Computational design of an apoptogenic protein that binds BCL-xL and MCL-1 simultaneously and potently.
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ABSTRACT:
SUBMITTER: Kim S
PROVIDER: S-EPMC9218148 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-x(L)/Bcl-2 dependence. Surprisingly, analysis of Mcl-1-dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-x(L) in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.
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Project description:Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques--yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis--to elucidate specificity determinants for binding to Bcl-x(L)versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x(L) selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x(L), Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x(L)-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x(L) binders.
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Project description:Informatics and computational design methods were used to create new molecules that could potentially bind antiapoptotic proteins, thus promoting death of cancer cells. Apoptosis is a cellular process that leads to the death of damaged cells. Its malfunction can cause cancer and poor response to conventional chemotherapy. After being activated by cellular stress signals, proapoptotic proteins bind antiapoptotic proteins, thus allowing apoptosis to go forward. An excess of antiapoptotic proteins can prevent apoptosis. Designed molecules that mimic the roles of proapoptotic proteins can promote the death of cancer cells. The goal of our study was to create new putative mimetics that could simultaneously bind several antiapoptotic proteins. Five new small molecules were designed that formed stable complexes with BCL-2, BCL-XL, and MCL-1 antiapoptotic proteins. These results are novel because, to our knowledge, there are not many, if any, small molecules known to bind all three proteins. Drug-likeness studies performed on the designed molecules, as well as previous experimental and preclinical studies on similar agents, strongly suggest that the designed molecules may indeed be promising drug candidates. All five molecules showed "drug-like" properties and had overall drug-likeness scores between 81% and 96%. A single drug based on these mimetics should cost less and cause fewer side effects than a combination of drugs each aimed at a single protein. Computer-based molecular design promises to accelerate drug research by predicting potential effectiveness of designed molecules prior to laborious experiments and costly preclinical trials.
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Project description:An estimated 40,000 deaths will be attributed to breast cancer in 2016, underscoring the need for improved therapies. Evading cell death is a major hallmark of cancer, driving tumor progression and therapeutic resistance. To evade apoptosis, cancers use antiapoptotic Bcl-2 proteins to bind to and neutralize apoptotic activators, such as Bim. Investigation of antiapoptotic Bcl-2 family members in clinical breast cancer datasets revealed greater expression and more frequent gene amplification of MCL1 as compared with BCL2 or BCL2L1 (Bcl-xL) across three major molecular breast cancer subtypes, Luminal (A and B), HER2-enriched, and Basal-like. While Mcl-1 protein expression was elevated in estrogen receptor α (ERα)-positive and ERα-negative tumors as compared with normal breast, Mcl-1 staining was higher in ERα+ tumors. Targeted Mcl-1 blockade using RNAi increased caspase-mediated cell death in ERα+ breast cancer cells, resulting in sustained growth inhibition. In contrast, combined blockade of Bcl-2 and Bcl-xL only transiently induced apoptosis, as cells rapidly acclimated through Mcl-1 upregulation and enhanced Mcl-1 activity, as measured in situ using Mcl-1/Bim proximity ligation assays. Importantly, MCL1 gene expression levels correlated inversely with sensitivity to pharmacologic Bcl-2/Bcl-xL inhibition in luminal breast cancer cells, whereas no relationship was seen between the gene expression of BCL2 or BCL2L1 and sensitivity to Bcl-2/Bcl-xL inhibition. These results demonstrate that breast cancers rapidly deploy Mcl-1 to promote cell survival, particularly when challenged with blockade of other Bcl-2 family members, warranting the continued development of Mcl-1-selective inhibitors for targeted tumor cell killing.Implications: Mcl-1 levels predict breast cancer response to inhibitors targeting other Bcl-2 family members, and demonstrate the key role played by Mcl-1 in resistance to this drug class. Mol Cancer Res; 15(3); 259-68. ©2016 AACR.
