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

Abstract

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.

Shotgun Kinetic Target-Guided Synthesis Approach Enables the Discovery of Small-Molecule Inhibitors against Pathogenic Free-Living Amoeba Glucokinases

“…(Figure S1) The thio acids were prepared as fluorenylmethyl thioesters from corresponding carboxylic acids, then subsequently deprotected with 5% DBU in situ prior to carrying out the templation experiments; as previously described . Carboxylic acids and sulfonyl azides used in this study were either commercially available or synthesized as previously reported . The investigation of Nf Glck-templated N -acylsulfonamide ( SZTA ) formation commenced with the incubation of fragments in a multicombinatorial fashion in the presence and absence of the parasitic enzyme.…”

“…The origin of the fragments used in this study may help to explain these findings. Our library was initially curated to target Bcl-X L and thus was designed to bind the large, hydrophobic interfaces found in protein–protein interactions. ,, Expansion of this library to target another PPI, Mcl-1, has unsurprisingly led to a larger, more hydrophobic fragment library than would be expected for targeting enzymes . This is exemplified by average c Log P and MW values of 6.3 and 606 Da, respectively, for the 12 compounds.…”

“…The fragments used in the KTGS experiment were a random selection of 38 sulfonyl azides ( SZ1–SZ38 ) and 45 thio acids ( TA1–TA45 ) from our in-house fragment library developed from our previous work. 47 ( Figure S1 ) The thio acids were prepared as fluorenylmethyl thioesters from corresponding carboxylic acids, then subsequently deprotected with 5% DBU in situ prior to carrying out the templation experiments; as previously described. 19 Carboxylic acids and sulfonyl azides used in this study were either commercially available or synthesized as previously reported.…”

“… 19 Carboxylic acids and sulfonyl azides used in this study were either commercially available or synthesized as previously reported. 47 The investigation of Nf Glck-templated N -acylsulfonamide ( SZTA ) formation commenced with the incubation of fragments in a multicombinatorial fashion in the presence and absence of the parasitic enzyme. Sets of 5 thio acids were incubated with all 38 sulfonyl azides, giving rise to 190 possible distinct SZTA products per well and 1710 combinations in total.…”

“…In fact, it has even been stated that coupling partners cannot be chosen at random and must be selected based on hits originating from docking studies and/or high-throughput screens. 25 Here, we describe the first shotgun KTGS approach performed on N. fowleri glucokinase ( Nf Glck) for the identification of pFLA Glck inhibitors using our previously demonstrated 47 multifragment sulfo-click strategy with LC–MS/MS detection. We use the term “shotgun” to highlight that the selection of coupling partners was made at random and was not guided by computational methods or high-resolution X-ray crystal structures.…”