In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras–effector interaction

• Published in: Proceedings of the National Academy of Sciences - PNAS 2013-05, Vol.110 (20), p.8182-8187
• Main Authors: Shima, Fumi, Yoshikawa, Yoko, Ye, Min, Araki, Mitsugu, Matsumoto, Shigeyuki, Liao, Jingling, Hu, Lizhi, Sugimoto, Takeshi, Ijiri, Yuichi, Takeda, Azusa, Nishiyama, Yuko, Sato, Chie, Muraoka, Shin, Tamura, Atsuo, Osoda, Tsutomu, Tsuda, Ken-ichiro, Miyakawa, Tomoya, Fukunishi, Hiroaki, Shimada, Jiro, Kumasaka, Takashi, Yamamoto, Masaki, Kataoka, Tohru
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; antineoplastic activity; antineoplastic agents; Antineoplastic Agents - pharmacology; Apoptosis; Binding sites; Biological Sciences; Cancer; Cell Line, Transformed; Cell Line, Tumor; colorectal neoplasms; Computational Biology - methods; Crystal structure; Drug Design; genes; Glutathione Transferase - metabolism; Guanosine Triphosphate - chemistry; Heterologous transplantation; Humans; Inhibitory concentration 50; Mice; Mice, Nude; mitogen-activated protein kinase; Models, Molecular; Molecular Conformation; Molecular interactions; Molecular structure; Molecules; Mutation; Neoplasm Transplantation; NIH 3T3 Cells; nuclear magnetic resonance spectroscopy; oncogene proteins; Oncogenes; Oncology; oral administration; Pharmacology; Protein Binding; Protein Conformation; ras Proteins - antagonists & inhibitors; ras Proteins - metabolism; Signal Transduction; Tumors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1217730110

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras–type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H- ras ᴳ¹²ⱽ–transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras ᴳ¹²ⱽ gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP ᵀ³⁵S, exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.