Unlike other methods for docking ligands to the rigid 3D structure of a known protein receptor, Glide approximates a complete systematic search of the conformational, orientational, and positional space of the docked ligand In this search, an initial rough positioning and scoring phase that dramatically narrows the search space is followed by torsionally flexible energy optimization on an OPLS-AA nonbonded potential grid for a few hundred surviving candidate poses The very best candidates are further refined via a Monte Carlo sampling of pose conformation; in some cases, this is crucial to obtaining an accurate docked pose Selection of the best docked pose uses a model energy function that combines empirical and force-field-based terms Docking accuracy is assessed by redocking ligands from 282 cocrystallized PDB complexes starting from conformationally optimized ligand geometries that bear no memory of the correctly docked pose Errors in geometry for the top-ranked pose are less than 1 A in nearly ha

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Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy.

Signal transduction by receptors with tyrosine kinase activity

Mutations in codon 12, 13, or 61 of one of the three ras genes, H-ras, K-ras, and N-ras, convert these genes into active oncogenes. Rapid assays for the detection of these point mutations have been developed recently and used to investigate the role mutated ras genes play in the pathogenesis of human tumors. It appeared that ras gene mutations can be found in a variety of tumor types, although the incidence varies greatly. The highest incidences are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%); in thyroid tumors (50%); and in myeloid leukemia (30%). For some tumor types a relationship may exist between the presence of a ras mutation and clinical or histopathological features of the tumor. There is some evidence that environmental agents may be involved in the induction of the mutations.

https://cancerres.aacrjournals.org/content/canres/49/17/4682.full.pdf

ras Oncogenes in Human Cancer: A Review

The mevalonate pathway produces isoprenoids that are vital for diverse cellular functions, ranging from cholesterol synthesis to growth control. Several mechanisms for feedback regulation of low-density-lipoprotein receptors and of two enzymes involved in mevalonate biosynthesis ensure the production of sufficient mevalonate for several end-products. Manipulation of this regulatory system could be useful in treating certain forms of cancer as well as heart disease.

Regulation of the mevalonate pathway.

Glide's ability to identify active compounds in a database screen is characterized by applying Glide to a diverse set of nine protein receptors. In many cases, two, or even three, protein sites are employed to probe the sensitivity of the results to the site geometry. To make the database screens as realistic as possible, the screens use sets of “druglike” decoy ligands that have been selected to be representative of what we believe is likely to be found in the compound collection of a pharmaceutical or biotechnology company. Results are presented for releases 1.8, 2.0, and 2.5 of Glide. The comparisons show that average measures for both “early” and “global” enrichment for Glide 2.5 are 3 times higher than for Glide 1.8 and more than 2 times higher than for Glide 2.0 because of better results for the least well-handled screens. This improvement in enrichment stems largely from the better balance of the more widely parametrized GlideScore 2.5 function and the inclusion of terms that penalize ligand−protei...

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Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening.

We report that residues Lys-16 and Asp-119 play critical roles in the guanine nucleotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha). Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding. The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10. Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed. By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of [Ala119]Ha to induce changes characteristic of cellular transformation was much greater than that of normal Ha and similar to that of the oncogenic [Val12, Thr59]Ha. In this assay, [Asn16]Ha and [Val12, Asn16, Thr59]Ha were similar in potency to normal Ha. In yeast cells, Ha proteins with reduced nucleotide affinity exert a dominant temperature-dependent lethality that is avoided by the coexpression of the activated yeast ras gene [Ala18, Val19]RAS2. We interpret the biological consequences of reducing the nucleotide affinity of ras proteins in terms of two opposing factors: a growth-promoting effect, resulting from an increase in the GDP-GTP exchange rate, and a growth-limiting effect, resulting from an increase in the nucleotide-free ras protein species.

https://www.pnas.org/content/pnas/83/4/952.full.pdf

Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects

To identify the amino acid residues of the Harvey (Ha) ras-encoded protein that are involved in protein-protein interactions, we have created a series of mutant Ha-ras proteins. In particular, amino acid substitutions have been introduced within two regions, residues 32-42 and 61-80, that are conserved among ras proteins from different species. We observed that amino acid substitutions at positions 35, 36, 38, 40, and, to a lesser extent, 39 and 78 reduce the biological potency of Ha-ras protein in both mammalian and Saccharomyces cerevisiae cells, without noticeably affecting the known intrinsic biochemistry of these proteins. The reduction of in vivo activity for these mutant ras proteins correlates with their reduced ability to stimulate yeast adenylate cyclase. The ras-protein-neutralizing antibody Y13-259 binds to six residues: Glu-63, Ser-65, Ala-66, Met-67, Gln-70, and Arg-73. Single substitutions for these residues reduce Y13-259 antibody binding by at least a factor of 1000 but do not significantly affect biological activity. These data are discussed in terms of the model for Ha-ras protein based on the structure of the elongation factor EF-Tu-GDP complex.

Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21

In cytosolic extracts of bovine brain, we detected ras GTPase activating protein (GAP) activity that stimulated the GTP hydrolytic activity of normal c-Ha-ras p21 but not that of the oncogenic [Val12]p21 variant. GAP was purified 19,500-fold by a five-column procedure involving DEAE-Sephacel, Sepharose 6B, orange dye and green dye matrices, and Mono Q resins. A single major protein band of 125 kDa was observed on NaDodSO4/polyacrylamide gels that correlated with the elution of GAP activity on Mono Q. Purified GAP was devoid of inherent GTP hydrolytic activity, suggesting that it was a regulator of ras intrinsic GTPase activity. Under submaximal velocity conditions, the second-order rate constant of GTP hydrolysis at 24 degrees C for p21-GTP + GAP (4.5 X 10(6) M-1.sec-1) was at least 1000-fold greater than that for [Val12]p21-GTP + GAP (less than 3 X 10(3) M-1.sec-1).

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Purification of ras GTPase activating protein from bovine brain

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

The yeast Saccharomyces cerevisiae contains two functional homologues of the ras oncogene family, RAS1 and RAS2. These genes are required for growth, and all evidence indicates that this essential function is the activation of adenylate cyclase. In contrast, ras in mammalian cells does not appear to influence adenylate cyclase activity. To clarify the relation between ras function in yeast and in higher eukaryotes, and the role played by yeast RAS in growth control, it is necessary to identify functions acting upstream of RAS in the adenylate cyclase pathway. The evidence presented here indicates that CDC25, identified by conditional cell cycle arrest mutations, encodes such an upstream function.

https://science.sciencemag.org/content/sci/235/4793/1218.full.pdf

Jackson B. Gibbs

Papers

Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects

Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21

Purification of ras GTPase activating protein from bovine brain

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae.