The combination of Curcumin and Doxorubicin on targeting PI3K/AKT/mTOR signaling pathway: an in vitro and molecular docking study for inhibiting the survival of MDA-MB-231.

The process of tumorigenesis is highly associated with the disruption of cell-cycle regulators and derangement of various signaling pathways, which end up with the inhibition of apoptosis and hyper-activation of survival pathways. The PI3K medicated AKT/mTOR pathway is the widely explained mechanism for cancer cell survival which causes the overexpression of MDM2 and downregulates the p53-BAX mediated apoptotic pathway. Curcumin (CUR), the phyto-compound, derived from Curcuma longa is currently being focused on for its anticancer activities against breast cancer cells, MDA-MB-231, not only because of its minimal cytotoxicity against healthy cells (HEK293) but also because it synergistically sensitizes the activity of Doxorubicin (DOXO) in lower doses, which can be a promising source for complementary drug development. This study aims to investigate the combinatorial effect of CUR and DOXO on PI3K/AKT/mTOR pathway proteins by sequential molecular docking analysis and MD simulation studies. The lower binding affinity of the sequentially docked protein-ligand complex proves the increasing binding affinity of CUR and DOXO in the combinatorial dose. The mRNA expressions of different genes of this pathway are observed and quantified using rt-qPCR, where the decreasing fold change (2-∆∆Ct) indicates the suppression of the AKT/mTOR pathway after co-treatment of CUR and DOXO against MDA-MB-231 cells. These in silico and in vitro findings can be a new horizon for further in vitro and clinical trials of breast cancer treatment. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00231-2.

The recent advancements in circRNA research: From biogenesis to therapeutic interventions.

Circular RNAs (circRNAs) belong to the genre of long non-coding RNAs that are formed by special back-splicing events and are currently the molecule of interest for studies globally due their involvement in various ailments like diabetes, neurodegenerative disorders, cardio-vascular diseases and cancers. These class of highly stable RNAs participate in diverse cellular functionalities including microRNA (miRNA) sponging, ceRNA (competing endogenous RNA) activity or via exhibiting RNA binding protein (RBP) interactions. They are also known to regulate cancer progression both positively and negatively through various biological pathways such as, modulating the cell cycle and apoptotic pathways, epigenetic regulation, and translational and/or transcriptional regulations etc. Given its significance, a variety of computational tools and dedicated databases have been created for the identification, quantification, and differential expression of such RNAs in combination with sequencing approaches. In this review, we provide a comprehensive analysis of the numerous computational tools, pipelines, and online resources developed in recent years for the detection and annotation of circRNAs. We also summarise the most recent findings regarding the characteristics, functions, biological processes, and involvement of circRNAs in diseases. The review emphasises the significance of circRNAs as potential disease biomarkers and new treatment targets.

Variation in glucose metabolism under acidified sodium nitrite mediated nitrosative stress in Saccharomyces cerevisiae.

AIMS: The work aimed to understand the important changes during glucose metabolism in Saccharomyces cerevisiae under acidified sodium nitrite (ac.NaNO2 ) mediated nitrosative stress. METHODS AND RESULTS: Confocal microscopy and fluorescence-activated cell sorting analysis were performed to investigate the generation of reactive nitrogen and oxygen species, and redox homeostasis under nitrosative stress was also characterized. Quantitative PCR analysis revealed that the expression of ADH genes was upregulated under such condition, whereas the ACO2 gene was downregulated. Some of the enzymes of the tricarboxylic acid cycle were partially inhibited, whereas malate metabolism and alcoholic fermentation were increased under nitrosative stress. Kinetics of ethanol production was also characterized. A network analysis was conducted to validate our findings. In the presence of ac.NaNO2 , in vitro protein tyrosine nitration formation was checked by western blotting using pure alcohol dehydrogenase and aconitase. CONCLUSIONS: Alcoholic fermentation rate was increased under stress condition and this altered metabolism might be conjoined with the defence machinery to overcome the nitrosative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work of this kind where the role of metabolism under nitrosative stress has been characterized in S. cerevisiae and it will provide a base to develop an alternative method of industrial ethanol production.

Protein-protein interaction and in silico mutagenesis studies on IL17A and its peptide inhibitor.

