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Clamp loaders ensure processive DNA replication by loading the toroidal shaped sliding clamps onto the DNA. The sliding clamps serve as a platform for the attachment of polymerases and several other proteins associated with the regulation of various cellular processes. Clamp loaders are fascinating as nanomachines that engage in protein-protein and protein-DNA interactions. The loading mechanism of the clamp around dsDNA at the atomic level has not yet been fully explored. We performed microsecond timescale molecular dynamics simulations to reveal the dynamics of two different intermediate complexes involved in loading of the clamps around DNA. We conducted various time-dependent MD-driven analyses including the highly robust Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) calculations to observe changes in the structural elements of the clamp loader-clamp-DNA complexes in open and closed states. Our studies revealed the structural consequences of ATP hydrolysis events at different subunits of the clamp loader. This study would help in a better understanding of the clamp loading mechanism and would allow tackling various complications that might arise due to irregularities in this process.

Dengue is a prominent viral disease transmitted by mosquitoes to humans that affects mainly tropical and subtropical countries worldwide. The global spread of dengue virus (DENV) is mainly occurred by Aedes aegypti and Aedes albopictus mosquitoes. The dengue virus serotypes-2 (DENV-2) is a widely prevalent serotype of DENV, that causes the hemorrhagic fever and bleeding in the mucosa, which can be fatal. In the life cycle of DENV-2, a structural capsid (DENV-2 C) protein forms the nucleocapsid assembly and bind to the viral progeny RNA. For DENV-2 maturation, the nucleocapsid is a vital component. We used virtual ligand screening to filter out the best in-house synthesized acridinedione analogs (DSPD molecules) that could efficiently bind to DENV-2 C protein. The molecular docking and dynamics simulations studies were performed to analyze the effect of DSPD molecules on DENV-2 C protein after binding. Our findings showed that DSPD molecules strongly interacted with DENV-2 C protein, as evident from molecular interactions and several time-dependent molecular dynamics-driven analyses. Moreover, this study was also supported by the thermodynamic binding free energy and steered molecular dynamics simulations. Therefore, we intend to suggest that the DSPD3 molecule could be used as a potential therapeutic molecule against dengue complications as compared to the cocrystallized inhibitor ST-148. However, further studies are required to demonstrate the ability of DSPD3 to induce DENV-2 C tetramer formation.

BRD3-BD2 protein belongs to the bromodomain and extra-terminal domain (BET) protein family.

Objective and design: Serratiopeptidase a multifaceted therapeutic enzyme renowned for its anti-inflammatory, analgesic, anti-biofilm, fibrinolytic and anti-edemic properties. It is vital to uncover more about the assets of such efficacious enzyme in order to facilitates their contribution in all health-related issues, notably inflammatory ailments. The current study sought to determine whether serratiopeptidase would disintegrate bradykinin related peptides (BRPs) from wasp venom in the same manner as it does with human bradykinin. Methods: To accomplish this objective, we used molecular modeling, docking, MD simulation, MMG/PBSA along with the SMD simulations. Results: We docked selected BRPs on to the binding pocket of wild and previously identified mutant (N412D) of serratiopeptidase. Based on their docked scores, top two BRPs were selected and their conformational behavior was analyzed employing molecular dynamics studies. Additionally, thermodynamics end-state energy analysis reported that both the complexes exhibited higher stability and analogous ∆G values when compared to the reference complex. Further, to understand the unbinding mechanism, we condemned external pulling force on both peptides and observed that BRP-7 peptide was tightly anchored and laid out the highest pulling force to get detach from the active pocket of serratiopeptidase. Conclusion: The current study endorses up the current findings and paves the way for serratiopeptidase to be used as an anti-angioedemic as well as fixed dose combination in hypotensive drugs.

