarian cancer

ObjectInosine 5′-monophosphate dehydrogenase type II (IMPDH2), as an oncogene, is reported to be involved in tumor formation and progression. However, the role of IMPDH2 in ovarian cancer remains unclear. Present study is aimed to investigate the expression and clinical significance of IMPDH2 in ovarian cancer. MethodsThe mRNA and protein levels of IMPDH2 were measured in 126 ovarian cancer and matched adjacent normal tissues by quantificational real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. Then, the association of IMPDH2 with clinicalpathological characters and prognosis was further evaluated. ResultsSignificant higher mRNA levels of IMPDH2 were observed in ovarian cancer compared with those in normal tissues (P < 0.001). IHC results shown the high-expression rate of IMPDH2 in ovarian cancer was 56.3%, which was obviously higher compared with that in normal tissues (23.8%, P < 0.0001). Moreover, IMPDH2 high-expression significantly correlated with tumor types and Federation International of Gynecology and Obstetrigue (FIGO) stages in ovarian cancer (P < 0.05). IMPDH2 overexpression predicted poorer prognosis and could serve as an independent prognostic factor. ConclusionsIMPDH2 is highly expressed in ovarian cancer and correlates with prognosis, which may serve as a potential prognostic biomarker.

IMPDH1/YB-1 Positive Feedback Loop Assembles Cytoophidia and Represents a Therapeutic Target in Metastatic Tumors.

Recently, cytoophidium, a nonmembrane-bound intracellular polymeric structure, has been shown to exist in various organisms, including tumor tissues, but its function and mechanism have not yet been examined. Examination of cytoophidia-assembled gene inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthetase (CTPS) mRNA levels showed that only IMPDH1 levels were significantly higher in the clear cell renal cell carcinoma (ccRCC). IMPDH1 was positively correlated with the metastasis-related gene Y-box binding protein 1 (YB-1) and served as an independent prognostic factor in ccRCC. Kaplan-Meier analysis indicated that patients with tumors that expressed high IMPDH1 levels had a shorter overall survival (OS) and disease-free survival (DFS). Furthermore, detection of cytoophidia by immunofluorescence staining in ccRCC tissues showed that IMPDH1-assembled cytoophidia are positively associated with tumor metastasis. Mechanistically, IMPDH1 and YB-1 formed an autoregulatory positive feedback loop: IMPDH1 maintained YB-1 protein stabilization; YB-1 induced IMPDH1 expression by binding to the IMPDH1 promoter motif. Functionally, IMPDH1-assembled cytoophidia physically interacted with YB-1 and translocated YB-1 into the cell nucleus, thus correlating with ccRCC metastasis. Our findings provide the first solid theoretical rationale for targeting the IMPDH1/YB-1 axis to improve metastatic renal cancer treatment.

In silico Metabolic Pathway Analysis Identifying Target Against Leishmaniasis – A Kinetic Modeling Approach

The protozoan Leishmania donovani, from trypanosomatids family is a deadly human pathogen responsible for causing Visceral Leishmaniasis. Unavailability of proper treatment in the developing countries has served as a major threat to the people. The absence of vaccines has made treatment possibilities to rely solely over chemotherapy. Also, reduced drug efficacy due to emerging resistant strains magnifies the threat. Despite years of formulations for an effective drug therapy, complexity of the disease is also unfortunately increasing. Absence of potential drug targets has worsened the scenario. Therefore exploring new therapeutic approach is a priority for the scientific community to combat the disease. One of the most reliable ways to alter the adversities of the infection is finding new biological targets for designing potential drugs. An era of computational biology allows identifying targets, assisting experimental studies. It includes sorting the parasite’s metabolic pathways that pins out proteins essential for its survival. We have directed our study towards a computational methodology for determining targets against L. donovani from the “purine salvage” pathway. This is a mainstay pathway towards the maintenance of purine amounts in the parasitic pool of nutrients proving to be mandatory for its survival. This study represents an integration of metabolic pathway and Protein-Protein Interactions analysis. It consists of incorporating the available experimental data to the theoretical methods with a prospective to develop a kinetic model of Purine salvage pathway. Simulation data revealed the time course mechanism of the enzymes involved in the synthesis of the metabolites. Modeling of the metabolic pathway helped in marking of crucial enzymes. Additionally, the PPI analysis of the pathway assisted in building a static interaction network for the proteins. Topological analysis of the PPI network through centrality measures (MCC and Closeness) detected targets found common with Dynamic Modeling. Therefore our analysis reveals the enzymes ADSL (Adenylosuccinate lyase) and IMPDH (Inosine-5′-monophosphate dehydrogenase) to be important having a central role in the modeled network based on PPI and kinetic modeling techniques. Further the available three dimensional structure of the enzyme “ADSL” aided towards the search for potential inhibitors against the protein. Hence, the study presented the significance of integrating methods to identify key proteins which might be putative targets against the treatment of Visceral Leishmaniasis and their potential inhibitors.

