Identification of potent BRD4-BD1 inhibitors using classical and steered molecular dynamics based free energy analysis.

In the present work a combination of traditional and steered molecular dynamics based techniques were employed to identify potential inhibitors against the human BRD4 protein (BRD4- BD1); an established drug target for multiple illnesses including various malignancies. Quinoline derivatives that were synthesized in-house were tested for their potential as new BRD4-BD1 inhibitors. Initially molecular docking experiments were performed to determine the binding poses of BRD4-BD1 inhibitors. To learn more about the thermodynamics of inhibitor binding to the BRD4-BD1 active site, the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) free energy calculations were conducted afterwards. The findings of the MM-PBSA analysis were further reinforced by performing steered umbrella sampling simulations which revealed crucial details about the binding/unbinding process of the most potent quinoline derivatives at the BRD4-BD1 active site. We report a novel quinoline derivative which can be developed into a fully functional BRD4-BD1 inhibitor after experimental validation. The identified compound (4 g) shows better properties than the standard BRD4-BD1 inhibitors considered in the study. The study also highlights the crucial role of Gln78, Phe79, Trp81, Pro82, Phe83, Gln84, Gln85, Val87, Leu92, Leu94, Tyr97, Met105, Cys136, Asn140, Ile146 and Met149 in inhibitor binding. The study provides a possible lead candidate and key amino acids involved in inhibitor recognition and binding at the active site of BRD4-BD1 protein. The findings might be of significance to medicinal chemists involved in the development of potent BRD4-BD1 inhibitors.

Bioactives from medicinal herb against bedaquiline resistant tuberculosis: removing the dark clouds from the horizon.

Tuberculosis is a contagious bacterial ailment that primarily affects the lungs and is brought on by the bacterium Mycobacterium tuberculosis (MTB). An antimycobacterial medication called bedaquiline (BQ) is specified to treat multidrug-resistant tuberculosis (MDR-TB). Despite its contemporary use in clinical practice, the mutations (D32 A/G/N/V/P) constrain the potential of BQ by causing transitions in the structural conformation of the atpE subunit-c after binding. In this study, we have taken the benzylisoquinoline alkaloids from thalictrum foliolosum due to its antimicrobial activity reported in prior literature. We used an efficient and optimized structure-based strategy to examine the wild type (WT) and mutated protein upon molecule binding. Our results emphasize the drastic decline in BQ binding affinity of mutant and WT atpE subunit-c complexes compared to thalirugidine (top hit) from thalictrum foliolosum. The decrease in BQ binding free energy is due to electrostatic energy because nearly every atom in a macromolecule harbors a partial charge, and molecules taking part in molecular recognition will interact electrostatically. Similarly, the high potential mean force of thalirugidine than BQ in WT and mutant complexes demonstrated the remarkable ability to eradicate mycobacteria efficiently. Furthermore, the Alamar blue cell viability and ATP determination assay were performed to validate the computational outcomes in search of novel antimycobacterial. Upon closer examination of the ATP determination assay, it became apparent that both BQ and thalirugidine showed similar reductions in ATP levels at their respective MICs, presenting a potential common mechanism of action.

Understanding the Role of Activation Loop Mutants in Drug Efficacy for FLT3-ITD.

The type III receptor tyrosine kinase FLT3 is a pivotal kinase for hematopoietic progenitor cell regulation, with significant implications in acute myeloid leukemia (AML) through mutations like internal tandem duplication (ITD). This study delves into the structural intricacies of FLT3, the roles of activation loop mutants, and their interaction with tyrosine kinase inhibitors. Coupled with this, the research leverages molecular contrastive learning and protein language modeling to examine interactions between small molecule inhibitors and FLT3 activation loop mutants. Utilizing the ConPLex platform, over 5.7 million unique FLT3 activation loop mutants-small molecule pairs were analyzed. The binding free energies of three inhibitors were assessed, and cellular apoptotic responses were evaluated under drug treatments. Notably, the introduction of the Xepto50 scoring system provides a nuanced metric for drug efficacy. The findings underscore the modulation of molecular interactions and cellular responses by Y842 mutations in FLT3-KD, highlighting the need for tailored therapeutic approaches in FLT3-ITD-related malignancies.

