Structure Based Design of Non-Natural Peptidic Macrocyclic Mcl-1 Inhibitors

Abstract

Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compound 1, a 1.5 μM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compound 1 bound to Mcl-1 in a β-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approximately 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to additional potency improvements. The use of protein-ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compound 26 is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 μM and Bcl-xL at >99 μM, and induces cleaved caspase-3 in MV4-11 cells with an IC₅₀ of 3 μM after 6 h.

Keywords: Mcl-1; macrocycles; peptides; protein−protein interactions.

Figure 1

Crystal structure of compound 1 (cyan) bound to Mcl-1 (purple) overlaid with 6-chloro-3-[3-(4-chloro-3,5-dimethylphenoxy)propyl]-1H-indole-2-carboxylic acid (yellow) bound to Mcl-1 (not shown); PDB 5KU9 and 4HW2. Surface colors: red, solvent exposed; green, hydrophobic; magenta, polar.

Figure 2

Crystal structure of compound 21 (pink) bound to Mcl-1 (orange); PDB 5MEV (2.94 Å). Distances in Å.

Figure 3

Crystal structure of compound 29 (gray) bound to Mcl-1 (brown); PDB 5MES (2.24 Å). Distances in Å.