Going beyond Binary: Rapid Identification of Protein–Protein Interaction Modulators Using a Multifragment Kinetic Target-Guided Synthesis Approach

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