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Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo , S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours. S63845 specifically inhibits MCL1 and induces tumour cell death in vitro and in vivo in diverse cancer-derived cell lines with an acceptable safety margin. MCL1 protein as a possible anti-cancer target These authors report the discovery and characterization of a novel inhibitor of the anti-apoptotic pro-survival protein MCL1, which is expressed by multiple tumour types. The compound, termed S63845, activates the BAX/BAK-dependent mitochondrial apoptotic pathway and shows efficacy in several solid tumour models, suggesting that inhibition of MCL1 could be a viable anti-cancer strategy, alone or in combination with other anti-cancer drugs.

MCL1, a BCL2 relative, is critical for the survival of many cells. Its turnover is often tightly controlled through both ubiquitin-dependent and -independent mechanisms of proteasomal degradation. Several cell stress signals, including DNA damage and cell cycle arrest, are known to elicit distinct E3 ligases to ubiquitinate and degrade MCL1. Another trigger that drives MCL1 degradation is engagement by NOXA, one of its BH3-only protein ligands, but the mechanism responsible has remained unclear. From an unbiased genome-wide CRISPR-Cas9 screen, we discovered that the ubiquitin E3 ligase MARCH5, the ubiquitin E2 conjugating enzyme UBE2K, and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome—specifically when MCL1 is engaged by NOXA. This mechanism of degradation also required the MCL1 transmembrane domain and distinct MCL1 lysine residues to proceed, suggesting that the components likely act on the MCL1:NOXA complex by associating with it in a specific orientation within the mitochondrial outer membrane. MTCH2 has not previously been reported to regulate protein stability, but is known to influence the mitochondrial localization of certain key apoptosis regulators and to impact metabolism. We have now pinpointed an essential but previously unappreciated role for MTCH2 in turnover of the MCL1:NOXA complex by MARCH5, further strengthening its links to BCL2-regulated apoptosis.

DNA double-strand breaks (DSBs) are mainly repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) pathways. Here, we showed that myeloid cell leukemia sequence 1 (Mcl-1) acts as a functional switch in selecting between HR and NHEJ pathways. Mcl-1 was cell cycle-regulated during HR, with its expression peaking in S/G2 phase. While endogenous Mcl-1 depletion reduced HR and enhanced NHEJ, Mcl-1 overexpression resulted in a net increase in HR over NHEJ. Mcl-1 directly interacted with the dimeric Ku protein complex via its Bcl-2 homology 1 and 3 (BH1 and BH3) domains, which are required for Mcl-1 to inhibit Ku-mediated NHEJ. Mcl-1 also promoted DNA resection mediated by the Mre11 complex and HR-dependent DSB repair. Using the Mcl-1 BH1 domain as a docking site, we identified a small molecule, MI-223, that directly bound to BH1 and blocked Mcl-1-stimulated HR DNA repair, leading to sensitization of cancer cells to hydroxyurea- or olaparib-induced DNA replication stress. Combined treatment with MI-223 and hydroxyurea or olaparib exhibited a strong synergy against lung cancer in vivo. This mechanism-driven combination of agents provides a highly attractive therapeutic strategy to improve lung cancer outcomes.

The use of an aryl boronic acid carbonyl warhead to target a noncatalytic lysine side chain enables the development of covalent inhibitors against the anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1). Targeted covalent inhibition of disease-associated proteins has become a powerful methodology in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here we used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to our knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chemistry strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochemical and cell-based assays. We identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biological phenomena.

Significance Specific protein–protein interactions are abundant in, and essential for, cellular life. In contrast to the well-studied docking of two already folded proteins, it has been recently established that many proteins are disordered and unfolded in the absence of their partner protein, but appear folded once bound. Must these initially disordered proteins transiently fold in isolation before binding their partners? We examine a small disordered protein and find that interactions with its (already structured) partner protein are what cause the relatively unstructured protein to fold. Thus, the requirement for one protein to fold is not an obstacle for reliable, fast association between two proteins. This result offers some explanation for the abundance of similar protein–protein interactions throughout biology. Protein–protein interactions are at the heart of regulatory and signaling processes in the cell. In many interactions, one or both proteins are disordered before association. However, this disorder in the unbound state does not prevent many of these proteins folding to a well-defined, ordered structure in the bound state. Here we examine a typical system, where a small disordered protein (PUMA, p53 upregulated modulator of apoptosis) folds to an α-helix when bound to a groove on the surface of a folded protein (MCL-1, induced myeloid leukemia cell differentiation protein). We follow the association of these proteins using rapid-mixing stopped flow, and examine how the kinetic behavior is perturbed by denaturant and carefully chosen mutations. We demonstrate the utility of methods developed for the study of monomeric protein folding, including β-Tanford values, Leffler α, Φ-value analysis, and coarse-grained simulations, and propose a self-consistent mechanism for binding. Folding of the disordered protein before binding does not appear to be required and few, if any, specific interactions are required to commit to association. The majority of PUMA folding occurs after the transition state, in the presence of MCL-1. We also examine the role of the side chains of folded MCL-1 that make up the binding groove and find that many favor equilibrium binding but, surprisingly, inhibit the association process.

