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Why is it so hard to develop RAS inhibitors?
What is Ras?Ras is any protein part of the Ras superfamily of proteins related in structure. Ras is a ubiquitous protein expressed in all cells in our body.  Ras proteins are part of a group of proteins termed the small GTPase class. Ras proteins are general involved in cell growth, differentiation, and survival. As you can see this makes the Ras superfamily of proteins the perfect candidate for contributing to cancer growth (unregulated proliferation and growth of cells). Ras specifically is involved in roughly 30% of human cancers[1].Ras proteins are part of complex, multifaceted signal transduction pathways that eventually lead to a these growth and proliferation signals which are transmitted via the nucleus.What exactly does signal transduction involve?Signal transduction is essentially the transmission of a signal from the outside of a cell to the nucleus inside. This signal results in alterations in cell metabolism, gene transcription (protein expression), and/or cell shape.There is generally a large amplification in signal as the agonist (binding signal) initially either binds on the outside of a cell membrane (as is the case for large hydrophilic molecules that will not be able to cross the hydrophobic phospholipid cell membrane) or on the inside of the cell (as is the case for hydrophobic steroid molecules that can cross the membrane with ease). This agonist causes a signal to be transmitted all the way to the nucleus. A signal agonist can cause an amplification in signal up to a million fold with ease.These pathways can be executed through a myriad of different cell receptors and pathways, some with higher levels of complexities and amplifications than others. For the purpose of our discussion, there are two main types of receptors: GPCR (G-protein coupled receptors) and RTK (receptor tyrosine kinases). The latter are much more heavily involved in growth, cell differentiation, etc. Both are involved in Ras activation.After the initial agonist binding to the receptor there is a few steps that are required for this signal to be transmitted which different slightly based on GPCR/RTK activation:Ligand -- Receptor -- Transducer/Effector -- Effector -- 2nd Messenger/Protein Kinase1) Ligand (agonist) binds to receptor. This can be done with an extra or intracellular receptor as previously described depending on the hydrophilic or hydrophobic characteristics of the ligand. In GPCRs this is a simple protein receptor, but with RTKs there can be an enzymatic activation of two protein receptors that causes them to link together and activate themselves and form one enzymatically linked receptor.2) Receptor sends the message to the transducer/effector, which then binds some sort of messenger to send the signal along. This is usually done by the phosphorylation of a protein that will carry this message forward. The point is that an activation must occur. Ras is activated by a protein called GEF and inactivated by a protein called GAP. This activation is rerather quickly in normal circumstances. In GPCRs this involves activation of a three subunit protein, whereas in RTKs it involves adaptor proteins that recruit the next protein required in the pathway.3) The transducer/effector (in this case Ras) sends the message to the last step of the pathway before the signal reaches the nucleus, the protein kinase pathway. This pathway (in this case the Raf/MEK/ERK pathway, also known as the MAP KKK pathway) will transduce the signal to the nucleus.Where do the problems arise?As you know, Ras is a small GTPase class of protein, and is a part of the signal transduction pathway which activates the protein kinase pathway and leads to the signal ultimately being transmitted. This activation is initially carried out through the GPCR or RTK and is turned off through a negative feedback loop from the products of the Ras pathway at the receptor/adaptor level. This normally stops the activation of the transducer/effector Ras.The problem arises when mutations in Ras occur - and they occur quite often in cancer patients. Once a mutation in Ras has occurred, it becomes permanently turned on and the pathway does not cease to stop. Even if the adaptor proteins or receptors receive the signal to stop activity it does not matter because Ras itself has mutated to decrease its own GTPase activity. As the exchange of GTP for GDP and the subsequent GTP hydrolysis stops due to the mutation, GTP stays bound to the Ras protein and unregulated cell proliferation and growth ensues. Not only does GTPase activity decrease, but the Ras protein loses sensitivity to GAP (one of the regular inhibitors of Ras)[2][3][4] and is exposed to a larger quantity of GEF (one of the regular activators of Ras through GTP binding)[5][6]. Thus, we can say that the problems arise through 3 main mechanisms:1) Mutations in the Ras protein itself that lead to decrease GTPase activity, and therefore a constantly bound GTP molecule and permanent activation of Ras.2) Overactivation of the wild-type protein that leads to the production of GEF.3) Loss of