RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Modelos Biológicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Leucemia/patologia , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Linfoma/patologia , Masculino , Camundongos , Modelos Moleculares , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Pirimidinas/administração & dosagem , Tiofenos/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Following the identification of thieno[2,3-d]pyrimidine-based selective and potent inhibitors of MCL-1, we explored the effect of core swapping at different levels of advancement. During hit-to-lead optimization, X-ray-guided S-N replacement in the core provided a new vector, whose exploration led to the opening of the so-called deep-S2 pocket of MCL-1. Unfortunately, the occupation of this region led to a plateau in affinity and had to be abandoned. As the project approached selection of a clinical candidate, a series of core swap analogues were also prepared. The affinity and cellular activity of these compounds showed a significant dependence on the core structure. In certain cases, we also observed an increased and accelerated epimerization of the atropoisomers. The most potent core replacement analogues showed considerable in vivo PD response. One compound was progressed into efficacy studies and inhibited tumor growth.
RESUMO
FBXO25 is one of the 68 human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of s-phase-kinase associated protein 1, really interesting new gene-box 1, Cullin 1, and F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FAND). Combining two-step affinity purification followed by MS with a classical two-hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, beta-actin, physically interacts through its N-terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FAND, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions.
Assuntos
Proteínas F-Box/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Actinas/análise , Actinas/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Proteínas F-Box/análise , Proteínas F-Box/química , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/química , Proteoma/químicaRESUMO
Reactive oxygen species oxidize proteins and modulate the proteasomal system in muscle-wasting cancer cachexia. On day 5 (D5), day 10 (D10), and day 14 (D14) after tumor implantation, skeletal muscle was evaluated. Carbonylated proteins and thiobarbituric acid reactive substances were measured. Chemiluminescence was employed for lipid hydroperoxide estimation. Glutathione, superoxide dismutase, and total radical antioxidant capacity were evaluated. The proteasomal system was assessed by mRNA atrogin-1 expression. Increased muscle wasting, lipid hydroperoxide, and superoxide dismutase, and decreased glutathione levels and total radical antioxidant capacity, were found on D5 in accordance with increased mRNA atrogin-1 expression. All parameters were significantly modified in animals treated with α-tocopherol. The elevation in aldehylde levels and carbonylated proteins observed on D10 were reversed by α-tocopherol treatment. Oxidative stress may trigger signal transduction of the proteasomal system and cause protein oxidation. These pathways may be associated with the mechanism of muscle wasting that occurs in cancer cachexia.
Assuntos
Carcinoma 256 de Walker/patologia , Músculo Esquelético/patologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Análise de Variância , Animais , Carcinoma 256 de Walker/metabolismo , Glutationa/metabolismo , Masculino , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Improving survival outcomes in adult B-cell acute lymphoblastic leukemia (B-ALL) remains a clinical challenge. Relapsed disease has a poor prognosis despite the use of tyrosine kinase inhibitors (TKIs) for Philadelphia chromosome positive (Ph+ ALL) cases and immunotherapeutic approaches, including blinatumomab and chimeric antigen receptor T cells. Targeting aberrant cell survival pathways with selective small molecule BH3-mimetic inhibitors of BCL-2 (venetoclax, S55746), BCL-XL (A1331852), or MCL1 (S63845) is an emerging therapeutic option. We report that combined targeting of BCL-2 and MCL1 is synergistic in B-ALL in vitro. The combination demonstrated greater efficacy than standard chemotherapeutics and TKIs in primary samples from adult B-ALL with Ph+ ALL, Ph-like ALL, and other B-ALL. Moreover, combined BCL-2 or MCL1 inhibition with dasatinib showed potent killing in primary Ph+ B-ALL cases, but the BH3-mimetic combination appeared superior in vitro in a variety of Ph-like ALL samples. In PDX models, combined BCL-2 and MCL1 targeting eradicated ALL from Ph- and Ph+ B-ALL cases, although fatal tumor lysis was observed in some instances of high tumor burden. We conclude that a dual BH3-mimetic approach is highly effective in diverse models of high-risk human B-ALL and warrants assessment in clinical trials that incorporate tumor lysis precautions.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteínas Proto-Oncogênicas c-bcl-2 , Adulto , Linfócitos B , Linhagem Celular Tumoral , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.
