Isoforms Of Inosine Monophosphate Dehydrogenase Research Articles

IMPDH2 forms spots at branching sites and distal ends of astrocyte stem processes.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in the de novo GTP biosynthesis pathway. Recent studies suggest that IMPDH2, an isoform of IMPDH, can localize to specific subcellular compartments under certain conditions and regulate site-specific GTP availability and small GTPase activity in invasive cancer cells. However, it is unclear whether IMPDH2 plays a site-specific regulatory role in subcellular functions in healthy cells. In this study, we focused on brain cells and examined the localization pattern of IMPDH2. We discovered that IMPDH2 forms localized spots in the astrocytes of the adult mouse hippocampus. Further analysis of spot distribution in primary astrocyte cultures revealed that IMPDH2 spots are predominantly localized on branching sites and distal ends of astrocyte stem processes. Our findings suggest a potential unidentified role for IMPDH2 and GTP synthesis specifically at specialized nodes of astrocyte branches.

Identification of a potent dual-function inhibitor for hIMPDH isoforms by computer-aided drug discovery approaches.

Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme in de novo biosynthesis of purine nucleotides. Due to this important role, it is a great target to drug discovery for a wide range of activities, especially immunosuppressant in heart and kidney transplantation. Both human IMPDH isoforms are expressed in stimulated lymphocytes. In addition to the side effects of existing drugs, previous studies have mainly focused on the type II isoform. In this study, virtual screening and computer-aided approaches were employed to identify potential drugs with simultaneous inhibitory effects on both human IMPDH isoforms. After Re-docking, Double-step docking, and identification of virtual hits based on the PLANTS scoring function, drug-likeness and ADME-Tox assessments of the topmost ligands were performed. Following further evaluation, the best ligand was selected and, in complex with both isoforms, simulated in monomeric and tetrameric forms using molecular dynamics to evaluate its stability and binding pattern. The results showed a potential drug candidate [(S)-N-(3-hydroxy-1-(4-hydroxyphenyl) propyl)-2-(3-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl) acetamide] with a high inhibitory effect on the two human IMPDH isoforms. This drug-like inhibitor could potentially serve as an immunosuppressant to prevent transplant rejection response by inhibiting B- and T-lymphocyte proliferation. In addition, its effect can be evaluated in various therapeutic targets in which IMPDH is known as a therapeutic target, especially in Covid-19 patients.

ANKRD9 is associated with tumor suppression as a substrate receptor subunit of ubiquitin ligase

BackgroundHuman ANKRD9 (ankyrin repeat domain 9) expression is altered in some cancers. MethodsWe tested genetic association of ANKRD9 with gastric cancer susceptibility and examined functional association of ANKRD9 with altered proliferation of MKN45 gastric cancer cells. We then identified ANKRD9-binding partners in HEK 293 embryonic kidney cells using quantitative proteomics, western blotting and complex reconstitution assays. We finally demonstrated ANKRD9's role of recognizing substrates for ubiquitination using in vitro ubiquitylation assay. ResultsANKRD9 is associated with cancer susceptibility in a comparison of single-nucleotide polymorphisms between 1092 gastric cancer patients and 1206 healthy controls. ANKRD9 depletion accelerates tumor progression by increasing cellular proliferation, piling up, and anchorage-independent growth of MKN45 cells. We discovered that ANKRD9 is a ubiquitin ligase substrate receptor subunit and has an anti-proliferative activity. ANKRD9 associates with CUL5 (not CUL2), ELOB, ELOC, and presumably RNF7 subunits, which together assemble into a cullin-RING superfamily E3 ligase complex. ANKRD9 belongs to the ASB family of proteins, which are characterized by the presence of ankyrin repeats and a SOCS box. In addition to its interactions with the other E3 ligase subunits, ANKRD9 interacts with two isoforms of inosine monophosphate dehydrogenase (IMPDH). These IMPDH isoforms are cognate substrates of the ANKRD9-containing E3 enzyme, which ubiquitinates them for proteasomal degradation. Their ubiquitination and turnover require the presence of ANKRD9. ConclusionANKRD9, a previously unidentified E3 substrate receptor subunit, functions in tumor suppression by recognizing the oncoprotein IMPDH isoforms for E3 ubiquitination and proteasomal degradation.

