RESUMO
The ubiquitination of the hypoxia-inducible factor-1α (HIF-1α) is mediated by interacting with the von Hippel-Lindau protein (VHL), and is associated with cancer, chronic anemia, and ischemia. VHL, an E3 ligase, has been reported to degrade HIF-1 for decades, however, there are few successful inhibitors currently. Poor understanding of the binding pocket and a lack of in-depth exploration of the interactions between two proteins are the main reasons. Hence, we developed an effective strategy to identify and design new inhibitors for protein-protein interaction targets. The hydroxyproline (Hyp564) of HIF-1α contributed the key interaction between HIF-1α and VHL. In this study, detailed information of the binding pocket were explored by alanine scanning, site-directed mutagenesis and molecular dynamics simulations. Interestingly, we found the interaction(s) between Y565 and H110 played a key role in the binding of VHL/HIF-1α. Based on the interactions, 8 derivates of VH032, 16a-h, were synthesized by introducing various groups bounded to H110. Further assay on protein and cellular level exhibited that 16a-h accessed higher binding affinity to VHL and markable or modest improvement in stabilization of HIF-1α or HIF-1α-OH in HeLa cells. Our work provides a new orientation for the modification or design of VHL/HIF-1α protein-protein interaction inhibitors.
Assuntos
Desenho de Fármacos , Hidroxiprolina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Proteína Supressora de Tumor Von Hippel-Lindau/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Hidroxiprolina/síntese química , Hidroxiprolina/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Researches have revealed several stressors, which could activate unfolded protein response (UPR) in cells. However, the survival or death pathway was determined by the duration of UPR exposure. Based on the UPR mediated death pathway, our study was aimed to investigate role of UPR on c-Jun N-terminal kinase (JNK)/activator protein-1 (Ap-1) signal transduction in diindolylmethane (DIM) treated ovarian cancer cell lines. Activation of UPR proteins, UPR mediated apoptotic signaling proteins and expression level of EpCAM, JNK, Ap-1, Caspase-3 and Bcl-2 were measured. Protein and gene expression, transcription factor activity, and protein phosphorylation were measured using standard molecular biology techniques. Our results demonstrated DIM treatment had significantly increased the expression of Endoplasmic reticulum (ER) stress regulators such as Bip, IRE1, CHOP and activation of UPR related apoptotic proteins in ovarian cancer cells. Decreased expression of EpCAM and activity of AP-1 transcription factor were observed in DIM treated cells. The pharmacologic inhibitors of the JNK signal transduction pathway, suggest that the impact of EpCAM expression on AP-1 transcription factor activity is mediated through the JNK pathway. Taken together, these results suggest that UPR mediated JNK/Ap-1 signal transduction has a significant role in the regulation of apoptosis in human ovarian cancer cells, and is a potential molecular target to enhance sensitivity of ovarian cancer to chemotherapy.