RESUMO
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.
RESUMO
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 (Kd =0.38 µM by ITC; IC50 =0.16 µM by ELISA), as well as nonphosphorylated Bcl-2 (npBcl-2; Kd =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.
Assuntos
Apoptose/efeitos dos fármacos , Descoberta de Drogas , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Células HEK293 , Células HL-60 , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
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-1H-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.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Biomimética/métodos , Desenho de Fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neoplasias/metabolismo , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/síntese química , Proteínas Proto-Oncogênicas/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desenho Assistido por Computador , Relação Dose-Resposta a Droga , Células HL-60 , Humanos , Concentração Inibidora 50 , Células K562 , Células MCF-7 , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/patologia , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de TempoRESUMO
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 (Ki 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.
Assuntos
Acridinas/química , Proteínas Reguladoras de Apoptose/química , Materiais Biomiméticos/química , Proteínas de Membrana/química , Peptídeos/química , Proteínas Proto-Oncogênicas/química , Acridinas/síntese química , Proteína 11 Semelhante a Bcl-2 , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/metabolismo , Simulação de Acoplamento Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
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.
Assuntos
Modelos Biológicos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ressonância de Plasmônio de Superfície , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Etoposídeo/química , Etoposídeo/farmacologia , Humanos , Cinética , Ligantes , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Células Tumorais CultivadasRESUMO
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 LD50 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.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/metabolismo , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fragmentos de Peptídeos , Fosforilação , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Paclitaxel is an alternative chemotherapeutic agent for chronic myelogenous leukemia (CML) when primary or secondary resistance of tyrosine kinase inhibitors (TKI) is emerging, because paclitaxel could bypass the apoptotic deficiencies linked to p53 and fas ligand pathways in CML. However, high levels of Bcl-2 family proteins in CML could resist paclitaxel-induced apoptosis. Herein, we utilized two BH3 mimetics ABT-737 and S1 to study the potential of BH3 mimetics in combination with paclitaxel in treatment of CML cells and illustrated the mechanism by which BH3 mimetics synergize with paclitaxel. As a single agent, S1 could induce apoptosis in CML-derived cell line K562, whereas ABT-737 was largely ineffective. However, both of the two agents could efficiently synergize with paclitaxel through intrinsic apoptosis pathway. By using Bcl-2 siRNA, Bcl-XL siRNA or Mcl-1 siRNA, we found although each of the three members exhibited activities to block paclitaxel-induced apoptosis, Mcl-1 was the determinant for the synergistic effect between paclitaxel and ABT-737 or S1. Furthermore, paclitaxel/ABT737 synergized to drastically upregulate Bim to displace Bak from Mcl-1, whereas S1 directly binds Mcl-1 to release both Bim and Bak. As such, ABT-737 and S1 sensitized CML to paclitaxel by Mcl-1 inhibition, indirect inhibition through Bim antagonizing Mcl-1, or direct inhibition through binding to Mcl-1 itself. Finally, activation of JNK/Bim pathway was identified as the apical mechanism for ABT-737/paclitaxel synergism. Together, our results demonstrated potent synergy between BH3 mimetics and paclitaxel in the killing of CML cells and revealed an important role for Mcl-1 in mediating synergism by these agents.