Method for Quantitative Protein-Ligand Affinity Measurements in Compound Mixtures

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• 作者：D. Allen Annis ; Gerald W. Shipps ; Jr. ; Yongqi Deng ; Janeta Popovici-Mü ; ller ; M. Arshad Siddiqui ; Patrick J. Curran ; Matthew Gowen ; William T. Windsor
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：June 15, 2007
• 年：2007
• 卷：79
• 期：12
• 页码：4538 - 4542
• 全文大小：166K
• 年卷期：v.79,no.12(June 15, 2007)
• ISSN：1520-6882

文摘

This manuscript describes an affinity selection-massspectrometry (AS-MS) method for quantitative protein-ligand binding affinity (K_d) measurements in large compound libraries. The ability of a titrant ligand to displacea target-bound library member-as measured by MS-reveals the affinity ranking of the mixture componentrelative to "internal affinity calibrants", compounds ofknown affinity for the target. This technique does notrequire that the precise concentration of each ligand isknown; therefore, unpurified products of mixture-basedcombinatorial synthesis may be used for affinity optimization and developing structure-activity relationships. Themethod is demonstrated for a series of ligands to theimportant oncology target CDK2 that were discovered byAS-MS screening of combinatorial libraries against thebasal form of the protein. AS-MS displacement curves forselect hits were acquired over a range of compoundconcentrations, confirming that binding affinity measurement results are concentration-insensitive. These hitswere evaluated in pools of purified compounds to verifythe method's applicability to hit triage in large chemicallibraries. The method was further tested using unpurified,mixture-based combinatorial libraries of >1000 compounds, yielding results that mirror those obtained frommixtures of purified compounds. The technique was thenused to identify optimized CDK2 ligands from compoundmixtures, quantitatively measure their affinities, andestablish structure-activity relationships among thesedrug leads.