Highly selective inhibition of IMPDH2 provides the basis of antineuroinflammation therapy

Inosine monophosphate dehydrogenase (IMPDH) of human is an attractive target for immunosuppressive agents. Currently, small-molecule inhibitors do not show good selectivity for different IMPDH isoforms (IMPDH1 and IMPDH2), resulting in some adverse effects, which limit their use. Herein, we used a small-molecule probe specifically targeting IMPDH2 and identified Cysteine residue 140 (Cys140) as a selective druggable site. On covalently binding to Cys140, the probe exerts an allosteric regulation to block the catalytic pocket of IMPDH2 and further induces IMPDH2 inactivation, leading to an effective suppression of neuroinflammatory responses. However, the probe does not covalently bind to IMPDH1. Taken together, our study shows Cys140 as a druggable site for selectively inhibiting IMPDH2, which provides great potential for development of therapy agents for autoimmune and neuroinflammatory diseases with less unfavorable tolerability profile.

Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms

We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

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

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

The Association of the Polymorphisms of IMPDH1 Gene and Acute Graft-Versus-Host Disease After Related and Unrelated Hematopoietic Stem Cell Transplantation

AbstractAbstract 2377 Background:Mycophenolate mofetil (MMF) has been widely used in the prophylaxis and treatment of graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Inosine monophosphate dehydrogenase (IMPDH) is the target of mycophenolic acid (MPA), the active metabolite of MMF. IMPDH is the key enzyme in the de-novo synthesis of nucleotides and induces the rate-limiting step in this synthesis. There are two isoforms of IMPDH, IMPDH1 is constructively expressed in all cell types, whereas IMPDH2 is only expressed in particular cell types. The proliferation of lymphocytes depends on the synthesis of nucleotides by IMPDH, whereas other types of cells have a salvage pathway for the synthesis of nucleotides. This makes MPA a drug that specifically inhibits the proliferation of the lymphocytes. Interindividual variability in IMPDH activity has been observed in healthy volunteers as well as transplant patients. The considerable variability in baseline IMPDH activity and MPA response may logically be under the control of genetic variation within the IMPDH gene or in gene expression. Analysis of genetic variants could provide the explantation for the variability of IMPDH activity and MMF response in transplant patients. The single nucleotide polymorphism (SNP) of IMPDH1 gene has recently reported to be relevant to acute rejection in renal transplant patients receiving MMF. There are no data about the impact of the polymorphisms of IMPDH1 gene on the outcome of allo-HSCT. The objective of this study was to investigate IMPDH1 genetic variants in allo-HSCT patients and to retrospectively look for the association of these polymorphisms with aGVHD. Methods:The entire study population consisted of 240 consecutive pairs of transplant recipients and their donors who were transplanted from 2001 to 2009 in our Center, including 138 pairs of recipients and their unrelated donors and 102 pairs of recipients and their HLA-identical sibling donors. Both in the unrelated and sibling transplantation cohorts, the patients received the same GVHD prophylaxis consisting of cyclosporin A, a short-term methotrexate and MMF. Genomic DNA was extracted from peripheral blood samples obtained from recipients and donors before transplantation. Four SNPs of IVS7 +125 G>A (rs2278293), IVS8-106 G>A (rs2278294), Exon15 1572 G>A (rs2228075) and 5' flanking region C>T (rs714510) in IMPDH1 gene were analyzed by Multiplex SnaPshot. Results:(1) The IMPDH1 IVS8 -106 G/G genotypes in recipients were significantly associated with a higher incidence of aGVHD than recipients with other genotypes either in the unrelated transplantation cohort or in the sibling transplantation cohort (in the unrelated cohort: 83.3% vs 63.9%, P=0.048; in the sibling cohort: 47.6% vs17.3%, P=0.008). Furthermore, in the unrelated transplantation cohort, the IMPDH1 IVS8 -106 G/G genotypes in recipients were also associated with a higher incidence of grades II-IV aGVHD (63.3% vs 38.0%, P=0.021). However donor IMPDH1 IVS8 -106 genotype had no significant influence on the incidence of aGVHD. (2) In the combined cohort, multivariate analysis confirmed that recipients with the IVS8 -106 G/G genotype were significantly associated with higher risk of developing aGVHD (RR=2.018, 95%CI: 1.354–3.009, P=0.001). Other three variables associated with the risk of aGVHD were myeloablative conditioning (RR=3.309, 95%CI: 1.538–7.121, P=0.002), donor female and recipient male (RR=1.679, 95%CI: 1.139–2.475, P=0.009), and unrelated donor (RR=4.633, 95%CI: 2.934–7.315, P<0.001). (3) The genotypes of IVS7 +125, Exon15 1572 and 5' flanking region were not found to be associated with the risk of aGVHD. Conclusions:These results, which is the first report of IMPDH1 gene polymorphic features of Chinese population with the risk of aGVHD, suggest an interaction of the recipient IMPDH1 IVS8 -106 genotypes on the risk of aGVHD. These results are helpful for predicting allo-HSCT outcome, monitoring MMF therapy on an individual patient basis. Disclosures:No relevant conflicts of interest to declare.