Nitric oxide disrupts bacterial cytokinesis by poisoning purine metabolism.

Cytostasis is the most salient manifestation of the potent antimicrobial activity of nitric oxide (NO), yet the mechanism by which NO disrupts bacterial cell division is unknown. Here, we show that in respiring Escherichia coli, Salmonella, and Bacillus subtilis, NO arrests the first step in division, namely, the GTP-dependent assembly of the bacterial tubulin homolog FtsZ into a cytokinetic ring. FtsZ assembly fails in respiring cells because NO inactivates inosine 5'-monophosphate dehydrogenase in de novo purine nucleotide biosynthesis and quinol oxidases in the electron transport chain, leading to drastic depletion of nucleoside triphosphates, including the GTP needed for the polymerization of FtsZ. Despite inhibiting respiration and dissipating proton motive force, NO does not destroy Z ring formation and only modestly decreases nucleoside triphosphates in glycolytic cells, which obtain much of their ATP by substrate-level phosphorylation and overexpress inosine 5'-monophosphate dehydrogenase. Purine metabolism dictates the susceptibility of early morphogenic steps in cytokinesis to NO toxicity.

Pharmacodynamic assessment of mycophenolic acid in resting and activated target cell population during the first year after renal transplantation.

AimsTo explore the pharmacodynamics of mycophenolic acid (MPA) through inosine monophosphate dehydrogenase (IMPDH) capacity measurement and purine levels in peripheral blood mononuclear cells (PBMC) longitudinally during the first year after renal transplantation (TX).MethodsPBMC were isolated from renal recipients 0–4 days prior to and 6–9 days, 5–7 weeks and 1 year after TX (before and 1.5 hours after dose). IMPDH capacity and purine (guanine and adenine) levels were measured in stimulated and nonstimulated PBMC.ResultsTwenty‐nine patients completed the follow‐up period, of whom 24 received MPA. In stimulated PBMC, the IMPDH capacity (pmol 10−6 cells min−1) was median (interquartile range) 127 (95.8–147) before TX and thereafter 44.9 (19.2–93.2) predose and 12.1 (4.64–23.6) 1.5 hours postdose across study days after TX. The corresponding IMPDH capacity in nonstimulated PBMC was 5.71 (3.79–6.93), 3.35 (2.31–5.62) and 2.71 (1.38–4.08), respectively. Predose IMPDH capacity in nonstimulated PBMC increased with time, reaching pre‐TX values at 1 year. In stimulated PBMC, both purines were reduced before (median 39% reduction across days after TX) and after (69% reduction) dose compared to before TX. No alteration in the purine levels was observed in nonstimulated PBMC. Patients needing dose reductions during the first year had lower pre‐dose IMPDH capacity in nonstimulated PBMC (1.87 vs 3.00 pmol 10−6 cells min−1, P = .049) at 6–9 days.ConclusionThe inhibitory effect of MPA was stronger in stimulated PBMC. Nonstimulated PBMC became less sensitive to MPA during the first year after TX. Early IMPDH capacity appeared to be predictive of dose reductions.

Cryo-EM structures demonstrate human IMPDH2 filament assembly tunes allosteric regulation.