Superoxide dismutases (SODs) protect the cells by catalyzing the dismutation of harmful superoxide radicals (O2•-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Here, a Cu, Zn SOD (WT) from a high altitude plant (Potentilla atrosanguinea) was engineered by substituting a conserved residue proline to glycine at position 61 (P61G). The computational analysis showed higher structural flexibility and clusters in P61G than WT. The P61G exhibited moderately higher catalytic efficiency (Km = 0.029 μM, Vmax = 1488) than WT protein (Km = 0.038 μM, Vmax = 1290.11). P61G showed higher thermostability as revealed from residual activity (72.25% for P61G than 59.31% for WT after heating at 80 °C for 60 min), differential calorimetry scanning and CD-spectroscopic analysis. Interestingly, the P61G mutation also resulted in enhanced tolerance to H2O2 inactivation than WT protein. The finding on enhancing the biophysico-chemical properties by mutating conserved residue could stand as an example to engineer other enzymes. Also, the reported mutant can be exploited in food and pharmaceutical industries.

The enzyme Phosphodiesterase 10A (PDE10A) plays a regulatory role in the cAMP/protein kinase A (PKA) signaling pathway by means of hydrolyzing cAMP and cGMP. PDE10A emerges as a relevant pharmacological drug target for neurological conditions such as psychosis, schizophrenia, Parkinson's, Huntington’s disease, and other memory-related disorders. In the current study, we subjected a set of 1,2,3-triazoles to be explored as PDE10A inhibitors using diverse computational approaches, including molecular docking, classical molecular dynamics (MD) simulations, Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) calculations, steered MD, and umbrella sampling simulations. Molecular docking of cocrystallized ligands papaverine and PFJ, along with a set of in-house synthesized molecules, suggested that molecule 3i haded the highest binding affinity, followed by 3h and 3j. Furthermore, the structural stability studies using MD and MM-PBSA indicated that the 3h and 3j formed stable complexes with PDE10A. The binding free energy of −240.642 kJ/mol and −201.406 kJ/mol was observed for 3h and 3j, respectively. However, the cocrystallized ligands papaverine and PFJ exhibited comparitively higher binding free energy values of −202.030 kJ/mol and −138.764 kJ/mol, respectively. Additionally, steered MD and umbrella sampling simulations provided conclusive evidence that the molecules 3h and 3j could be exploited as promising candidates to target PDE10A. Communicated by Ramaswamy H. Sarma

The quick widespread of the coronavirus and speedy upsurge in the tally of cases demand the fast development of effective drugs. The uridine-directed endoribonuclease activity of nonstructural protein 15 (Nsp15) of the coronavirus is responsible for the invasion of the host immune system. Therefore, developing potential inhibitors against Nsp15 is a promising strategy. In this concern, the in silico approach can play a significant role, as it is fast and cost-effective in comparison to the trial and error approaches of experimental investigations. In this study, six turmeric derivatives (curcuminoids) were chosen for in silico analysis. The molecular interactions, pharmacokinetics, and drug-likeness of all the curcuminoids were measured. Further, the stability of Nsp15-curcuminoids complexes was appraised by employing molecular dynamics (MD) simulations and MM-PBSA approaches. All the molecules were affirmed to have strong interactions and pharmacokinetic profile. The MD simulations data stated that the Nsp15-curcuminoids complexes were stable during simulations. All the curcuminoids showed stable and high binding affinity, and these curcuminoids could be admitted as potential modulators for Nsp15 inhibition.

An ecdysone receptor (EcR) is a nuclear receptor protein, a prime target for pesticide development processes due to its involvement in the growth and development of insects. The activation of EcRs leads to hasty and premature development of insects, thereby preventing them from destroying the crops. The high cost, environmental toxicity, and resistance towards the currently available insecticides have resulted in a growing demand for the development of economical and environment-friendly pesticides with unique insecticidal activity. Several reports on resistance towards conventional pesticides like ponasterone A and RH-5849 were reported in recent years. In this study, the plant derived (C. deodara), in-house synthesized molecules with benzosuberene scaffolds were screened for their ability to interact with the EcRs of both Hemiptera and Coleoptera. The preliminary analysis by molecular docking showed that some of our molecules exhibited excellent binding with the target protein. Moreover, molecular dynamics simulations, binding free energy estimations, and umbrella sampling simulations were conducted to validate the docking results. Our analysis revealed Com-7 and Com-3 as potential EcR agonists to target Hemiptera and Coleoptera, respectively. Also, Com-8 and Com-12 could be used as lead compounds against the EcR of Coleoptera.

