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.

Identification of selective protein–protein interaction inhibitors using efficient in silico peptide-directed ligand design

“…1). 19 In silico screening of the small molecule fragments then allows the identification of peptide/small molecule hybrids with restored affinity for the target site. The restoration of binding affinity implies that the small molecule fragment in some way emulates the peptide section it has replaced.…”

In Silico Peptide-Directed Ligand Design Complements Experimental Peptide-Directed Binding for Protein-Protein Interaction Modulator Discovery

“…The library was also evaluated to predict ADME properties, 30 to help ensure synthetic effort was most likely to generate results. The top 10 results were chosen for synthesis following published procedures with CuSO4 and sodium ascorbate in a tBuOH/H2O mix, 24 followed by purification with reverse phase HPLC. Synthesised compounds were evaluated in the FITC-NoxaB Mcl-1 competitive binding fluorescence anisotropy (FA) assay (Table 1, Figure 2A).…”

“…Synthesised compounds were evaluated in the FITC-NoxaB Mcl-1 competitive binding fluorescence anisotropy (FA) assay (Table 1, Figure 2A). 24 The NoxaB peptide (AAQLRRIGDKVNLRQKLLN) was employed as a positive control and to ensure the FA assay was performing adequately. Surprisingly, of the ten compounds prepared eight (80%) demonstrated binding in our assay, with an IC50 < 100 μM.…”

“…This very high hit rate demonstrates another example of the power of peptide-directed ligand design for PPI modulators. Analysis of the compounds shows that four compounds were identified by experimental peptide-directed binding (1, 2, 4 & 6), 24 with five new Mcl-1 binders identified with the in silico approach. Three of the top four binders were identified by the experimental screen, suggesting both methods are likely to find the most efficacious compounds.…”

In Silico Peptide-Directed Ligand Design Complements Experimental Peptide-Directed Binding for Protein-Protein Interaction Modulator Discovery

“…Our report of in silico peptide-directed ligand design 28 demonstrated a higher efficiency at discovering small molecule inhibitors of PPIs when compared to the analogous experimental peptide-directed binding. 24 The in silico method required the preparation of only 20 compounds to obtain a 50% hit rate of compounds which demonstrated an IC50 < 100 μM in in vitro protein fluorescence anisotropy assays. The experimental method revealed 54% of the small molecule compounds prepared demonstrated an IC50 < 100 μM, slightly higher than the in silico only route, but also required the preparation of 60 peptide-small molecule hybrids, adding time and cost.…”