Annona muricata Linn or usually identified as soursop is a potential anticancer plant that has been widely reported to contain valuable chemopreventive agents known as annonaceous acetogenins. The antiproliferative and anticancer activities of this tropical and subtropical plant have been demonstrated in cell culture and animal studies. A. muricata L. exerts inhibition against numerous types of cancer cells, involving multiple mechanism of actions such as apoptosis, a programmed cell death that are mainly regulated by Bcl-2 family of proteins. Nonetheless, the binding mode and the molecular interactions of the plant’s bioactive constituents have not yet been unveiled for most of these mechanisms. In the current study, we aim to elucidate the binding interaction of ten bioactive phytochemicals of A. muricata L. to three Bcl-2 family of antiapoptotic proteins viz. Bcl-2, Bcl-w and Mcl-1 using an in silico molecular docking analysis software, Autodock 4.2. The stability of the complex with highest affinity was evaluated using MD simulation. We compared the docking analysis of these substances with pre-clinical Bcl-2 inhibitor namely obatoclax. The study identified the potential chemopreventive agent among the bioactive compounds. We also characterized the important interacting residues of protein targets which involve in the binding interaction. Results displayed that anonaine, a benzylisoquinoline alkaloid, showed a high affinity towards the Bcl-2, thus indicating that this compound is a potent inhibitor of the Bcl-2 antiapoptotic family of proteins.

B-cell lymphoma-2 (Bcl-2) family proteins are fundamental regulators of intrinsic cell apoptosis, and overexpression of apoptotic proteins (Bcl-2 and Mcl-1) is a characteristic of many haematological malignancies. Thus, it is necessary to discover novel inhibitors to treat leukemia. In the current study, we synthesized a series of quercetagetin derivatives (compounds 2a–2t, 3a–3j and 4a–4g) and evaluated their anticancer activities on four leukemia cells (U937, K562, K562R and KG-1). Among those synthesized derivatives, compounds 2a exhibited the best antiproliferative activity (IC50 = 0.276, 0.159, 0.312 and 0.271 µM to U937, K562, K562R and KG-1, respectively). In addition, 2a induced apoptosis in K562 and markedly arrested the cell cycle G2/M phase of K562. The Western blot assay showed that 2a is a potential inhibitor that can effectively suppress the expression of Bcl-2 and Mcl-1. The molecular docking study predicted that 2a had firm interactions with the active pockets of Bcl-2 and Mcl-1. Finally, in silico pharmacokinetic evaluation of 2a indicated its potential as an anti-leukemia drug lead in the future.

Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome B act complex shows that the resistance mutations cluster in a pocket surrounding the branch point adenosine, suggesting a competitive mode of action. Collectively, we propose that PHF5A–SF3B1 forms a central node for binding to these splicing modulators. A number of natural occurring small-molecule splicing modulators are known. Here, the authors combine chemogenomic, structural and biochemical methods and show that these compounds also target the spliceosome-associated protein PHF5A and propose a potential modulator binding site in the PHF5A–SF3B1 complex.

Identification of an allosteric mechanism disrupting the antiapoptotic BH3 binding activity of MCL-1 offers a new approach for targeting the apoptotic resistance of human cancers. MCL-1 is an antiapoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. Like BCL-2, MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of proapoptotic BCL-2 family members, a mechanism harnessed by cancer cells to establish formidable apoptotic blockades. Although drugging the BH3-binding groove has been achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small-molecule covalent modification of C286 at a new interaction site distant from the BH3-binding groove. Our structure–function analyses revealed that the BH3 binding capacity of MCL-1 and its suppression of BAX are impaired by molecular engagement, a phenomenon recapitulated by C286W mutagenic mimicry in vitro and in mouse cells. Thus, we characterize an allosteric mechanism for disrupting the antiapoptotic BH3 binding activity of MCL-1, informing a new strategy for disarming MCL-1 in cancer.

Anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1), are often overexpressed in cancer, which aids tumor growth and treatment resistance. As a result, these proteins are excellent candidates for novel anticancer drugs. Within this study a virtual library of betuline derivatives was built and screened for possible Bcl-2, Bcl-XL, and Mcl-1 inhibitors. For every target, molecular docking simulations were performed using two different engines (AutoDock Vina and Glide). The ligands that most frequently appeared among the top candidates were shortlisted after comparing the top-20 hits from both docking scoring functions. To assess binding stability, five of these promising compounds were chosen and run through 100 ns molecular dynamics (MD) simulations in complex with every target protein. Key persistent intermolecular contacts were identified from MD contact frequency histograms, and stability was evaluated using root-mean-square deviation (RMSD) profiles of protein–ligand complexes following equilibration. Additionally, Prime MM-GBSA binding energies (ΔG_bind) for the 15 docked complexes were computed, and ligand efficiency was reported. Two substances, BOxNaf1 and BT3, stood out among the screened derivatives as the most stable binders to all three Bcl-2 family targets according to the dual docking and MD analysis approach. When the MM-GBSA and RMSF/rGyr data are considered alongside docking and MD stability, BOxNaf1 and BOxPhCl1 emerge as the most compelling dual/multi-target candidates, whereas BT3, though MD stable, shows weaker MM-GBSA energetics and is retained as a lower-priority backup chemotype.