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>

Efficient Ligand Discovery Using Sulfur(VI) Fluoride Reactive Fragments

“…This allowed for screening in a D2B format as crude reaction mixtures, circumventing the requirement for purification and thus accelerating reactive fragment library screens. 29 A succinimide-activated (OSu) amide coupling was employed using DMSO and N-ethylmorpholine (NEM) as the solvent and base, respectively. 29 The conditions were initially trialled on a panel of 12 diverse aminefunctionalised fragments by addition of SF reactive moieties (1a, 2a, and 3a) in both dry DMSO, and DMSO:water (9:1) to assess robustness to hydrolysis under the reaction conditions.…”

“…29 A succinimide-activated (OSu) amide coupling was employed using DMSO and N-ethylmorpholine (NEM) as the solvent and base, respectively. 29 The conditions were initially trialled on a panel of 12 diverse aminefunctionalised fragments by addition of SF reactive moieties (1a, 2a, and 3a) in both dry DMSO, and DMSO:water (9:1) to assess robustness to hydrolysis under the reaction conditions. Reactions with OSu esters 2a and 3a afforded good conversion to the desired products and tolerance of 10% water, while meta-substituted OSu ester 1a gave poorer conversions due to hydrolysis of the SF group to the sulfonic acid (see Fig.…”

“…Such iteration-based screens allow for faster design-make-test cycles in contrast to traditional methods in early-stage drug discovery, leading to the more efficient identification of potent and selective probes. 29 To explore whether any alternative sulfonamide-containing fragments would give high modification yields at a faster rate, a new library was designed. For this, 96 amine-functionalised fragment analogues of hits 2b-e were selected, coupled to OSu ester 2a, and the resultant library was incubated with CAII for 1 h prior to analysis by intact protein LC-MS.…”

“…This strategy employed a high-throughput chemistry direct-tobiology (HTC-D2B) workflow, providing an expedient and accessible method for the rapid and iterative generation of SF reactive fragment libraries. [27][28][29]…”

Efficient large-scale exploration of fragment hit progression by exploiting binding-site purification of actives (B-SPA) through combining multi-step array synthesis and HT crystallography.

“…29 Additionally, progress has been made in recent years to developing low-cost HTS platforms 32 for the synthesis, purification and analysis of compounds. [33][34][35][36] However, it appears that the scale of compound exploration must be at least an order of magnitude larger than can be supported by these evolutions on typical early discovery budgets. For instance, Gao et al 37 have used "on-the-fly" nanoscale library syntheses by acoustic dispensing to generate large arrays.…”