Inosine monophosphate dehydrogenase (IMPDH) mediates the first committed step in guanine nucleotide biosynthesis and plays important roles in cellular proliferation and the immune response. IMPDH reversibly polymerizes in cells and tissues in response to changes in metabolic demand. Self-assembly of metabolic enzymes is increasingly recognized as a general mechanism for regulating activity, typically by stabilizing specific conformations of an enzyme, but the regulatory role of IMPDH filaments has remained unclear. Here, we report a series of human IMPDH2 cryo-EM structures in both active and inactive conformations. The structures define the mechanism of filament assembly, and reveal how filament-dependent allosteric regulation of IMPDH2 makes the enzyme less sensitive to feedback inhibition, explaining why assembly occurs under physiological conditions that require expansion of guanine nucleotide pools. Tuning sensitivity to an allosteric inhibitor distinguishes IMPDH from other metabolic filaments, and highlights the diversity of regulatory outcomes that can emerge from self-assembly.

P584 Use of mycophenolate mofetil in inflammatory bowel disease: results from the ENEIDA registry

Abstract Background Mycophenolate mofetil (MMF) is an immunomodulatory drug that inhibits T and B cells, through a reversible inhibition of inositol monophosphate dehydrogenase. MMF is commonly used for prophylaxis of organ rejection after transplant. Studies to evaluate its use in inflammatory bowel disease (IBD) are limited especially after the appearance of biological treatments (BT). The aim of this study was to evaluate the efficacy and safety of MMF in IBD. Methods IBD patients who had received MMF were identified in the ENEIDA registry (prospective database of GETECCU). Patients with Crohn′s disease (CD) or Ulcerative colitis (UC) in whom oral MMF was prescribed for this condition were evaluated. Demographic and IBD clinical data were collected. Clinical activity was assessed through Harvey-Bradshaw Index (HBI) and partial Mayo score (pMS); C-reactive protein (CRP) and faecal calprotectin (FC) were reviewed at baseline (at MMF starting), 3 and 6 months and at the end of follow-up (at MMF stopping or the last visit while on MMF). Adverse events (AEs) during MMF treatment were documented. Statistical analyses were performed by descriptive statistics and non-parametric tests. Results Between June 1999 and November 2018 a total of 83 patients received MMF (mean age 36.4 (SD12.3) years; 52F/31M; 66 CD and 17 UC). Indication for MMF use was maintenance of remission (50%), induction of remission (44%) and post-surgical prophylaxis (6%). Mean MMF dose used was 1269.8 (SD 741) mg/day. In 61% of cases, systemic steroids were administered starting MMF and it was used concomitantly to BT in 27% of patients. In CD, statistically significant differences in HBI at 6 months (mean 5.9 (SD 4.8), p = 0.04) and at the end of follow-up (mean 5.7 (SD 5), p = 0.014) compared with baseline (mean 7.6 (SD 4.3)) were observed. In UC, statistically significant differences in pMS at 6 months (mean 2.1 (SD 2.7), p = 0.018) and at the end of follow-up (mean 1.8 (SD 2.6), p = 0.003) compared with baseline (mean 4.8 (SD 2.5)) were determined. No significant differences in CRP or CF values during follow-up were observed. Concomitant use of BT was significantly associated with clinical response (OR 1.36 95% CI 1.08–1.73). MMF was maintained for a median time of 28.9 months (IQR 20.4–37.5) and was stopped in 84% of patients due to lack of response (50%), loss of response (17%) and remission (15%). Overall, 23% of patients presented MMF-related AEs (60% abdominal pain). MMF was withdrawn in 11% of patients due to AEs. Conclusion MMFshows a clinical benefit in the long term in both CD and UC patients with a favourable safety profile. MMF could be a treatment option alone or in combination with biologics in patients with IBD in clinical practice.

Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients

Due to the development of immunosuppressants, the focus in transplanted patients has shifted from short-term to long-term survival as well as a better adjustment of these drugs in order to prevent over- and under-immunosuppression. Mycophenolic acid (MPA) is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) and approved for prophylaxis of acute rejection after kidney, heart, and liver transplantation, where it has become a part of the standard therapy. Targeting inosine monophosphate IMPDH activity as a surrogate pharmacodynamic marker of MPA-induced immunosuppression may allow a more accurate assessment of efficacy and aid in limiting toxicity in liver transplanted patients. Assess IMPDH-inhibition in liver transplant recipients and its impact on biliary/infectious complications, acute cellular rejection (ACR) and liver dependent survival. This observational cohort study comprises 117 liver transplanted patients that were treated with mycophenolate mofetil (MMF) for at least 3 months. Blood samples (BS) were collected and MPA serum level and IMPDH activity were measured before (t(0)), 30minutes (t(30)) and 2h after (t(120)) MMF morning dose administration. Regarding MPA, we assessed the area under the curve (AUC). Patients were prospectively followed up for one year and assessed for infectious and biliary complications, episodes of ACR and liver dependent survival. The MPA levels showed a broad interindividual variability at t(0) (2.0±1.8ng/ml), t(30) (12.7±9.0ng/ml) and t(120) (7.5±4.3ng/ml). Corresponding IMPDH activity was at t(o) (23.2±9.5 nmol/h/mg), at t(30) (16.3±8.8 nmol/h/mg) and t(120) (18.2±8.7 nmol/h/mg). With regard to MPA level we found no correlation with infectious or biliary complications within the follow-up period. Patients with baseline IMPDH(a) below the median had significant more viral infections (6 (10.2%) vs. 17 (29.3%); P=0.009) with especially more cytomegalovirus (CMV) infections (1 (3.4%) vs. 6 (21.4%); P=0.03)). Furthermore, patients with baseline IMPDH(a) above the median developed more often non-anastomotic biliary strictures (8 (13.6%) vs. 1 (1.7%), P=0.03). We found the group reaching the combined clinical endpoint of death and re-transplantation showing significantly lower MPA baseline values (t(0) 0.9±0.7 vs. 2.1±1.8μg/ml Mann-Whitney-U: P=0.02). We calculated a simplified MPA(AUC) with the MPA level at baseline, 30 and 120minutes after MPA administration. Whereas we found no differences with regard to baseline characteristics at entry into the study patients with MPA (AUC) below the median experienced significantly more often the combined clinical endpoint (12.1% (7/58) vs. 0.0% (0/57); P=0.002) and had a reduced actuarial re-transplantation-free survival (1.0 year vs. 0.58 years; Log-rank: P=0.007) during the prospective one-year follow-up period. In univariate and multivariate analysis including gender, age, BMI, ACR, MPA (AUC) and IMPDH(a) only BMI, MPA (AUC) and IMPDH(a) were independently associated with reduced actuarial re-transplantation-free survival. MPA-levels and IMPDH-activity in liver transplanted patients allows individual risk assessment. Patients with higher IMPDH inhibition acquire more often viral infections. Insufficient IMPDH inhibition is associated with development of non-anastomotic bile duct strictures and reduced re-transplantation-free survival.

Optimization Strategies for Purification of Mycophenolic Acid Produced by Penicillium brevicompactum.

The microbial fermentation of Penicillium brevicompactum produces secondary metabolite mycophenolic acid (MPA), which exhibits antifungal, antiviral, antibacterial, and antitumor activity. It is also a potent, selective, non-competitive, and reversible inhibitor of the human inosine monophosphate dehydrogenase (IMPDH). This study is an attempt to optimize the MPA production through a fermentation process using Penicillium brevicompactum and its further purification process optimization. In the batch fermentation process, the maximum concentration of MPA (1.84g/L) was attained in a 3.7L stirred tank reactor. Response surface methodology (RSM) using central composite design (CCD) was employed as a statistical tool to investigate the effect of pH, the volume of eluent and flow rate of the mobile phase on MPA purification process. Under optimum conditions, the experimental yield was observed to be 84.12%, which matched well with the predictive yield of 84.42%. High-performance liquid chromatography (HPLC) and Fourier-transform infrared spectroscopy (FTIR) analysis of the fermented product was carried out to confirm the presence of mycophenolic acid. The MPA purification was done by using column chromatography technique. The purification of broth involved mycophenolic acid extraction by selecting different solvents on the basis of polarity and the extraction efficiency of solvent. Various solid support materials were used for MPA purification in column chromatography. The MPA recovery through alumina column was observed to be 84.12% under the optimum conditions, which was maximum elution as compared with other support materials. The optimized purification process yielded pure MPA crystals.

