Identification of Potent Leads for Human cAMP Dependent Protein Kinase Catalytic Subunit Alpha: A Strategic Application of Virtual Screening for Cancer Therapeutics

The advancement in therapeutic applications focused on specific macromolecular compounds of deregulated cell signaling pathways bestowed novel approach to design the ligands as drug molecules against several life threatening diseases such as Cancer. In humans, protein kinase A is one of the important kinases those were involved in cell signaling mechanism. cAMP, G-proteins and ATP molecules were required for activation of protein kinase A (PKA), upon activation, PKA catalytic subunits (PRKACA,PRKACB and PRKACG) undergoes many cellular functions like cell proliferations, cell cycle regulation, and survival of cells through acting on many substrates. Overexpression of extracellular cAMP dependent protein kinase A catalytic subunits (PRKACA) causes severe tumorgenesis in different organs (prostate gland, breast, lungs and pancreas) leading to cancer. High throughput virtual screening was implemented herein to identify the potent leads for human PRKACA that stimulates chronic form of cancers. In silico functional and phylogenetic analysis of PRKACA protein provided enough evidences towards its cancer stimulating nature. The human PRKACA crystal structure in complex with inhibitor ‘796’ (PDB ID: 2GU8) was optimized in Maestro v9.0 and the amino acid residues constituting inhibitor interaction site were determined. Fifteen published inhibitors were selected including HA1077, Flavopiridol, Roscovitine, MLN-518, PP2 and Gleevec which were already in clinical trials for high throughput screening at Ligand.Info database. An in house library of 5388 compounds was designing from the above screening procedure were prepared in LigPrep for molecular docking with human PRKACA. Maestro Glide docking from lesser to higher stringency towards minor steric classes were applied subsequently to the prepared ligand dataset against a grid around centroid of the identified inhibitor interaction site of human PRKACA and 21 lead molecules with good docking scores were obtained. Lead ‘1’ (Leptosidin) with relatively least docking score (-11.02 Kcal/mol) compared to other 20 lead molecules and 15 published inhibitors delineates it as potentially the best competitive inhibitor among all. The promising inhibitory activity of Leptosidin is further supported from analysis of binding orientations of human PRKACA- Leptosidin complex deciphering the Lead 1 blocks the active site residues Thr51, Glu121, Val123, Glu127 and Thr183 by forming hydrogen bond. Thus, Leptosidin could be futuristic perspective chemical compound to design drug molecule against human PRKACA in numerous cancers, however, further in vitro and in vivo studies were required to verify the computational strategic prediction of PKA holoenzyme against cancer therapeutics

In Silico Analysis To Explore Novel Inhibitors For Human Proto-oncogene Tyrosine Protein Kinase Src

The first oncogene and the first non receptor tyrosine kinase, Src, plays a key role in cell morphology, motility, proliferation and survival. Over expression of Src kinase activity disrupts the RAS pathway in signaling pathway, where it loses its ability to hydrolyse GTP and thus, leads to cancer. A wide range of evidences indicated that Src-signaling was important in the oncogenesis of prostate cancer and other tumours. Src-signaling is involved in androgen-induced proliferation of prostate cancer in cancer tissue of patients having castration-refractory prostate cancer. Once prostate cancer becomes castration-resistant, bone metastases become significant problem for which treatment options are limited. As Src is involved in multiple signaling pathways, central to prostate cancer development, progression, and metastasis, in addition to normal and pathologic osteoclast activities, Src inhibition becomes a valid therapeutic strategy for investigation. Existing Src kinase inhibitors are less efficient towards prostate cancer and bone metastasis. Hence an in silico work was carried out to identify novel potent inhibitors. Three published inhibitors of human Src kinase those are currently under clinical trial such as AZDO530, bosutinib and dasatinib were subjected to high-throughput screening from more than million entries of Ligand.Info Meta-Database, based on the assumption that small molecules with similar structure have similar pharmacological properties. The ligand dataset of 1152 generated through this approach were prepared using LigPrep to generate possible conformations of each ligand molecule, and at the same time duplicate conformers, conformations with reactive functional group and ADME violaters were rejected. The human Src kinase co-crystal structure with AZDO530 was analyzed to find the inhibitor binding site. The crystal structure was optimized and energy was minimized applying OPLS force field in Maestro v9.0. Glide 5.5 docking was performed to predict the binding orientation of prepared ligand molecule into a grid of 20 x 20 x 20 Å created around the centroid of optimized human Src kinase. Ten lead molecules with good binding affinity with human Src were identified. In silico pharmacokinetics study for these ten lead molecules had shown no ADME violation. Analysis of Lead ‘1’ - human Src docking complex had revealed a XP Gscore of -11.56 kcal/mol with highly stabilized hydrogen bond network with Ala390, Asn391, Lys295, W543 and W640 and good Van der Waals interactions. The docking complex coincides well with the native co-crystallized human Sac and inhibitor AZDO530 complex. Thus, Fisetin, identified as Lead ‘1’ in the present study would be highly useful for developing potential drug molecules for treatment of advanced prostate cancer