Mycophenolate pharmacokinetics and pharmacodynamics in belatacept treated renal allograft recipients – a pilot study

BackgroundMycophenolic acid (MPA) is widely used as part of immunosuppressive regimens following allograft transplantation. The large pharmacokinetic (PK) and pharmacodynamic (PD) variability and narrow therapeutic range of MPA provide a potential for therapeutic drug monitoring. The objective of this pilot study was to investigate the MPA PK and PD relation in combination with belatacept (2nd generation CTLA4-Ig) or cyclosporine (CsA).MethodsSeven renal allograft recipients were randomized to either belatacept (n = 4) or cyclosporine (n = 3) based immunosuppression. Samples for MPA PK and PD evaluations were collected predose and at 1, 2 and 13 weeks posttransplant. Plasma concentrations of MPA were determined by HPLC-UV. Activity of inosine monophosphate dehydrogenase (IMPDH) and the expressions of two IMPDH isoforms were measured in CD4+ cells by HPLC-UV and real-time reverse-transcription PCR, respectively. Subsets of T cells were characterized by flow cytometry.ResultsThe MPA exposure tended to be higher among belatacept patients than in CsA patients at week 1 (P = 0.057). Further, MPA concentrations (AUC0–9 h and C0) increased with time in both groups and were higher at week 13 than at week 2 (P = 0.031, n = 6). In contrast to the postdose reductions of IMPDH activity observed early posttransplant, IMPDH activity within both treatment groups was elevated throughout the dosing interval at week 13. Transient postdose increments were also observed for IMPDH1 expression, starting at week 1. Higher MPA exposure was associated with larger elevations of IMPDH1 (r = 0.81, P = 0.023, n = 7 for MPA and IMPDH1 AUC0–9 h at week 1). The maximum IMPDH1 expression was 52 (13–177)% higher at week 13 compared to week 1 (P = 0.031, n = 6). One patient showed lower MPA exposure with time and did neither display elevations of IMPDH activity nor IMPDH1 expression. No difference was observed in T cell subsets between treatment groups.ConclusionThe significant influence of MPA on IMPDH1 expression, possibly mediated through reduced guanine nucleotide levels, could explain the elevations of IMPDH activity within dosing intervals at week 13. The present regulation of IMPDH in CD4+ cells should be considered when interpreting measurements of IMPDH inhibition.

Characterisation of inosine monophosphate dehydrogenase expression during retinal development: Differences between variants and isoforms

