Abstract 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 BH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl‐1. In contrast, Bim BH3 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.
Promotion of Mcl-1 protein binding to the ubiquitin ligase Mule to catalyze its ubiquitination could enhance an apoptotic signaling pathway in cancer cells. In their Communication on page 14250 ff. Z. Zhang and co-workers identify a compound that zips these two proteins together by driving a non-helical QRN motif on Mc1-1 to adopt a helical conformation. Promotion of Mcl-1 protein binding to the ubiquitin ligase Mule to catalyze its ubiquitination could enhance an apoptotic signaling pathway in cancer cells. In their Communication on page 14250 ff. Z. Zhang and co-workers identify a compound that zips these two proteins together by driving a non-helical QRN motif on Mc1-1 to adopt a helical conformation. Lipid Bilayers Regulatory RNAs Two-Dimensional Materials
Abstract 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 1 H, 15 N 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 μ M , respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.
Abstract 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 BH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl‐1. In contrast, Bim BH3 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.
Eine verstärkte Bindung von Mcl-1 mit Mule könnte dessen Ubiquitinierung katalysieren und einen apoptotischen Signalweg in Krebszellen verstärken. In ihrer Zuschrift auf S. 14462 ff. identifizieren Z. Zhang und Mitarbeiter eine Verbindung, die diese beiden Proteine aneinander bindet, indem sie ein nichthelikales QRN-Motiv auf Mcl-1 in eine helikale Konformation überführt. Eine verstärkte Bindung von Mcl-1 mit Mule könnte dessen Ubiquitinierung katalysieren und einen apoptotischen Signalweg in Krebszellen verstärken. In ihrer Zuschrift auf S. 14462 ff. identifizieren Z. Zhang und Mitarbeiter eine Verbindung, die diese beiden Proteine aneinander bindet, indem sie ein nichthelikales QRN-Motiv auf Mcl-1 in eine helikale Konformation überführt. Lipid-Doppelschichten Regulatorische RNA Zweidimensionale Materialien
Background and Purpose Although the ongoing clinical trials of ABT‐263 and ABT‐199 in chronic lymphocytic leukaemia (CLL) have indicated that BH3 mimetics hold considerable promise, understanding the mechanism of CLL resistance to BH3 mimetics remains a challenge. Experimental Approach The LD 50 values of ABT‐737, ABT‐263 and ABT‐199 in a number of primary CLL cells from 40 patients, were determined. The levels of Bcl‐2 family proteins, including phosphorylated Bcl‐2 (pBcl‐2) and their interactions were measured by immunoblotting and co‐immunoprecipitation. In vitro binding assays were performed by isothermal titration calorimetry and ELISA. BH3 profiling in isolated mitochondria was analysed. Key Results The ratio of (Mcl‐1 + pBcl‐2) to Bcl‐2 expression provided the most significant predictive marker for the cytotoxic potential of ABT‐737, ABT‐263 and ABT‐199 in the panel of CLL samples. Mechanistically, pBcl‐2 inhibited the effects of the ABT compounds on the displacement of Bax and Bim from Bcl‐2, thereby suppressing mitochondrial apoptosis. The ABT compounds exhibited 100–300‐fold lower binding affinity to the glutamic acid, phosphomimetic, mutant of Bcl‐2 (T69E, S70E and S87E; EEE‐Bcl‐2). BH3 peptides exhibited different rank orders of binding affinities to full‐length WT‐Bcl‐2 and full‐length EEE‐Bcl‐2. Conclusions and Implications Our study suggested that a structural alteration in the BH3‐binding groove was induced by phosphorylation of Bcl‐2. Our data also provided a framework to overcome resistance of CLL cells to the ABT compounds by combining pBcl‐2 kinase inhibitors with the ABT compounds.
We have previously reported a small‐molecule two‐face Bim BH3 mimetic, 2,3‐dihydroxy‐6‐(4‐isopropylphenylthio)anthracene‐9,10‐dione ( 1 ). Herein, we linked a polyphenol fragment, which was deconstructed from compound 1 , with a drug‐derived building block gained from computer‐aided molecular design. 2‐Phenyl‐1 H ‐benzo[ d ]imidazole as a new scaffold for two‐face Bim mimetics was developed; based on this, a series of Mcl‐1/Bcl‐2 dual inhibitors were obtained. The most potent compound 6d binds to Mcl‐1 and Bcl‐2 with K i values of 127 and 607 nM, respectively, and effectively induces apoptosis in a dose‐dependent, mechanism‐based manner in multiple cancer cell lines.
Abstract 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 μ M by ITC; IC 50 =0.16 μ M by ELISA), as well as nonphosphorylated Bcl‐2 (npBcl‐2; K d =0.38 μ M ; IC 50 =0.12 μ M ). However, ABT‐737 and S1 had much weaker affinities to EEE‐Bcl‐2 (IC 50 =1.43 and >10 μ M , respectively), compared with npBcl‐2 (IC 50 =0.011 and 0.74 μ M , respectively). The allosteric effect on BH3 binding groove by Bcl‐2 phosphorylation in the loop region was illustrated for the first time.
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