Assuntos
Antineoplásicos/química , Descoberta de Drogas/métodos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Animais , Antineoplásicos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HCT116 , Células HeLa , Humanos , Camundongos , Camundongos SCID , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.
Assuntos
Biomimética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Animais , Antineoplásicos/farmacologia , Quimioterapia Combinada , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fragmentos de Peptídeos , Proteínas Proto-Oncogênicas , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Atrogin-1/MAFbx/FBXO32 is a muscle-specific ubiquitin-ligase (E3) that is dramatically increased in atrophying muscle. Here, we have investigated the functional relationship between atrogin-1 and FBXO25 which shares 65% amino acid identity. Using a RT-PCR, we demonstrated that FBXO25 is highly expressed in brain, kidney, and intestine, whereas atrogin-1 expression is largely restricted to striate muscle. FBXO25 was shown here to contain a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the major components of SCF-type E3s. In addition, the productive SCF complex containing FBXO25 showed ubiquitin ligase activity. We investigated the differential expression of atrogin-1 and FBXO25 in fasted and dexamethasone-treated mice and also in rats with streptozotocin-induced diabetes. Although the atrogin-1 was strongly induced in muscle in all three models, no changes were observed in the expression of FBXO25. Therefore, here we have shown that FBXO25 is a novel F-box protein analogous to atrogin-1, which is not involved in muscle atrophy. Further functional studies should elucidate the exact role of FBXO25 in the ubiquitin-proteasome pathway.
Assuntos
Proteínas F-Box/metabolismo , Proteínas Musculares/metabolismo , Atrofia Muscular/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Proteínas F-Box/química , Proteínas F-Box/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/química , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Ligases SKP Culina F-Box/química , Alinhamento de Sequência , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/químicaRESUMO
The development of BH3 mimetics, which antagonize prosurvival proteins of the BCL-2 family, represents a potential breakthrough in cancer therapy. Targeting the prosurvival member MCL-1 has been an area of intense interest because it is frequently deregulated in cancer. In breast cancer, MCL-1 is often amplified, and high expression predicts poor patient outcome. We tested the MCL-1 inhibitor S63845 in breast cancer cell lines and patient-derived xenografts with high expression of MCL-1. S63845 displayed synergistic activity with docetaxel in triple-negative breast cancer and with trastuzumab or lapatinib in HER2-amplified breast cancer. Using S63845-resistant cells combined with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) technology, we identified deletion of BAK and up-regulation of prosurvival proteins as potential mechanisms that confer resistance to S63845 in breast cancer. Collectively, our findings provide a strong rationale for the clinical evaluation of MCL-1 inhibitors in breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Amplificação de Genes , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Pirimidinas/uso terapêutico , Receptor ErbB-2/genética , Tiofenos/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Docetaxel , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Lapatinib , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Análise de Sobrevida , Taxoides/farmacologia , Taxoides/uso terapêutico , Tiofenos/farmacologia , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/patologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15(-/-) bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation.
Assuntos
Linhagem da Célula , Citocinas/metabolismo , Células Eritroides/citologia , Células Eritroides/metabolismo , Animais , Diferenciação Celular , Citocinas/deficiência , Eritroblastos/metabolismo , Células Eritroides/enzimologia , Eritropoetina/metabolismo , Globinas/metabolismo , Interferons/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosfolipase C gama/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Baço/citologia , Baço/metabolismo , Ubiquitinação , Ubiquitinas/deficiência , Ubiquitinas/metabolismoRESUMO
Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.
Assuntos
Androgênios/fisiologia , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Proteínas Ligases SKP Culina F-Box/biossíntese , Testosterona/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/biossíntese , Androgênios/deficiência , Animais , Western Blotting , Masculino , Camundongos , Microscopia Confocal , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/efeitos dos fármacos , Orquiectomia , Tamanho do Órgão/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Motivo Tripartido , Ubiquitina/metabolismoRESUMO
Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.