Analysis of RAS Subfamily involved in Cancer via in silico Approach and Designing of Potent Drug against Cancer

Author Name(s): *Anushree Srivastava, Amita Kashyap
Author Email: anushree.srivastava02@gmail.com

Abstract

RAS subfamily belongs to a class of proteins called small GTPase, which are a group of hydrolase enzymes that can bind (‘on state’) and hydrolyze (‘off state’) guanosine triphosphate (GTP). Hence, RAS proteins act as binary molecular switches and play a consequential role in intracellular signal transduction which regulates processes such as actin cytoskeletal integrity, proliferation, differentiation, cell adhesion, apoptosis, and cell migration. Upon mutation oncogenic forms of RAS proteins beget overactive signaling inside the cell, which ultimately leads to cancer. Therefore, discerning the functionality of RAS proteins, and the identification of inhibitors that block the mutated RAS proteins, may lead a successful step towards cancer therapy. The present bio-computational analysis was performed using web-based tools and servers. Multiple sequence alignment of selected human RAS subfamily proteins with other homologous sequences revealed highly conserved regions. The present work determined the physico-chemical properties of selected RAS proteins such as their hydrophilic nature; alpha–helical structure; close evolutionary relationship with higher vertebrates. Functional sites and domains within the protein were analyzed and the Protein-protein interaction study was made that revealed the functional association of RAS proteins with several effector proteins. The target site on the receptor protein was predicted based on the mutation analysis and the best binding site was selected. Comparative analysis of the binding affinity between the receptor protein and final screened chemical ligands was performed using Docking analysis, which determined Trimetrexate as the best ligand acting against mutation in HRAS, NRAS, KRAS and DIRAS1 proteins due to its lowest docking energy. The present work supports Trimetrexate as potential chemical compound to block oncogenic RAS activity.

Keywords

Cancer, Drug-designing, Proteomic analysis, RAS subfamily proteins.

Introduction

Cancer is abnormal growth of cells caused by multiple changes in gene expression leading to deregulated balance of cell proliferation and cell death, ultimately evolving into population of cells that can invade tissues and metastasize to distant sites causing significant morbidity. The characteristic that delineates the malignant cancer from a benign tumor are the abilities to invade locally to spread to regional lymph nodes and to metastasize to distant organs in the body. The kind of genes that are involved are oncogenes, which when activated lead to deregulated cell proliferation and the tumor suppressor genes would become inactivated or deleted, producing a loss of cell’s check and balance in controlling cell proliferation and differentiation. The single most common, if not universal, trait that occurs in all cancers is genetic drift, or the ability of cells to lose the stringent requirement for precise DNA replication and to acquire the ability to undergo sequential progressive changes in their genomes, through mutations, gene rearrangements and gene deletions. This has sometimes been called the acquisition of “mutator phenotype” [1] RAS Proteins and Cancer: RAS protein family members belong to a class of protein called small GTPase, and are involved in cellular signal transduction and in cell growth, differentiation, and survival. Mutations in RAS genes can cause unintended and overactive signaling inside the cell; as these signals result in cell growth and division, overactive RAS signaling can ultimately lead to cancer[2] RAS oncogenes can be activated by point mutations so that the GTPase reaction can no longer be stimulated by GAP – this increases the half life of active RAS-GTP mutants[3] Constitutively active RAS (RASD) is one which contains mutations that prevent GTP hydrolysis, thus locking RAS in a permanently ‘On’ state. Mutations in the RAS family of proto-oncogenes are very common, being found in 20% to 30% of all human tumors [4]. High rates of KRAS-activating missense mutations have been detected in non–small cell lung cancer (15 to 20% of tumors) [5], colon adenomas (40%) [6] and pancreatic adenocarcinomas (95%) [7], making it the single most common mutationally activated human oncoprotein. In some tumors, HRAS- or NRAS-activating mutations are also seen. More than half of the most malignant thyroid tumors, characterized as poorly differentiated or undifferentiated, harbor a mutation in KRAS, HRAS, or NRAS [8]. In addition to mutational activation, RAS genes are amplified or over expressed in some tumors [9]. Other mechanisms leading to RAS over activation in tumor cells include the deletion of genes encoding negative regulators (for example NF1, a GAP for RAS, in neurological tumors) [10-13] and over expression of positive regulators (such as SOS1, a GEF for RAS, in renal cancer cells) [14]. Analysis of RASSF2 protein expression in a series of human lung tumor cell lines shows that the protein is frequently down-regulated. RASSF2 inhibits the growth
of tumor cells, and its growth-inhibitory properties are enhanced by activated KRAS. RASSF2 promotes both cell cycle arrest and apoptosis; consequently, its down-regulation may play a key role in the development of cancer [15]. Down-regulation of DIRAS1 protein was significantly correlated with Esophageal Squamous Cell Carcinoma (ESCC) lymph node metastasis, which was associated with its function in inhibiting cell migration and invasion by down-regulation of MMP-2 and MMP-9 via ERK- and p38-dependent signaling pathway. Findings suggest that DIRAS1 plays an important suppressive role in the development and progression of ESCC [16]. Therefore, the importance of aberrant RAS protein function in human cancers may be greater than expected [17], and makes it an object of study.

Conclusion

The present analysis entails 5 members of RAS subfamily: HRAS, NRAS, KRAS, RASSF2, and DIRAS1, selected from NCBI database. HRAS, NRAS, KRAS show high conservation which suggests their functional similarity, while DIRAS1 is relatively distinct subgroup as concluded from the overall analysis. Sequence alignment revealed that RASSF2 is most divergent among selected members. From the present analysis it can be concluded that selected human RAS proteins have high degree of homology with higher vertebrates. Physicochemical properties of RAS proteins presented significant data which will avail in protein purification to further unveil the dimensions of RAS functionality. Secondary structure revealed that all 4 protein are largely alpha helical. RAS-domain is found to be the functional domain, conserved in all 4 proteins, and responsible for GTP-hydrolysis. Phosphorylation, glycosylation, myristoylation, prenylation, and ATP/GTP binding
sites are critical to the normal RAS functioning which are found to be conserved in all four proteins. From the present in sillico drug designing, it can be concluded that Trimetrexate can be an effective inhibitor for RAS oncogenic activity, which will in turn arrest the RAS over expression and metastasis of cancerous cells. It can be further incorporated into drug development proceedings and clinical trials.

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