Computerized Protein Modeling and Molecular Docking Analysis of Human Proto Oncogene Tyrosine Protein Kinase YES for Discovery of Novel Lead Molecules

Human proto-oncogene tyrosine-protein kinase YES (YES) is a non receptor kinase belongs to Src family. This gene lies in close proximity to thymidylate synthase gene on chromosome 18, and a corresponding pseudogene has been found on chromosome 22. In hepatocellular carcinoma and colorectal carcinoma elevated human YES activity was observed. Inhibitors of human YES reported till date are in clinical trials and associated with several side effects. The present study was mainly aimed in homology modeling of human YES and discovery of novel lead molecules that inhibit YES kinase more efficiently with fewer side effects. Virtual screening and docking techniques were applied to identify novel lead molecule of YES kinase. As there was no reported human YES crystal structural data, the three dimensional structure of human YES was constructed based on template structure (PDB ID: 2H8H) obtained through homology search using MODELLER 9V7. The model was refined, energy minimized and assessed through PROCHECK. Active site residues of human YES were identified from the homology model in complex with template ligand AZD0530 and were further confirmed using CASTp. Five published inhibitors of YES family (Dasatinib, Bosutinib, SU6656, AZD0530 and CGP77675) were identified through literature search. High throughput virtual screening method at Ligand.Info was applied for these five inhibitors to establish a library of 1932 structural analogs. LigPrep was used to generate possible conformations of each ligand molecules from structural analog library. The ligand duplicates conformers, ligands having reactive functional group and poor ADME properties were rejected from the prepared dataset. Glide 5.5 was used to generate a grid box by picking the active site residues of human YES protein. Through sequential applications of stringent mode glide docking procedures from Glide HTVS to SP to XP respectively, 13 potential inhibitors were proposed. The docking complexes of each inhibitor with human YES protein were analyzed and lead ‘1’ molecule was identified to have higher binding affinity to human YES protein (XP Gscore: -12.07 Kcal/mol) compared to existing published inhibitors and other 12 lead molecules. The lead ‘1’ - human YES docking complex was highly stabilized through hydrogen bond network with amino acid residues Thr348, Asp358, Asp414 and Phe415. Moreover, from the results obtained we could decipher that lead ‘1’ molecule can be raised into potential inhibitors after binding assays, substantiated experimental investigations and passing several phases of clinical trials

In silico identification of potential inhibitors for human aurora kinase b

Cell cycle progression through mitosis and meiosis involves regulation by serine/threonine kinases from the aurora family. Aurora kinase b (Aurkb) is mainly involved in the proper segregation of chromosomes during mitosis as well as meiosis. However, over expression of Aurkb leads to the unequal distribution of genetic information creating aneuploid cells, a hallmark of cancer. Thus, Aurkb can be used as an effective molecular target for computer-aided drug discovery against cancer. Existing Aurkb inhibitors are less efficient, hence an in silico work was carried out to identify novel potent inhibitors. Three published inhibitors azd1152, zm447439 and N-(4-{[6-methoxy-7-(3-morpholin-4-ylpropoxy) quinazolin- 4-yl] amino} phenyl) benzamide were subjected to high throughput virtual screening of over 1 million entries from a ligand info meta database, to generate a 1161 compound library. The crystal structure was optimized and energy was minimized applying an OPLS force field in Maestro v9.0. Molecular docking using Glide was performed to predict the binding orientation of the prepared ligand molecule into a grid of 20*20*20 Å created around the centroid of the optimized human Aurkb protein. Nine lead molecules with good binding affinity with human Aurkb were identified. In silico pharmacokinetics study for these nine lead molecules has shown no ADME violation. Analysis of lead ‘1’- human Aurkb docking complex has revealed a XP Gscore of -10.20 kcal/mol with a highly stabilized hydrogen bond network with Asp218 and Ala157 and good Van der wall interactions. The docking complex coincides well with the native co- crystallized human Aurkb and inhibitor zm447439 complex. Thus, lead 1 would be highly useful for developing potential drug molecules for the treatment of cancer

Docking-based virtual screening for the exploration of potential antagonists for human IGFBP6

