No abstract

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.

The expansion factors for the mean-square end-to-end distance and radius of gyration are studied for the helical worm-like (HW) chain with the excluded-volume effects incorporated in the Yamakawa–Stockmayer scheme. In this scheme, approximate closed expressions for them are expressed in terms of the excluded-volume parameter z and the first-order perturbation coefficient K(L) as a function of the total contour length L of the chain. The ring-closure probabilities necessary for the evaluation of K(L) are evaluated by a slight modification of the method previously developed for the Kratky–Porod (KP) worm-like chain. The actual evaluation is carried out for the values of the HW model parameters in their limited ranges, and also for the KP chain. It is then numerically shown that K(L) for the HW chain is approximately equal to that for the KP chain. From a simple analysis, this conclusion may be expected to be generally correct, so that the expansion factors for the HW chain may be expressed in terms of the simple K(L) for the KP chain. The derived closed expressions for them may be regarded as valid for any ordinary flexible chains of arbitrary length.

In order to examine the correlation between stiffness and excluded-volume effects in polymer chains, polymethylene-like rotational isomeric state chains with excluded volume are simulated and their mean-square radii of gyration are evaluated by Monte Carlo methods. The present results and also experimental data for ordinary flexible chains may well be explained by the Kratky–Porod worm-like chain, or more generally the helical worm-like chain, with the excluded-volume effects incorporated in the Yamakawa–Stockmayer scheme. For typical stiff chains with excluded volume, however, there is clear disagreement between their theory and experiment, and the problem remains unsolved.

Small-angle X-ray scattering profiles for an amylose fragment (maltoheptaose) in aqueous solution were observed and compared with the theoretical profiles calculated for an ensemble of chain conformations generated by molecular dynamics simulations and Monte Carlo simulations. The Monte Carlo results based on the disaccharide conformation energy map obtained without explicitly considering surrounding water molecules were in satisfactory agreement with the experimental results, provided that the effective dielectric constant was set to four. In contrast, the results of the fully solvated molecular dynamics simulations performed using the Cff91, Cff, Gromos, Glycam93, and Glycam99 force-fields were unexpectedly different from each other. Among them, Cff91 gave most satisfactory agreement with experiment, but the other fields yielded conformations that were somewhat or highly extended. It was also shown that recently developed Glycam99 is a significant improvement over Glycam 93. The representative snapshots of the two successful simulations resembled the regular helical structure reported by Goldsmith et al. (J. Mol. Biol. 1982, 156, 411). The source of the large force-field dependence was investigated by examining the various Ramachandran-like plots for the glycosidic torsion angles. For comparison, similar plots of ab initio energy for maltose (i.e., a fragment with two glucose units) were also calculated at the Hartree-Fock level, although in a simplified manner. These plots suggest that the extended conformation arises from too strong a preference for a certain rotational isomeric state of the glycosidic linkage. A procedure to remedy this over-preference can be devised, although a need of further elaboration of the force-field is indicated. The significance of force-fields is also illustrated in modeling a cyclodextrin composed of 14 glucose units.