RESUMO
Tissue repair and regenerative medicine address the important medical needs to replace damaged tissue with functional tissue. Most regenerative medicine strategies have focused on delivering biomaterials and cells, yet there is the untapped potential for drug-induced regeneration with good specificity and safety profiles. The Hippo pathway is a key regulator of organ size and regeneration by inhibiting cell proliferation and promoting apoptosis. Kinases MST1 and MST2 (MST1/2), the mammalian Hippo orthologs, are central components of this pathway and are, therefore, strong target candidates for pharmacologically induced tissue regeneration. We report the discovery of a reversible and selective MST1/2 inhibitor, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide (XMU-MP-1), using an enzyme-linked immunosorbent assay-based high-throughput biochemical assay. The cocrystal structure and the structure-activity relationship confirmed that XMU-MP-1 is on-target to MST1/2. XMU-MP-1 blocked MST1/2 kinase activities, thereby activating the downstream effector Yes-associated protein and promoting cell growth. XMU-MP-1 displayed excellent in vivo pharmacokinetics and was able to augment mouse intestinal repair, as well as liver repair and regeneration, in both acute and chronic liver injury mouse models at a dose of 1 to 3 mg/kg via intraperitoneal injection. XMU-MP-1 treatment exhibited substantially greater repopulation rate of human hepatocytes in the Fah-deficient mouse model than in the vehicle-treated control, indicating that XMU-MP-1 treatment might facilitate human liver regeneration. Thus, the pharmacological modulation of MST1/2 kinase activities provides a novel approach to potentiate tissue repair and regeneration, with XMU-MP-1 as the first lead for the development of targeted regenerative therapeutics.
Assuntos
Fator de Crescimento de Hepatócito/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Sulfonamidas/farmacologia , Acetaminofen/toxicidade , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colite/induzido quimicamente , Colite/prevenção & controle , Cristalização , Fator de Crescimento de Hepatócito/química , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/transplante , Ensaios de Triagem em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lesão Pulmonar/tratamento farmacológico , Camundongos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/química , Medicina Regenerativa , Serina-Treonina Quinase 3 , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/química , Pesquisa Translacional BiomédicaRESUMO
The role of the unfolded protein response (UPR) in tissue homeostasis remains largely unknown. Here we find that loss of Mst1/2, the mammalian Hippo orthologues, or their regulator WW45, leads to a remarkably enlarged endoplasmic reticulum (ER) size-associated UPR. Intriguingly, attenuation of the UPR by tauroursodeoxycholic acid (TUDCA) diminishes Mst1/2 mutant-driven liver overgrowth and tumorigenesis by promoting nuclear exit and degradation of Hippo downstream effector Yap. Yap is required for UPR activity and ER expansion to alleviate ER stress. During the adaptive stage of the UPR, PERK kinase-eIF2α axis activates Yap, while prolonged ER stress-induced Hippo signalling triggers assembly of the GADD34/PP1 complex in a negative feedback loop to inhibit Yap and promote apoptosis. Significantly, the deregulation of UPR signals associated with Yap activation is found in a substantial fraction of human hepatocellular carcinoma (HCC). Thus, we conclude Yap integrates Hippo and UPR signalling to control liver size and tumorigenesis.
Assuntos
Carcinogênese/patologia , Neoplasias Hepáticas/patologia , Fígado/crescimento & desenvolvimento , Fígado/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Resposta a Proteínas não Dobradas , Fator 6 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Carcinogênese/efeitos dos fármacos , Proteínas de Ciclo Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Hep G2 , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Humanos , Neoplasias Hepáticas/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Tamanho do Órgão/efeitos dos fármacos , Fosfoproteínas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina-Treonina Quinase 3 , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Proteínas de Sinalização YAPRESUMO
The transcriptional coactivator Yes-associated protein (YAP) plays an important role in organ-size control and tumorigenesis. However, how Yap gene expression is regulated remains unknown. This study shows that the Ets family member GABP binds to the Yap promoter and activates YAP transcription. The depletion of GABP downregulates YAP, resulting in a G1/S cell-cycle block and increased cell death, both of which are substantially rescued by reconstituting YAP. GABP can be inactivated by oxidative mechanisms, and acetaminophen-induced glutathione depletion inhibits GABP transcriptional activity and depletes YAP. In contrast, activating YAP by deleting Mst1/Mst2 strongly protects against acetaminophen-induced liver injury. Similar to its effects on YAP, Hippo signaling inhibits GABP transcriptional activity through several mechanisms. In human liver cancers, enhanced YAP expression is correlated with increased nuclear expression of GABP. Therefore, we conclude that GABP is an activator of Yap gene expression and a potential therapeutic target for cancers driven by YAP.