Guanine Nucleotide Biosynthesis Research Articles

Combining multi-omics analysis to identify host-targeted targets for the control of Brucella infection.

Human infections caused by Brucella (called brucellosis) are among the most common zoonoses worldwide with an estimated 500,000 cases each year. Since chronic Brucella infections are extremely difficult to treat, there is an urgent need for more effective therapeutics. As a facultative intracellular bacterium, Brucella is strictly parasitic in the host cell. Here, we performed proteomic and transcriptomic and metabolomic analyses on Brucella infected patients, mice and cells that provided an extensive "map" of physiological changes in brucellosis patients and characterized the metabolic pathways essential to the response to infection, as well as the associated cellular response and molecular mechanisms. This is the first report utilizing multi-omics analysis to investigate the global response of proteins and metabolites associated with Brucella infection, and the data can provide a comprehensive insight to understand the mechanism of Brucella infection. We demonstrated that Brucella increased nucleotide synthesis in the host, consistent with increased biomass requirement. We also identified IMPDH2, a key regulatory complex that controls nucleotide synthesis during Brucella infection. Pharmacological targeting of IMPDH2, the rate-limiting enzyme in guanine nucleotide biosynthesis, efficiently inhibits B. abortus growth both in vitro and in vivo. Through screening a library of natural products, we identified oxymatrine, an alkaloid obtained primarily from Sophora roots, is a novel and selective IMPDH2 inhibitor. In further in vitro bacterial inhibition assays, oxymatrine effectively inhibited the growth of B. abortus, which was impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. This moderately potent, structurally novel compound may provide clues for further design and development of efficient IMPDH2 inhibitors and also demonstrates the potential of natural compounds from plants against Brucella.

IMPDH2: a new gene associated with dominant juvenile-onset dystonia-tremor disorder

The aetiology of dystonia disorders is complex, and next-generation sequencing has become a useful tool in elucidating the variable genetic background of these diseases. Here we report a deleterious heterozygous truncating variant in the inosine monophosphate dehydrogenase gene (IMPDH2) by whole-exome sequencing, co-segregating with a dominantly inherited dystonia-tremor disease in a large Finnish family. We show that the defect results in degradation of the gene product, causing IMPDH2 deficiency in patient cells. IMPDH2 is the first and rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides, a dopamine synthetic pathway previously linked to childhood or adolescence-onset dystonia disorders. We report IMPDH2 as a new gene to the dystonia disease entity. The evidence underlines the important link between guanine metabolism, dopamine biosynthesis and dystonia.

Coordinated Formation of IMPDH2 Cytoophidium in Mouse Oocytes and Granulosa Cells.

Inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme catalyzing de novo biosynthesis of guanine nucleotides, aggregates under certain circumstances into a type of non-membranous filamentous macrostructure termed “cytoophidium” or “rod and ring” in several types of cells. However, the biological significance and underlying mechanism of IMPDH assembling into cytoophidium remain elusive. In mouse ovaries, IMPDH is reported to be crucial for the maintenance of oocyte–follicle developmental synchrony by providing GTP substrate for granulosa cell natriuretic peptide C/natriuretic peptide receptor 2 (NPPC/NPR2) system to produce cGMP for sustaining oocyte meiotic arrest. Oocytes and the associated somatic cells in the ovary hence render an exciting model system for exploring the functional significance of formation of IMPDH cytoophidium within the cell. We report here that IMPDH2 cytoophidium forms in vivo in the growing oocytes naturally and in vitro in the cumulus-enclosed oocytes treated with IMPDH inhibitor mycophenolic acid (MPA). Inhibition of IMPDH activity in oocytes and preimplantation embryos compromises oocyte meiotic and developmental competences and the development of embryos beyond the 4-cell stage, respectively. IMPDH cytoopidium also forms in vivo in the granulosa cells of the preovulatory follicles after the surge of luteinizing hormone (LH), which coincides with the resumption of oocyte meiosis and the reduction of IMPDH2 protein expression. In cultured COCs, MPA-treatment causes the simultaneous formation of IMPDH cytoopidium in cumulus cells and the resumption of meiosis in oocytes, which is mediated by the MTOR pathway and is prevented by guanosine supplementation. Therefore, our results indicate that cytoophidia do form in the oocytes and granulosa cells at particular stages of development, which may contribute to the oocyte acquisition of meiotic and developmental competences and the induction of meiosis re-initiation by the LH surge, respectively.

