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

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.

Design and synthesis of 8-octyl-benzolactam-V9, a selective activator for protein kinase C epsilon and eta.

Conventional (alpha, betaI, betaII, gamma) and novel (delta, epsilon, eta, theta) protein kinase C (PKC) isozymes are main targets of tumor promoters, such as phorbol esters and indolactam-V (ILV). We have recently found that 1-hexyl derivatives of indolinelactam-V (2, 3), in which the indole ring of ILV was replaced with the indoline ring, showed a binding preference for novel PKCs over conventional PKCs. To develop a new ILV analogue displaying increased synthetic accessibility and improved binding selectivity for novel PKCs, we have designed 8-octyl-benzolactam-V9 (4), a simple analogue without the pyrrolidine moiety of 2 and 3. Compound 4 showed significant binding selectivity for isolated C1B domains of novel PKCs. Moreover, 4 translocated PKC epsilon and eta from the cytoplasm to the plasma membrane of HeLa cells at 1 microM, whereas other PKC isozymes did not respond even at 10 microM. These results indicate that 4 could be a selective activator for PKC epsilon and eta.

Indolactam-V is involved in the CH/pi interaction with Pro-11 of the PKCdelta C1B domain: application for the structural optimization of the PKCdelta ligand.

The CH/pi interaction between the indole ring of indolactam-V (IL-V) and the hydrogen atom at position 4 of Pro-11 of the PKCdelta C1B domain was evaluated using the mutant peptide of the PKCdelta C1B domain, in which the CH/pi interaction was inhibited by substitution of the hydrogen atom with a fluorine atom. IL-V showed about a 10 times lower binding affinity to the mutant peptide compared to the wild-type peptide, suggesting that the CH/pi interaction could play a pivotal role in the binding of IL-V to the PKCdelta C1B domain. On the other hand, benzolactam-V8 (BL-V8), with the benzene ring instead of the indole ring of IL-V, might lack the CH/pi interaction. The low binding affinity of BL-V8 could be enhanced by the effective formation of the CH/pi interaction as exemplified by the synthesis of naphtholactam-V8 (NL-V8).

Synthesis and binding selectivity of 7- and 15-decylbenzolactone-V8 for the C1 domains of protein kinase C isozymes.

Benzolactone-V8 (4) is a lactone analogue of the artificial tumor promoter benzolactam-V8 (1). To investigate the effect of hydrophobic substituents at positions 7 and 15 of 4 on binding selectivity for protein kinase C (PKC) isozymes, 7- and 15-decylbenzolactone-V8 (7, 8) were synthesized and their binding affinities for synthetic PKC isozyme C1 peptides were examined. Compound 8 showed moderate selectivity for novel PKC isozymes similar to 9-decylbenzolactone-V8 (5), while 7 was less selective. Compounds 7 and 8 showed no significant selectivity among novel PKC isozymes unlike 8-decylbenzolactone-V8 (6). These results indicate that the introduction of a hydrophobic substituent at position 8 of 4 is most effective in the development of PKC epsilon- and PKCeta-selective binders.