Analiza DNK oštećenja izazvanog tiazofurinom u humanim ćelijama pune krvi primenom in vitro komet testa

Objective. Inosine 5'-monophosphate dehydrogenase (IMPDH) activity in cancer cells is increased. Tiazofurin selectively inhibits the activity of IMPDH, and it has been granted for the treatment of different cancers and new viral diseases. Its widespread use was limited because exposure to tiazofurin under certain circumstances was found to have a higher frequency of severe non-hematologic toxicity. Therefore, the objective of this study was to examine genotoxic action and inducement of DNA damage of tiazofurin using the comet assay. Methods. The ability of tiazofurin to induce DNA damage was evaluated using single-cell gel electrophoresis (SCGE) technique/comet assay. Human whole blood cells were exposed to three final concentrations of tiazofurin (1µM/mL, 2 µM/mL, and 5 µM/mL) for 30 min in vitro. Results. Our results indicate that tiazofurin produced a significant level of DNA damage on whole blood cells after 30 min of exposure vs. control. All tested concentrations were significantly comet-forming, in a concentration-dependent manner. Conclusion. Our investigation on the tiazofurin-treated cells and their relationship to the formation of DNA damage demonstrated that the genotoxic effect was induced after exposure to tiazofurin under described conditions.

The ability of Mycophenolate Mofetil to induce chromosome aberration and DNA damage in Mus Musculus Mice

Mycophenolate mofetil (MMF, cellcept) is widely used in maintenance immunosuppressive therapy for prevention refractory rejection in sold organ transplant recipients. MMF is orally used, and rapidly metabolized to its active constituent Mycophenolic acid. MPA is an inhibitor of Inosine Monophosphate Dehydrogenase ΙΙ (IMPDH ΙΙ) in lymphocyte causing in reduction in intracellular guanine nucleotide pools and leads to inhibition of lymphocyte proliferation. MMF is considered to be an effective and safe immunosuppressive agent compared with other medicins. MMF lacks the nephrotoxicity. But it has important side effects, gastrointestinal and heamatological adverse effects are the most common. The results of this study show the high significant in total chromosome aberrations in treatment groups compared with control. Also there is high significant differences in total damage DNA in treatment groups than control. In conclusion, MMF may has a high genotoxicity by induced chromosome aberration and DNA damage.

Analysis of polysaccharide hydrolases secreted by Aspergillus flavipes FP-500 on corn cobs and wheat bran as complex carbon sources

Aspergillus flavipes FP-500 is a Mexican native strain that has been reported as a good producer of xylanases and pectinases; therefore, it promises a strong impact on biotechnology. To provide an overview of protein secretion by A. flavipes, we carried out a comparative proteome analysis of extracellular proteins in liquid cultures with two heterogeneous agro-industrial residues; corn cob (CC) and wheat bran (WB), as carbon sources. Extracellular proteins obtained from both cultures were identified using MS/MS spectrometry. We identified 134 proteins, which were classified into four groups: glycosyl hydrolases (GH), esterases/proteases, miscellaneous proteins, and unidentified proteins. Around 50% of the total proteins identified were GH such as xylanases, β-xylosidases, β-galactosidases, cellulolytic enzymes like β-glucosidase, endoglucanases, and cellobiohydrolases. From this family, a core of 22 (16%) of the proteins identified were found in both substrates, CC and WB, whereas 30% and 54% were unique for CC and WB, respectively. In the esterases/proteases group, proteases, lipases and esterases like feruloylesterases, and acetyl-xylanesterase were identified. Proteins with diverse functions such as monophosphate dehydrogenase or N-acetylglucosaminidase were present. Here, we present strong evidences indicating that the composition and heterogeneity of the used carbon source determine the specific set of protein secreted by the fungus.

Abstract B135: Discovery of BGS2019, a highly potent and selective covalent inhibitor of GMPS using IMTACTM platform