In mammals there are two ubiquitous, catalytically indistinguishable isoforms of inosine monophosphate dehydrogenase and mutations in the type I isoform, but not type II, cause retina-specific disorders. We have characterised the spatio-temporal expression of these proteins during development of the rat retina and performed functional investigations of the recently described retinal type I variants. Inosine monophosphate dehydrogenase was present in all immature cells throughout the retina during embryonic and neonatal development. Following eye opening and cell differentiation its distribution was restricted to the photoreceptors and bipolar cells, becoming prominent in Müller cells with aging. Type II was present in early, developing retinae whilst type I was undetectable. An isoform switch occurred around P10, after which the type I variants, type Iα and type Iγ, were the major forms. Functional investigations indicate type Iγ has greater catalytic activity compared with other variants and isoforms. Finally, all forms of type I show an increased propensity to form intracellular macrostructures compared to type II and these structures appear to be regulated in response to changing intracellular GTP levels. Collectively these data demonstrate that (i) type I does not play a role in early retinal development, (ii) type Iγ has greater activity and (iii) there are differences between type I and type II isoforms. These observations are consistent with the aetiology of retinitis pigmentosa and raise the possibility that programmed expression of specific inosine monophosphate dehydrogenase proteins may have arisen to meet the requirements of the cellular environment.

Probing Binding Requirements of Type I and Type II Isoforms of Inosine Monophosphate Dehydrogenase with Adenine-Modified Nicotinamide Adenine Dinucleotide Analogues

Novel tiazofurin adenine dinucleotide (TAD) analogues 25-33 containing a substituent at C2 of the adenine ring have been synthesized as inhibitors of the two isoforms of human IMP-dehydrogenase. The 2-ethyl TAD analogue 33 [Ki = 1 nM (type I), Ki = 14 nM (type II)] was found to be the most potent. It did not inhibit three other cellular dehydrogenases up to 50 microM. Mycophenolic adenine bis(phosphonate)s containing a 2-phenyl (37) or 2-ethyl group (38), were prepared as metabolically stable compounds, both nanomolar inhibitors. Compound 38 [Ki = 16 nM (type I), Ki = 38 nM (type II)] inhibited proliferation of leukemic K562 cells (IC50 = 1.1 microM) more potently than tiazofurin (IC50 = 12.4 microM) or mycophenolic acid (IC50 = 7.7 microM).

Real-Time PCR Determination of IMPDH1 and IMPDH2 Expression in Blood Cells

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide synthesis and is implicated in cell cycle control. Inhibition of this enzyme is associated with immunosuppressive, antiviral, and antitumor activity. IMPDH basal activity increases after initiation of immunosuppressive therapy. A real-time reverse-transcription PCR assay was developed and validated for mRNA quantification of the 2 human IMPDH isoforms. Target gene expressions were normalized to the geometric mean of 3 housekeeping genes. Assay utility was tested by analyzing patient samples and cultured cells exposed to immunosuppressive drugs such as the IMPDH inhibitor mycophenolic acid. The assay was linear over 6 logs of cDNA input and demonstrated specific quantification of IMPDH1 and IMPDH2 expression in cultured cells and patient samples. Limits of detection and quantification were 10 and 10(3) copies of cDNA per reaction, respectively. Within-run and total between-day CVs were <15% for normalized expression. Changes in IMPDH1 and 2 expression were observed in patient samples after initiation of an immunosuppressive regimen that included calcineurin inhibitors, mycophenolate mofetil, and steroids. This assay can be used to study the regulation of IMPDH expression and the involvement of the enzymes in immunological and malignant proliferative conditions. This may contribute to the processes of drug development and to the establishment of monitoring strategies for treatment effect and disease activity.

Forced Expression of the “IY” Mutant Inosine Monophosphate Dehydrogenase II Results in Physiologically Significant Resistance to Mycophenolic Acid In Vitro.

