Stoichiometric Selection of Tight-Binding Inhibitors by Wild-Type and Mutant Forms of Malarial (Plasmodium falciparum) Dihydrofolate Reductase

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• 作者：Sumalee Kamchonwongpaisan ; Jarunee Vanichtanankul ; Bongkoch Tarnchompoo ; Jirundon Yuvaniyama ; Supannee Taweechai ; and Yongyuth Yuthavong
• 刊名：Analytical Chemistry
• 出版年：2005
• 出版时间：March 1, 2005
• 年：2005
• 卷：77
• 期：5
• 页码：1222 - 1227
• 全文大小：102K
• 年卷期：v.77,no.5(March 1, 2005)
• ISSN：1520-6882

文摘

A simple method for screening combinatorial and otherlibraries of inhibitors of malarial (Plasmodium falciparum) dihydrofolate reductase (PfDHFR) has beendeveloped, based on the affinities of the inhibitors withthe enzyme. In the presence of limiting amounts of theenzyme, a number of inhibitors in the library were boundto extents reflecting the relative binding affinities. Following ultrafiltration and guanidine hydrochloride treatmentto release bound inhibitors, the amounts of free andbound inhibitors could be determined by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The differences in the patternsreflected the binding of high-affinity components compared with the other members in the library. A goodcorrelation was found between the inhibition constants (K_ivalues) and the extent of binding of inhibitors to wild-type,double (C59R+S108N) and quadruple mutant (N51I+C59R+S108N+I164L) of PfDHFR, as well as humanDHFR. In addition to identifying lead components of thelibraries with high affinities (low K_i values) and stabilities(low k_off rates), this simple method also provides analternative way for quickly and accurately calculatingenzyme binding affinities of inhibitors in combinatorialchemical libraries.