MEK1 is an attractive target due to its role in selective extracellular-signal-regulated kinase phosphorylation, which plays a pivotal role in regulating cell proliferation. Another benefit of targeting the MEK protein is its unique hydrophobic pocket that can accommodate highly selective allosteric inhibitors. To date, various MEK1 inhibitors have reached clinical trials against several cancers, but they were discarded due to their severe toxicity and low efficacy. Thus, the development of allosteric inhibitors for MEK1 is the demand of the hour. In this in-silico study, molecular docking, long-term molecular dynamics (5 µs), and molecular mechanics Poisson-Boltzmann surface area analysis were undertaken to address the potential of quinolines as allosteric inhibitors. We selected four reference MEK1 inhibitors for the comparative analysis. The drug-likeness and toxicity of these molecules were also examined based on their ADMET and Toxicity Prediction by Komputer Assisted Technology profiles. The outcome of the analysis revealed that the quinolines (4m, 4o, 4s, and 4n) exhibited better stability and binding affinity while being nontoxic compared to reference inhibitors. We have reached the conclusion that these quinoline molecules could be checked by experimental studies to validate their use as allosteric inhibitors against MEK1.

Aminoarylbenzosuberene (AAB) molecules were chosen for in silico analysis to develop effective and more competent 11β-hydroxysteroid dehydrogenase (11β-HSD1) protein inhibitors.

The proliferating cell nuclear antigen (PCNA) has emerged as a promising candidate for the development of novel cancer therapeutics. PCNA is a nononcogenic mediator of DNA replication that regulates a diverse range of cellular functions and pathways through a comprehensive list of protein-protein interactions. The hydrophobic binding pocket on PCNA offers an opportunity for the development of inhibitors to target various types of cancers and modulate protein-protein interactions. In the present study, we explored the binding modes and affinity of molecule I1 (standard molecule) with the previously suggested dimer interface pocket and the hydrophobic pocket present on the frontal side of the PCNA monomer. We also identified potential lead molecules from the library of in-house synthesized 3-methylenisoindolin-1-one based molecules to inhibit the protein-protein interactions of PCNA. Our results were based on robust computational methods, including molecular docking, conventional, steered, and umbrella sampling molecular dynamics simulations. Our results suggested that the standard inhibitor I1 interacts with the hydrophobic pocket of PCNA with a higher affinity than the previously suggested binding site. Also, the proposed molecules showed better or comparable binding free energies as calculated by the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach and further validated by enhanced umbrella sampling simulations. In vitro and in vivo methods could test the computationally suggested molecules for advancement in the drug discovery pipeline.

Abscisic acid (ABA) although complicated and expensive to produce, plays an important role in signalling responsible for regulation of developmental manifestations such as seed maturation and surviving through stress conditions. Hence, development of cost effective molecules with minimal side effects that mimic the functions of ABA is the need of the hour. In this agreement, we screened a series of 27 in-house synthesized 3-methyleneisoindolin-1-one molecules over three ABA receptors (PYR1, PYL1, and PYL3). The commercial ABA agonist Pyrabactin was taken as a standard ligand in this study. The top three molecules for each receptor were selected and further evaluated to estimate the dynamical contribution and complex stability via Molecular Mechanics-Poisson Boltzmann surface area calculations. Two molecules (Mol26 and Mol25) showed higher binding free energy and stable complex conformation for PYL3 in comparison to Pyrabactin. This study revealed the structural basis of the binding mechanism of 3-methyleneisoindolin-1-one molecules with ABA receptors. Mol26 and Mol25 were identified for the development of specific PYL3 agonists with a vast potential in agriculture to accentuate the ABA like action in plants.