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Project description:Malignant melanoma is one of the most difficult cancers to treat due to its resistance to chemotherapy. Despite recent successes with BRAF inhibitors and immune checkpoint inhibitors, many patients do not respond or become resistant to these drugs. Hence, alternative treatments are still required. Due to the importance of the BCL-2-regulated apoptosis pathway in cancer development and drug resistance, it is of interest to establish which proteins are most important for melanoma cell survival, though the outcomes of previous studies have been conflicting. To conclusively address this question, we tested a panel of established and early passage patient-derived cell lines against several BH3-mimetic drugs designed to target individual or subsets of pro-survival BCL-2 proteins, alone and in combination, in both 2D and 3D cell cultures. None of the drugs demonstrated significant activity as single agents, though combinations targeting MCL-1 plus BCL-XL, and to a lesser extent BCL-2, showed considerable synergistic killing activity that was elicited via both BAX and BAK. Genetic deletion of BFL-1 in cell lines that express it at relatively high levels only had minor impact on BH3-mimetic drug sensitivity, suggesting it is not a critical pro-survival protein in melanoma. Combinations of MCL-1 inhibitors with BRAF inhibitors also caused only minimal additional melanoma cell killing over each drug alone, whilst combinations with the proteasome inhibitor bortezomib was more effective in multiple cell lines. Our data show for the first time that therapies targeting specific combinations of BCL-2 pro-survival proteins, namely MCL-1 plus BCL-XL and MCL-1 plus BCL-2, could have significant benefit for the treatment of melanoma.
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Project description:Inhibition of anti-apoptotic Mcl-1 is a promising anticancer strategy to overcome the survival and chemoresistance of a broad spectrum of human cancers. We previously reported on the identification of a natural product marinopyrrole A (1) that induces apoptosis in Mcl-1-dependent cells through Mcl-1 degradation. Here, we report the design and synthesis of novel marinopyrrole-based analogs and their evaluation as selective inhibitors of Mcl-1 as well as dual Mcl-1/Bcl-xL inhibitors. The most selective Mcl-1 antagonists were 34, 36 and 37 with 16-, 13- and 9-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding, respectively. Among the most potent dual inhibitors is 42 which inhibited Mcl-1/Bim and Bcl-xL/Bim binding 15-fold (IC50 = 600 nM) and 33-fold (500 nM) more potently than (±)-marinopyrrole A (1), respectively. Fluorescence quenching, NMR analysis and molecular docking indicated binding of marinopyrroles to the BH3 binding site of Mcl-1. Several marinopyrroles potently decreased Mcl-1 cellular levels and induced caspase 3 activation in human breast cancer cells. Our studies provide novel "lead" marinopyrroles for further optimization as selective Mcl-1 inhibitors and dual Mcl-1 and Bcl-xL inhibitors.
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Project description:Although Bcl-xL and Mcl-1, two antideath Bcl-2 members, have similar, flexible binding sites, they can achieve high binding selectivity to endogenous binding partners and synthetic small-molecule inhibitors. Here, we employed molecular dynamic (MD) simulations and hotspot analysis to investigate the conformational flexibility of these proteins and their binding hotspots at the binding sites. Backbone flexibility analyses indicate that the highest degree of flexibility in Mcl-1 is the ?4 helical segment as opposed to the ?3 helix in Bcl-xL among four helical segments in their binding sites. Furthermore, common and unique binding hotspots at both proteins were identified using small-molecule probes. These analyses can aid the design of potent and specific small-molecule inhibitors for these proteins.
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Project description:Most patients with chronic lymphocytic leukemia (CLL) initially respond to targeted therapies, but eventually relapse and develop resistance. Novel treatment strategies are therefore needed to improve patient outcomes. Here, we performed direct drug testing on primary CLL cells and identified synergy between eight different mitogen-activated protein kinase kinase (MEK) inhibitors and the B-cell lymphoma 2 (Bcl-2) antagonist venetoclax. Drug sensitivity was independent of immunoglobulin heavy-chain gene variable region (IGVH) and tumor protein p53 (TP53) mutational status, and CLL cells from idelalisib-resistant patients remained sensitive to the treatment. This suggests that combined MEK/Bcl-2 inhibition may be an option for high-risk CLL. To test whether sensitivity could be detected in other B-cell malignancies, we performed drug testing on cell line models of CLL (n = 4), multiple myeloma (MM; n = 8), and mantle cell lymphoma (MCL; n = 7). Like CLL, MM cells were sensitive to the MEK inhibitor trametinib, and synergy was observed with venetoclax. In contrast, MCL cells were unresponsive to MEK inhibition. To investigate the underlying mechanisms of the disease-specific drug sensitivities, we performed flow cytometry-based high-throughput profiling of 31 signaling proteins and regulators of apoptosis in the 19 cell lines. We found that high expression of the antiapoptotic proteins myeloid cell leukemia-1 (Mcl-1) or B-cell lymphoma-extra large (Bcl-xL) predicted low sensitivity to trametinib + venetoclax. The low sensitivity could be overcome by combined treatment with an Mcl-1 or Bcl-xL inhibitor. Our findings suggest that MEK/Bcl-2 inhibition has therapeutic potential in leukemia and myeloma, and demonstrate that protein expression levels can serve as predictive biomarkers for treatment sensitivities.
