ABSTRACT: Kinetic target-guided synthesis (KTGS) is a powerful screening approach that enables identification of small moleculemodulators for biomolecules. While many KTGS variants have emerged, a majority of the examples suffer from limited throughputand a poor signal/noise ratio, hampering reliable hit detection. Herein, we present our optimized multifragment KTGS screeningstrategy that tackles these limitations. This approach utilizes selected reaction monitoring liquid chromatography tandem massspectrometry for hit detection, enabling the incubation of 190 fragment combinations per screening well. Consequentially, ourfragment library was expanded from 81 possible combinations to 1710, representing the largest KTGS screening library assembled todate. The expanded library was screened against Mcl-1, leading to the discovery of 24 inhibitors. This work unveils the true potentialof KTGS with respect to the rapid and reliable identification of hits, further highlighting its utility as a complement to the existingrepertoire of screening methods used in drug discovery.
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