Small Heat Shock Protein (sHsp22.98) from Trialeurodes vaporariorum Plays Important Role in Apple Scar Skin Viroid Transmission.

Trialeurodes vaporariorum, commonly known as the greenhouse whitefly, severely infests important crops and serves as a vector for apple scar skin viroid (ASSVd). This vector-mediated transmission may cause the spread of infection to other herbaceous crops. For effective management of ASSVd, it is important to explore the whitefly's proteins, which interact with ASSVd RNA and are thereby involved in its transmission. In this study, it was found that a small heat shock protein (sHsp) from T. vaporariorum, which is expressed under stress, binds to ASSVd RNA. The sHsp gene is 606 bp in length and encodes for 202 amino acids, with a molecular weight of 22.98 kDa and an isoelectric point of 8.95. Intermolecular interaction was confirmed through in silico analysis, using electrophoretic mobility shift assays (EMSAs) and northwestern assays. The sHsp22.98 protein was found to exist in both monomeric and dimeric forms, and both forms showed strong binding to ASSVd RNA. To investigate the role of sHsp22.98 during ASSVd infection, transient silencing of sHsp22.98 was conducted, using a tobacco rattle virus (TRV)-based virus-induced gene silencing system. The sHsp22.98-silenced whiteflies showed an approximate 50% decrease in ASSVd transmission. These results suggest that sHsp22.98 from T. vaporariorum is associated with viroid RNA and plays a significant role in transmission.

Dispersion-corrected DFT calculations and umbrella sampling simulations to investigate stability of Chrysin-cyclodextrin inclusion complexes.

The study of inclusion complexes of Chrysin (ChR) with three forms of cyclodextrins (CDs) α-, ß-, and γ-CD was accomplished to examine the stability of ChR inside the central cavities of CDs. The aim of study was to identify the most suitable form of CD to improve the hydro-solubility of poorly soluble ChR bioactive molecule. Microsecond timescale molecular dynamics (MD) simulations were performed on four inclusion complexes (α-CD/ChR, ß-CD/ChR, and two conformations of γ-CD/ChR) to examine the dynamics of ChR inside the cavity of CDs. The first conformation of γ-CD/ChR inclusion complex (γ-CD1/ChR) was identified to possess the highest affinity between host and guest molecule on the basis of binding energy calculated by employing Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) and umbrella sampling simulations. To further strengthen the claims of classical and biased MD studies, Our own N-layered Integrated molecular Orbital and Molecular mechanics (ONIOM) (wB97XD/6-311+g(d,p):pm7) calculations were performed on the selected inclusion complexes. The ONIOM based complexation energy reaffirmed that ChR had highest affinity for the γ-CD1 host molecule. Further, the non-covalent interaction analysis was conducted using Multiwfn software on QM-optimized inclusion complexes with wB97XD/6-311+G(d,p) model chemistry, revealing non-covalent interactions between ChR and CDs. This atomic level information helped us to gain better insights into critical atoms of ChR and CD that participated in intermolecular interactions and identify γ-CD as a suitable host molecule for improving the hydro-solubulity of ChR. The structural insights would help to derive new derivatives of γ-CD with better host capacity.

Effect of co-pigments on anthocyanins of Rhododendron arboreum and insights into interaction mechanism.

The impact of intermolecular copigmentation between five phenolic acids, two flavonoid and three amino acids with R. arboreum anthocyanins (ANS) and its isolated cyanidin-3-O-monoglycosides were investigated through experimental and theoretical approach. On addition of different copigments, phenolic acid induced strong hyperchromic (0.26-0.55 nm) and bathochromic shift (6.6-14.2 nm). The color intensity and stability of ANS with, storage at 4 °C & 25 °C, sunlight, oxidation and heat were evaluated by chromaticity, anthocyanin content, kinetic and structural simulation analysis. The strongest copigmentation reaction was observed with narningin (NA) and also showed high thermostability and highest half-life i.e. 3.39 h-1.24 h at 90-160 °C. The cyanidin-3-O-monoglycosides were analysed for their copigmentation effect and observations revealed that NA displayed best copigmentation effect to cyanidin-3-O-arabinoside (B) followed by cyanidin-3-O-galactoside (A), and cyanidin-3-O-rhamnoside (C). Additionally, structural simulation and steered molecular dynamics insights NA is the most favourable co-pigment involving π-π stacking and H-bonding.