Nonmyeloablative conditioning regimens followed by allogeneic hematopoietic cell transplantation (HSCT) rely on pre and post transplant immunosuppression to overcome host-versus-graft reactions and control graft-versus-host-disease (GVHD). Patients with less than 50% of donor T cells by day 14 after transplant have a higher risk of graft rejection and relapse than those with more than 50% donor chimerism (Blood 2004; 104(8):2254). However, with increasing donor T-cell chimerism the risk of acute GVHD increases. Immunosuppression may inhibit the beneficial graft-versus-infection and graft-versus-leukemia effects mediated by mature donor T cells. Genetically modifying T cells with a drug resistance gene is one way to confer protection from the effects of the immunosuppressive agents, retain donor T cell function, and increase donor T cell chimerism by day 14. Mycophenolate mofetil (MMF) has been used in the post transplant setting to decrease the severity of GVHD and reduce the risk of host vs. graft reaction leading to graft failure. The active metabolite of MMF, mycophenolic acid (MPA), inhibits the enzyme inosine monophosphate dehydrogenase (IMPDH). The type II isoform of IMPDH is the rate-limiting enzyme in de novo guanosine synthesis and is preferentially expressed in activated T and B cells which lack the salvage pathway for guanosine synthesis. A number of investigators have identified IMPDH II mutants that have altered MPA binding capacity and normal guanosine synthetic activity ex vivo (BBA 2002; 1594:27, JBC. 1997; 272:961, BBA. 1994; 1217:156). We have generated six mutants of IMPDH II based on in vitro data and x-ray crystallography of the MPA binding site and transduced them into a human T cell line (Jurkat), a canine large granular lymphocyte cell line (CLGL-90) and a fibroblast cell line (NIH3T3) using oncoretroviral transduction. The mutants include: S276A (Amut), S276F (Fmut), T333I + S351Y (IYmut), L30F + Q277R (FRmut), Q277R + A462T (RTmut), and S276Y (Ymut). We generated bicistronic (IRES containing) retroviral vectors that expressed either HA-tagged wild type human IMPDH II (WT) or an HA-tagged IMPDH mutant and EGFP. We show that forced expression of WT will increase the MPA IC50 3 fold (IC50=3uM) compared to non-transduced cells. Forced expression of only the IY mutant in all cell lines tested was able to transfer resistance to MPA in excess of WT (IC50>50uM). This is clinically relevant since MPA concentrations measured after transplant in patients on MMF are around 8uM (Blood. 2005; 106(13):4381). To show that resistance was related to the expression of the mutant enzyme we correlated GFP by FACS with HA-IMPDH II expression by western blot analysis, and correlated this with resistance to MPA in vitro. This was true regardless of selection in MPA or transduction and cell sorting for >90% GFP positive cells. Analysis of IMPDH activity in lysates of transduced cells is currently underway. This is the first report where forced expression only of the IY mutant provides a clear enhanced resistance to MPA in vitro compared to cells over expressing wild type IMPDH II. Currently, we are generating bicistronic maloney based oncoretroviral vectors which carry a suicide gene (CD34-TK) and the IMPDH-HA IY mutant which will allow us to pharmacologically modulate the level of donor T cells after allogeneic stem cell transplantation.

Mycophenolic Acid Is a Potent Inhibitor of Angiogenesis

Angiogenesis is necessary for the vascularization of a tumor, providing essential nourishment for tumor growth, and the progression and metastasis of cancer cells.1 Although tremendous efforts and resources have been dedicated to discovering angiogenesis inhibitors, very few candidates have been approved by FDA for therapeutic applications. Currently, Avastin (bevacizumab), a humanized monoclonal antibody, and Macugen (Pegaptanib), a pegylated oligonucleotide aptamer, both directed against vascular endothelial growth factor (VEGF) pathway, are two of the approved drugs for clinical use. There is still a great need to develop small molecule drugs for antiangiogenesis purpose. One approach to broaden the discovery platform is to identify antiangiogenic activities from existing drugs that have well defined toxicity and pharmacokinetics. Once a known drug is demonstrated to inhibit angiogenesis, it would move into the clinic trails more rapidly. Because most known drugs have well defined structures and defined biological targets, it is also possible to further improve the compound structure or select the target as a new entry point for angiogenesis-based studies and therapies. Currently there are more than 2000 known drugs that can be used for re-purposing applications. It would be desirable to have a single in vivo assay allowing rapid screening of a large number of compounds for new activities such as angiogenesis inhibition. Recently, the zebrafish has been shown to be a useful model organism for this type of approach.2,3 Zebrafish blood vessels form by angiogenic sprouting and appear to use the same pathways necessary for blood vessel growth in mammals.4 Using transgenic embryos expressing GFP in vessels, the embryonic blood vessels in live zebrafish can be directly …

Identification of Type 1 Inosine Monophosphate Dehydrogenase as an Antiangiogenic Drug Target

To rapidly discover clinically useful angiogenesis inhibitors, we created and screened a library of existing drugs for inhibition of endothelial cell proliferation. Mycophenolic acid (MPA), an immunosuppressive drug, was found to potently inhibit endothelial cell proliferation in vitro and block tumor-induced angiogenesis in vivo. Using RNA interference, we found that knockdown of one of the two known isoforms of inosine monophosphate dehydrogenase (IMPDH-1) is sufficient to cause endothelial cell cycle arrest.

