The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer

Abstract

The development of selective inhibitors for discrete anti-apoptotic BCL-2 family proteins implicated in pathologic cell survival remains a formidable but pressing challenge. Such precisely tailored compounds would serve as molecular probes and targeted therapies to study and treat human diseases driven by specific anti-apoptotic blockades. In particular, MCL-1 has emerged as a major resistance factor in human cancer. By screening a library of stabilized alpha-helix of BCL-2 domains (SAHBs), we determined that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor. X-ray crystallography and mutagenesis studies defined key binding and specificity determinants, including the capacity to harness the hydrocarbon staple to optimize affinity while preserving selectivity. MCL-1 SAHB directly targets MCL-1, neutralizes its inhibitory interaction with pro-apoptotic BAK and sensitizes cancer cells to caspase-dependent apoptosis. By leveraging nature's solution to ligand selectivity, we generated an MCL-1-specific agent that defines the structural and functional features of targeted MCL-1 inhibition.

Figure 1

Identification of an MCL-1-selective BH3 domain. (a) A panel of Stabilized Alpha-Helix of BCL-2 domains (SAHBs) was designed based on the BH3 domains of pro- and anti-apoptotic BCL-2 family members. A pair of crosslinking non-natural amino acids (X) were substituted at the indicated i, i+4 position of the non-interacting helical surface and “stapled” by ruthenium-catalyzed olefin metathesis. To optimize the activity of the Grubbs’ ruthenium catalyst, sulfur-containing methionines were replaced with norleucines, which are designated by the letter B. (b) Dissociation constants for the binding of fluorescently labeled SAHBs to MCL-1ΔNΔC were determined by fluorescence polarization assay (FPA) and nonlinear regression analysis. (c) Among the SAHBs that bound MCL-1ΔNΔC with high affinity, only MCL-1 SAHB_A displayed a potent and exclusive interaction with MCL-1ΔNΔC, as evidenced by FPA performed with FITC-MCL-1 SAHB_A against a broad panel of anti-apoptotic targets. Data are mean and s.d. for experiments performed in at least triplicate.

Figure 2

Binding and specificity determinants of the MCL-1 BH3 helix. (a) A panel of sequential alanine mutants (alanine scan) of FITC-MCL-1 SAHB_A was generated for FPA binding analysis, revealing key residues within the core BH3 sequence required for high affinity MCL-1ΔNΔC binding. Glutamate mutagenesis was also performed to evaluate the contribution of native alanine and glycine residues to MCL-1ΔNΔC binding. *, K_D >10 μM. (b) A single point mutation of V220F eliminated the MCL-1 specificity of MCL-1 SAHB_A, conferring binding affinity to both MCL-1ΔNΔC and BCL-X_LΔC, as demonstrated by FPA. (c) Sampling a variety of staple positions along the α-helical surface revealed disruption of MCL-1ΔNΔC binding only by the G217,Q221 staple (MCL-1 SAHB_C), which is located at the hydrophobic binding interface. MCL-1 SAHB_D exhibited the strongest binding activity (K_D, 10 nM), with 4-fold improvement over the parental MCL-1 SAHB_A. Data are mean and s.d. for experiments performed in at least triplicate.

Figure 3

Crystal structure of the MCL-1 SAHB_D/MCL-1ΔNΔC complex. (a) MCL-1 SAHB_D engages MCL-1ΔNΔC at the canonical BH3 binding groove of anti-apoptotic proteins, as determined by x-ray crystallography at 2.32-Å resolution (PDB 3MK8). Hydrophobic interactions at the binding interface are reinforced by a complementary polar interaction network that involves MCL-1 SAHB_D residues R214 and D218 and MCL-1ΔNΔC residues S255, D256, N260, and R263. The side chains of hydrophobic, positively charged, negatively charged and hydrophilic residues are colored yellow, blue, red and green, respectively. (b) The core BH3 residues L213, V216, G217 and V220 of MCL-1 SAHB_D make direct contact with a hydrophobic cleft at the surface of MCL- 1ΔNΔC. (c) The hydrocarbon staple, bearing an olefin in the cis conformation, contributes additional hydrophobic contacts at the perimeter of the core interaction site.

Figure 4

MCL-1 SAHB_D dissociates the inhibitory MCL-1/BAK complex in vitro and in situ, and sensitizes BAK-dependent mitochondrial cytochrome c release. (a) MCL-1 SAHBs effectively prevent sequestration of the BAK BH3 helix by MCL-1ΔNΔC, as demonstrated by competition FPA. N.D., no detected displacement. (b) MCL-1 SAHB_D dose-responsively sensitized BID BH3-induced and BAK-dependent mitochondrial apoptosis, as measured by cytochrome c release assay performed on wild type and Bak^−/− mitochondria. (c) An OPM2 multiple myeloma cellular lysate was incubated with the indicated biotinylated MCL-1 SAHB_D constructs in the presence of ultraviolet light, followed by streptavidin-based purification, stringent washing to remove non-covalent binders, elution, and MCL-1 western analysis. The photoreactive MCL-1 pSAHB_D, generated by replacing L210 with a benzophenone-bearing non-natural amino acid (Bpa), directly crosslinked to native MCL-1 within the cellular lysate, whereas no covalent crosslinking was observed for MCL-1 SAHB_D, which lacked the photoreactive benzophenone moiety. (d) The native interaction between BAK and MCL-1 was dose-responsively disrupted by treatment of OPM2 cells with MCL-1 SAHB_D, as assessed by MCL-1 immunoprecipitation and BAK western analysis. Binding and cytochrome c release data are mean and s.d. for experiments performed in at least triplicate. Vehicle, deionized water.

Figure 5

Selective MCL-1 targeting by MCL-1 SAHB_D sensitizes death receptor signaling and induces caspase-dependent cancer cell apoptosis. (a) Jurkat T-cell leukemia and (b) OPM2 cells were exposed to MCL-1 SAHB_D singly and in combination with low dose death receptor agonists TRAIL and Fas ligand (FasL) in the presence or absence of the pan-caspase inhibitor, z-VAD. Cell viability measured by MTT assay at 24 hours revealed dose-responsive and caspase-dependent apoptosis sensitization of Jurkat (TRAIL and FasL) and OPM2 (TRAIL) cells by MCL-1 SAHB_D. The capacity of MCL-1 SAHB_D to sensitize (c) Jurkat and (d) OPM2 cells to death receptor stimuli correlated with dose-responsive activation of caspase 3/7, as measured by luminescence of DEVD-cleaved substrate. Data are mean and s.d. for experiments performed in at least triplicate. Vehicle, deionized water.