GAP function due to the decreased sensitivity of the Ras protein.So why can't we inhibit Ras once it has mutated?Pharmaceutical companies have been spending billions of dollars in trying to inhibit Ras once it has been mutated. However, these companies have had only minimal success, if any at all. The first approach has been taken in regards to the disrupting the Ras to GTP binding, but none have succeeded.1) Using competitive inhibitors for the enzymatic active site of Ras. This actually backfired because any molecule that could function to do this would actually further impede any potential GTPase activity. This inactivity gives Ras its oncogenic activity in the first place. Regardless, the only molecules that have been found to achieve this function are clostridial cytotoxins (a type of bacterial toxin), and these toxins work enzymatically to create a Ras protein that is covalently modified, causing Ras to become resistant to GAP entirely. Using an molecule to do the opposite, creating an agonist, would be very difficult (restoring GAP sensitivity or GTPase activity) and it has been shown that it may not be feasible at all[7].2) Developing a drug in attempt to displace GTP from Ras. This has proven a dead end road due despite its promising nature. Due to the high kinetic affinity between the binding of Ras to GTP, the ATP binding site drug competitors on the market cannot compete (micro molar vs nano molar affinities). Disrupting the Ras to GTP high affinity binding has been characterized as "undruggable" in a recent 2022 study[8]. There is still enthusiastic chatter in this arena but so far no one has further succeeded in this task.Some have also tried to disrupt the upstream GEF activator protein (SOS in the previous image)[9][10]. However, nothing yet has been developed to show major promise.This has left us with the absence of an obvious molecule on the level of Ras to target. The next focus has been involved in the construction of Ras itself.Post-translation covalent protein modifications are essential to the formation of Ras, and so it has been suggested that it may be a good target to go after the tools involved in those modifications - specifically farnesyl transferase. Theoretically, disruption of the proteins post-translational modifications would result in a non-functional protein. Furthermore, it was hypothesized that the presence of inactive Ras would act as an inhibitor to previously active Ras signalling through the sequestering of molecules to the proper sub cellular locations involved in its signalling pathways.This approach has involved using farnesyl transferase inhibitors (FTIs). A couple of these FTIs actually made it all the way to phase 3 clinic trials after showing promising results before it was shown to lack the anticipated anticancer activity[11].So why did this not work? Most likely because they drugs were developed using a mutated version of an isoform of Ras. The belief was that all isoforms of Ras would respond similarly, but that unfortunately is not the case. It has been shown that the active in vivo isoforms of Ras become geranylgeranylated in cells in the presence of FTIs, which allows them to bypass the action of the FTIs and develop fully. Nothing further has been developed in this area, but it is possible that FTIs will produce benefits in the future.Another focus has been on Ras at the expression level. In other words, scientists have been trying to reduce the expression of particularly harmful isoforms.Targets for this approach include promoters of specific isoforms of Ras such as the guanine-rich sequence forming a G-quadruplex structure which has been confirmed in human DNA[12]. Many drugs are already available on the market which can bind and stabilize these G-quadruplex DNA structures and can regulate expression of Ras in vitro. However, regulatory activity of these structures can be negative or positive in vivo depending on the isoform of Ras. Also the sequences that are compatible with these structures are widespread within human DNA. Therefore it is unlikely that this will pra proper mechanism for stopping Ras.Pieces of regulatory RNA called microRNAs (specifically microRNA-622) are also being tested which have been shown to decrease specific isoforms of Ras. It is unknown how efficacious this path will turn out to beThe last major focus has been with the protein kinase pathway. Inactivating the MAP KKK pathway you have previously learned about would cause the Ras signal to cease before it could cause any damage. A major problem with this approach is that it is so far downstream from the source of the problem.Since the oncogenic signal from Ras is transmitted through multiple pathways it has been speculated that this will not prove to be very effective[14][15][16][17][18][19][20]. Despite this, FDA approval of one of these inhibitors (trametinib) has been granted. Another MAP KKK inhibitor, MEK162, has shown promising results also in melanoma patients[13].For this approach to be effective the MAP KKK signally pathway would have to predominate the signal transduction pathway from Ras. Another problem with this approach is that resistance