Enzymes最新文献

The Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS, Salirasib®) interferes with Ras membrane interactions that are crucial for Ras-dependent signaling and cellular transformation. FTS had been successfully evaluated in clinical trials of cancer patients. Interestingly, its effect is mediated by targeting Ras chaperones that serve as key coordinators for Ras proper folding and delivery, thus offering a novel target for cancer therapy. The development of new FTS analogs has revealed that the specific modifications to the FTS carboxyl group by esterification and amidation yielded compounds with improved growth inhibitory activity. When FTS was combined with additional therapeutic agents its activity toward Ras was significantly augmented. FTS should be tested not only in cancer but also for genetic diseases associated with abnormal Ras signaling, as well as for various inflammatory and autoimmune disturbances, where Ras plays a major role. We conclude that FTS has a great potential both as a safe anticancer drug and as a promising immune modulator agent.

The Ras-like superfamily of low molecular weight GTPases is made of five major families (Arf/Sar, Rab, Ran, Ras, and Rho), highly conserved across evolution. This is in keeping with their roles in basic cellular functions (endo/exocytosis, vesicular trafficking, nucleocytoplasmic trafficking, cell signaling, proliferation and apoptosis, gene regulation, F-actin dynamics), whose alterations are associated with various types of diseases, in particular cancer, neurodegenerative, cardiovascular, and infectious diseases. For these reasons, Ras-like pathways are of great potential in therapeutics and identifying inhibitors that decrease signaling activity is under intense research. Along this line, guanine exchange factors (GEFs) represent attractive targets. GEFs are proteins that promote the active GTP-bound state of GTPases and represent the major entry points whereby extracellular cues are converted into Ras-like signaling. We previously developed the yeast exchange assay (YEA), an experimental setup in the yeast in which activity of a mammalian GEF can be monitored by auxotrophy and color reporter genes. This assay was further engineered for medium-throughput screening of GEF inhibitors, which can readily select for cell-active and specific compounds. We report here on the successful identification of inhibitors against Dbl and CZH/DOCK-family members, GEFs for Rho GTPases, and on the experimental setup to screen for inhibitors of GEFs of the Arf family. We also discuss on inhibitors developed using virtual screening (VS), which target the GEF/GTPase interface with high efficacy and specificity. We propose that using VS and YEA in combination may represent a method of choice for identifying specific and cell-active GEF inhibitors.

Ras is a hub protein in signal transduction pathways leading to the control of cell proliferation, migration, and survival and a major target for drug discovery due to the presence of its mutants in about 20% of human cancers. Yet, the discovery of small molecules that can directly interfere with its function has been elusive in spite of intense efforts. This is most likely due to its highly flexible nature and the lack of a well-ordered active site. This chapter contains a discussion of our current understanding of conformational states in Ras-GTP, with focus on a recently discovered allosteric switch mechanism that may promote intrinsic hydrolysis of GTP in the presence of Raf. We discuss the manner in which small molecules are known to affect the equilibrium of states in Ras-GTP and suggest novel strategies to go forward in the search for inhibitors of this master signaling protein.

Oncogenic mutations in the Ras (rat sarcoma) protein lead to a permanent activation of the Ras pathway and are found in approximately 30% of all human tumors. During signal transduction, Ras is transiently activated by GTP binding and interacts with effector proteins such as Raf kinase. Ras complexed with GTP (T) occurs in at least two conformational states, states 1(T) and 2(T), where state 2(T) represents the true effector-interaction state and state 1(T) has only a low affinity for effectors. Stabilization of state 1(T) by small molecules such as metal-cyclens can reduce the affinity for effectors and thus it can lead to an interruption of the signal transduction chain. Metal-cyclens bind inside the nucleotide-binding pocket to GTP, shifting the conformational equilibrium of Ras toward state 1(T). In contrast, Zn(2+)-BPA (bis(2-picolyl)amine) binds outside the nucleotide-binding pocket but nevertheless allosterically stabilizes state 1(T) and thus inhibits Raf interaction. It shows a higher affinity for the oncogenic mutant Ras(G12V) than for wild type in contrast to other compounds such as Zn(2+)-cyclen.

