In vitro and in vivo characterization of AS2643361, a novel and highly potent inosine 5'-monophosphate dehydrogenase inhibitor

Inosine 5'-monophosphate (IMP) dehydrogenase is a critical target in solid organ transplantation. To this end, the development of mycophenolate mofetil (MMF) represents a major advance in transplant medicine. Here, we investigated the in vitro and in vivo pharmacological effects of a novel IMP dehydrogenase inhibitor, AS2643361, in several immunological and non-immunological models. The in vitro inhibitory activity of AS2643361 on immune cell and endothelial cell proliferation and on antibody production from lipopolysaccharide-stimulated B cells, was significantly more potent than that of mycophenolic acid, the active form of MMF, despite the similar potency of these compounds on IMP dehydrogenase. In a rat heterotopic cardiac transplant model, monotherapy using orally administered AS2643361 at 10 or 20mg/kg/day prolonged the median graft survival time from 6 to 16 and 19days, respectively. In dinitrophenol-lipopolysaccharide stimulated rats, oral administration of AS2643361 at 2.5, 5 or 10mg/kg/day resulted in suppression of antibody production. In vivo antibody production against alloantigen was also suppressed by AS2643361 treatment at 5 or 10mg/kg/day. Furthermore, treatment with AS2543361 effectively inhibited balloon injury induced-intimal thickening, which is a major cause of late allograft loss. Overall, the in vivo activity of AS2643361 was over two-fold more potent than that of MMF. In addition, gastrointestinal toxicity, considered a dose-limiting factor for MMF, was reduced with AS2643361 treatment. These results suggest AS2643361 has higher potency and less toxicity than MMF, making it a potential candidate for treatment of acute and chronic rejection in transplant medicine.     

次黄嘌呤核苷酸脱氢酶抑制剂产生菌的筛选及aquayamycin的结构鉴定

筛选次黄嘌呤核苷酸脱氢酶(IMPDH)抑制剂及其产生菌,为新的抗癌药物和免疫抑制药物的研发提供先导化合物。对菌株进行发酵培养,通过以IMPDH为靶点的高通量筛选模型获得微生物活性代谢产物及其阳性菌种,采用16s rDNA序列构建阳性菌株的系统发育进化树,综合波谱解析确定化合物结构,并利用相关细胞对化合物进行活性评价。结果鉴定N05WA-1324A产生菌菌株为链霉菌属菌株,m/z 486,分子式为C₂₅H₂₆O₁₀,为aquayamycin。该化合物具有较强的IMPDH抑制活性,IC50为18.1μmol/L;对T淋巴细胞有很强的抑制活性,在2.5μmol/L浓度下能抑制99.8%的细胞增殖;同时对人结肠癌细胞株SW-620和人乳腺癌细胞株MDA-MB-231具有较强的增殖抑制活性,IC50分别为8.6和23.3μmol/L。N05WA-1324A具有很强的IMPDH和免疫抑制活性为国内外首次报道,在细胞水平上的活性评价显示其具有抗癌药物和免疫抑制药物的开发潜力。     

Mycophenolic acid inhibits inosine 5'-monophosphate dehydrogenase and suppresses immunoglobulin and cytokine production of B cells

Mycophenolic acid (MPA) reversibly inhibits inosine 5'-monophosphate dehydrogenase (IMPDH), an enzyme involved in the de novo synthesis of guanine nucleotides. Previously, mycophenolate mofetil (MMF), the pro-drug of MPA, was shown to exert beneficial effects on the systemic lupus erythematosus (SLE)-like disease in MRLlpr/lpr mice. In this study, MPA's immunomodulating effects in vitro on the B cell hybridoma MAR 18.5 were investigated. The cells were exposed for MPA at either 1 or 10 microM for 24 h, and the levels of immunoglobulins, cytokines and lactate dehydrogensase in supernatants were measured. The frequency of immunoglobulin producing cells and the proliferation and viability of the cells was also investigated. MPA exposure reduced the frequency of immunoglobulin producing cells, decreased the levels of immunoglobulins and cytokines in the supernatants, and decreased the cell proliferation. MPA was slightly cytotoxic as indicated by increased lactate dehydrogenase (LDH) levels and reduced viability. All MPA-induced effects were totally reversed by the addition of guanosine to the cultures. Thus, since activated B lymphocytes play a central role in lupus and our results show that B cells are targets for MPA, we propose that direct effects on B cells may be an important mechanism for the ameliorating effects of MMF in SLE.     

