ALARM NMR: A Rapid and Robust Experimental Method To Detect Reactive False Positives in Biochemical Screens

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• 作者：Jeffrey R. Huth ; Renaldo Mendoza ; Edward T. Olejniczak ; Robert W. Johnson ; Darlene A. Cothron ; Yaya Liu ; Claude G. Lerner ; Jun Chen ; and Philip J. Hajduk
• 刊名：Journal of the American Chemical Society
• 出版年：2005
• 出版时间：January 12, 2005
• 年：2005
• 卷：127
• 期：1
• 页码：217 - 224
• 全文大小：174K
• 年卷期：v.127,no.1(January 12, 2005)
• ISSN：1520-5126

文摘

High-throughput screening (HTS) of large compound collections typically results in numeroussmall molecule hits that must be carefully evaluated to identify valid drug leads. Although several filteringmechanisms and other tools exist that can assist the chemist in this process, it is often the case that costlysynthetic resources are expended in pursuing false positives. We report here a rapid and reliable NMR-based method for identifying reactive false positives including those that oxidize or alkylate a protein target.Importantly, the reactive species need not be the parent compound, as both reactive impurities andbreakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactivemolecules by nuclear magnetic resonance) and is based on monitoring DTT-dependent ¹³C chemical shiftchanges of the human La antigen in the presence of a test compound or mixture. Extensive validation hasbeen performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity.This included comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing acollection of more than 3500 compounds containing HTS hits from 23 drug targets. The data show thatcurrent in silico filtering tools fail to identify more than half of the compounds that can act via reactivemechanisms. Significantly, we show how ALARM NMR data has been critical in identifying reactivecompounds that would otherwise have been prioritized for lead optimization. In addition, a new filteringtool has been developed on the basis of the ALARM NMR data that can augment current in silico programsfor identifying nuisance compounds and improving the process of hit triage.