Oncogenic mutant K-Ras is highly prevalent in multiple human tumors. Despite significant efforts to directly target Ras activity, no K-Ras-specific inhibitors have been developed and taken into the clinic. Since Ras proteins must be anchored to the inner leaflet of the plasma membrane (PM) for full biological activity, we devised a high-content screen to identify molecules with ability to displace K-Ras from the PM. Here we summarize the biochemistry and biology of three classes of compound identified by this screening method that inhibit K-Ras PM targeting: staurosporine and analogs, fendiline, and metformin. All three classes of compound significantly abrogate cell proliferation and Ras signaling in K-Ras-transformed cancer cells. Taken together, these studies provide an important proof of concept that blocking PM localization of K-Ras is a tractable therapeutic target.

Inhibition of oncogenic Ras activation through small molecules is a promising approach to the pharmacologic treatment of human tumors. A common strategy to block Ras activation and signal transduction is based on molecules that interfere with the guanine exchange factors (GEF)-promoted nucleotide exchange. We developed several generations of small molecules active in inhibiting Ras activation at low micromolar concentrations. Some of these compounds are more active on cell lines expressing oncogenic Ras than on normal cells and are therefore good hit compounds for anticancer drug development. The molecules belonging to the last generation are soluble in water and allowed the identification of binding site on Ras by means of NMR experiments in deuterated water. The experimentally-determined Ras-binding site comprises residues belonging to the α-2 helix and the β-3 strand of the central β-sheet in the Switch 2 region. Synthetic molecules bind Ras in a region belonging to the more extended Ras/GEF-binding site, and a possible mechanism of Ras inhibition by these compounds can be the blockade of GEF-mediated nucleotide exchange.

MCP compounds were developed with the idea to inhibit RAS/RAF interaction. They were identified by carrying out high-throughput screens of chemical compounds for their ability to inhibit RAS/RAF interaction in the yeast two-hybrid assay. A number of compounds including MCP1, MCP53, and MCP110 were identified as active compounds. Their inhibition of the RAS signaling was demonstrated by examining RAF and MEK activities, phosphorylation of ERK as well as characterizing their effects on events downstream of RAF. Direct evidence for the inhibition of RAS/RAF interaction was obtained by carrying out co-IP experiments. MCP compounds inhibit proliferation of a wide range of human cancer cell lines. Combination studies with other drugs showed that MCP compounds synergize with MAPK pathway inhibitors as well as with microtubule-targeting chemotherapeutics. In particular, a strong synergy with paclitaxel was observed. Efficacy to inhibit tumor formation was demonstrated using mouse xenograft models. Combination of MCP110 and paclitaxel was particularly effective in inhibiting tumor growth in a mouse xenograft model of colorectal carcinoma.

The Ras superfamily G-proteins are monomeric proteins of approximately 21kDa that act as a molecular switch to regulate a variety of cellular processes. The structure of the Ras superfamily G-proteins, their regulators as well as posttranslational modification of these proteins leading to their membrane association have been elucidated. The Ras superfamily G-proteins interact at their effector domains with their downstream effectors via protein-protein interactions. Mutational activation or overexpression of the Ras superfamily G-proteins has been observed in a number of human cancer cases. Over the years, a variety of approaches to inhibit the Ras superfamily G-proteins have been developed. These different approaches are discussed in this volume.

Small G proteins of the Rho family and their activators the guanine nucleotide exchange factors (RhoGEFs) regulate essential cellular functions and their deregulation has been associated with an amazing variety of human disorders, including cancer, inflammation, vascular diseases, and mental retardation. Rho GTPases and RhoGEFs therefore represent important targets for inhibition, not only in basic research but also for therapeutic purposes, and strategies to inhibit their function are actively being sought. Our lab has been very active in this field and has used the peptide aptamer technology to develop the first RhoGEF inhibitor, using the RhoGEF Trio as a model. Trio function has been described mainly in cell motility and axon growth in the nervous system via Rac1 GTPase activation, but recent findings suggest it to play also a role in the aggressive phenotype of various cancers, making it an attractive target for drug discovery. The object of this chapter is to demonstrate that targeting a RhoGEF using the peptide aptamer technology represents a valid and efficient approach to inhibit cellular processes in which Rho GTPase activity is upregulated. This is illustrated here by the first description of a peptide inhibitor of the oncogenic RhoGEF Tgat, TRIP(E32G), which is functional in vivo. On a long-term perspective, these peptide inhibitors can also serve as therapeutic tools or as guides for the discovery of small-molecule drugs, using an aptamer displacement screen.