Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics
Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics
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DOI：
10.1021/jm060202r
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发表时间：
2006-08-10
影响因子：
7.3
通讯作者：
Kotra, Lakshmi P.
中科院分区：
文献类型：
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作者：
Poduch, Ewa;Bello, Angelica M.;Kotra, Lakshmi P.
一磷酸腺苷脱羧酶(ODCase)的抑制剂在RNA病毒、寄生虫和其他传染病中有应用。ODCase催化一磷酸鸟苷(OMP)的脱羧基，生成尿苷一磷酸(UMP)。根据底物OMP和ODCase抑制剂巴比妥酸单磷酸BMP中的亚结构体积，设计了新的抑制剂6-氨基-UMP和6-氰基-UMP。建立了一种新的等温滴定量热法(ITC)来研究ODCase的抑制动力学。通过监测一磷酸核苷脱羧反应中产生的热量来测量反应速率。测定了底物脱羧酶的动力学参数(k(CAT)=21 S(-1)，K-M=5mU M)和摩尔热(Delta H-APP=5kcal/mol)。6-氮杂-UMP和6-氰基-UMP的抑制常数(K-I)分别为12.4µM和29µM。6-氨基-UMP是一种有效的ODCase抑制剂，其抑制常数为840 nM。在这里，我们揭示了利用生物等构学原理设计的第一个ODCase抑制剂，以及一种用等温量热法研究酶抑制的新方法。
Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and K-M = 5 mu M) and the molar enthalpy (Delta H-app = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (K-i) were determined to be 12.4 mu M and 29 mu M for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.