IMPDH forms the cytoophidium in zebrafish

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.

Mechanism of Inhibitor Selectivity Revealed by Mutagenesis and pre‐Steady‐State Studies

Inosine 5’-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP, with concomitant reduction of NAD+ to NADH. This is the pivotal step for de novo guanine nucleotide biosynthesis. The structural and functional differences between mammalian and bacterial IMPDHs make this a promising antibiotic target. Selective and potent inhibitors of bacterial IMPDHs with different scaffolds, like A110 and C91, bind in the cofactor site. Despite the high conservation of this site, some inhibitors display a surprising preference for certain bacterial IMPDHs. The structural basis for this selectivity is unclear even though 16 crystal structures of IMPDH-inhibitor complexes have been solved. IMPDH contains an active site flap that is highly dynamic and disordered. Here, we hypothesize that transient interactions with the flap may account for varied inhibitor potencies. In this work, we report a mutagenesis study and pre-steady-state stopped-flow kinetic investigation of Bacillus anthracis IMPDH (BaIMPDH) aimed to determine the role of the flap in inhibitor selectivity. We introduced a L413A flap mutation in BaIMPDH and measured how inhibitor potencies changed. The results show some inhibitors (eg. A110) have the same potency towards both wild type and mutant enzymes, but other inhibitors (eg. C91) were found to have different potencies. To determine if the mutation changes the relative amount of inhibitor binding intermediates in catalytic cycle, we performed stopped-flow kinetic experiments to defined eleven microscopic rate constants and two equilibrium constants that characterize both the catalytic cycle and details of the inhibition mechanism. Together with steady-state initial rate studies, the results obtained for wild type BaIMPDH show that both A110 and C91 binds with high affinity predominantly to the covalent intermediate (E-XMP*) on the reaction pathway (Kd ≍ 50 nM for A110 and Kd ≍ 40 nM for C91). Only a weak binding interaction (Kd ≍ 1 μM) is observed between A110 and E•IMP, while C91 has a bit stronger interaction with E•IMP (Kd ≍ 0.5 μM). Further pre-steady-state stopped-flow kinetic investigation for BaIMPDH/L413A are in progress to determine the effects of this particular mutation on the microscopic rate constants that characterize the catalytic cycle and on the details of the inhibition mechanism.

IMPDH2 promotes cell proliferation and epithelial-mesenchymal transition of non-small cell lung cancer by activating the Wnt/β-catenin signaling pathway.

Inosine 5'-monophosphate dehydrogenase type II (IMPDH2) is an important enzyme involved in the biosynthesis of guanine nucleotides. Therefore, the present study aimed to investigate the potential and molecular mechanism of IMPDH2 in non-small cell lung cancer (NSCLC). Reverse transcription-quantitative PCR and immunohistochemistry were used to detect IMPDH2 expression levels in NSCLC tissues and cells. A Cell Counting Kit-8 assay, colony formation assay, flow cytometry, wound healing, Transwell assay, western blotting and immunofluorescence analyses were utilized to identify the effects of upregulated IMPDH2 levels on NSCLC cells. The expression levels of IMPDH2 have been discovered to be upregulated in several types of human cancer; however, the biological and clinical value of IMPDH2 in NSCLC remains unclear. The results of the present study revealed that the expression levels of IMPDH2 were significantly upregulated in NSCLC tissues. Furthermore, the genetic knockdown of IMPDH2 significantly hindered the proliferation, apoptosis, invasion, migration and epithelial-mesenchymal transition of NSCLC cells, whereas the overexpression of IMPDH2 achieved the opposite results. In addition, the results of the present study demonstrated that the inhibition of IMPDH2 inhibited the Wnt/β-catenin signaling pathway by decreasing the expression levels of Wnt3a and β-catenin, while increasing the expression levels of phosphorylated glycogen synthase kinase-3β in NSCLC cells. These findings of the present study indicated that IMPDH2 may promote NSCLC progression by activating the Wnt/β-catenin signaling pathway, which suggested that IMPDH2 may be a novel therapeutic target for patients with NSCLC.