Malaria remains a deadliest disease brought about by Plasmodium species, among one of these species, disease due to Plasmodium falciparum (Pf) is life-threatening. The structures of PfLDH and human LDH are very similar in terms of L-LDH activity, and their biological functions are also equivalent. Therefore, any treatment aiming blocking the functions of PfLDH can affect human LDH. Thus, the main objective of this study is to identify the molecule that exhibits selectivity towards PfLDH without a profound effect on human LDH. In this research, a set of 68 quinolines based molecules were used for molecular docking. From molecular docking, we selected molecules 3j, 4b, 4h, 4m based on their binding affinity, ligand efficiency, lipophilic ligand efficiency, and torsion with selectivity towards PfLDH. The stability of the docked molecules was compared to Chloroquine (reference inhibitor) by applying molecular dynamics simulations and molecular mechanics poisson boltzmann surface area calculations. All the selected molecules showed selectivity for PfLDH with stable dynamic behavior and high binding free energy in comparison to Chloroquine. After examining the molecular mechanics poisson boltzmann surface area ratio results, molecule 3j was reported as a potential and specific inhibitor for PfLDH with a novel mechanism of binding to PfLDH while the remaining molecules 4b, 4h, 4m could further be modified to be used as potent inhibitors against malarial infection.

Tobacco mosaic virus (TMV) coat protein (CP) self assembles in viral RNA deprived transgenic plants to form aggregates based on the physical conditions of the environment. Transgenic plants in which these aggregates are developed show resistance toward infection by TMV referred to as CP-MR. This phenomenon has been extensively used to protect transgenic plants against viral diseases. The mutants T42W and E50Q CP confer enhanced CP-MR as compared to the WT CP. The aggregates, when examined, show the presence of helical discs in the case of WT CP; on the other hand, mutants show the presence of highly stable non-helical long rods. These aggregates interfere with the accumulation of MP as well as with the disassembly of TMV in plant cells. Here, we explored an atomic level insight to the process of CP-MR through MD simulations. The subunit-subunit interactions were assessed with the help of MM-PBSA calculations. Moreover, classification of secondary structure elements of the protein also provided unambiguous information about the conformational changes occurring in the two chains, which indicated toward increased flexibility of the mutant protein and seconded the other results of simulations. Our finding indicates the essential structural changes caused by the mutation in CP subunits, which are critically responsible for CP-MR and provides an in silico insight into the effects of these transitions over CP-MR. These results could further be utilized to design TMV-CP-based small peptides that would be able to provide appropriate protection against TMV infection. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-020-00225-0) contains supplementary material, which is available to authorized users.

Highly regiospecific, copper salts-free and neat conditions have been demonstrated for the 1,3-dipolar Azide-Alkyne Cycloaddition (AAC) reactions under mechanochemical conditions. A group of structurally challenging alkynes and heterocyclic derivatives was efficiently implemented to achieve highly functionalized 1,4-disubstituted-1,2,3-triazoles in good to excellent yield by using the Cu beads without generation of unwanted byproducts. Furthermore, high-speed ball milling (HSBM) strategy has also been extended to the synthesis of commercially available pharmaceutical agent, Rufinamide, an antiepileptic drug (AED) and its analogues. The same strategy was also applied for the synthesis of Cl-derivative of Rufinamide. Analysis of the single crystal XRD data of the triazole was also performed for the final structural confirmation. The Cu beads can be easily recoverable from the reaction mixture and used for the further reactions without any special treatment.

The current global pandemic due to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has taken a substantial number of lives across the world. Although few vaccines have been rolled-out, a number of vaccine candidates are still under clinical trials at various pharmaceutical companies and laboratories around the world. Considering the intrinsic nature of viruses in mutating and evolving over time, persistent efforts are needed to develop better vaccine candidates. In this study, various immuno-informatics tools and bioinformatics databases were deployed to derive consensus B-cell and T-cell epitope sequences of SARS-CoV-2 spike glycoprotein. This approach has identified four potential epitopes which have the capability to initiate both antibody and cell-mediated immune responses, are non-allergenic and do not trigger autoimmunity. These peptide sequences were also evaluated to show 99.82% of global population coverage based on the genotypic frequencies of HLA binding alleles for both MHC class-I and class-II and are unique for SARS-CoV-2 isolated from human as a host species. Epitope number 2 alone had a global population coverage of 98.2%. Therefore, we further validated binding and interaction of its constituent T-cell epitopes with their corresponding HLA proteins using molecular docking and molecular dynamics simulation experiments, followed by binding free energy calculations with molecular mechanics Poisson–Boltzmann surface area, essential dynamics analysis and free energy landscape analysis. The immuno-informatics pipeline described and the candidate epitopes discovered herein could have significant impact upon efforts to develop globally effective SARS-CoV-2 vaccines.