Regulation of inosine monophosphate dehydrogenase type I and type II isoforms in human lymphocytes

The enzyme inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo biosynthesis of guanine nucleotides. Inhibition of IMPDH leads to immunosuppression by decreasing guanine nucleotides that are required for the proliferation of lymphocytes. IMPDH activity is mediated by two highly conserved isoforms, type I and type II. We have characterized the mRNA and protein expression of the two isoforms in a variety of human tissues, peripheral blood mononuclear cells (PBMCs), and selected cell lines to investigate their regulation. Type I mRNA was expressed in most tissues with high expression in PBMCs and low expression in thymus. IMPDH type II transcript was also detected in most tissues with low expression in spleen and PBMCs. In PBMCs, induction of both type I and type II mRNAs was observed within 12 hr of mitogenic stimulation. Using type-selective IMPDH antibodies, an increase in the levels of type I and type II proteins was observed after mitogenic stimulation. The effect of two IMPDH inhibitors, MPA and VX-497, was investigated on the expression of type I and type II isoforms. VX-497 is an orally bioavailable, potent and reversible inhibitor of IMPDH, with broad applicability in many viral and immune system-mediated diseases. MPA and VX-497 inhibit both isoforms of IMPDH in vitro. Prolonged treatment of lymphocytes with either VX-497 or MPA did not lead to an increase in type I or type II IMPDH protein levels. These results are discussed in the context of IMPDH being a target for immunosuppressive, anti-viral and anti-cancer therapy.

Apoptosis of superantigen-activated T cells induced by mycophenolate mofetil treatment.

Mycophenolate mofetil (MMF), an ester prodrug of mycophenolic acid (MPA), is a potent immunosuppressive agent used in clinical organ transplantation. MPA preferentially inhibits the type II isoform of inosine monophosphate dehydrogenase, depletes GTP, suppresses transfer of mannose and fucose to glycoproteins, and prevents lymphocyte proliferation in vivo. Whether MMF can also delete activated T cells in vivo by triggering an apoptotic signal was addressed in this study. To this end we analyzed the activity of MMF in mice injected with the bacterial superantigen staphylococcal enterotoxin B (SEB). Superantigens bind to MHC class II molecules without requirement for processing, and activate subsets of CD4+ and CD8+ T cells whose T cell receptor beta chains express Vbeta family-specific homologous sequences. This model that shares several features with direct allorecognition has the unique advantage of allowing a precise monitoring of activated T cells. BALB/c mice treated with MMF (100 mg/kg/ day) or vehicle were injected with SEB. Serum cytokines, CD4+ and CD8+ Vbeta8+ cells were monitored in blood and lymphoid tissues, and apoptosis was determined by externalization of membrane phosphatidyl serine, double strand DNA breaks, and expression of B220 antigen by Vbeta8+ cells. MMF treatment decreased tumor necrosis factor alpha, interferon gamma, and interleukin-10 secretion induced by SEB. It did not modify other early activation events (blast transformation, CD69 and CD25 expression) but completely inhibited SEB-induced expansion of Vbeta8+ cells by inducing apoptosis of SEB-reactive T cells. A similar effect was observed in CD95-ligand-deficient mice. Repeated SEB injections associated with MMF resulted in a marked decrease of CD8+ Vbeta8+ T cells. SEB-induced increase of Vbeta8+ thymocytes was not prevented by MMF treatment. Results obtained in this in vivo model suggest that MMF treatment may induce deletion of activated peripheral T cells and decrease early cytokine responses.

