V.I. Vernadsky Crimean Federal University

V.I. Vernadsk y Crimean Federal University. -t9ts..

Group members :. Rukhsar Bano Shah Alam Khan Md-Shadab Alam Varunraj Baskaran Sandeep Bharadhvaj Tejaswini Rangaswamy.

KRAS is a oncogene found on the short arm of chromosome 12. KRAS ( Kirsten rat sarcoma virus) is a gene that provides instructions for making a protein called K-Ras , a part of the RAS/MAPK pathway . The protein relays signals from outside the cell to the cell's nucleus. These signals instruct the cell to grow and divide ( proliferate ) or to mature and take on specialized functions ( differentiate ). It is called KRAS because it was first identified as a viral oncogene in the K irsten RA t S arcoma virus.The oncogene identified was derived from a cellular genome, so KRAS , when found in a cellular genome, is called a proto-oncogene ..

The K-Ras protein is a GTPase , a class of enzymes which convert the nucleotide guanosine triphosphate (GTP) into guanosine diphosphate (GDP) . In this way the K-Ras protein acts like a switch that is turned on and off by the GTP and GDP molecules. To transmit signals, it must be turned on by attaching (binding) to a molecule of GTP. The K-Ras protein is turned off (inactivated) when it converts the GTP to GDP. When the protein is bound to GDP, it does not relay signals to the cell's nucleus..

KRAS is a signal transducer protein , which plays an important role in various cellular signalling events such as in regulation of cell proliferation . It is a critical hub in the cell circuitry, as upon an upstream stimulus it transduces activating signals to several cellular signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway . KRAS cycles between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states . Only in the GTP-bound state, KRAS is able to bind and activate its effector proteins, such as RAF-kinases, PI3K and RalGDS . KRAS itself becomes activated when a guanosine exchange factor (GEF) protein displaces GDP from the nucleotide binding site , resulting eventually in GTP binding, as there is a higher intracellular concentration of GTP than GDP . Inactivation of the active KRAS occurs upon GTP hydrolysis to GDP..

KRAS is the most oncogenic with its 85% share of all mutated RAS proteins observed in cancer , . KRAS missense mutations are particularly frequent in the pancreatic, colorectal and lung cancers (COSMIC v.90) . In cancer, three mutation hotspots: G12, G13 and Q61 are observed in RAS genes. In this regard, KRAS differs from the NRAS and HRAS, as it is the only RAS isoform where the position 12 mutations are predominant . The G domain of KRAS, comprised of residues 1–166 , forms the basis of biological functionality of the GTPase proteins . This domain encompasses six beta-strands, forming the protein core, surrounded by five alpha-helices . In addition to the G domain, KRAS has a flexible C-terminal structural element, named the hypervariable region (HVR), which plays a crucial role in anchoring RAS to the membrane ..

Other important functional elements of KRAS are the switch-regions, so-called switch-I and switch-II. These switches form the binding interface for effector proteins, as well as for RAS regulators (GAPs and GEFs). To point out, several residue definitions are used for the switch regions in the literature, which are rather arbitrary, due to high intrinsic flexibility of these regions. For instance, in switch-II definitions, the beginning falls between residues 58–60 and ends among residues 67–76, excluding or including partially or fully the helix α2. Here, for the illustrative purposes only, a definition of residues 30–40 for switch-I, residues 58–72 for switch-II and residues 10–14 for P-loop is used .The mutation hotspots in cancer are located in P-loop or in switch-II ..

Switch-I Switch-II GDP p-loop $2 P-loop a2 ct5 IB6 ct4 61 1213 10 30 60 HVR 70 MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLLDILDTAG QEEYSAMRDQ 80 90 100 110 120 130 140 YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP 150 160 170 180 188 FIETSAKTRQ GVDDAFYTLV REIRKHKEKM SKDGKKKKKK SKTKCVIM.

The gene KRAS may undergo alternative splicing and thus result in two isoforms: KRAS4A and KRAS4B (also known as isoform 2A and 2B, respectively). These isoforms differ mainly in their HVR residues 167–189, but also residues 151, 153, 165 and 166 are dissimilar. Active KRAS signalling occurs at the membrane. In order to become associated to membrane, KRAS’ membrane anchoring HVR needs to undergo a few post-translational modifications . First, the C-terminal CAAX sequence (CVIM in KRAS4B) is farnesylated at C185, which is followed by proteolytic cleavage of the three terminal residues. Finally, the terminal carboxyl group of C185 is methylated. A polybasic region of the HVR, composed of multiple lysine residues, is also important for the membrane association . As KRAS4A does not contain this polybasic region, it is further palmitoylated at an additional cysteine residue C180 ..

. One of the most common drivers in human cancer is the mutant KRAS protein. Not so long ago KRAS was considered as an undruggable oncoprotein. After a long struggle, however, we finally see some light at the end of the tunnel as promising KRAS targeted therapies are in or approaching clinical trials. In recent years, together with the promising progress in RAS drug discovery, our understanding of KRAS has increased tremendously. This progress has been accompanied with a resurgence of publicly available KRAS structures, which were limited to nine structures less than ten years ago. Furthermore, the ever-increasing computational capacity has made biologically relevant timescales accessible, enabling molecular dynamics (MD) simulations to study the dynamics of KRAS protein in more detail at the atomistic level..

KRAS amplification : Amplification of the KRAS locus and corresponding protein expression was analyzed in 582 gastric adenocarcinomas employing fluorescence in-situ hybridization (FISH) and immunohistochemistry. Amplification status was correlated with clinico -pathological features, clinical outcome and molecular tumor data including a correlation to the TCGA subtypes of gastric carcinoma. KRAS gene can also be amplified in colorectal cancer. Tumors or cell lines harboring this genetic lesion are not responsive to EGFR inhibitors . Although KRAS amplification is an infrequent event in colorectal cancer, it might be responsible for precluding response to anti- EGFR treatment in some patients. Amplification of wild-type Kras has also been observed in ovarian,gastric , uterine, and lung cancers..

G12C mutation : One fairly frequent driver mutation is KRAS G12C which is adjacent a shallow binding site. This has allowed the development of electrophilic KRAS inhibitors that can form irreversible covalent bonds with nucleophilic sulfur atom of Cys-12 and hence selectively target KRAS G12C and leave wild-type KRAS untouched.Two KRAS G12C mutant covalent inhibitors have reached clinical testing: AMG 510 ( Amgen )and MRTX-849 ( Mirati Therapeutics ) while ARS-3248 ( Wellspring Biosciences / Janssen ) has received an investigational new drug (IND) approval to start clinical trials. An antisense oligonucleotide (ASO), AZD4785 ( AstraZeneca / Ionis Therapeutics ) targeting KRAS has completed a phase I study but was discontinued from further development because of insufficient knockdown of the target. G12D mutation : The most common KRAS mutuation is G12D; normally amino acid position 12 of the KRAS protein is glycine but in G12D it is occupied by aspartic acid. Until May 2019 there were no clinical trials targeting the mutuation . The first clinical trial was sponsored by the National Cancer Institute. There is another trial on G12D from Mirati which is seeking investigational new drug (IND) approval in H1:2021 to start clinical trials..

Switch-I Switch-I Switch-II Switch-II G12 G12.