Designed BH3 peptides with high affinity and specificity for targeting Mcl-1 in cells

Abstract

Mcl-1 is overexpressed in many cancers and can confer resistance to cell-death signaling in refractory disease. Molecules that specifically inhibit Mcl-1 hold potential for diagnosing and disrupting Mcl-1-dependent cell survival. We selected three peptides from a yeast-surface display library that showed moderate specificity and affinity for binding to Mcl-1 over Bfl-1, Bcl-xL, Bcl-2, and Bcl-w. Specificity for Mcl-1 was improved by introducing threonine at peptide position 2e. The most specific peptide, MS1, bound Mcl-1 with 40-fold or greater specificity over four other human Bcl-2 paralogs. In BH3 profiling assays, MS1 caused depolarization in several human Mcl-1-dependent cell lines with EC50 values of ∼3 μM, contrasted with EC50 values of >100 μM for Bcl-2-, Bcl-xL-, or Bfl-1-dependent cell lines. MS1 is at least 30-fold more potent in this assay than the previously used Mcl-1 targeting reagent NoxaA BH3. These peptides can be used to detect Mcl-1 dependency in cells and provide leads for developing Mcl-1 targeting therapeutics.

Figure 1

Comparison of Bim BH3 position 2e in structures of Bfl-1, Bcl-x_L, and Mcl-1. (a) Helix 4 is closer to Ala2e in the Bfl-1:Bim BH3 structure (light:dark purple, 2VM6) than in the Mcl-1:Bim BH3 structure (light:dark cyan, 2PQK), as shown by aligning the structures in PyMOL.^, (b) Threonine modeled at position 2e clashes (red disks) with helix 4 in the Bfl-1 complex (left) but does not clash significantly in the Mcl-1 complex (right). Mutations were modeled in PyMOL by selecting the most preferred backbone-dependent rotamer for Thr; all rotamers are predicted to clash on the Bfl-1 structure backbone. (c) Ala2e is angled further into the BH3 binding groove in the Bcl-x_L:Bim BH3 structure (light:dark green, 3FDL) than in the Mcl-1:Bim BH3 structure (light:dark cyan, 2PQK). (d) A salt bridge network that exists in the Bcl-x_L:Bim BH3 structure (green) between Glu129, Arg132, and Arg3b would be disrupted at the equivalent sites in Mcl-1:Bim BH3 structure 2PQK (white). Glutamate and arginine are shown modeled in the place of His252 and Ser255 in the 2PQK structure, with rotamers chosen to position the side chains as close as possible to the orientations in the 3FDL structure.

Figure 2

Heat map of the EC₅₀ values (peptide concentration in nM) for mitochondrial depolarization induced by engineered and native BH3 peptides in four cell lines. Engineered peptide names are shaded in gray. Each cell line is dependent upon a single Bcl-2 family member: Mcl-1, Bcl-2, Bcl-x_L, or Bfl-1 as determined by BH3 profiling with native BH3-only peptides (Supplementary Figure 3e–h). All experiments were performed at least three times. The inset shows an example titration curve; see Supplementary Figure 3 and Supplementary Table 3 for other curves and all EC₅₀ values with 95% confidence intervals.