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.

Ras family small GTPases assume two interconverting conformations, "inactive" state 1 and "active" state 2, in their GTPbound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5-(␤,␥-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the 31 P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.Small GTPases Ras (H-Ras, K-Ras, and N-Ras) are the products of the ras proto-oncogenes and presumed to be some of the most promising targets for anti-cancer drug development because of their high frequency of mutational activation in a variety of human cancers (1). Ras functions as a molecular switch by cycling between GTP-bound active and GDP-bound inactive forms in intracellular signaling pathways controlling cell growth and differentiation. Conversion between the GDPbound and the GTP-bound forms is controlled by guanine nucleotide exchange factors and GTPase-activating proteins (2, 3). Ras comprises the Ras family of small GTPases together with a number of its relatives, including Rap1, Rap2, R-Ras, R-Ras2/ TC1, M-Ras/R-Ras3, etc. (1). X-ray crystallographic and NMR analyses of H-Ras and Rap1A, alone or in complex with their effectors, revealed that the exchange of GTP for GDP results in allosteric conformational changes in two adjacent regions, termed switch I (residues 32-38) and switch II (residues 60 -75), and enables Ras to execute downstream signaling through direct interaction with its effectors, such as Raf kinases and phosphoinositide 3-kinases (2, 3). Recent 31 P NMR spectroscopic studies on Ras unveiled its novel structural feature, the conformational dynamics in the GTP-bound form (4). H-Ras and K-Ras in complex with Mg 2ϩ and a non-hydrolyzable GT...

Ras small GTPases undergo dynamic equilibrium of two interconverting conformations, state 1 and state 2, in the GTPbound forms, where state 2 is recognized by effectors, whereas physiological functions of state 1 have been unknown. Limited information, such as static crystal structures and 31 P NMR spectra, was available for the study of the conformational dynamics. Here we determine the solution structure and dynamics of state 1 by multidimensional heteronuclear NMR analysis of an HRasT35S mutant in complex with guanosine 5-(␤, ␥-imido)-triphosphate (GppNHp). The state 1 structure shows that the switch I loop fluctuates extensively compared with that in state 2 or H-Ras-GDP. Also, backbone 1 H, 15 N signals for state 2 are identified, and their dynamics are studied by utilizing a complex with c-Raf-1. Furthermore, the signals for almost all the residues of H-Ras⅐GppNHp are identified by measurement at low temperature, and the signals for multiple residues are found split into two peaks corresponding to the signals for state 1 and state 2. Intriguingly, these residues are located not only in the switch regions and their neighbors but also in the rigidly structured regions, suggesting that global structural rearrangements occur during the state interconversion. The backbone dynamics of each state show that the switch loops in state 1 are dynamically mobile on the picosecond to nanosecond time scale, and these mobilities are significantly reduced in state 2. These results suggest that multiconformations existing in state 1 are mostly deselected upon the transition toward state 2 induced by the effector binding.Small GTPases H-Ras, K-Ras, and N-Ras, collectively called Ras, are the products of the ras proto-oncogenes and function as molecular switches by cycling between the GTP-bound active and the GDP-bound inactive forms in intracellular signaling pathways controlling proliferation, differentiation, and apoptosis of cells. GTP hydrolysis on Ras is markedly stimulated by GTPase-activating proteins, whereas conversion from the GDP-bound form to the GTP-bound form is promoted by guanine nucleotide exchange factors (1, 2). Ras comprise the Ras family of small GTPases together with a number of its relatives including Rap1, Rap2, R-Ras, R-Ras2/TCL, M-Ras/RRas3, RalA, RalB, etc. (3). Structural studies of Ras showed that structural differences between the GDP-and GTP-bound forms universally exist in two flexible regions, called switch I (residues 32-38 in H-Ras) and switch II (residues 60 -75 in H-Ras) (1). GTP-sensitive orientation of the switch regions enables Ras to interact with their effectors such as Raf kinases and phosphoinositide 3-kinases (2). Recent 31 P NMR studies suggested that H-Ras in the nucleoside triphosphate form exists in equilibrium between two kinds of conformational states, state 1 and state 2, around the phosphate groups of GTP or its non-hydrolyzable analogues, GppNHp 3 and GTP␥S, bound to the protein (4 -6). This conformational heterogeneity has been commonly observed in a number of Ras homolo...

Although some members of Ras family small GTPases, including M-Ras, share the primary structure of their effector regions with Ras, they exhibit vastly different binding properties to Ras effectors such as c-Raf-1. We have solved the crystal structure of M-Ras in the GDP-bound and guanosine 5-(␤,␥-imido)triphosphate (Gpp(NH)p)-bound forms. The overall structure of MRas resembles those of H-Ras and Rap2A, except that M-Ras-Gpp(NH)p exhibits a distinctive switch I conformation, which is caused by impaired intramolecular interactions between Thr-45 (corresponding to Thr-35 of H-Ras) of the effector region and the ␥-phosphate of Gpp(NH)p. Previous 31 P NMR studies showed that HRas-Gpp(NH)p exists in two interconverting conformations, states 1 and 2. Whereas state 2 is a predominant form of H-Ras and corresponds to the "on" conformation found in the complex with effectors, state 1 is thought to represent the "off " conformation, whose tertiary structure remains unknown.31 P NMR analysis shows that free M-Ras-Gpp(NH)p predominantly assumes the state 1 conformation, which undergoes conformational transition to state 2 upon association with c-Raf-1. These results indicate that the solved structure of M-Ras-Gpp(NH)p corresponds to the state 1 conformation. The predominance of state 1 in M-Ras is likely to account for its weak binding ability to the Ras effectors, suggesting the importance of the tertiary structure factor in small GTPase-effector interaction. Further, the first determination of the state 1 structure provides a molecular basis for developing novel anti-cancer drugs as compounds that hold Ras in the state 1 "off " conformation.

Mammalian Ras proteins regulate multiple effectors including Raf, Ral guanine nucleotide dissociation stimulator (RalGDS), and phosphoinositide 3-kinase. In the nematode Caenorhabditis elegans, LIN-45 Raf has been identified by genetic analyses as an effector of LET-60 Ras. To search for other effectors in C. elegans, we performed a yeast two-hybrid screening for LET-60-binding proteins. The screening identified two cDNA clones encoding a phosphoinositide-specific phospholipase C (PI-PLC) with a predicted molecular mass of 210 kDa, designated PLC210. PLC210 possesses two additional functional domains unseen in any known PI-PLCs. One is the C-terminal Ras-associating domain bearing a structural homology with those of RalGDS and AF-6. This domain, which could be narrowed down to 100 amino acid residues, associated in vitro with human Ha-Ras in a GTP-dependent manner and competed with yeast adenylyl cyclase for binding Ha-Ras. The binding was abolished by specific mutations within the effector region of Ha-Ras. The other functional domain is the N-terminal CDC25-like domain, which possesses a structural homology to guanine nucleotide exchange proteins for Ras. These results strongly suggest that PLC210 belongs to a novel class of PI-PLC, which is a putative effector of Ras.