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.

IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma.

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer, glioblastoma (GBM). This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein, Nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in GBM reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of GBM cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for aberrant nucleolar function and increased anabolic processes in GBM, which constitutes a primary event in gliomagenesis.

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.

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.

Investigation of IMPDH2 overexpression in renal cell carcinoma (RCC) & 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) & 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 & 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 & cell maintenance in Gliobastoma Multiforme cell lines. Hypothesis: We are investigating the feasibility of using IMPDH2 expression in RCC as a diagnostic & 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 & interface. Tissue microarray (TMA) was constructed using the fully automated TMA Master. TMA sectioned and stained with H &E & 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) & staining intensity (SI) (Negative-0, Weak-1, Moderate-2, and Strong-3). The scoring pathologist was blinded to the clinical data & 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 & pathological variables including TNM stage, histologic grade (Fuhrman Grade) & oncologic outcomes including overall survival (OS) & 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 & may give insight to future personalized & targeted treatment strategies (IMPDH2 inhibitors like mycophenolate) for RCC.

Design, synthesis, and biological evaluation of Helicobacter pylori inosine 5'-monophosphate dehydrogenase (HpIMPDH) inhibitors.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes a crucial step in the biosynthesis of guanine nucleotides. Being a validated target for immunosuppressive, antiviral, and anticancer drug development, lately it has been exploited as a promising target for antimicrobial therapy. Extending our previous work on Mycobacterium tuberculosis IMPDH, GuaB2, inhibitor development, we screened a set of 23 new chemical entities (NCEs) with substituted flavone (Series 1) and 1,2,3-triazole (Series 2) core structures for their in vitro Helicobacter pylori IMPDH (HpIMPDH) and human IMPDH2 (hIMPDH2) inhibitory activities. All the NCEs possessed acceptable molecular, physicochemical, and toxicity property profiles. The ranges for HpIMPDH and hIMPDH2 inhibition were 9-99.9% and 16-57%, respectively, at 10 μM concentration. The most potent HpIMPDH inhibitor, 25c, exhibited IC50 value of 1.27 μM with no hIMPDH2 inhibitory activity. The moderately potent, structurally novel hit molecule, 25c, may serve as a lead for further design and development of highly potent HpIMPDH inhibitors.

Anfractuous assemblies of IMP dehydrogenase and CTP synthase: new twists on regulation?

CTP synthase (CTPS) and IMP dehydrogenase (IMPDH) catalyse the rate-limiting steps of de novo CTP and guanosine nucleotide biosynthesis, respectively, and form filament assemblies in response to inhibitors. A recent study explores the morphology and dynamics of these assemblies using fluorescence and super-resolution confocal microscopy with cell lines expressing CTPS1 and IMPDH2 fusion proteins. The formation and dismantling of mixed assemblies depends on nucleotide levels, suggesting a co-regulation function.

