Computational screening and structural analysis of Gly201Arg and Gly201Asp missense mutations in human cyclin-dependent kinase 4 protein.

The regulatory proteins, cyclins, and cyclin-dependent kinases (CDKs) control the cell cycle progression. CDK4 gene mutations are associated with certain cancers such as melanoma, breast cancer, and rhabdomyosarcoma. Therefore, understanding the mechanisms of cell cycle control and cell proliferation is essential in developing cancer treatment regimens. In this study, we obtained cancer-causing CDK4 mutations from the COSMIC database and subjected them to a series of in silico analyses to identify the most significant mutations. An overall of 238 mutations (119 missense mutations) retrieved from the COSMIC database were investigated for the pathogenic and destabilizing properties using the PredictSNP and iStable algorithms. Further, the amino acid position of the most pathogenic and destabilizing mutations were analyzed to understand the nature of amino acid conservation across the species during the evolution. We observed that the missense mutations G201R and G201D were more significant and the Glycine at position 201 was found to highly conserved. These significant mutations were subjected to molecular dynamics simulation analysis to understand the protein's structural changes. The results from molecular dynamics simulations revealed that both G201R and G201D of CDK4 are capable of altering the protein's native form. On comparison among the most significant mutations, G201R disrupted the protein structure higher than the protein with G201D.

Computational and structural investigation of Palmitoyl-Protein Thioesterase 1 (PPT1) protein causing Neuronal Ceroid Lipofuscinoses (NCL).

The Neuronal Ceroid Lipofuscinoses (NCL) are a group of progressive neurodegenerative disorders, associated with 14 Ceroid Lipofuscinosis Neuronal genes (CLN1-14). The mutations in the Palmitoyl-Protein Thioesterase 1 (PPT1) protein serve as one of the major reasons for the causative of NCL. The PPT1 involves degrading and modifying cysteine residues in proteins or peptides by removing thioester-linked fatty acyl groups like palmitate prefers acyl chains of 14-18 carbons in length. In this study, we have analyzed the impact of PPT1 mutations on the deleteriousness, stability, conservative nature of amino acid, and impact of mutations on the protein structure. We have also used molecular dynamics simulations using GROMACS to perceive the alteration in the dynamic behavior of the PPT1 at the residual level. In this study, we have retrieved 23 PPT1 mutations from the UniProt database, and these were subjected to a series of analyses using varied computer algorithms. From these analyses, out of 23 mutations, 16 mutations were identified as deleterious. Among 16, eight mutations were identified to destabilize the protein structure, and finally, two mutations (W38C and L222P) were found to be positioned in the highly conserved region. The structural impact study observed that the mutant proline could disrupt the alpha helix formed by the leucine at position 222. Finally, from the molecular dynamics simulations, we observed that due to the mutations (W38C and L222P), the protein had experienced higher deviation, fluctuation, and lower compactness. These structural changes elucidate that these mutations can impact the structure and function of the PPT1 protein.

Structure-Based Virtual Screening to Identify Novel Potential Compound as an Alternative to Remdesivir to Overcome the RdRp Protein Mutations in SARS-CoV-2.

The number of confirmed COVID-19 cases is rapidly increasing with no direct treatment for the disease. Few repurposed drugs, such as Remdesivir, Chloroquine, Hydroxychloroquine, Lopinavir, and Ritonavir, are being tested against SARS-CoV-2. Remdesivir is the drug of choice for Ebola virus disease and has been authorized for emergency use. This drug acts against SARS-CoV-2 by inhibiting the RNA-dependent-RNA-polymerase (RdRp) of SARS-CoV-2. RdRp of viruses is prone to mutations that confer drug resistance. A recent study by Pachetti et al. in 2020 identified the P323L mutation in the RdRp protein of SARS-CoV-2. In this study, we aimed to determine the potency of lead compounds similar to Remdesivir, which can be used as an alternative when variants of SARS-CoV-2 develop resistance due to RdRp mutations. The initial screening yielded 704 compounds that were 90% similar to the control drug, Remdesivir. On further evaluation through drugability and antiviral inhibition percentage analyses, we shortlisted 32 and seven compounds, respectively. These seven compounds were further analyzed for their molecular interactions, which revealed that all seven compounds interacted with RdRp with higher affinity than Remdesivir under native conditions. However, three compounds failed to interact with the mutant protein with higher affinity than Remdesivir. Dynamic cross-correlation matrix (DCCM) and vector field collective motions analyses were performed to identify the precise movements of docked complexes' residues. Furthermore, the compound SCHEMBL20144212 showed a high affinity for native and mutant proteins and might provide an alternative against SARS-CoV-2 variants that might confer resistance to Remdesivir. Further validations by in vitro and in vivo studies are needed to confirm the efficacy of our lead compounds for their inhibition against SARS-CoV-2.