could easily emergence with these inhibitors.There are other newer methods being developed of Ras, but it's unclear if any will prove to be useful at this point. Scientists are continually still working to stop Ras and will most likely continue to do so until they find a way as the benefits of such a discover would have a very high payoff for humanity.What happens if we do block Ras?There will still be complications even if we do find a way to simply block the actions of Ras, as Ras is involved in widespread functions in the human body.To give one example, it has been shown that Ras is part of an important pathway for synaptic remodelling in the human brain (the very process that underlies memory)[21]. Mutations in regulators of Ras in neutrons are found in patients with non syndromic mental retardation[22]. There is also evidence that the H-Ras isoform is involved in control of synaptic plasticity[23].This is just one more example in the challenges that we still have yet to face in blocking Ras.References:A. T. Baines, D. Xu, and C. J. Der, “Inhibition of Ras for cancer treatment: the search continues,” Future Medicinal Chemistry, vol. 3, no. 14, pp. 1787–1808, 2022. J. B. Gibbs, I. S. Sigal, M. Poe, and E. M. Scolnick, “Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules,” Proceedings of the National Academy of Sciences of the United States of America, vol. 81, no. 18, pp. 5704–5708, 1984.G. Bollag and F. McCormick, “Differential regulation of rasGAP and neurofibromatosis gene product activities,” Nature, vol. 351, no. 6327, pp. 576–579, 1991.U. Krengel, I. Schlichting, A. Scherer et al., “Three-dimensional strucutures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules,” Cell, vol. 62, no. 3, pp. 539–548, 1990.K. Zhang, A. G. Papageorge, and D. R. Lowy, “Mechanistic aspects of signaling through Ras in NIH 3T3 cells,” Science, vol. 257, no. 5070, pp. 671–674, 1992.R. R. Mattingly and I. G. Macara, “Phosphorylatlon-dependent activation of the Ras-GRF/CDC25(Mm) exchange factor by muscarinic receptors and G-protein?? subunits,” Nature, vol. 382, no. 6588, pp. 268–272, 1996.K. Scheffzek, M. R. Ahmadian, W. Kabsch et al., “The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic ras mutants,” Science, vol. 277, no. 5324, pp. 333–338, 1997.T. Tanaka and T. H. Rabbitts, “Interfering with RAS-effector protein interactions prevent RAS-dependent tumour initiation and causes stop-start control of cancer growth,” Oncogene, vol. 29, no. 45, pp. 6064–6070, 2010.T. Maurer, L. S. Garrenton, A. Oh et al., “Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 14, pp. 5299–5304, 2012.Q. Sun, J. P. Burke, J. Phan et al., “Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation,” Angewandte Chemie International Edition, vol. 51, pp. 6140–6143, 2012.A. M. Tsimberidou, C. Chandhasin, and R. Kurzrock, “Farnesyltransferase inhibitors: where are we now?” Expert Opinion on Investigational Drugs, vol. 19, no. 12, pp. 1569–1580, 2010.G. Biffi, D. Tannahill, J. McCafferty, and S. Balasubramanian, “Quantitative visualization of DNA G-quadruplex structures in human cells,” Nature Chemistry, vol. 5, pp. 182–186, 2013.P. A. Ascierto, D. Schadendorf, C. Berking et al., et al., “MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study,” The Lancet Oncology, vol. 14, pp. 249–256, 2013.K. R. Stengel and Y. Zheng, “Essential role of Cdc42 in Ras-induced transformation revealed by gene targeting,” PLoS ONE, vol. 7, Article ID e37317, 2012.N. Mitin, K. L. Rossman, and C. J. Der, “Signaling interplay in ras superfamily function,” Current Biology, vol. 15, no. 14, pp. R563–R574, 2005.A. V. Patel, D. Eaves, W. J. Jessen et al., et al., “Ras-driven transcriptome analysis identifies aurora kinase A as a potential malignant peripheral nerve sheath tumor therapeutic target,” Clinical Cancer Research, vol. 18, pp. 5020–5030, 2012.S. Eser, N. Reiff, M. Messer et al., et al., “Selective requirement of PI3K/PDK1 signaling for Kras oncogene-driven pancreatic cell plasticity and cancer,” Cancer Cell, vol. 23, pp. 406–420, 2013.H. Y. Chow, A. M. Jubb, J. N. Koch et al., et al., “p21-Activated kinase 1 is required for efficient tumor formation and progression in a Ras-mediated skin cancer model,” Cancer Research, vol. 72, pp. 5966–5975, 2012.R. E. Menard and R. R. Mattingly, “Cell surface receptors activate p21-activated kinase 1 via multiple Ras and PI3-kinase-dependent pathways,” Cellular Signalling, vol. 15, no. 12, pp. 1099–1109, 2003.Q. Li and R. R. Mattingly, “Restoration of E-cadherin cell-cell junctions requires both expression of E-cadherin and suppression of ERK MAP kinase activation in ras-transformed breast epithelial cells,” Neoplasia, vol. 10, no. 12, pp. 1444–1458, 2008.E. J. Weeber and J. D. Sweatt, “Molecular neurobiology of human cognition,” Neuron, vol. 33, no. 6, pp. 845–848, 2022. L. B. Rosen, D. D. Ginty, M. J. Weber, and M. E. Greenberg, “Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras,” Neuron, vol. 12, no. 6, pp. 1207–1221, 1994.K. A. Rauen, “HRAS and the Costello syndrome,” Clinical Genetics, vol. 71, no. 2, pp. 101–108, 2007.
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