Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl-1

Abstract

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.

Histidine-Covalent Stapled Alpha-Helical Peptides Targeting hMcl-1

“…The design of novel pharmacological tools targeting protein–protein interactions (PPIs) has long been regarded as a challenging task. To this end, when PPIs are mediated by an alpha-helix, helix stabilizing strategies have been proposed, some of which have led to agents that have entered clinical trials. − We recently reported on novel ligand discovery strategies to target PPIs that included mixture-based screening strategies, such as the HTS by NMR and related approaches. , More recently, we also focused on strategies to derive Lys-covalent PPI antagonists , using aryl-fluorosulfates or aryl-sulfonyl fluorides. In a recent application, we derived a novel BH3 peptide targeting covalently Lys 234 of the antiapoptotic Bcl-2 protein hMcl-1 …”

“…hMcl-1 is often overexpressed in cancer, where it confers resistance to chemotherapy or radiation. Small molecule inhibitors have emerged for hMcl-1, − and research is still ongoing in this target space. ,− In addition to small molecule inhibitors, as anticipated above, one approach that holds great therapeutic potential in targeting PPIs is the design of stapled alpha-helical peptides. , These could provide alternative strategies for advancing novel therapeutic agents or for the design of pharmacological tools for basic research and target validation studies. However, in targeting PPIs with stapled alpha-helices, potency, selectivity, solubility, and cell penetration are all issues that need to be addressed.…”

“… 1 − 7 We recently reported on novel ligand discovery strategies to target PPIs that included mixture-based screening strategies, such as the HTS by NMR and related approaches. 8 , 9 More recently, we also focused on strategies to derive Lys-covalent PPI antagonists 10 , 11 using aryl-fluorosulfates or aryl-sulfonyl fluorides. In a recent application, we derived a novel BH3 peptide targeting covalently Lys 234 of the antiapoptotic Bcl-2 protein hMcl-1.…”

“…Small molecule inhibitors have emerged for hMcl-1, 13 − 16 and research is still ongoing in this target space. 9 , 16 − 21 In addition to small molecule inhibitors, as anticipated above, one approach that holds great therapeutic potential in targeting PPIs is the design of stapled alpha-helical peptides. 1 , 7 These could provide alternative strategies for advancing novel therapeutic agents or for the design of pharmacological tools for basic research and target validation studies.…”