By means of limited proteolysis assay, three-dimensional NMR, X-ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl-2 homology 3 (BH3) domain of Mcl-1 has been identified as a conformational switch which controls Mcl-1 ubiquitination. Noxa binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl-1. In contrast, Bim binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl-1 inhibitor, which locates at the BH3 domain of Mcl-1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro-apoptotic partners, but also facilitates Mcl-1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl-1-dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.

Background and Purpose The biological significance of the multi‐site phosphorylation of Bcl‐2 at its loop region (T69, S70 and S87) has remained controversial for decades. This is a major obstacle for understanding apoptosis and anti‐tumour drug development. Experimental Approach We established a mathematical model into which a phosphorylation and de‐phosphorylation process of Bcl‐2 was integrated. Paclitaxel‐treated breast cancer cells were used as experimental models. Changes in the kinetics of binding with its critical partners, induced by phosphorylation of Bcl‐2 were experimentally obtained by surface plasmon resonance, using a phosphorylation‐mimicking mutant EEE‐Bcl‐2 (T69E, S70E and S87E). Key Results Mathematical simulations combined with experimental validation showed that phosphorylation regulates Bcl‐2 with different dynamics depending on the extent of Bcl‐2 phosphorylation and the phosphorylated Bcl‐2‐induced changes in binding kinetics. In response to Bcl‐2 homology 3 (BH3)‐only protein Bmf stress, Bcl‐2 phosphorylation switched from diminishing to enhancing the Bcl‐2 anti‐apoptotic ability with increased phosphorylation of Bcl‐2, and the turning point was 50% Bcl‐2 phosphorylation induced by 0.2 μM paclitaxel treatment. In contrast, Bcl‐2 phosphorylation enhanced the anti‐apoptotic ability of Bcl‐2 towards other BH3‐only proteins Bim, Bad and Puma, throughout the entire phosphorylation procedure. Conclusions and Implications The model could accurately predict the effects of anti‐tumour drugs that involve the Bcl‐2 family pathway, as shown with ABT‐199 or etoposide.

By means of limited proteolysis assay, three‐dimensional NMR, X‐ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl‐2 homology 3 (BH3) domain of Mcl‐1 has been identified as a conformational switch which controls Mcl‐1 ubiquitination. NoxaBH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl‐1. In contrast, BimBH3 binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl‐1 inhibitor, which locates at the BH3 domain of Mcl‐1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro‐apoptotic partners, but also facilitates Mcl‐1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl‐1‐dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.

No α-helical mimetic that exhibits Bcl-2/MDM2 dual inhibition has been rationally designed due to the different helicities of the α-helixes at their binding interfaces. Herein, we extracted a one-turn α-helix-mimicking ortho-triarene unit from o-phenylene foldamers. Linking benzamide substrates with a rotatable C-N bond, we constructed a novel semirigid pyramid-like scaffold that could support its two-turn α-helix mimicry without aromatic stacking interactions and could adopt the different dihedral angles of the key residues of p53 and BH3-only peptides. On the basis of this universal scaffold, a series of substituent groups were installed to capture the key residues of both p53TAD and BimBH3 and balance the differences of the bulks between them. Identified by FP, ITC, and NMR spectroscopy, a compound 6e (zq-1) that directly binds to Mcl-1, Bcl-2, and MDM2 with balanced submicromolar affinities was obtained. Cell-based experiments demonstrated its antitumor ability through Bcl-2/MDM2 dual inhibition simultaneously.

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Based on a known selective Mcl-1 inhibitor, 6-chloro-3-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1H-indole-2-carboxylic acid, we applied a fragment-based approach to obtain new molecules that extended into the p1 pocket of the BH3 groove and then exhibited binding selectivity for the Mcl-1 over the Bcl-2 protein. After we deconstructed the 1H-indole-2-carboxylic acid from the parental molecule, a benzenesulfonyl was substituted at the 1-position to adopt a geometry preferred for accessing the p1 pocket according to the binding mode of the parental molecule identified by X-ray crystallography. A linear relationship between the free energy of ligand binding (ΔG) and the count of non-hydrogen heavy atoms (HAC) was maintained during the molecular growing to occupy the p1 pocket. Finally, we not only obtained compound 12 with a 7.5-fold selectivity to Mcl-1 (K = 0.48 µM by fluorescence polarization) over Bcl-2 (K = 3.6 µM), but also provided evidence that additional occupation of the p1 pocket is more favorable for Mcl-1 than for Bcl-2 binding, and contributes more to Mcl-1 inhibition than occupation of the p2 pocket. Compound 12 exhibited a selective killing ability on Mcl-1-dependent cancer cells.

Pilose antler is a rare animal-sourced traditional Chinese medicine that has been demonstrated to cause a change in total immunity. Little is known about its specific effect on immune cells. In this study, the immunological modulatory effects of pilose antler extract on mice were investigated. Mice were gavaged daily for 7 days with pilose antler extracts, and peripheral blood was analysed by flow cytometry. Our results show that pilose antler extract exerted distinctive effects on the different immune cell types analysed. Pilose antler gavage transiently increased T cell percentage and decreased B cell percentage in peripheral blood. After treatment, the proliferation of killer and helper T cells was stimulated in the short term, but in the longer term, NK and memory T cell populations were increased. These results suggest that pilose antler treatment has both transient and long-term effects on the immune system in mice.ARTICLE HISTORY

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 (ΔEvdw ) and non-polar solvation energy (ΔGnp ) 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 Val(253) , leading to a narrowed BH3-binding groove to impede Puma(BH) (3) 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(BH) (3) complex is improved beyond its direct binding affinity toward Mcl-1 itself, and compound 3 exhibits much more efficiency to compete with Puma(BH) (3) than compound 2. Our study provides a new strategy to improve inhibitory activity on Mcl-1 based on the conformational dynamic change.