Design of potential drug-like candidates for cardiovascular therapy is of interest in recent years. Elevated level of IGFBP-6 leads to perilous diseases like atherosclerosis, high output cardiac failure and myocardial infarction. The current IGFBP-6 inhibitors available in clinical practice are not having satisfactory anti-cardiovascular effects, leaving room for further improvement. Therefore, with an aim to propose a potent inhibitor to arrest the cardiovascular disease caused by IGFBP-6, tools of computer-aided drug designing were used for virtual screening from small molecule databases. Accordingly, IGFBP-6 tertiary structure was solved through nuclear magnetic resonance techniques retrieved from the protein data bank. Fifteen IGFBP-6 inhibitors were acquired from literature databases and subsequent 2D searching protocol of Ligand.Info yielded 5759 structural analogs. The 3D structural conversion and multiple conformations for each compound were generated using LigPrep with constraints of ADME evaluation and toxicity assessments. The docking and scoring calculations were performed using Glide v5.7. The Glide extra precision (XP) docking had reported 138 leads and ranked based on XP Gscore. Six leads having better XP Gscore compared to current IGFBP-6 inhibitors were proposed as potential inhibitors. Chelidamic acid showed the highest XP Gscore (-7.094 Kcal/mol) with good pharmacological properties and molecular interaction with IGFBP-6. Therefore, chelidamic acid is proposed for rational drug design towards cardiovascular therapy

Functional annotation of human parvovirus b19 proteome and Molecular docking of VP1 protein with Teniposide

Parvovirus B19 is a common source of infection with a seroprevalence of 60-70% in the adult population. Human parvovirus causes several distinct clinical syndromes such as erythema, polyarthritis resembling rheumatoid arthritis, vasculitis, hydrops fetalis, myocarditis etc. Recent years have witnessed manifest increase in clinical knowledge of parvovirus B19-associated complications, diagnosis and treatment. Traditional immunosuppressive therapy being unsuccessful, anti-viral therapy might be worthy of consideration. Functional annotation of human parvoviral proteome may provide insights into role of viral proteome in its survival and pathogenic mechanisms. Availability of whole proteome in the open source aided functional annotation of protein using SVMprot. Protein functions common to both structural and non-structural proteins are metal -binding, lipid-binding, copper-binding, calcium-binding, zinc-binding, magnesium-binding, DNA-binding, RNA-binding, transmembrane and outer membrane group of proteins. VP1 protein of human parvovirus b19 being quiet important for its structural integrity and being non homologous to human proteome, was identified as an attractive molecular target for structure based drug discovery. 3D structure of target protein VP1protein is predicted based on VP2 protein from human parvovirus b19 using MODELLER9V6. The modeled structure is validated using PROCHECK. Binding sites were predicted in Discovery studio 2.0 and Teniposide was found to be best docked lead molecule among selected ligands those docked to the functionally important site 1 using LigandFit protocol. The docking complex shows teniposide forming strong hydrogen bond with serine 580 of modeled VP1 protein. The strong hydrogen bonding, hydrophobic interaction, Vander wall interaction marked by teniposide would affect the structural integrity of parvovirus B19 significantly. The detailed insight of structural, functional and interactive aspect of the protein is being studied would enable us delineate it as potential drug target. Also different docking poses generated through docking studies may be considered as a model for identifying drug candidate against parvovirus

In Silico Analysis and Identification of Potent Inhibitors for Human Serine/Threonine Protein Kinase PIM-3 Involved in Hepatic Cancer