Cyclin-dependent kinases (CDKs) are known for their vital role in regulating cell cycle progression through protein-kinase interactions. CDKs also help in regulating transcription and development of the central nervous system. Inhibition of CDKs is a very fundamental approach for drug discovery in areas of different types of cancers, Alzheimer's, and HIV infections. The present research focuses on finding a novel, potent, and specific natural inhibitors of CDK isoforms (CDK2, CDK5, CDK7, CDK9). Molecular docking, molecular dynamics (MD) simulations, and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) were carried out to get an in-depth understanding of protein-ligand interactions. Based on our molecular docking results, Ligands-3, 5, 14, and 16 were screened among 17 different Pyrrolone-fused benzosuberene compounds as potent and specific inhibitors without any cross-reactivity against different CDK isoforms. Analysis of MD simulations and MM-PBSA studies, revealed the binding energy profiles of all the selected complexes. Our selected ligands performed better than the standard inhibitor (Roscovitine). Ligands-3 and 14 show specificity for CDK7 and Ligands-5 and 16 were specific against CDK9. These ligands are expected to possess lower risk of side effects due to their natural origin. Moreover, the backbone structure of these ligands could also be exploited to develop specific inhibitors against other CDK isoforms.Communicated by Ramaswamy H. Sarma.

Superoxide dismutases (SODs) protect the cells by catalyzing the dismutation of harmful superoxide radicals (O

Graphical abstract, Highlights • Nsp15 is involved in viral replication and suppression of host immune response. • Three bioactive molecules of tea showed high binding affinity with Nsp15. • The molecules selected on basis of robust MD simulations and MMPBSA calculations. • Tea bioactive molecules could be developed as inhibitors of Nsp15 to fight the virus., Immensely aggravated situation of COVID-19 has pushed the scientific community towards developing novel therapeutics to fight the pandemic. Small molecules can possibly prevent the spreading infection by targeting specific vital components of the viral genome. Non-structural protein 15 (Nsp15) has emerged as a promising target for such inhibitor molecules. In this investigation, we docked bioactive molecules of tea onto the active site of Nsp15. Based on their docking scores, top three molecules (Barrigenol, Kaempferol, and Myricetin) were selected and their conformational behavior was analyzed via molecular dynamics simulations and MMPBSA calculations. The results indicated that the protein had well adapted the ligands in the binding pocket thereby forming stable complexes. These molecules displayed low binding energy during MMPBSA calculations, substantiating their strong association with Nsp15. The inhibitory potential of these molecules could further be examined by in-vivo and in-vitro investigations to validate their use as inhibitors against Nsp15 of SARS-CoV2.

Background and Aim A novel coronavirus, called the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been found to cause COVID-19 in humans and some other mammals. The nonstructural protein 16 (NSP16) of SARS-CoV-2 plays a significant part in the replication of viruses and suppresses the ability of innate immune system to detect the virus. Therefore, inhibiting NSP16 can be a secure path towards identifying a potent medication against SARS-CoV-2. Tea (Camellia sinensis) polyphenols have been reported to exhibit potential treatment options against various viral diseases. Methods We conducted molecular docking and structural dynamics studies with a set of 65 Tea bioactive compounds to illustrate their ability to inhibit NSP16 of SARS-CoV-2. Moreover, post-simulations end state thermodynamic free energy calculations were estimated to strengthen our results. Results and Conclusion Six bioactive tea molecules showed better docking scores than the standard molecule sinefungin. These results were further validated by MD simulations, where Theaflavin compound demonstrated lower binding free energy in comparison to the standard molecule sinefungin. The compound theaflavin could be considered as a novel lead compound for further evaluation by in-vitro and in-vivo studies., Graphical abstract Image 1

