Peptide-Directed Binding for the Discovery of Modulators of α-Helix-Mediated Protein-Protein Interactions: Proof-of-Concept Studies with the Apoptosis Regulator Mcl-1

Abstract

Targeting PPIs with small molecules can be challenging owing to large, hydrophobic binding surfaces. Herein, we describe a strategy that exploits selective α-helical PPIs, transferring these characteristics to small molecules. The proof of concept is demonstrated with the apoptosis regulator Mcl-1, commonly exploited by cancers to avoid cell death. Peptide-directed binding uses few synthetic transformations, requires the production of a small number of compounds, and generates a high percentage of hits. In this example, about 50 % of the small molecules prepared showed an IC₅₀ value of less than 100 μm, and approximately 25 % had IC₅₀ values below 1 μm to Mcl-1. Compounds show selectivity for Mcl-1 over other anti-apoptotic proteins, possess cytotoxicity to cancer cell lines, and induce hallmarks of apoptosis. This approach represents a novel and economic process for the rapid discovery of new α-helical PPI modulators.

Keywords: apoptosis; drug discovery; medicinal chemistry; protein-protein interactions; solid-phase synthesis.

Scheme 1

A) Concept of using peptide‐directed binding to target PPIs. B) Extended NoxaB peptide fragment that demonstrated restored affinity for Mcl‐1.

Scheme 2

Synthesis of the small‐molecule/peptide hybrids by SPPS and subsequent synthesis of small‐molecule peptide mimics. DIPEA=diisopropylethylamine, DMF=dimethylformamide, TFA=trifluoroacetic acid, TIPS=triisopropylsilane.

Figure 1

Representative titrations of compounds 18 (left) and 21 (right) on 5 nm FITC‐NoxaB peptide in the presence of 10 nm Mcl‐1 protein (red) or 5 nm FITC‐Bid peptide in the presence of 30 nm Bcl‐2 (yellow) and Bcl‐x_L (green), demonstrating no appreciable binding to Bcl‐2 and Bcl‐x_L.

Figure 2

Representative small molecules induce hallmarks of apoptosis. A) Increase of caspase‐3 activation in BxPC‐3 cells 4 h after treatment. B) Externalization of phosphatidylserine, a hallmark of apoptosis, was evaluated by exposing BxPC‐3 cells to compounds 18 (25 μm), 20 (5 μm), 21 (5 μm), and 23 (25 μm) for 4 h followed by annexin‐V‐FLUOS. Images were taken at 10× magnification with a GFP filter (top) and white light (bottom).