Discovery of novel human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2) inhibitors as potential anticancer agents

The enzyme inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotides, and thus regulates the guanine nucleotide pool required for cell proliferation. Of the two isoforms, human IMPDH type 2 (hIMPDH2) is a validated molecular target for potential immunosuppressive, antiviral and anticancer chemotherapy. In search of newer hIMPDH2 inhibitors as potential anticancer agents, three novel series (A: 5-aminoisobenzofuran-1(3H)-one, B: 3,4-dimethoxyaniline and C: benzo[d]-[1,3]dioxol-5-ylmethanamine) were synthesized and evaluated for in vitro and cell-based activities. A total of 37 molecules (29–65) were screened for their in vitro hIMPDH2 inhibition, with particular emphasis on establishing their structure–activity relationship (SAR) trends. Eight compounds (hits, 30, 31, 33–35, 37, 41 and 43) demonstrated significant enzyme inhibition (>70% @ 10 μM); especially the A series molecules were more potent than B series (<70% inhibition @ 10 μM), while C series members were found to be inactive. The hIMPDH2 IC50 values for the hits ranged from 0.36 to 7.38 μM. The hits displaying >80% hIMPDH2 inhibition (30, 33, 35, 41 and 43) were further assessed for their cytotoxic activity against cancer cell lines such as MDA-MB-231 (breast adenocarcinoma), DU145 (prostate carcinoma), U87 MG (glioblastoma astrocytoma) and a normal cell line, NIH-3T3 (mouse embryonic fibroblast) using MTT assay. Most of the compounds exhibited higher cellular potency against cancer cell lines and notably lower toxicity towards NIH-3T3 cells compared to mycophenolic acid (MPA), a prototypical hIMPDH2 inhibitor. Two of the series A hits (30 and 35) were evaluated in human peripheral blood mononuclear cells (hPBMC) assay and found to be better tolerated than MPA. The calculated/predicted molecular and physicochemical properties were satisfactory with reference to drug-likeness. The molecular docking studies clearly demonstrated crucial interactions of the hits with the cofactor-binding site of hIMPDH2, further providing critical information for refining the design strategy. The present study reports the design and discovery of structurally novel hIMPDH2 inhibitors as potential anticancer agents and provides a guide for further research on the development of safe and effective anticancer agents, especially against glioblastoma.

T cell activation triggers reversible inosine-5'-monophosphate dehydrogenase assembly.

T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca2+-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic inosine-5'-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca2+ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation.

Expanding Benzoxazole-Based Inosine 5'-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure-Activity As Potential Antituberculosis Agents.

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. Mycobacterium tuberculosis ( Mtb) inosine 5'-monophosphate dehydrogenase 2 ( MtbIMPDH2) is a promising yet controversial potential target. The inhibition of MtbIMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure-activity relationship (SAR) for the benzoxazole series of MtbIMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 μM can be achieved. The antibacterial activity of the most promising compound, 17b (Q151), is derived from the inhibition of MtbIMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of 17b, alleviating the concern that guanine salvage can protect Mtb in vivo. These findings suggest that MtbIMPDH2 is a vulnerable target for tuberculosis.

Combined Human Genome-wide RNAi and Metabolite Analyses Identify IMPDH as a Host-Directed Target against Chlamydia Infection

Chlamydia trachomatis (Ctr) accounts for >130million human infections annually. Since chronic Ctr infections are extremely difficult to treat, there is an urgent need for more effective therapeutics. As an obligate intracellular bacterium, Ctr strictly depends on the functional contribution of the host cell. Here, we combined a human genome-wide RNA interference screen with metabolic profiling to obtain detailed understanding of changes in the infected cell and identify druggable pathways essential for Ctr growth. We demonstrate that Ctr shifts the host metabolism toward aerobic glycolysis, consistent with increased biomass requirement. We identify key regulator complexes of glucose and nucleotide metabolism that govern Ctr infection processes. Pharmacological targeting of inosine-5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in guanine nucleotide biosynthesis, efficiently inhibits Ctr growth both invitro and invivo. These results highlight the potency of genome-scale functional screening for the discovery of drug targets against bacterial infections.