Background: Malaria remains a common life-threatening infectious disease across the globe due to the development of resistance by Plasmodium parasite against most antimalarial drugs. The situation demands new and effective drug candidates against Plasmodium. Objectives: The objective of this study is to design, synthesize and test novel quinoline based molecules against the malaria parasite. Methods: C2 and C8 modified quinoline analogs obtained via C-H bond functionalization approach were synthesized and evaluated for inhibition of growth of P. falciparum grown in human red blood cells using SYBR Green microtiter plate based screening. Computational molecular docking studies were carried out with top fourteen molecules using Autodoc software. Results: The biological evaluation results revealed good activity of quinoline-8-acrylate 3f (IC50 14.2 µM), and the 2-quinoline-α-hydroxypropionates 4b (IC50 6.5 µM), 4j (IC50 5.5 µM) and 4g (IC50 9.5 µM), against chloroquine sensitive Pf3D7 strain. Top fourteen molecules were screened also against chloroquine resistant Pf INDO strain and the observed resistant indices were found to lie between 1 and 7.58. Computational molecular docking studies indicated a unique mode of binding of these quinolines to Falcipain-2 and heme moiety, indicating these to be the probable targets of their antiplasmodial action. Conclusion: An important finding of our work is the fact that unlike Chloroquine which shows a resistance Index of 15, the resistance indices for the most promising molecules studied by us were about one indicating equal potency against drug sensitive and resistant strains of the malaria parasite.

Pyrrolone-fused benzosuberene (PBS) compounds were semi-synthesized from α,β,γ-Himachalenes extracted from the essential oil of Cedrus deodara following amino-vinyl-bromide substituted benzosuberenes as intermediates. These PBSs compounds classified as an attractive source of therapeutics. The α-isoform of PI3K which is a pivotal modulator of PI3K/AKT/mTOR signaling pathway, responsible for neurological disorders like epilepsy, found as a potential target molecule against these 17 semi-synthesized PBS compounds using in silico ligand-based pharmacophore mapping and target screening. The compounds screened using binding affinities, ADMET properties, and toxicity that were accessed by in silico docking simulations and pharmacokinetics profiling. Ultimately two compounds viz., PBS-8 and PBS-9 were selected for further in vivo evaluation using a zebrafish (Danio rerio) model of pentylenetetrazol (PTZ)-induced clonic convulsions. Additionally, gene expression studies performed for the genes of the PI3K/AKT/mTOR pathway which further validated our results. In conclusion, these findings suggested that PBS-8 is a promising candidate that could bedeveloped as a potential antiepileptic.

Aurora A is a mitotic serine/threonine kinase protein that is a proposed target of the first-line anticancer drug design. It has been found to be overexpressed in many human cancer cells, including hematological, breast, and colorectal. Here, we focus on a particular somatic mutant S155R of Aurora kinase A protein, whose activity decreases because of loss of interaction with a TPX2 protein that results in ectopic expression of the Aurora kinase A protein, which contributes chromosome instability, centrosome amplification, and oncogenic transformation. The primary target of this study is to select a drug molecule whose binding results in gaining S155R mutant interaction with TPX2. The computational methodology applied in this study involves mapping of hotspots (for uncompetitive binding), virtual screening, protein-ligand docking, postdocking optimization, and protein-protein docking approach. In this study, we screen and validate ZINC968264, which acts as a potential molecule that can improve the loss of function occurred because of mutation (S155R) in Aurora A. Our approaches pave a suitable path to design a potential drug against physiological condition manifested because of S155R mutant in Aurora A.