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Project description:It has been widely accepted that mitochondria-dependent apoptosis initiates when select BH3-only proteins (BID, BIM, etc.) directly engage and allosterically activate effector proteins BAX/BAK. Here, through reconstitution of cells lacking all eight pro-apoptotic BH3-only proteins, we demonstrate that all BH3-only proteins primarily target the anti-apoptotic BCL-2 proteins BCL-xL/MCL-1, whose simultaneous suppression enables membrane-mediated spontaneous activation of BAX/BAK. BH3-only proteins' apoptotic activities correlate with affinities for BCL-xL/MCL-1 instead of abilities to directly activate BAX/BAK. Further, BID and BIM do not distinguish BAX from BAK or accelerate BAX/BAK activation following inactivation of BCL-xL/MCL-1. Remarkably, death ligand-induced apoptosis in cells lacking BH3-only proteins and MCL-1 is fully restored by BID mutants capable of neutralizing BCL-xL, but not direct activation of BAX/BAK. Taken together, our findings provide a "Membrane-mediated Permissive" model, in which the BH3-only proteins only indirectly activate BAX/BAK by neutralizing the anti-apoptotic BCL-2 proteins, and thus allowing BAX/BAK to undergo unimpeded, spontaneous activation in the mitochondrial outer membrane milieu, leading to apoptosis initiation.
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Project description:During neurogenesis, proliferating neural precursor cells (NPC) exit the cell cycle and differentiate into postmitotic neurons. The proteins that regulate cell survival through the stages of differentiation, however, are still poorly understood. Here, we examined the roles of the anti-apoptotic Bcl-2 proteins, Mcl-1 and Bcl-xL, in promoting survival as cells progress through the stages of neurogenesis in the mouse embryonic central nervous system. We used Nestin-mediated, nervous system-specific conditional deletion of mcl-1, bcl-x or both to identify their distinct and overlapping roles. Individual conditional deletion of mcl-1 (MKO) and bcl-x (BKO) suggested sequential roles in promoting cell survival during developmental neurogenesis. In the MKO embryo, apoptosis begins at embryonic day 10 (E10) in the proliferating NPC population throughout the entire developing nervous system. In the BKO embryo, apoptosis begins later at E11 within the postmitotic neuron populations. In the double (mcl-1 and bcl-x) conditional knockout (DKO), cell death extended throughout both proliferating and non-proliferating cell populations resulting in embryonic lethality at E12, earlier than in either the MKO or BKO. Apoptotic cell death of the entire central nervous system in the DKO demonstrates that both genes are necessary for cell survival during developmental neurogenesis. To determine whether Mcl-1 and Bcl-xL have overlapping anti-apoptotic roles during neurogenesis, we examined the impact of gene dosage. Loss of a single bcl-x allele in the MKO embryo exasperated apoptotic cell death within the NPC population revealing a novel anti-apoptotic role for Bcl-xL in proliferating NPCs. Cells were rescued from apoptosis in both the MKO and BKO embryos by breeding with the Bax null mouse line indicating that Mcl-1 and Bcl-xL have a common pro-apoptotic target during developmental neurogenesis. Taken together, these findings demonstrate that Mcl-1 and Bcl-xL are the two essential anti-apoptotic Bcl-2 proteins required for the survival of the developing mammalian nervous system.
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