Mycophenolate mofetil and its mechanisms of action

Mycophenolate mofetil (MMF, CellCept®) is a prodrug of mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH). This is the rate-limiting enzyme in de novo synthesis of guanosine nucleotides. T- and B-lymphocytes are more dependent on this pathway than other cell types are. Moreover, MPA is a fivefold more potent inhibitor of the type II isoform of IMPDH, which is expressed in activated lymphocytes, than of the type I isoform of IMPDH, which is expressed in most cell types. MPA has therefore a more potent cytostatic effect on lymphocytes than on other cell types. This is the principal mechanism by which MPA exerts immunosuppressive effects.Three other mechanisms may also contribute to the efficacy of MPA in preventing allograft rejection and other applications. First, MPA can induce apoptosis of activated T-lymphocytes, which may eliminate clones of cells responding to antigenic stimulation. Second, by depleting guanosine nucleotides, MPA suppresses glycosylation and the expression of some adhesion molecules, thereby decreasing the recruitment of lymphocytes and monocytes into sites of inflammation and graft rejection. Third, by depleting guanosine nucleotides MPA also depletes tetrahydrobiopterin, a co-factor for the inducible form of nitric oxide synthase (iNOS). MPA therefore suppresses the production by iNOS of NO, and consequent tissue damage mediated by peroxynitrite.CellCept® suppresses T-lymphocytic responses to allogeneic cells and other antigens. The drug also suppresses primary, but not secondary, antibody responses. The efficacy of regimes including CellCept® in preventing allograft rejection, and in the treatment of rejection, is now firmly established. CellCept® is also efficacious in several experimental animal models of chronic rejection, and it is hoped that the drug will have the same effect in humans.

Crystal structure of human type II inosine monophosphate dehydrogenase: implications for ligand binding and drug design.

Inosine monophosphate dehydrogenase (IMPDH) controls a key metabolic step in the regulation of cell growth and differentiation. This step is the NAD-dependent oxidation of inosine 5' monophosphate (IMP) to xanthosine 5' monophosphate, the rate-limiting step in the synthesis of the guanine nucleotides. Two isoforms of IMPDH have been identified, one of which (type II) is significantly up- regulated in neoplastic and differentiating cells. As such, it has been identified as a major target in antitumor and immunosuppressive drug design. We present here the 2.9-A structure of a ternary complex of the human type II isoform of IMPDH. The complex contains the substrate analogue 6-chloropurine riboside 5'-monophosphate (6-Cl-IMP) and the NAD analogue selenazole-4-carboxamide adenine dinucleotide, the selenium derivative of the active metabolite of the antitumor drug tiazofurin. The enzyme forms a homotetramer, with the dinucleotide binding at the monomer-monomer interface. The 6 chloro-substituted purine base is dehalogenated, forming a covalent adduct at C6 with Cys-331. The dinucleotide selenazole base is stacked against the 6-Cl-IMP purine ring in an orientation consistent with the B-side stereochemistry of hydride transfer seen with NAD. The adenosine end of the ligand interacts with residues not conserved between the type I and type II isoforms, suggesting strategies for the design of isoform-specific agents.

Mycophenolate mofetil--clinical and experimental experience.

Mycophenolate mofetil (MMF) is the first drug approved for the prevention of renal allograft rejection in the United States in the last 10 years. MMF is the morpholinoethyl ester of mycophenolic acid (MPA) and is a selective, reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH), an enzyme that is critical for the production of guanine monophosphate. MPA is a potent inhibitor of IMPDH, particularly the type II isoform. Compared with other cell types, lymphocytes appear to be more sensitive to inhibition of the type II isoform of IMPDH. This decreases the metabolism of guanine nucleotides, which are necessary for cell function. MMF is rapidly converted to MPA, which is the pharmacologically active drug. MPA is highly bound to serum albumin, and recent evidence suggests that the pharmacologic activity of MPA is a function of the unbound drug. Recent studies in human clinical transplantation have demonstrated the efficacy of this compound in renal transplantation. A major clinical study with > 1,499 patients demonstrated a 50% reduction in the incidence of acute rejection when compared with azathioprine or placebo control. The primary side effects in these studies were leukopenia, gastrointestinal problems, and cytomegalovirus disease. MMF represents a major advance in immunosuppression for renal transplant recipients. Ongoing studies are being performed in other types of solid organ transplantation.