A direct-to-biology high-throughput chemistry approach to reactive fragment screening.

Ross P Thomas,David J Fallon,Stephen Besley,Nicholas C O Tomkinson,Emma K Grant,Peter Pogány,Michael M Hann,Francesca Zappacosta,Rachel E Heap,David House,Jacob T Bush

doi:10.1039/d1sc03551g

Abstract

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines ‘direct-to-biology’ high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn2+ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design–make–test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.

Highlights

Human genetics and functional genomic studies are providing insights into potential therapeutic targets on an unprecedented scale.[1,2,3,4,5] Validation of these targets in early-stage discovery relies on the rapid identi cation of tool molecules that can inform on the mechanism of action best suited to therapeutic intervention.[6]
The approach would involve the synthesis of whole libraries of reactive fragments in a 384-well plate-based format followed by direct screening of the crude products with a protein target.[33,34,35,36,37,38,39]
It was anticipated that the diazirine OSu-ester 1 could be added to a plate containing fragment amines to afford the desired high-throughput chemistry (HTC)-PhABits in sufficient purity to allow direct screening against targets of interest

Summary

Introduction

Human genetics and functional genomic studies are providing insights into potential therapeutic targets on an unprecedented scale.[1,2,3,4,5] Validation of these targets in early-stage discovery relies on the rapid identi cation of tool molecules that can inform on the mechanism of action best suited to therapeutic intervention.[6]. The approach would involve the synthesis of whole libraries of reactive fragments in a 384-well plate-based format followed by direct screening of the crude products with a protein target.[33,34,35,36,37,38,39] Crucially, the intact protein LC-MS screening method provides a quality control for the chemical identity of any hit fragments, as determined by the observed protein mass shi . HTC was subsequently used to synthesise and screen a second-generation library of 100 hit analogues to improve hit rate and crosslinking yield.

Results

Conclusion