Phloretin and phlorizin mitigates inflammatory stress and alleviate adipose and hepatic insulin resistance by abrogating PPARγ S273-Cdk5 interaction in type 2 diabetic mice.

AIMS: The rising prevalence of type 2 diabetes mellitus (T2DM) and accompanying insulin resistance is alarming globally. Natural and synthetic agonists of PPARγ are potentially attractive candidates for diabetics and are known to efficiently reverse adipose and hepatic insulin resistance, but related side effects and escalating costs are the causes of concern. Therefore, targeting PPARγ with natural ligands is advantageous and promising approach for the better management of T2DM. The present research aimed to assess the antidiabetic potential of phenolics Phloretin (PTN) and Phlorizin (PZN) in type 2 diabetic mice. MAIN METHODS: In silico docking was performed to check the effect of PTN and PZN on PPARγ S273-Cdk5 interactions. The docking results were further validated in preclinical settings by utilizing a mice model of high fat diet-induced T2DM. KEY FINDINGS: Computational docking and further MD-simulation data revealed that PTN and PZN inhibited the activation of Cdk5, thereby blocking the phosphorylation of PPARγ. Our in vivo results further demonstrated that PTN and PZN administration significantly improved the secretory functions of adipocytes by increasing adiponectin and reducing inflammatory cytokine levels, which ultimately reduced the hyperglycaemic index. Additionally, combined treatment of PTN and PZN decreased in vivo adipocyte expansion and increased Glut4 expression in adipose tissues. Furthermore, PTN and PZN treatment reduced hepatic insulin resistance by modulating lipid metabolism and inflammatory markers. SIGNIFICANCE: In summary, our findings strongly imply that PTN and PZN are candidates as nutraceuticals in the management of comorbidities related to diabetes and its complications.

Profiling the disintegration of BRPs released by massive wasp stings using serratiopeptidase: An in-silico insight.

Serratiopeptidase is 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 facilitate 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. To accomplish this objective, we docked selected BRPs onto the binding pocket of wild and previously identified mutant (N412D) of serratiopeptidase. Based on their docked scores, the 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 identical ΔG values when compared to the reference complex. Further, we condemned the external pulling forces on both peptides to observe the force needed in the disassociation process to endorse the binding affinity findings in terms of unbinding mechanism. This analysis suggested that BRP-7 (Wasp kinin PMM1) peptide was tightly anchored and laid out the highest pulling force to get detach from the active pocket of serratiopeptidase in contrast to the BRP-6 peptide. The current study endorses up the present findings and paves the way for serratiopeptidase to be used as an anti-angioedemic peptidase as well as a fixed-dose combination (FDC) in hypotensive drugs.

A comparative study on inclusion complex formation between formononetin and ß-cyclodextrin derivatives through multiscale classical and umbrella sampling simulations.

Formononetin, a naturally occurring isoflavone exhibits a wide range of therapeutic applications including antioxidant, anti-tumor, antiviral, anti-diabetic and neuroprotective activities. However, the low hydro-solubility of formononetin has limited its prospective use in cosmetic, neutraceutical and pharmaceutical industries. Cyclodextrins (CDs), especially ß-CD and its derivatives have emerged as promising agents to improve the water solubility of poorly hydrosoluble compounds by the formation of inclusion complexes. We employed multiscale (1000 ns) explicit solvent and umbrella sampling molecular dynamics (MD) simulations to study the interactions and thermodynamic parameters of inclusion complex formation between formononetin and five most commonly used ß-CD derivatives. Classical MD simulations revealed two possible binding conformations of formononetin inside the central cavity of hydroxypropyl-ß-CD (HP-ß-CD), randomly methylated-ß-CD (ME-ß-CD), and sulfobutylether-ß-CD (SBE-ß-CD). The binding conformation with the benzopyrone ring of formononetin inside the central cavity of ß-CD derivatives was more frequent than the phenyl group occupying the hydrophobic cavity. These interactions were supported by a variety of non-bonded contacts including hydrogen bonds, pi-lone pair, pi-sigma, and pi-alkyl interactions. Formononetin showed favorable end-state MD-driven thermodynamic binding free energies with all the selected ß-CD derivatives, except succinyl-ß-CD (S-ß-CD). Furthermore, umbrella sampling simulations were used to investigate the interactions and thermodynamic parameters of the host-guest inclusion complexes. The SBE-ß-CD/formononetin inclusion complex showed the lowest binding energy signifying the highest affinity among all the selected host-guest inclusion complexes. Our study could be used as a standard for analyzing and comparing the ability of different ß-CD derivatives to enhance the hydro-solubility of poorly soluble molecules.

