RESUMO
Small interfering RNAs (siRNAs) can be utilized not only as functional biological research tools but also as therapeutic agents. For the clinical use of siRNA as drugs, various chemical modifications have been used to improve the activity of siRNA drugs, and further chemical modifications are expected to improve the utility of siRNA therapeutics. As the 5' nucleobase of the guide strand affects the interaction between an siRNA and AGO2 and target cleavage activity, structural optimization of this specific position may be a useful strategy for improving siRNA activity. Here, using the in silico model of the complex between human AGO2 MID domain and nucleoside monophosphates, we screened and synthesized an original adenine-derived analog, 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine), that fits better than the natural nucleotide bases into the MID domain of AGO2. Introduction of the 6-mCEPh-purine analog at the 5'-end of the siRNA guide strand significantly enhanced target knockdown activity in both cultured cell lines and in vivo animal models. Our findings can help expand strategies for rationally optimizing siRNA activity via chemical modifications of nucleotide bases.
Assuntos
Adenina/farmacologia , Proteínas Argonautas/genética , Interferência de RNA/efeitos dos fármacos , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/agonistas , Complexo de Inativação Induzido por RNA/agonistas , Adenina/análogos & derivados , Adenina/síntese química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Animais , Apolipoproteína B-100/antagonistas & inibidores , Apolipoproteína B-100/sangue , Apolipoproteína B-100/química , Apolipoproteína B-100/genética , Proteínas Argonautas/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Colesterol/sangue , Células HeLa , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Masculino , Metilação , Camundongos , Camundongos Knockout , Modelos Moleculares , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Uridina Monofosfato/química , Uridina Monofosfato/metabolismoRESUMO
The Hippo signaling pathway regulates cell fate and organ development. In the Hippo pathway, transcriptional enhanced associate domain (TEAD) which is a transcription factor is activated by forming a complex with yes-associated protein 1 (YAP1) or transcriptional coactivator with PDZ-binding motif (TAZ, also called WWTR1). Hyper-activation of YAP1/TAZ, leading to the activation of TEAD, has been reported in many cancers, including malignant pleural mesothelioma (MPM). Therefore, the YAP1/TAZ-TEAD complex is considered a novel therapeutic target for cancer treatment. However, few reports have described YAP1/TAZ-TEAD inhibitors, and their efficacy and selectivity are poor. In this study, we performed a high-throughput screening of a neurofibromin 2 (NF2)-deficient MPM cell line and a large tumor suppressor kinase 1/2 (LATS1/2)-deficient non-small-cell lung cancer cell line using a transcriptional reporter assay. After screening and optimization, K-975 was successfully identified as a potent inhibitor of YAP1/TAZ-TEAD signaling. X-ray crystallography revealed that K-975 was covalently bound to an internal cysteine residue located in the palmitate-binding pocket of TEAD. K-975 had a strong inhibitory effect against protein-protein interactions between YAP1/TAZ and TEAD in cell-free and cell-based assays. Furthermore, K-975 potently inhibited the proliferation of NF2-non-expressing MPM cell lines compared with NF2-expressing MPM cell lines. K-975 also suppressed tumor growth and provided significant survival benefit in MPM xenograft models. These findings indicate that K-975 is a strong and selective TEAD inhibitor with the potential to become an effective drug candidate for MPM therapy.