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.

Self-maintenance of the shoot apical meristem (SAM), from which aerial organs are formed throughout the life cycle, is crucial in plant development. Class I Knotted1-like homeobox (KNOX) genes restrict cell differentiation and play an indispensable role in maintaining the SAM. However, the mechanism that positively regulates their expression is unknown. Here, we show that expression of a rice (Oryza sativa) KNOX gene, Oryza sativa homeobox1 (OSH1), is positively regulated by direct autoregulation. Interestingly, loss-of-function mutants of OSH1 lose the SAM just after germination but can be rescued to grow until reproductive development when they are regenerated from callus. Double mutants of osh1 and d6, a loss-of-function mutant of OSH15, fail to establish the SAM both in embryogenesis and regeneration. Expression analyses in these mutants reveal that KNOX gene expression is positively regulated by the phytohormone cytokinin and by KNOX genes themselves. We demonstrate that OSH1 directly binds to five KNOX loci, including OSH1 and OSH15, through evolutionarily conserved cis-elements and that the positive autoregulation of OSH1 is indispensable for its own expression and SAM maintenance. Thus, the maintenance of the indeterminate state mediated by positive autoregulation of a KNOX gene is an indispensable mechanism of self-maintenance of the SAM.

Gene expression throughout the reproductive process in rice (Oryza sativa) beginning with primordia development through pollination/fertilization to zygote formation was analyzed. We analyzed 25 stages/organs of rice reproductive development including early microsporogenesis stages with 57,381 probe sets, and identified around 26,000 expressed probe sets in each stage. Fine dissection of 25 reproductive stages/organs combined with detailed microarray profiling revealed dramatic, coordinated and finely tuned changes in gene expression. A decrease in expressed genes in the pollen maturation process was observed in a similar way with Arabidopsis and maize. An almost equal number of ab initio predicted genes and cloned genes which appeared or disappeared coordinated with developmental stage progression. A large number of organ-/stage-specific genes were identified; notably 2,593 probe sets for developing anther, including 932 probe sets corresponding to ab initio predicted genes. Analysis of cell cycle-related genes revealed that several cyclin-dependent kinases (CDKs), cyclins and components of SCF E3 ubiquitin ligase complexes were expressed specifically in reproductive organs. Cell wall biosynthesis or degradation protein genes and transcription factor genes expressed specifically in reproductive stages were also newly identified. Rice genes homologous to reproduction-related genes in other plants showed expression profiles both consistent and inconsistent with their predicted functions. The rice reproductive expression atlas is likely to be the most extensive and most comprehensive data set available, indispensable for unraveling functions of many specific genes in plant reproductive processes that have not yet been thoroughly analyzed.

In flowering plants, knotted1-like homeobox (KNOX) transcription factors play crucial roles in establishment and maintenance of the shoot apical meristem (SAM), from which aerial organs such as leaves, stems, and flowers initiate. We report that a rice (Oryza sativa) KNOX gene Oryza sativa homeobox1 (OSH1) represses the brassinosteroid (BR) phytohormone pathway through activation of BR catabolism genes. Inducible overexpression of OSH1 caused BR insensitivity, whereas loss of function showed a BR-overproduction phenotype. Genome-wide identification of loci bound and regulated by OSH1 revealed hormonal and transcriptional regulation as the major function of OSH1. Among these targets, BR catabolism genes CYP734A2, CYP734A4, and CYP734A6 were rapidly upregulated by OSH1 induction. Furthermore, RNA interference knockdown plants of CYP734A genes arrested growth of the SAM and mimicked some osh1 phenotypes. Thus, we suggest that local control of BR levels by KNOX genes is a key regulatory step in SAM function.