Protein-protein interactions of Interleukin-17 (IL17) play vital role in the autoimmune and inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, and psoriasis. Potent therapeutics for these diseases could be developed by blocking or modulating these interactions through biologics, peptide inhibitors and small molecule inhibitors. Unlike biologics, peptide inhibitors are cost effective and can be orally available. Peptide inhibitors do not require a binding groove as that of small molecules either. Therefore, crystal structure of IL17A in complex with a high affinity peptide inhibitor (HAP) (1-IHVTIPADLWDWIN-14) is investigated with an aim to find hot spots that could improve its potency. An in silico mutagenesis strategy was implemented using FoldX PSSM to scan for positions tolerant to amino acid substitution. Three positions T4, A7, and N14 showed improved stability when mutated with 'F/M/Y', 'P' and 'F/M/Y', respectively. A set of 31 mutant peptides are designed through combinations of these tolerant mutations using Build Model application of FoldX. Binding affinity and interactions of 31 peptides are assessed through protein-peptide docking and binding free energy calculations. Two peptides namely, P1 ("1-IHVTIPPDLWDWIY-14") and P2 ("1-IHVMIPPDLWDWIF-14") showed better binding affinity to IL17A dimerization site compared to HAP. Interactions of P1, P2 and HAP are also analyzed through 100 ns molecular dynamics simulations using GROMACS v5.0. The results revealed that the P2 peptide likely to offer better potency compared to HAP and P1. Therefore, the P2 peptide can be synthesized to develop oral therapies for autoimmune and inflammatory diseases with further experimental evaluations. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02856-y.

In silico discovery and evaluation of phytochemicals binding mechanism against human catechol-O-methyltransferase as a putative bioenhancer of L-DOPA therapy in Parkinson disease.

Levodopa (L-DOPA) therapy is normally practised to treat motor pattern associated with Parkinson disease (PD). Additionally, several inhibitory drugs such as Entacapone and Opicapone are also cosupplemented to protect peripheral inactivation of exogenous L-DOPA (~80%) that occurs due to metabolic activity of the enzyme catechol-O-methyltransferase (COMT). Although, both Entacapone and Opicapone have U.S. Food and Drug Administration approval but regular use of these drugs is associated with high risk of side effects. Thus, authors have focused on in silico discovery of phytochemicals and evaluation of their effectiveness against human soluble COMT using virtual screening, molecular docking, drug-like property prediction, generation of pharmacophoric property, and molecular dynamics simulation. Overall, study proposed, nine phytochemicals (withaphysalin D, withaphysalin N, withaferin A, withacnistin, withaphysalin C, withaphysalin O, withanolide B, withasomnine, and withaphysalin F) of plant Withania somnifera have strong binding efficiency against human COMT in comparison to both of the drugs i.e., Opicapone and Entacapone, thus may be used as putative bioenhancer in L-DOPA therapy. The present study needs further experimental validation to be used as an adjuvant in PD treatment.

Kinome analyses of Candida albicans, C. parapsilosis and C. tropicalis enable novel kinases as therapeutic drug targets in candidiasis.

Candida spp. have attracted considerable attention as they cause serious human diseases in immunocompromised individuals. The genomes of the pathogenic Candida spp. have been sequenced, but systemic characterizations of their kinomes are yet to be reported. As in various eukaryotes, the protein kinases play crucial regulatory roles in pathogenicity of Candida. Increased frequency of antifungal resistance in Candida spp. requires significant attention to explore novel therapeutic molecules for their control. The present in-silico study involves novel bioinformatics strategies to identify the kinase proteins and their potential drug targets with the purpose to combat fungal infections. The study reports 103, 107 and 106 kinase proteins from 3 Candida spp., C. albicans, C. parapsilosis and C. tropicalis, respectively. Moreover, 79 common kinase proteins were identified, of which 54 proteins play essential roles in Candida spp. and 42 proteins were human non-homologues. Among the essential and human non-homologous protein kinases, 9 were found to be common essential human non-homologues, of which 6 are uniquely present in Candida. These 6 protein kinases namely, Hsl1, Npr1, Ptk2, Kin2, Ksp1 and orf19.3854 (CAALFM_CR06040WA) are involved in various molecular and cellular processes regulating virulence or pathogenicity. Further, these 6 kinases are prioritized as potential drug targets and explored for discovering new lead compounds against candidiasis. The drug repurposing approach for these 6 kinases show 13 approved drugs and investigational compounds that might play substantial inhibitory roles during combating candidiasis.

Prediction of the differentially expressed circRNAs to decipher their roles in the onset of human colorectal cancers.