Computational analysis of protein-ligand interaction by targeting a cell cycle restrainer.

BACKGROUND AND OBJECTIVE: The cyclin-dependent kinases 4/6 (CDK4/6) are among the most crucial controllers of the cell cycle, and their abnormal activity may induce uncontrolled cell multiplication, leading to cancers. The FDA currently approved three CDK4/6 inhibitors, however, they are associated with a variety of side effects. Thus it is required to design/develop novel potent and safe CDK4/6 inhibitors. METHODS: In the present work, we furnished an integrated in-silico approach followed by steered molecular dynamics (SMD) simulations to identify molecules that can be developed into novel CDK4/6 inhibitors. RESULTS: Out of thirty-two 3-methyleneisoindolin-1-one molecules we selected top three M18, M24, and M32 molecules as potential drug candidates based on their respective interaction energies. According to the robust 250 ns MD simulations and thermodynamic free energy, M24 was the best molecule in comparison to palbociclib. In SMD, M24 required ∼205.587 kJ/mol/nm external pulling force, while palbociclib needed ∼160.97 kJ/mol/nm to dissociate from the binding pocket of the CDK4. CONCLUSIONS: The high pulling force required for M24 dissociation from the binding site denotes stronger binding with CDK4. Therefore, M24 offers the possibility of a critical starting structure in developing effective CDK4 inhibitors.

Structure restoration and aggregate inhibition of V30M mutant transthyretin protein by potential quinoline molecules.

Transthyretin (TTR) is a tetrameric protein found in human plasma and cerebrospinal fluid that functions as a transporter of thyroxine (T4) and retinol. A mutation resulting in the substitution of valine to methionine at position 30 (V30M) is the most common mutation that destabilizes the tetramer structure of TTR protein resulting in a fatal neuropathy known as TTR amyloidosis. The V30M TTR-induced neuropathy can be inhibited through stabilization of the TTR tetramer by the binding of small molecules. We accessed the potential of in-house synthesized quinoline molecules to stabilize the V30M TTR structure and analyzed the impact of protein-ligand interactions through molecular docking, molecular dynamics (MD) simulations, steered MD, and umbrella sampling simulations. This study revealed that the binding of quinoline molecules reverted back the structural changes including the residual flexibility, changes in secondary structural elements, and also restored the alterations in the electrostatic surface potential induced by the V30M mutation. Further, the top-most 4G and 4R molecules were compared with an FDA-approved drug (Tafamidis) and a reference quinoline molecule 14C. Here, we intend to suggest that the quinoline molecules could revert the structural changes, cease tetramer dissociation, prevent abnormal oligomerization and therefore could be developed as an effective therapeutics against TTR amyloidosis.

Identification of potential mutational hotspots in serratiopeptidase to address its poor pH tolerance issue.

Serratiopeptidase is the multifunctionality metalloendopeptidase extensively employed in biopharmaceutical and industrial biotechnology. Despite its poor pH tolerance, serratiopeptidase must withstand the highly acidic environment of the gastrointestinal tract to be used as a potent anti-inflammatory and analgesic medication. In earlier studies, post-translational deamination related mutations showed alteration in the net charge of protein's surface. Therefore, the current study aimed to enhance the acid resistance of serratiopeptidase via implementing computational interventions to screen out the most stable mutational hotspot. The methodology used in this study is as follows: (a) Higher accessibility to surface (b) 4 Å away from active site region to avoid interference with its proteolytic activity, and (c) By converting non-conserved amide residues to acidic residues. A docking study has been conducted to establish the substrate specificity and binding affinity to native and mutant proteins. The docking outcomes were then validated using molecular dynamic simulations to clarify each mutant's molecular stability and conformation while preserving their activity. The results showed that N412D is the best-screened mutant with negative electrostatic potential that can alter the overall charge on the protein's surface with increased H+ ions. Alteration in overall charge leads to protein surface more acidic that causes a common ion effect in stomach pH and act as a buffer which could stabilize the serratiopeptidase amid extreme pH.Communicated by Ramaswamy H. Sarma.