Hit discovery of Mycobacterium tuberculosis inosine 5′-monophosphate dehydrogenase, GuaB2, inhibitors

Tuberculosis remains a global concern. There is an urgent need of newer antitubercular drugs due to the development of resistant forms of Mycobacterium tuberculosis (Mtb). Inosine 5′-monophosphate dehydrogenase (IMPDH), guaB2, of Mtb, required for guanine nucleotide biosynthesis, is an attractive target for drug development. In this study, we screened a focused library of 73 drug-like molecules with desirable calculated/predicted physicochemical properties, for growth inhibitory activity against drug-sensitive MtbH37Rv. The eight hits and mycophenolic acid, a prototype IMPDH inhibitor, were further evaluated for activity on purified Mtb-GuaB2 enzyme, target selectivity using a conditional knockdown mutant of guaB2 in Mtb, followed by cross-resistance to IMPDH inhibitor-resistant SRMV2.6 strain of Mtb, and activity on human IMPDH2 isoform. One of the hits, 13, a 5-amidophthalide derivative, has shown growth inhibitory potential and target specificity against the Mtb-GuaB2 enzyme. The hit, 13, is a promising molecule with potential for further development as an antitubercular agent.

Inosine 5'-Monophosphate Dehydrogenase (IMPDH) as a Potential Target for the Development of a New Generation of Antiprotozoan Agents.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is a metabolic enzyme that catalyzes the critical step in guanine nucleotide biosynthesis, and thus is at the center of cell growth and proliferation. However, although this enzyme has been exploited as potential target for the development of immunosuppressive, anticancer, and antiviral agents, the functional importance of IMPDH as a promising antiprotozoan drug target is still in its infancy mainly because of the availability of alternative nucleotides metabolic pathways in many of these parasites. This situation suggests that the inhibition of IMPDH might have little to no effect on the survival of protozoan parasites. As a result, no IMPDH inhibitor is currently commercially available or has advanced to clinical trials as a potential antiprotozoan drug. Nevertheless, recent advances toward the development of selective inhibitors of the IMPDH enzyme from Crystosporidium parvum as potential drug candidates against cryptosporidiosis should revive further investigations of this drug target in other protozoa parasites. The current review examines the chemical structures and biological activities of reported protozoan's IMPDH inhibitors. SciFinder was used to broadly pinpoint reports published on the topic in the chemical literature, with no specific time frame. Opportunities and challenges towards the development of inhibitors of IMPDH enzymes from protozoa parasites as potential chemotherapies toward the respective diseases they cause are also discussed.

Preclinical activity of FF-10501-01, a novel inosine-5′-monophosphate dehydrogenase inhibitor, in acute myeloid leukemia

BackgroundFF-10501-01 is a selective inosine monophosphate dehydrogenase (IMPDH) inhibitor that has shown activity in cancer cell lines. We studied whether FF-10501-01 is effective in targeting a variety of hypomethylating agent (HMA)-sensitive and −resistant acute myelogenous leukemia (AML) cell lines. MethodsWe treated multiple cell lines (including HMA-resistant cells) with FF-10501-01 and analyzed proliferation, apoptosis, and cell cycle status. We also assessed HMA-FF-10501-01 combinations and the ability of extracellular guanosine to rescue cell proliferation in FF-10501-01-treated cells. We performed high-performance liquid chromatography (HPLC) to study guanine nucleotide levels in treated and untreated cells. Finally, we studied the effects of FF-10501-01 in fresh peripheral blood cells taken from AML patients. ResultsFF-10501-01 showed a strong dose-dependent effect on proliferation and induced apoptosis at approximately 30μM. The effects of FF-10501-01 treatment on cell cycle status were variable, with no statistically significant trends. Guanosine rescued proliferation in FF-10501-01-treated cells, and HPLC results showed significant decreases in phosphorylated guanosine levels in MOLM13 cells. FF-10501-01 effectively reduced proliferation at concentrations of 300μM and above in 3 primary AML samples. ConclusionsFF-10501-01 effectively induces AML cell death and reduces AML peripheral blood cell proliferation by targeting guanine nucleotide biosynthesis regardless of HMA resistance status.