Circular RNAs belong to the class of endogenous long non-coding RNAs that play important roles in many physiological processes including tumorigenesis. One such process is the onset of colorectal cancers (CRC) which is one of the most prevalent cancers in the world. However, the involvement of the circRNAs in CRC progression is still obscure. In this study, we screened the differentially expressed circRNAs in CRC by taking 10 pairs of tumor and non-tumor transcriptomic data. Datasets were downloaded from EBI ENA database and differential expression analysis was performed. For functional characterization and pathway enrichment of differentially expressed circRNAs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed. Interactions with miRNAs and RNA binding proteins (RBPs) were predicted using miRanda, miRTarBase and starBase tools respectively. Our results identified total of 122 differentially expressed circRNAs in CRC onset, including 85 upregulated and 37 downregulated. GO and KEGG analyses revealed these circRNAs to be involved in many tumorigenic pathways. In addition, we predicted many miRNA and RBP targets of significantly expressed circRNAs that could exhibit the functional role in CRC progression. Combined analyses of miRanda, miRTarBase and KEGG pathway suggested that the possibly affected genes by circRNA-miRNA sponge to be associated with many cancer related pathways. From our findings we concluded 16 novel differentially expressed circRNAs that could play important roles in carcinogenesis of CRC. Our findings provide new insights in circRNA research and could therefore be useful in the development of potential biomarker and therapeutic approaches for CRC.

Network analysis of hyphae forming proteins in Candida albicans identifies important proteins responsible for pathovirulence in the organism.

Candida albicans causes two types of major infections in humans: superficial infections, such as skin and mucosal infection, and life-threatening systemic infections, like airway and catheter-related blood stream infections. It is a polymorphic fungus with two distinct forms (yeast and hyphal) and the morphological plasticity is strongly associated with many disease causing proteins. In this study, 137 hyphae associated proteins from Candida albicans (C. albicans) were collected from different sources to create a Protein-Protein Interaction (PPI) network. Out of these, we identified 18 hub proteins (Hog1, Hsp90, Cyr1, Cdc28, Pkc1, Cla4, Cdc42, Tpk1, Act1, Pbs2, Bem1, Tpk2, Ras1, Cdc24, Rim101, Cdc11, Cdc10 and Cln3) that were the most important ones in hyphae development. Ontology and functional enrichment analysis of these proteins could categorize these hyphae associated proteins into groups like signal transduction, kinase activity, biofilm formation, filamentous growth, MAPK signaling etc. Functional annotation analysis of these proteins showed that the protein kinase activity to be essential for hyphae formation in Candida. Additionally, most of the proteins from the network were predicted to be localized on cell surface or periphery, suggesting them as the main protagonists in inducing infections within the host. The complex hyphae formation phenomenon of C. albicans is an attractive target for exploitation to develop new antifungals and anti-virulence strategies to combat C. albicans infections. We further tried to characterize few of the most crucial proteins, especially the kinases by their sequence and structural prospects. Therefore, through this article an attempt to understand the hyphae forming protein network analysis has been made to unravel and elucidate the complex pathogenesis processes with the principal aim of systems biological research involving novel Bioinformatics strategies to combat fungal infections.

Protective role of epigallocatechin-3-gallate in NADPH oxidase-MMP2-Spm-Cer-S1P signalling axis mediated ET-1 induced pulmonary artery smooth muscle cell proliferation.

The signalling pathway involving MMP-2 and sphingosine-1-phosphate (S1P) in endothelin-1 (ET-1) induced pulmonary artery smooth muscle cell (PASMC) proliferation is not clearly known. We, therefore, investigated the role of NADPH oxidase derived O2.--mediated modulation of MMP2-sphingomyeline-ceramide-S1P signalling axis in ET-1 induced increase in proliferation of PASMCs. Additionally, protective role of the tea cathechin, epigallocatechin-3-gallate (EGCG), if any, in this scenario has also been explored. ET-1 markedly increased NADPH oxidase and MMP-2 activities and proliferation of bovine pulmonary artery smooth muscle cells (BPASMCs). ET-1 also caused significant increase in sphingomyelinase (SMase) activity, ERK1/2 and sphingosine kinase (SPHK) phosphorylations, and S1P level in the cells. EGCG inhibited ET-1 induced increase in SMase activity, ERK1/2 and SPHK phosphorylations, S1P level and the SMC proliferation. EGCG also attenuated ET-1 induced activation of MMP-2 by inhibiting NADPH oxidase activity upon inhibiting the association of the NADPH oxidase components, p47phox and p67phox in the cell membrane. Molecular docking study revealed a marked binding affinity of p47phox with the galloyl group of EGCG. Overall, our study suggest that ET-1 induced proliferation of the PASMCs occurs via NADPH oxidase-MMP2- Spm- Cer-S1P signalling axis, and EGCG attenuates ET-1 induced increase in proliferation of the cells by inhibiting NADPH oxidase activity.