An insight from computational approach to explore novel, high-affinity phosphodiesterase 10A inhibitors for neurological disorders.

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.

Mechanochemical Approach towards Multi-Functionalized 1,2,3-Triazoles and Anti-Seizure Drug Rufinamide Analogs Using Copper Beads.

Highly regiospecific, copper-salt-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, the high-speed ball milling (HSBM) strategy has also been extended to the synthesis of the commercially available pharmaceutical agent, Rufinamide, an antiepileptic drug (AED) and its analogues. The same strategy was also applied for the synthesis of the 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 are easily recoverable from the reaction mixture and used for the further reactions without any special treatment.

Inhibition of nonstructural protein 15 of SARS-CoV-2 by golden spice: A computational insight.

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.

A ricin-based peptide BRIP from Hordeum vulgare inhibits Mpro of SARS-CoV-2.

COVID-19 pandemic caused by SARS-CoV-2 led to the research aiming to find the inhibitors of this virus. Towards this world problem, an attempt was made to identify SARS-CoV-2 main protease (Mpro) inhibitory peptides from ricin domains. The ricin-based peptide from barley (BRIP) was able to inhibit Mpro in vitro with an IC50 of 0.52 nM. Its low and no cytotoxicity upto 50 µM suggested its therapeutic potential against SARS-CoV-2. The most favorable binding site on Mpro was identified by molecular docking and steered molecular dynamics (MD) simulations. The Mpro-BRIP interactions were further investigated by evaluating the trajectories for microsecond timescale MD simulations. The structural parameters of Mpro-BRIP complex were stable, and the presence of oppositely charged surfaces on the binding interface of BRIP and Mpro complex further contributed to the overall stability of the protein-peptide complex. Among the components of thermodynamic binding free energy, Van der Waals and electrostatic contributions were most favorable for complex formation. Our findings provide novel insight into the area of inhibitor development against COVID-19.

Theaflavin 3-gallate inhibits the main protease (Mpro) of SARS-CoV-2 and reduces its count in vitro.

The main protease (Mpro) of SARS-CoV-2 has been recognized as an attractive drug target because of its central role in viral replication. Our previous preliminary molecular docking studies showed that theaflavin 3-gallate (a natural bioactive molecule derived from theaflavin and found in high abundance in black tea) exhibited better docking scores than repurposed drugs (Atazanavir, Darunavir, Lopinavir). In this study, conventional and steered MD-simulations analyses revealed stronger interactions of theaflavin 3-gallate with the active site residues of Mpro than theaflavin and a standard molecule GC373 (a known inhibitor of Mpro and novel broad-spectrum anti-viral agent). Theaflavin 3-gallate inhibited Mpro protein of SARS-CoV-2 with an IC50 value of 18.48 ± 1.29 µM. Treatment of SARS-CoV-2 (Indian/a3i clade/2020 isolate) with 200 µM of theaflavin 3-gallate in vitro using Vero cells and quantifying viral transcripts demonstrated reduction of viral count by 75% (viral particles reduced from Log106.7 to Log106.1). Overall, our findings suggest that theaflavin 3-gallate effectively targets the Mpro thus limiting the replication of the SARS-CoV-2 virus in vitro.

Kutkin, iridoid glycosides enriched fraction of Picrorrhiza kurroa promotes insulin sensitivity and enhances glucose uptake by activating PI3K/Akt signaling in 3T3-L1 adipocytes.

BACKGROUND: 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. PURPOSE: 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. METHODS: 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. RESULTS: In silico analysis revealed the binding affinities of major constituents of KT (P-I& P-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. CONCLUSION: 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.