We have recently reported on Lys-covalent agents that, based on aryl-sulfonyl fluorides, were designed to target binding site Lys 311 in the X-linked inhibitor of apoptosis protein (XIAP). Similar to XIAP, melanoma-IAP (ML-IAP), a less well-characterized IAP family protein, also presents a lysine residue (Lys 135), which is in a position equivalent to that of Lys 311 of XIAP. On the contrary, two other members of the IAP family, namely, cellular-IAPs (cIAP1 and cIAP2), present a glutamic acid residue in that position. Hence, in the present work, we describe the derivation and characterization of the very first potent ML-IAP Lys-covalent inhibitor with cellular activity. The agent can be used as a pharmacological tool to further validate ML-IAP as a drug target and eventually for the development of ML-IAP-targeted therapeutics.

We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein−protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein−protein interactions.