Abstract Guanosine monophosphate synthetase (GMPS) is a glutamine amidotransferase catalyzing amination of xanthosine monophosphate (XMP) to form GMP. It is a enzyme involved in de novo purine biosynthesis. T and B lymphocytes, and certain cancer cells, such as malignant melanoma are dependent on de novo synthesis of purine nucleotide for activation and proliferation. GMPS is an attractive target for drug discovery and development to treat autoimmune diseases, organ transplant rejection, and cancers. Mycophenolate acid (MPA), an active ingredient of Mycophenolate Mofetil (Cellcept) has been used for organ transplantation and autoimmune diseases. MPA is a natural product which inhibits inosine monophosphate dehydrogenase (IMPDH), an enzyme upstream of GMPS. Using IMTACTM (Isobaric Mass Tagged Affinity Characterization) platform to perform global proteome ligand screen in live cancer cells, a highly potent and highly selective irreversible inhibitor against GMPS was discovered. Here we report the identification, in vitro characterization and in vivo evaluation of this molecule BGS2019. Target engagement of BGS2019 was determined by In-Gel-Fluorescent assay in cell with an EC50 of 15.6nM. It inhibited GMPS enzymatic activity with an IC50 of 6nM. The mechanism of BGS2019 in cell-based assays was tested, which showed the accumulation of IMP (the substrate for GMPS) and the reduction of GMP, GDP and GTP, upon BGS2019 treatment. BGS2019 was evaluated for its cell growth inhibition activity in several cancer cell lines and it suppressed cancer cell growth with IC50 values from 30nM to 100nM. In vivo administration of BGS2019 was well tolerated in mice and exhibited tumor suppression in animal model. Citation Format: Hang Chen, Heather Ha, Robert Stanley, Cindy Huang, Qian Cai, Irene Yuan, Ping Cao. Discovery of BGS2019, a highly potent and selective covalent inhibitor of GMPS using IMTACTM platform [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B135. doi:10.1158/1535-7163.TARG-19-B135

Abstract B134: IMPDH1 and IMPDH2 as therapeutic targets in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) tumors are dependent on purine biosynthesis for proliferation and survival and relapsed ALL is particularly associated with transcriptional up-regulation of this pathway. Increased dependency on purine biosynthesis highlights IMPDH (inosine monophosphate dehydrogenase), the rate-limiting enzyme in guanosine production, as a potential therapeutic target for the treatment of ALL. To assess the role of IMPDH1 and IMPDH2 in ALL development and progression, we generated NOTCH1-induced leukemias from Impdh1 knockout and Impdh2 conditional knockout mice. In addition, we have completed whole exome sequencing and RNAseq profiling of 12 pairs of ALL xenografts corresponding to matched diagnostic and relapsed ALL samples and have performed dose response assays with the IMPDH inhibitor mizoribine. Additionally and towards the identification of synthetic lethal genetic vulnerabilities with IMPDH inhibition in ALL we have performed a genome wide CRISPR screen in REH ALL cells with the IMPDH inhibitor mizoribine. Overall, these studies highlight the therapeutic potential for IMPDH inhibitors for the treatment of ALL. Citation Format: Chelsea Dieck, Koichi Oshima, Alberto Ambesi-Impiombato, Adolfo Ferrando. IMPDH1 and IMPDH2 as therapeutic targets in acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B134. doi:10.1158/1535-7163.TARG-19-B134

A structural determinant of mycophenolic acid resistance in eukaryotic inosine 5'-monophosphate dehydrogenases.

Mycophenolic acid (MPA) is a potent natural product inhibitor of fungal and other eukaryotic inosine 5'-monophosphate dehydrogenases (IMPDHs) originally isolated from spoiled corn silage. MPA is produced by the filamentous fungi Penicillium brevicompactum, which contains two IMPDHs, PbIMPDHA and PbIMPDHB, both of which are MPA-resistant. The MPA binding sites of these enzymes are identical to MPA-sensitive IMPDHs, so the structural determinants of resistance are unknown. Here we show that a single residue, Ser267, accounts for the MPA resistance of PbIMPDHA. Substitution of Ser267 with Ala, the residue most commonly found in this position in eukaryotic IMPDHs, makes PbIMPDHA sensitive to MPA. Conversely, Aspergillus nidulans IMPDH becomes MPA-resistant when the analogous Ala residue is substituted with Ser. These substitutions have little effect on the catalytic cycles of either enzyme, suggesting the fitness costs are negligible despite the strong conservation of Ala at this position. Intriguingly, while only 1% of fungal IMPDHs contain Ser or Thr at position 267, these residues are found in the IMPDHs from several Aspergillus species that grow at the low temperatures also favored by Penicillium. Perhaps Ser/Thr267 is an evolutionary signature of MPA exposure.

Covalent inactivation of Mycobacterium thermoresistibile inosine-5′-monophosphate dehydrogenase (IMPDH)

Inosine-5′-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in nucleotide biosynthesis. Because of its critical role in purine biosynthesis, IMPDH is a drug design target for immunosuppressive, anticancer, antiviral and antimicrobial chemotherapy. In this study, we use mass spectrometry and X-ray crystallography to show that the inhibitor 6-Cl-purine ribotide forms a covalent adduct with the Cys-341 residue of Mycobacterium thermoresistibile IMPDH.

