Barnes B J, Eakin A E, Izydore R A, Hall I H
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360, USA.
Biochemistry. 2000 Nov 14;39(45):13641-50. doi: 10.1021/bi0004044.
Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo purine biosynthesis. IMPDH activity results from expression of two isoforms. Type I is constitutively expressed and predominates in normal resting cells, while Type II is selectively up-regulated in neoplastic and replicating cells. Inhibitors of IMPDH activity selectively targeting the Type II isoform have great potential as cancer chemotherapeutic agents. For this study, an expression system was developed which yields 35-50 mg of soluble, purified recombinant Type I and II protein from 1 L of bacteria. In addition, three 1,5-diazabicyclo[3.1.0]hexane-2,4-diones were synthesized and shown to act as specific inhibitors of human recombinant Type II IMPDH. The agents are competitive inhibitors with respect to the endogenous substrate IMP and K(i) values range from 5 to 44 microM but were inactive as inhibitors of the Type I isoform at concentrations ranging from 0.5 to 500 microM. IC(50) values for recombinant Type II inhibition were determined and compared to IC(50) values obtained from Molt-4 cell extracts of IMPDH. Cytotoxicity assays revealed that the compounds inhibited Molt-4 leukemia growth with ED(50) values of 3.2-7.6 microM. Computational docking studies predict that the compounds bind to IMPDH in the IMP-binding site, although interactions with residues differ from those previously determined to interact with bound IMP. While all residues predicted to interact directly with the bound compounds are conserved in the Type I and Type II isoforms, sequence divergence within a helix adjacent to the active site may contribute to the observed selectivity for the human Type II isoform. These compounds represent the first class of selective IMPDH Type II inhibitors which may serve as lead compounds for the development of isoform-selective cancer chemotherapy.
肌苷5'-单磷酸脱氢酶（IMPDH）是嘌呤从头合成途径中的限速酶。IMPDH的活性源于两种同工型的表达。I型同工型组成性表达，在正常静息细胞中占主导地位，而II型同工型在肿瘤细胞和增殖细胞中选择性上调。选择性靶向II型同工型的IMPDH活性抑制剂作为癌症化疗药物具有巨大潜力。在本研究中，开发了一种表达系统，该系统可从1升细菌中产生35 - 50毫克可溶性、纯化的重组I型和II型蛋白。此外，合成了三种1,5 - 二氮杂双环[3.1.0]己烷 - 2,4 - 二酮，并证明它们可作为人重组II型IMPDH的特异性抑制剂。这些试剂是针对内源性底物肌苷5'-单磷酸（IMP）的竞争性抑制剂，其抑制常数（Ki）值范围为5至44微摩尔，但在0.5至500微摩尔的浓度范围内对I型同工型无抑制活性。测定了重组II型抑制的半数抑制浓度（IC50）值，并与从Molt - 4细胞提取物中获得的IMPDH的IC50值进行比较。细胞毒性试验表明，这些化合物以3.2 - 7.6微摩尔的半数有效剂量（ED50）值抑制Molt - 4白血病细胞的生长。计算对接研究预测，这些化合物在IMP结合位点与IMPDH结合，尽管与残基的相互作用与先前确定的与结合的IMP相互作用的情况不同。虽然预测与结合的化合物直接相互作用的所有残基在I型和II型同工型中都是保守的，但活性位点相邻螺旋内的序列差异可能导致观察到的对人II型同工型的选择性。这些化合物代表了第一类选择性II型IMPDH抑制剂，可作为开发同工型选择性癌症化疗的先导化合物。