The structure of inosine 5'-monophosphate dehydrogenase and the design of novel inhibitors

The enzyme IMPDH is a homotetramer of approximately 55 kDa subunits and consists of a (beta/alpha)(8) barrel core domain and a smaller subdomain. The active site has binding pockets for the two substrates IMP and NAD. The enzymatic reaction of oxidation of IMP to XMP proceeds through a covalent mechanism involving an active site cysteine residue.This enzyme is a target for immunosuppressive agents because it catalyzes a key step in purine nucleotide biosynthesis which is important for the proliferation of lymphocytes. Several X-ray structures of inhibitors bound to IMPDH have been published. The uncompetitive IMPDH inhibitor MPA is the active metabolite of the immunosuppressive agent mycophenolate mofetil (CellCept(R)) which is approved for the prevention of acute rejection after kidney and heart transplantation. The bicyclic ring system of MPA packs underneath the hypoxanthine ring of XMP*, thereby trapping this covalent intermediate of the enzymatic reaction. Ribavirin monophosphate, the active metabolite of the antiviral agent ribavirin, is a substrate mimic of IMP. The structure of the two inhibitors 6-Cl-IMP and SAD binding in the IMP and NAD pockets of IMPDH, respectively, gives information for the binding mode of the di-nucleotide cofactor to the enzyme.At Vertex Pharmaceuticals a structure-based drug design program for the design of IMPDH inhibitors was initiated. Several new lead compound classes unrelated to other IMPDH inhibitors were found. Integrating structural information into an iterative drug-design process led to the design of VX-497. VX-497 is a potent uncompetitive enzyme inhibitor of IMPDH. The phenyl-oxazole moiety of the molecule packs underneath XMP*, analogous to MPA. VX-497 also makes several new interactions that are not observed in the binding of MPA. VX-497 is a potent immunosuppressive agent in vitro and in vivo. A Phase I clinical trial has been successfully concluded and the compound is currently in Phase II trials in psoriasis and hepatitis C. The rapid progress from initiation of the drug design program to a compound entering clinical trials illustrates the power of structure-based drug design to accelerate the drug discovery process.The structural information on IMPDH has also significantly increased our knowledge about the mechanistic details of this fascinating enzyme.     

A novel series of non-nucleoside inhibitors of inosine 5'-monophosphate dehydrogenase with immunosuppressive activity.

Inhibitors of inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) are effective immunosuppressive drugs that may also have additional potential applications as antitumour and antimicrobial agents. The clinical value of the most potent and specific inhibitor of IMPDH, mycophenolic acid, is limited by its rapid metabolism in vivo to an inactive glucuronide derivative. There is, therefore, a considerable incentive to develop structurally novel, preferably non-nucleoside, inhibitors with greater metabolic stability than mycophenolic acid. Here, we describe a high throughput screen for inhibitors of IMPDH, which facilitated the discovery of a single novel non-nucleoside inhibitor from a collection of approximately 80,000 compounds. The inhibitor is a pyridazine, which, like mycophenolic acid, exerts uncompetitive inhibition of IMPDH. Analysis of the enzyme kinetics suggests that the inhibitory action of the pyridazine is similar to that of mycophenolic acid, which involves trapping of a covalent intermediate formed during the conversion of IMP to xanthosine monophosphate. Chemical modification of the lead compound resulted in pyridazine derivatives with enhanced potency against IMPDH and guanine nucleotide synthesis in cultured cells in vitro and also against guanine nucleotide synthesis in the mouse spleen in vivo. One of the compounds was available in sufficient quantity to demonstrate highly effective immunosuppressive activity in a model of delayed type hypersensitivity in mice. To our knowledge, the novel pyridazines described in this report represent the first non-nucleoside uncompetitive inhibitors of IMPDH with immunosuppressive activity since the discovery of the inhibitory activity of mycophenolic acid and its derivatives thirty years ago.     

Development and application of a high-performance liquid chromatography-based assay for determination of the activity of inosine 5'-monophosphate dehydrogenase in whole blood and isolated mononuclear cells

With the objective of pharmacodynamic monitoring of the immunosuppressive efficacy of mycophenolate mofetil (MMF) (CellCept, Hoffman-LaRoche, Grenzach-Wyhlen, Germany), a method for determination of the inosine monophosphate dehydrogenase (IMPDH) activity in whole blood cell (WBC) lysates and mononuclear cells (MNCs) was developed. The assay is based on the incubation of WBC lysates or lysed MNCs in the presence of supplemented inosine 5'-monophosphate (IMP) and nicotimamide adenine dinucleotide (NAD). The formation of xanthosine 5'-monophosphate (XMP) was determined by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The analytical method was validated, and the obtained data demonstrated that the amount of XMP in WBC and MNC lysates can be reliably determined by this method. Under assay conditions the rate of XMP formation remained constant within the incubation period of 60 minutes and a quantification of product formation at 30 and 60 minutes proved to be sufficient to reliably characterize the IMPDH activity. Applications of this assay with whole blood indicated extremely high IMPDH-activities in samples from patients with renal transplant receiving MMF. IMPDH monitoring within 10 hours after administration of the morning dose demonstrated a marked enzyme inhibition between 2 hours and 3 hours postdosing, but the activities returned to predose levels within one dose interval. The analysis of isolated cell fractions indicated that the IMPDH-activity is predominantly located in erythrocytes. The contribution of MNCs to the whole blood activity remained below 10%. In order to simulate the in vivo exposure of MNCs to mycophenolic acid, an "erythrocyte- and platelet-free" whole blood was reconstituted by resuspension of isolated MNCs with plasma. This strategy allowed for the reliable measurement of IMPDH activity in the target cells of immunosuppression.     