PIM3 belongs to a family of proto oncogenes that encode serine/threonine protein kinases in human. Pim-3 is involved in cell cycle progression, suppression of apoptosis and proliferation of human hepatoma cell lines. During normal cell cycle progression, Bcl-2 associated death promoter (BAD protein) inactivates Bcl-2 and Bcl-xL there by promoting apoptosis. However, phosphorylation of BAD protein by Pim-3 leaves Bcl-2 free to inhibit Bax-triggered apoptosis. Thus, designing inhibitors against Pim-3 would stop BAD phosphorylation, hence would be highly useful for development of novel means of cancer therapeutics. Computer aided drug designing approach was followed here to explore lead molecules targeting human Pim-3. For this, proteomic nature and phylogeny of human Pim-3 was studied. A homology model of human Pim-3 was generated based on crystal structure of human Pim-1 (PDB ID: 1XWS) using Modeller9v7. Pim-1 inhibitor ‘BI1’ was incorporated into the 3D model and further energy minimized by applying OPLS force field in Maestro v9.0. The stereochemistry assessment of the model had revealed a highly reliable model with 94.8% of residues in the most favored region and no residues in disallowed region of Ramachandran plot. The human Pim-3 3D model inhibitor (BI1) complex was analyzed to locate active site residues such as Leu44, Gly45, Phe49, Val52, Ala65, Lys67, Glu89, Ile104, Leu120, Glu121, Arg122, Pro123, Glu171, Leu174, Ile185 and Asp186. These active site residues were further confirmed using SiteMap. BI1 and two published Pim family inhibitors (Imidazo[1,2-b]pyridazine, SGI-1776) structural analogs were searched from more than one million entries of Ligand.Info Metadatabase and an in house library of 1171 compounds was prepared. Maestro 9.0 virtual screening protocol was used to check binding affinity of 1171 compounds into Pim-3 active site through sequential application of flexible Glide HTVS (Virtual High Throughput Screening), SP and XP method respectively. Fifteen lead molecules identified in the present study were proposed as potential drug molecules against human Pim-3. Lead‘1’ was observed to have higher binding affinity towards human Pim-3 with lowest XP Gscore (-11.90 kcal/mol). The docking complex was observed to be highly stabilized through formation of hydrogen bond with Glu124 and Van der Waal interactions with active site residues. From the present study, it can be concluded that the generated model of human Pim-3 can be used for further studies and lead ‘1’ structure would be highly useful for designing novel inhibitors against hepatic cancer

Envelope protein as molecular target for YFV (Yellow fever virus) drug discovery

Yellow fever (YF), a mosquito-borne viral haemorrhagic fever, is one of the most lethal viral diseases. Despite the availability of vaccines, yellow fever virus (YFV) strikes an estimated 2, 00,000 persons world-wide each year and causes 30,000 deaths approximately. There are no approved antiviral therapies for the treatment of YFV disease in humans. YFV 17D strain RNA genome is of 10,862 nucleotides, which encodes three structural proteins (C, PrM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Identification of different protein functions facilitates a mechanistic understanding of YFV infection and opens novel means for drug development. Functional assignment of complete YFV proteome was done through support Vector machine (SVM). Significant functions of YFV Envelope (E) protein are Transmembrane, Aptamer-binding protein, Coat protein, Zinc-binding, Manganese-binding and Metal-binding etc. The E protein being non homologous to human and important in receptor binding, hemagglutination of erythrocytes at acidic pH, induction of the protective immune response, and involvement in an intraendosomal acid-catalyzed fusion step necessary for infection was selected as a potent molecular target against YFV drug discovery. 3D structure of target protein E protein is predicted using MODELLER9V6 and validated through PROCHECK analysis. The Binding site tool of Discovery Studio 2.0 was used to find sites all over protein. Heparin was identified as an important ligand against the molecular target and was docked to the functionally important binding site using LigandFit protocol. The YFV E protein -Heparin docking complex associated with strong hydrogen bonding with SER 483, GLN 443 and ALA 441 residues. The strong docking interaction may impede the infection causes due to association of YFV E protein and Core membrane. Further, the predicted model can be used as reference towards designing candidate drugs against YFV

In silico identification of potential inhibitors for human aurora kinase b

Cell cycle progression through mitosis and meiosis involves regulation by serine/threonine kinases from the aurora family. Aurora kinase b (Aurkb) is mainly involved in the proper segregation of chromosomes during mitosis as well as meiosis. However, over expression of Aurkb leads to the unequal distribution of genetic information creating aneuploid cells, a hallmark of cancer. Thus, Aurkb can be used as an effective molecular target for computer-aided drug discovery against cancer. Existing Aurkb inhibitors are less efficient, hence an in silico work was carried out to identify novel potent inhibitors. Three published inhibitors azd1152, zm447439 and N-(4-{[6-methoxy-7-(3-morpholin-4-ylpropoxy) quinazolin- 4-yl] amino} phenyl) benzamide were subjected to high throughput virtual screening of over 1 million entries from a ligand info meta database, to generate a 1161 compound library. The crystal structure was optimized and energy was minimized applying an OPLS force field in Maestro v9.0. Molecular docking using Glide was performed to predict the binding orientation of the prepared ligand molecule into a grid of 20*20*20 Å created around the centroid of the optimized human Aurkb protein. Nine lead molecules with good binding affinity with human Aurkb were identified. In silico pharmacokinetics study for these nine lead molecules has shown no ADME violation. Analysis of lead ‘1’- human Aurkb docking complex has revealed a XP Gscore of -10.20 kcal/mol with a highly stabilized hydrogen bond network with Asp218 and Ala157 and good Van der wall interactions. The docking complex coincides well with the native co- crystallized human Aurkb and inhibitor zm447439 complex. Thus, lead 1 would be highly useful for developing potential drug molecules for the treatment of cancer