Exploration and validation of diphosphate-based Plasmodium LytB inhibitors using computational approaches.

LytB or IspH is an indispensable enzyme and a suitable drug target of Plasmodium falciparum that participate in isoprenoid biosynthesis of nonmevalonate pathway (MEP). Recently, we have investigated the structural dynamics of Plasmodium LytB and proposed some novel diphosphate-based inhibitors using molecular modeling and docking studies. Here, we have tried to characterize those previously screened molecules by quantitative structure activity relationships and pharmacophore-based analyses, as well as validated the dynamics of their interactions with LytB protein. Five total compounds having PubChem CID 516, 125696, 165275, 448012, and 9921431 were predicted with significant inhibitory activity by quantitative structure activity relationships and pharmacophore models. Again, the molecular dynamics simulation results showed that these five compounds are able to form stable complexes with the receptor through many direct and water mediated interactions. The binding free energies calculated by Poisson-Boltzmann surface area method resulted within the range between -99.77 and - 43.74 kcal/mol, which favoured their profound inhibitory affinity. Residues of LytB like His41, His74, Ser222, Ser223, and Asn224 in LytB were the main protagonists in contributing the majority of interaction energies to the ligands. Finally, the ADMET, toxicity, and drug-likeness scores also affirm these compounds to be considered for further development of new antimalarial inhibitor in the future.

Probing the mechanism of SIRT1 activation by a 1,4-dihydropyridine.

The NAD+-dependent deacetylase SIRT1 plays important roles in several physiological processes such as transcription, genome stability, stress responses, and aging. Due to its diverse role in metabolisms, SIRT1 has emerged as a potential therapeutic target in many human disorders such as type II diabetes, cardiovascular and neurodegenerative diseases, and cancer. Recent studies have reported that modulation of SIRT1 activity by phenolic activators like resveratrol and some 1,4-dihydropyridines (1,4-DHPs) can inhibit tumor growth by promoting apoptosis in cancer cells. However, the mechanism of SIRT1 activation is still not clear. In this report, we have tried to elucidate the mechanism of SIRT1 activation from studies on its interaction with a synthetic 1,4-DHP derivative (DHP-8; 3,5-diethoxy carbonyl-4-(4-nitrophenyl)-2,6-dimethyl-1,4-dihydropyridine) using molecular modeling, docking, simulation, and free energy analyses. Owing to the absence of full-length human SIRT1 structure, multi-template based modeling approach was opted followed by docking of DHP-8 at its allosteric site. In presence of DHP-8, the overall conformation of SIRT1 was found to be more stable (especially at its substrate binding sites) with a large structural variation at its N-terminal domain while bound to substrate p53 or p53-W. Determination of the MM/PBSA free energy indicated that the binding of DHP-8 to SIRT1 significantly increased the binding affinity of SIRT1 to its substrate p53-W as well as to NAD+. Overall, this study depicts the atomistic detailed mechanism for the direct activation of SIRT1 by a 1,4-DHP. This would serve to develop new SIRT1 activators for future therapeutic perspectives.

Analysis of interleukin 23 and 7G10 interactions for computational design of lead antibodies against immune-mediated inflammatory diseases.

Wealth of structural data on theurapeutic targets in complex with monoclonal antibodies (mAbs) and advances in molecular modeling algorithms present exciting opportunities in the field of novel biologic design. Interleukin 23 (IL23), a well-known drug target for autoimmune diseases, in complex with mAb 7G10 offers prospect to design potent lead antibodies by traversing the complete epitope-paratope interface. Herein, key interactions aiding antibody-based neutralization in IL23-7G10 complex are resolute through PyMOL, LigPlot+, Antibody i-Patch, DiscoTope and FoldX. Six amino acids Ser31, Val33, Asn55, Lys59 in heavy chain and His34, Ser93 in light chain are subjected to in silico mutagenesis with residues Met, Trp, Ile, Leu and Arg. A set of 431 mutant macromolecules are outlined. Binding affinities of these molecules with IL23 are estimated through protein-protein docking by employing ZDOCK, ClusPro and RosettaDock. Subsequently, the macromolecules revealed comparable result with 7G10 are cross validated through binding free-energy calculations by applying Molecular Mechanics/Poisson Boltzman Surface Area method in CHARMM. Thirty nine designed theoretical antibodies showed improved outcome in all evaluations; from these, top 10 molecules showed at least nine unit better binding affinity compared to the known mAb. These molecules have the potential to act as lead antibodies. Subsequent molecular dynamics simulations too favored prospective of best ranked molecule to have therapeutic implications in autoimmune and inflammatory diseases. Abbreviations: IL23: interleukin 23; IL17: interleukin17; Ab: antibody; Ag: antigen; mAbs: monoclonal antibodies; STAT3: signal transducer and activator of transcription 3; STAT4: signal transducer and activator of transcription 4; PDB: protein databank; MM/PBSA: molecular mechanics Poisson-Boltzmann surface area; Ag-Ab: antigen- antibody complex; SPC/E: extended simple point charge; SD: steepest descents; PME: particle mesh ewald; dG: binding free energies; Fv: variable fragment.