Genetic ablation of purine salvage in Cryptosporidium parvum reveals nucleotide uptake from the host cell

The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrheal disease and an important contributor to early-childhood mortality. Waterborne outbreaks occur frequently, even in countries with advanced water treatment capabilities, and there is currently no fully effective treatment. Nucleotide pathways are attractive targets for antimicrobial development, and several laboratories are designing inhibitors of these enzymes as potential treatment for Cryptosporidium infections. Here we take advantage of newly available molecular genetics for Cryptosporidium parvum to investigate nucleotide biosynthesis by directed gene ablation. Surprisingly, we found that the parasite tolerates the loss of classical targets including dihydrofolate reductase-thymidylate synthase (DHFR-TS) and inosine monophosphate dehydrogenase (IMPDH). We show that thymidine kinase provides a route to thymidine monophosphate in the absence of DHFR-TS. In contrast, only a single pathway has been identified for C. parvum purine nucleotide salvage. Nonetheless, multiple enzymes in the purine pathway, as well as the adenosine transporter, can be ablated. The resulting mutants are viable under normal conditions but are hypersensitive to inhibition of purine nucleotide synthesis in their host cell. Cryptosporidium might use as-yet undiscovered purine transporters and salvage enzymes; however, genetic and pharmacological experiments led us to conclude that Cryptosporidium imports purine nucleotides from the host cell. The potential for ATP uptake from the host has significant impact on our understanding of parasite energy metabolism given that Cryptosporidium lacks oxidative phosphorylation and glycolytic enzymes are not constitutively expressed throughout the parasite life cycle.

Purine-Metabolising Enzymes and Apoptosis in Cancer.

The enzymes of both de novo and salvage pathways for purine nucleotide synthesis are regulated to meet the demand of nucleic acid precursors during proliferation. Among them, the salvage pathway enzymes seem to play the key role in replenishing the purine pool in dividing and tumour cells that require a greater amount of nucleotides. An imbalance in the purine pools is fundamental not only for preventing cell proliferation, but also, in many cases, to promote apoptosis. It is known that tumour cells harbour several mutations that might lead to defective apoptosis-inducing pathways, and this is probably at the basis of the initial expansion of the population of neoplastic cells. Therefore, knowledge of the molecular mechanisms that lead to apoptosis of tumoural cells is key to predicting the possible success of a drug treatment and planning more effective and focused therapies. In this review, we describe how the modulation of enzymes involved in purine metabolism in tumour cells may affect the apoptotic programme. The enzymes discussed are: ectosolic and cytosolic 5′-nucleotidases, purine nucleoside phosphorylase, adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, and inosine-5′-monophosphate dehydrogenase, as well as recently described enzymes particularly expressed in tumour cells, such as deoxynucleoside triphosphate triphosphohydrolase and 7,8-dihydro-8-oxoguanine triphosphatase.

Anti-Tumor Potential of IMP Dehydrogenase Inhibitors: A Century-Long Story.

The purine nucleotides ATP and GTP are essential precursors to DNA and RNA synthesis and fundamental for energy metabolism. Although de novo purine nucleotide biosynthesis is increased in highly proliferating cells, such as malignant tumors, it is not clear if this is merely a secondary manifestation of increased cell proliferation. Suggestive of a direct causative effect includes evidence that, in some cancer types, the rate-limiting enzyme in de novo GTP biosynthesis, inosine monophosphate dehydrogenase (IMPDH), is upregulated and that the IMPDH inhibitor, mycophenolic acid (MPA), possesses anti-tumor activity. However, historically, enthusiasm for employing IMPDH inhibitors in cancer treatment has been mitigated by their adverse effects at high treatment doses and variable response. Recent advances in our understanding of the mechanistic role of IMPDH in tumorigenesis and cancer progression, as well as the development of IMPDH inhibitors with selective actions on GTP synthesis, have prompted a reappraisal of targeting this enzyme for anti-cancer treatment. In this review, we summarize the history of IMPDH inhibitors, the development of new inhibitors as anti-cancer drugs, and future directions and strategies to overcome existing challenges.