Appendage- and\nScaffold-Diverse Electrophilic and\nPhotoreactive Probes for Integrated Phenotypic Screening–Target\nIdentification Campaigns via a Minimalist Bifunctional Isocyanide

One of the grand challenges in chemical biology is identifying\na small-molecule modulator for all proteins within a proteome. To\nexpand the variety and number of ligandable proteins for drug discovery,\nthe objective of this study was to synthesize and evaluate the protein\ntarget profiles of electrophilic and photoreactive fully functionalized\nsmall-molecule probes (FFSMPs) featuring increased scaffold-, appendage-,\nand protein-reactive functional group (PRFG) diversity. FFSMPs contain:\n(1) a protein-binding motif, (2) an electrophilic or photoreactive\nPRFG for target protein capture, and (3) a terminal alkyne for click\nchemistry-based proteomic applications. These compounds can be directly\napplied in phenotypic screening programs to identify ligand–protein\npairs in cells unbiasedly. Herein, we highlight 17 examples from 34\nstructurally diverse FFSMPs featuring five electrophiles, three photoreactive\ngroups, and 15 chemical scaffolds. Essential to the synthesis of the\nFFSMPs was a new minimalist bifunctional isocyanide in an “isocyanide-based\nmulticomponent reaction–Boc deprotection–arming”\nsynthetic sequence. To the best of our knowledge, this is the first\nreport concerning the preparation of appendage- and scaffold-diverse\nFFSMPs for integrated phenotypic screening-target identification campaigns\nwith the ability to examine either electrophilic or photoreactive\nPRFGs. In contrast, the status quo for such studies has been appendage-diverse\nFFSMPs comprised of a single chemical scaffold and a single PRFG,\nwhich limits efficient target protein capture and/or chemical space\nsampling significantly in the quest for discovering new drug targets\nand/or compounds with novel mechanisms of action. Phenotypic screening\nof the electrophilic members of our library identified several FFSMPs\nwith potent antiproliferative activity against MCF10CA1a breast cancer\ncells. One of these FFSMPs (Compound <b>4a</b>) covalently targeted\nand potently inhibited protein disulfide isomerase A1 (PDIA1). This\nstudy supports the continued use of minimalist bifunctional isocyanides\nas valuable building blocks for preparing structurally diverse FFSMPs\nfor integrated phenotypic screening–target identification campaigns.