A Direct-to-Biology High-Throughput Chemistry Approach to Reactive Fragment Screening

Abstract

<p>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 <a>new medicines. Here, we report a screening platform that combines ‘direct-to-biology’ high-throughput chemistry (D2B-HTC) with photoreactive covalent fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates. Screening the HTC-PhABit library with </a>carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn<sup>2+</sup> 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.</p>

Reactive fragments targeting carboxylate residues employing direct to biology, high-throughput chemistry

“…1c). 42 This methodology employs 384-well plate-based amide couplings of amine fragments with an activated succinimide ester bearing the reactive functionality (1, Fig. 1c).…”

“…To generate the ACT-PhABit library, we selected 546 amine fragments that afforded high conversion to the corresponding amides in a HTC-PhABit library. 42 These included primary unhindered-, primary hindered-and secondary cyclic-alkylamines (ESI † section 3.1) (for library properties see Fig. S1 †).…”

“…S1 †). 42 Amines were plated as singletons in 384-well plates (10 mM DMSO) followed by addition of a DMSO solution of OSu ester 1 and N-ethylmorpholine (NEM). The plates were sealed and incubated at room temperature for 24 h to afford the crude ACT-PhABits.…”

“…Previously we have reported the screening of diazirine containing fragments for the discovery of ligands for proteins of interest. 14,42 A challenge with this approach can be the low crosslinking yields achieved with carbenes, and it was anticipated that the nitrilimine furnished by the photoactivation of the ACT reactive group may offer improved crosslinking yields.…”

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome

“…8,9 More recently, reactive fragment-based screening platforms have been developed for streamlined and robust detection of hits, both with purified proteins of interest and in proteome-wide screening, which offers a route to expand the liganded proteome (Figure 1Aiii). [10][11][12][13][14][15][16] Existing reactive approaches have traditionally utilised cysteine-targeting electrophiles, exploiting the enhanced nucleophilicity of these residues to enable quantitative modification, and often inhibition, of target proteins (Figure 1Bi). [17][18][19] There are, however, a limited number of protein pockets that contain an accessible cysteine.…”