Gliomas are lethal and aggressive form of brain tumors with resistance to conventional radiation and cytotoxic chemotherapies; inviting continuous efforts for drug discovery and drug delivery. Interestingly, small molecule hybrids are one such pharmacophore that continues to capture interest owing to their pluripotent medicinal effects. Accordingly, we earlier reported synthesis of potent Styryl-cinnamate hybrids (analogues of Salvianolic acid F) along with its plausible mode of action (MOA). We explored iTRAQ-LC/MS-MS technique to deduce differentially expressed landscape of native & phospho-proteins in treated glioma cells. Based on this, Protein-Protein Interactome (PPI) was looked into by employing computational tools and further validated in vitro. We hereby report that the Styryl-cinnamate hybrid, an analogue of natural Salvianolic acid F, alters key regulatory proteins involved in translation, cytoskeleton development, bioenergetics, DNA repair, angiogenesis and ubiquitination. Cell cycle analysis dictates arrest at G0/G1 stage along with reduced levels of cyclin D; involved in G1 progression. We discovered that Styryl-cinnamate hybrid targets glioma by intrinsically triggering metabolite-mediated stress. Various oncological circuits alleviated by the potential drug candidate strongly supports the role of such pharmacophores as anticancer drugs. Although, further analysis of SC hybrid in treating xenografts or solid tumors is yet to be explored but their candidature has gained huge impetus through this study. This study equips us better in understanding the shift in proteomic landscape after treating glioma cells with SC hybrid. It also allows us to elicit molecular targets of this potential drug before progressing to preclinical studies.

Therapeutic failure and drug resistance are common sequelae to insulin resistance associated with type 2 diabetes mellitus (T2DM). Consequently, there is an unmet need of alternative strategies to overcome insulin resistance associated complications.To demonstrate whether Kutkin (KT), iridoid glycoside enriched fraction of Picrorhiza kurroa extract (PKE) has potential to increase the insulin sensitivity vis à vis glucose uptake in differentiated adipocytes.Molecular interaction of KT phytoconstituents, picroside-I (P-I)picroside- II (P-II) with peroxisome proliferator-activated receptor gamma (PPARγ), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) were analyzed in silico. Cellular viability and adipogenesis were determined by following 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium bromide (MTT) assay and Oil Red-O staining. Further, ELISA kit based triglycerides and diacylglycerol-O-Acyltransferase-1 (DGAT1) were assessed in differentiated adipocytes. ELISA based determination were performed to check the levels of adiponectin and tumor necrosis factor alpha (TNF-α). However, Flow cytometry and immunofluorescence based assays were employed to measure the glucose uptake and glucose transporter 4 (glut4) expression in differentiated adipocytes, respectively. Further to explore the targeted signaling axis, mRNA expression levels of PPARγ, CCAAT/enhancer binding protein α (CEBPα), and glut4 were determined using qRT-PCR and insulin receptor substrate-1 (IRS-1), Insulin receptor substrate-2 (IRS-2), PI3K/Akt, AS160, glut4 followed by protein validation using immunoblotting in differentiated adipocytes.In silico analysis revealed the binding affinities of major constituents of KT (P-IP-II) with PPARγ/PI3K/Akt. The enhanced intracellular accumulation of triglycerides with concomitant activation of PPARγ and C/EBPα in KT treated differentiated adipocytes indicates augmentation of adipogenesis in a concentration-dependent manner. Additionally, at cellular level, KT upregulated the expression of DAGT1, and decreases fatty acid synthase (FAS), and lipoprotein lipase (LPL), further affirmed improvement in lipid milieu. It was also observed that KT upregulated the levels of adiponectin and reduced TNFα expression, thus improving the secretory functions of adipocytes along with enhanced insulin sensitivity. Furthermore, KT significantly promoted insulin mediated glucose uptake by increasing glut4 translocation to the membrane via PI3/Akt signaling cascade. The results were further validated using PI3K specific inhibitor, wortmannin and findings revealed that KT treatment significantly enhanced the expression and activation of p-PI3K/PI3K and p-Akt/Akt even in case of treatment with PI3K inhibitor wortmannin alone and co-treatment with KT in differentiated adipocytes and affirmed that KT as activator of PI3K/Akt axis in the presence of inhibitor as well.Collectively, KT fraction of PKE showed anti-diabetic effects by enhancing glucose uptake in differentiated adipocytes via activation of PI3K/Akt signaling cascade. Therefore, KT may be used as a promising novel natural therapeutic agent for managing T2DMand to the best of our knowledge, this is the first report, showing the efficacy and potential molecular mechanism of KT in enhancing insulin sensitivity and glucose uptake in differentiated adipocytes.