Riluzole-induced apoptosis in osteosarcoma is mediated through Yes-associated protein upon phosphorylation by c-Abl Kinase.

Our lab has previously demonstrated Riluzole to be an effective drug in inhibiting proliferation and inducing apoptosis in both human and mouse osteosarcoma. Yes-associated protein is a transcription co-activator, known to be involved in cell proliferation or apoptosis depending on its protein partner. In the present study we investigated the role of YAP in apoptosis in osteosarcoma, we hypothesized that YAP may be activated by Riluzole to induce apoptosis in osteosarcoma. By knocking down the expression of YAP, we have demonstrated that Riluzole failed to induce apoptosis in YAP deficient osteosarcoma cells. Riluzole caused translocation of YAP from the cytoplasm to the nucleus, indicating YAP's role in apoptosis. Both Riluzole-induced phosphorylation of YAP at tyrosine 357 and Riluzole-induced apoptosis were blocked by inhibitors of c-Abl kinase. In addition, knockdown of c-Abl kinase prevented Riluzole-induced apoptosis in LM7 cells. We further demonstrated that Riluzole promoted interaction between YAP and p73, while c-Abl kinase inhibitors abolished the interaction. Subsequently, we demonstrated that Riluzole enhanced activity of the Bax promoter in a luciferase reporter assay and enhanced YAP/p73 binding on endogenous Bax promoter in a ChIP assay. Our data supports a novel mechanism in which Riluzole activates c-Abl kinase to regulate pro-apoptotic activity of YAP in osteosarcoma.

Application of Structure-based Methods to Analyze Resistance Mutations for Chemically Diverse Non-Nucleoside Reverse Transcriptase Inhibitors.

BACKGROUND: Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are used in combination with antiretroviral therapy to suppress viral loads in HIV patients. The chemical design of NNRTIs has changed in recent years in response to resistance-associated mutations (RAMs) and resistance. NNRTIs are chemically diverse compounds that bind an allosteric site of HIV RT. Resistance- associated mutations (RAMs) identified in HIV patients are associated with NNRTI resistance. RAMs confer amino acid changes that alter both structural and physiochemical properties of the allosteric site. Ultimately, these changes reduce NNRTI affinity. Previously, we used a combination of computational and experimental methods to analyze and validate RAMs for 3 diarylpyrimidine (DAPY) NNRTIs. OBJECTIVE: The objective of this study is to apply these methods to other chemically diverse, non- DAPY NNRTIs. MATERIALS AND METHODS: We selected MIV-150 (experimental microbicide) and doravirine for this study. A computational and molecular modeling strategy was used to evaluate the effects of RAMs. Calculated changes in drug affinity and stability (ΔS + ΔA) were used to determine overall resistance levels: susceptible, low, intermediate, and high. The ΔS + ΔA values for K101P suggest that this mutation confers intermediate/high-level resistance to MIV-150, but remains susceptible to doravirine. Based on the determined resistance levels, we analyzed the models and used Molecular Dynamics (MD) to compare the interactions of MIV-150/doravirine with RT wild-type (WT) and RT (K101P). From MD, we found that key interactions were lost with RT (K101P), but were retained with doravirine. To experimentally validate our findings, we conducted a fluorescence-based reverse transcription assay for MIV-150 with RT (WT) and RT (K101P). IC50 values determined in assays showed a 101-fold change in potency for MIV-150, but essentially no change for doravirine. RESULTS: Our computational and experimental results are also consistent with antiviral data reported in the literature. CONCLUSION: We believe that this approach is effective for analyzing mutations to determine resistance profiles for chemically diverse NNRTIs in development.