Rational design of proteolytically stable, cell-permeable peptide-based selective Mcl-1 inhibitors

Abstract

Direct chemical modifications provide a simple and effective means to "translate" bioactive helical peptides into potential therapeutics targeting intracellular protein-protein interactions. We previously showed that distance-matching bisaryl cross-linkers can reinforce peptide helices containing two cysteines at the i and i+7 positions and confer cell permeability to the cross-linked peptides. Here we report the first crystal structure of a biphenyl-cross-linked Noxa peptide in complex with its target Mcl-1 at 2.0 Å resolution. Guided by this structure, we remodeled the surface of this cross-linked peptide through side-chain substitution and N-methylation and obtained a pair of cross-linked peptides with substantially increased helicity, cell permeability, proteolytic stability, and cell-killing activity in Mcl-1-overexpressing U937 cells.

Figure 1

Crystal structure of mouse Mcl-1 in complex with Bph-cross-linked Noxa-BH3 peptide 2. (a) Overall complex structure. The peptide and the side chains of three canonical hydrophobic residues of peptide 2, Leu-4 (h2), Ile-7 (h3) and Val-11 (h4), are colored in deep teal. The two flexible loops, Gly-1733Gly-187 (in the front; shown in dashed line) and Leu-2163Val-224 (in the back; not shown), are disordered in the electron density maps. (b) A stereo view of the superimposition of Bph-cross-linked peptide 2 (yellow-colored stick model) with mNoxa-BH3 peptide (green-colored stick model) as seen in 2JM6. The BH3-binding pocket of mMcl-1 is rendered in surface model.

Figure 2

CD spectra of the Bph-cross-linked and linear Noxa peptides and their calculated percent helicity values. The peptides were dissolved in ACN/H₂O (1:1) for a final concentration of 50 μM. The percent helicity was calculated based on [θ]₂₂₂ value.

Figure 3

Flow cytometry analysis of HeLa cells after treatment with 10 μM fluorescein-labeled peptides, Fluo-Noxa, Fluo-2, Fluo-5, and Fluo-8. (a) Structure of Fluo-8; (b) Representative flow cytometry histogram at 37°C and 4°C; (c) Bar graph showing normalized relative fluorescence: filled bar = 37°C; open bar = 4°C.

Figure 4

Proteolytic stability of the linear and Bph-cross-linked Noxa peptides in the presence of (a) chymotrypsin (ChT), (b) trypsin, and (c) mouse serum. A zoom-in view of the degradation plot for the linear Noxa peptide is shown on the right in (c). The calculated half-life (t_1/2) values for the various peptides are given in (d).