Inhibition of interactions between Mcl-1 and proapoptotic proteins is considered to be a therapeutic strategy to induce apoptosis in cancer cells. Here, we adopted molecular dynamics simulation with molecular mechanics–Poisson Boltzmann/surface area method (MM-PB/SA) to study the inhibition mechanism of three Mcl-1 inhibitors, compounds 1, 2 and 3. Analysis of energy components shows that the better binding free energy of compound 3 than compounds 1 and 2 is attributable to the van der Waals energy (ΔE_vdw) and non-polar solvation energy (ΔG_np) upon binding. In addition to the excellent agreement with previous experimentally determined affinities, our simulation results further show a bend of helix 4 on Mcl-1 upon compound 3 binding, which is driven by hydrophobic interaction with residue Val²⁵³, leading to a narrowed BH3-binding groove to impede Puma^BH3 binding. The computational result is consistent with our competitive isothermal titration calorimetry (ITC) assays, which shows that the competitive ability of compound 3 toward Mcl-1/Puma^BH3 complex is improved beyond its direct binding affinity toward Mcl-1 itself, and compound 3 exhibits much more efficiency to compete with Puma^BH3 than compound 2. Our study provides a new strategy to improve inhibitory activity on Mcl-1 based on the conformational dynamic change.

Although the role of Bcl-2 phosphorylation is still under debate, it has been identified in a resistance mechanism to BH3 mimetics, for example ABT-737 and S1. We identified an S1 analogue, S1-16, as a small-molecule inhibitor of pBcl-2. S1-16 efficiently kills EEE-Bcl-2 (a T69E, S70E, and S87E mutant mimicking phosphorylation)-expressing HL-60 cells and high endogenously expressing pBcl-2 cells, by disrupting EEE-Bcl-2 or native pBcl-2 interactions with Bax and Bak, followed by apoptosis. In vitro binding assays showed that S1-16 binds to the BH3 binding groove of EEE-Bcl-2 (K_d=0.38 μM by ITC; IC₅₀=0.16 μM by ELISA), as well as nonphosphorylated Bcl-2 (npBcl-2; K_d=0.38 μM; IC₅₀=0.12 μM). However, ABT-737 and S1 had much weaker affinities to EEE-Bcl-2 (IC₅₀=1.43 and >10 μM, respectively), compared with npBcl-2 (IC₅₀=0.011 and 0.74 μM, respectively). The allosteric effect on BH3 binding groove by Bcl-2 phosphorylation in the loop region was illustrated for the first time.

The design of a cross-acridine scaffold mimicking the i, i+3, i+5, and i+7 residues distributed over a two-face, two-turn α-helix is described. Docking studies and 2D ¹H,¹⁵N HSQC NMR spectroscopy provide compelling evidence that compound 3 d accurately reproduces the arrangement of four hotspots in the Bim BH3 peptide to permit binding to the Mcl-1 and Bcl-2 proteins (K_i 0.079 and 0.056 μM, respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.

The use of small molecule B-cell lymphoma 2 homology domain 3 mimetics to neutralize the B-cell lymphoma 2 protein is an attractive strategy for cancer treatment due to its ability to cause targeted cell apoptosis. We have previously reported the design and optimization of a series of B-cell lymphoma 2 homology domain 3-mimetics, called compounds 1–6. In this study, we evaluated the optimization of B-cell lymphoma 2 homology domain 3-mimetics from a thermodynamic perspective. Understanding the thermodynamic parameters of B-cell lymphoma 2 homology domain 3-mimetics plays a critical role in the development of B-cell lymphoma 2 small-molecule inhibitors. The thermodynamic parameters for the interactions of these compounds with the myeloid cell leukemia sequence 1 protein were obtained using isothermal titration calorimetry. Owing to compounds 1–6 overcoming enthalpy–entropy compensation, the affinities of them improved gradually. Toward binding to the myeloid cell leukemia sequence 1 protein, compound 6 was deemed optimal with an obtained K_d value of 238 nm, which is a 10⁴-fold improvement compared with 1. Analysis of the enthalpy and −TΔS efficiencies showed that ligand efficiencies with respect to molecular size are correlated with the enthalpic efficiencies. Notably, an enthalpy gain of 4.65 kcal/mol identified that an additional hydrogen bond is formed by 2 with myeloid cell leukemia sequence 1 compared with compound 1. For the first time, hydrogen bonding between a small-molecule inhibitor of B-cell lymphoma 2 was demonstrated experimentally.

Small molecule S1 is a pan-BH3 mimetic that can bind antiapoptotic Bcl-2, Bcl-xL and Mcl-1 proteins. Herein, different Bcl-2 member expression cancer cell lines (NCI-H345, MCF-7, SMMC-7721 and Hela) and cells deficient in Bax and/or Bak by shRNA were used to unravel the cascade of events by which S1 promotes apoptosis compared with Bcl-2/Bcl-xL inhibitor ABT-737. We identified that S1 exhibited broader antitumour spectrum than ABT-737 through disruption of more Bcl-2 interactions including Mcl-1/Bak interaction. Moreover, the individual and combined roles of Bax and Bak in S1-induced apoptosis were revealed. Our results showed that S1 induced a Bak-mediated apoptosis. Bak played a predominant role in either S1 or ABT-737-induced apoptosis through the cooperation with Bax on the formation of large oligomers on mitochondrial membrane.