Modes of action of hypoxanthine, inosine and inosine 5'-monophosphate on cyclic nucleotide phosphodiesterase from bovine brain

A purified bovine brain cyclic nucleotide phosphodiesterase catalyzed the hydrolysis of both cyclic AMP and cyclic GMP. Alternative substrate inhibition experiments indicated that cyclic AMP and cyclic GMP were hydrolyzed by the same enzyme and that they shared a common binding site. Inosine, inosine 5'-monophosphate and hypoxanthine competitively inhibited the hydrolysis of cyclic AMP and cyclic GMP by bovine brain cyclic nucleotide phosphodiesterase. This inhibition of cyclic nucleotide hydrolysis by the purines was affected by the pH of the mixture. The inhibition constants of inosine, inosine 5'-monophososphate and hypoxanthine when inhibiting enzymatic hydrolysis of cyclic AMP were 0.36 ± 0.05, 1.3 ± 0.2 and 3.2 ± 0.5 mM, respectively. With cyclic GMP as substrate, the inhibition constants were 0.50 ± 0.09, 1.8 ± 0.2 and 4.5 ± 0.7 mM for inosine, inosine 5'-monophosphate and hypoxantine respectively. The per cent inhibition by inosine, inosine 5'-monophosphate or hypoxanthine of the cyclic nucleotide phosphodiesterase activity was not altered by the addition of calmodulin (calcium-dependent protein activator of cyclic nucleotide phosphodiesterase) to the enzyme. The effect of calmodulin was not changed by these purine inhibitors. These results suggest that the binding site of calmodulin differed from that of the inhibitors and from that of cyclic AMP and cyclic GMP.     

Studies on the mechanism of action of benzamide riboside: a novel inhibitor of IMP dehydrogenase

Benzamide is a well known inhibitor of poly(ADP-ribose)polymerase, an enzyme involved in DNA repair. However, benzamide exhibited neuotoxicity in animals and hence, in the hope of overcoming this problem, benzamide riboside (BR) was synthesized. Our mechanism of action studies on BR suggested that the agent was being metabolized to its 5'-monophosphate and then to its NAD analogue (BAD, benzamide adenine dinucleotide) that inhibits Inosine 5'-monophosphate dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme of the branched purine nucleotide synthetic pathway that provides guanylates including GTP and dGTP. There are two isoforms of IMPDH, type I that is constitutively present in all cells, and type II that is inducible and is present in highly proliferating cells such as cancer. Ongoing studies with BR analogues suggest that they are more selective in inhibiting IMPDH type II. Our studies have characterized the metabolites of BR, especially its NAD analogue, BAD, by synthesizing this active metabolite by enzymatic means, and identifying its structure by NMR and mass spectrometry. We have partially purified IMPDH from tumor cells and have examined the kinetics of inhibition of IMPDH by BAD. We have also compared biochemical and cytotoxic activities of BR with tiazofurin and selenazofurin, that share similar mechanisms of action with BR. Our studies demonstrated that 2-3-fold more BAD is formed compared to TAD and SAD, the active metabolites of tiazofurin and selenazofurin, respectively. BR has demonstrated potent cytotoxic activity in a diverse group of human tumor cells, specifically more active in sarcomas and CNS neoplasms compared to tiazofurin or selenazofurin. Future in vivo animal studies should set a stage for determining its effectiveness in clinical Phase I studies.     

KRIBB3, a novel microtubule inhibitor, induces mitotic arrest and apoptosis in human cancer cells

KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) inhibited cancer cell growth in vitro and in vivo. Flow cytometry studies showed that KRIBB3 caused cell cycle arrest at the G(2)/M phase and subsequent apoptosis. This was confirmed as accumulation of Cyclin B1 and cleavage of poly(ADP-ribose) polymerase (PARP) were detected. While transient inhibition by KRIBB3 led to reversible mitotic arrest, prolonged exposure to KRIBB3-induced apoptosis. Co-immunoprecipitation experiments showed that KRIBB3 initially induced association of inhibitory Mad2 with p55CDC (mammalian homologue of CDC20), an activator of APC/C (anaphase-promoting complex/cyclosome), suggesting that the mitotic spindle checkpoint was activated by KRIBB3. However, the level of this inhibitory complex of Mad2 with p55CDC was gradually decreased 24 h after KRIBB3 treatment, and was hardly detectable after 48 h, indicating some slipping of the mitotic checkpoint. Consistent with these observations, KRIBB3 activated the mitotic spindle checkpoint by disrupting the microtubule cytoskeleton. KRIBB3 was proven to be a tubulin inhibitor using in vitro polymerization assays and in vivo indirect immunofluorescence staining. The temporal pattern of Bax activation by KRIBB3 was similar to PARP cleavage, suggesting that Bax is a mediator of KRIBB3-dependent apoptosis. Furthermore, when KRIBB3 was administered intraperitoneally into nude mice at 50 mg/kg or 100 mg/kg, it inhibited 49.5 or 70.3% of tumor growth, respectively. These results suggest that KRIBB3 is a good drug candidate for cancer therapy.     

Computational insights into the inhibition of inosine 5'-monophosphate dehydrogenase by mycophenolic acid analogs: three-dimensional quantitative structure-activity relationship and molecular docking studies

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a key enzyme in the de novo synthesis of guanosine nucleotides. It is considered as an important target in the quest for drugs in the immunosuppressive, antiviral, antibacterial, and anticancer therapeutic areas. Herein, we report the 3D-QSAR analyses using comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA) and docking on mycophenolic acid derivates for the first time. We obtained cross-validated q(2) value of 0.805 for CoMFA and 0.620 for CoMSIA, while the non-cross-validated r(2) values for them were 0.969 and 0.935, respectively. Based on the CoMFA and CoMSIA contour maps and docking analyses, some key structural factors responsible for inhibitory activity were revealed. The results obtained from this study could be used for the rational design of potent inhibitors against IMPDH.     

Effect of the inosine 5′-monophosphate dehydrogenase inhibitor BMS-566419 on renal fibrosis in unilateral ureteral obstruction in rats

Chronic allograft nephropathy (CAN) is a major cause of late allograft loss. One morphological characteristic of CAN is renal interstitial fibrosis. Mycophenolate mofetil (MMF), the inosine 5′-monophosphate dehydrogenase (IMPDH) inhibitor, has been reported to attenuate the progression of renal interstitial fibrosis. However, the question of whether the newly synthesized IMPDH inhibitors with structures different from MMF have an antifibrotic effect remains unanswered. We evaluated the antifibrotic effects of BMS-566419, a chemically synthesized IMPDH inhibitor, using an experimental rat model, unilateral ureteral obstruction (UUO), in comparison with those of MMF. Expression levels of monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-beta1 (TGF-β1), which play important roles in UUO-induced renal fibrosis, were also investigated to determine the mechanism by which BMS-566419 affects the progression of renal fibrosis. After 14 days of UUO, interstitial fibrosis was frequently observed in the renal cortex of rats administered vehicle control. BMS-566419 by oral administration showed a significant and dose-dependent suppressive effect on UUO-induced renal fibrosis in histopathological experiments. BMS-566419 treatment also decreased collagen content, as indicated by hydroxyproline concentration, and the expression of collagen type 1 mRNA. BMS-566419 also decreased the expression of mRNA for both MCP-1 and TGF-β1. The antifibrotic effects of treatment with BMS-566419 at 60 mg/kg seemed comparable to those with MMF at 40 mg/kg. These results suggest that BMS-566419 and other chemically synthesized IMPDH inhibitors have beneficial pharmacological effects similar to those of MMF, and are potential pharmaceutical candidates in the treatment of fibrotic renal disease, including CAN.     

BCH, an inhibitor of system L amino acid transporters, induces apoptosis in cancer cells

PURPOSE: L-Type amino acid transporter 1 (LAT1) is highly expressed in cancer cells to support their continuous growth and proliferation. We have examined the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), an inhibitor of system L amino acid transporters, and the mechanism by which BCH suppresses cell growth in cancer cells. METHODS: The effect of BCH and the mechanism of BCH on cell growth suppression in cancer cells were examined using amino acid transport measurement, MTT assay, DNA fragmentation analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunoblotting. RESULTS: BCH inhibited L-leucine transport in a concentration-dependent manner, and it inhibited cell growth in a time-dependent manner in KB human oral epidermoid carcinoma cells, Saos2 human osteogenic sarcoma cells and C6 rat glioma cells. The formation of a DNA ladder was observed, and the number of TUNEL-positive cells was increased with BCH treatment. Furthermore, the proteolytic processing of caspase-3 in KB and C6 cells and of caspase-7 in KB, Saos2 and C6 cells was increased by BCH treatment. CONCLUSION: These results suggest that the inhibition of LAT1 activity by BCH leads to apoptotic cancer cell death by inducing intracellular depletion of neutral amino acids necessary for cancer cell growth.     

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.