Novel 1,4-dihydropyridine induces apoptosis in human cancer cells through overexpression of Sirtuin1.

1,4-Dihydropyridines (1,4-DHPs) are important as a class of heterocyclic compounds that exhibit wide range of biological actions. Many of its derivatives are already characterized as medicinally important drugs and used worldwide. In this study, we have screened some novel Hantzsch 1,4-DHP compounds using both in silico (QSAR and Pharmacophore) and in vitro (cytotoxic screening). 1,4-DHP showed selective cytotoxicity against five human cancerous cell lines; A375, A549, HeLa, HepG2 and SH-SY5Y but limited effect towards normal skin keratinocyte (HaCaT), lung fibroblast (WL-38) and healthy peripheral blood mononuclear cells. In A375 and HepG2 cells, one of the 1,4-DHP derivative (DHP-8) was found to inhibit cell proliferation, and simultaneously increased the apoptotic population as well as mitochondrial membrane depolarization. Furthermore, the mitochondrial signal was triggered with the activation of cleaved Caspase9, Caspase3 and PARP. The treatment with DHP-8 also increased the expression level of SIRT1, subsequently decreasing the level of pAKTser473 and survivin. Reduced pAKTser473 expression led to decrease the phosphorylated inactive form of GSK3ßser9 and as a result, proteasomal degradation of Mcl-1 occurred in both the cell lines. Here, we suggest that the apoptotic effect of DHP-8 in A375 and HepG2 cells was mediated by AKT and survivin pathways through SIRT1 activation. The involvement of DHP-8 in SIRT1 activation was further verified by co-treatment of nicotinamide with DHP-8 in both A375 and HepG2 cells. Overall, this study emphasizes the possible potential and therapeutic role of DHP-8 in skin and liver cancer.

Role of curcumin in PLD activation by Arf6-cytohesin1 signaling axis in U46619-stimulated pulmonary artery smooth muscle cells.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to produce phosphatidic acid (PA) which in some cell types play a pivotal role in agonist-induced increase in NADPH oxidase-derived [Formula: see text]production. Involvement of ADP ribosylation factor (Arf) in agonist-induced activation of PLD is known for smooth muscle cells of systemic arteries, but not in pulmonary artery smooth muscle cells (PASMCs). Additionally, role of cytohesin in this scenario is unknown in PASMCs. We, therefore, determined the involvement of Arf and cytohesin in U46619-induced stimulation of PLD in PASMCs, and the probable mechanism by which curcumin, a natural phenolic compound, inhibits the U46619 response. Treatment of PASMCs with U46619 stimulated PLD activity in the cell membrane, which was inhibited upon pretreatment with SQ29548 (Tp receptor antagonist), FIPI (PLD inhibitor), SecinH3 (inhibitor of cytohesins), and curcumin. Transfection of the cells with Tp, Arf-6, and cytohesin-1 siRNA inhibited U46619-induced activation of PLD. Upon treatment of the cells with U46619, Arf-6 and cytohesin-1 were translocated and associated in the cell membrane, which were not inhibited upon pretreatment of the cells with curcumin. Cytohesin-1 appeared to be necessary for in vitro binding of GTPγS with Arf-6; however, addition of curcumin inhibited binding of GTPγS with Arf-6 even in the presence of cytohesin-1. Our computational study suggests that although curcumin to some extent binds with Tp receptor, yet the inhibition of Arf6GDP to Arf6GTP conversion appeared to be an important mechanism by which curcumin inhibits U46619-induced increase in PLD activity in PASMCs.

Role of catechins on ET-1-induced stimulation of PLD and NADPH oxidase activities in pulmonary smooth muscle cells: determination of the probable mechanism by molecular docking studies.

The treatment of human pulmonary artery smooth muscle cells with ET-1 stimulates the activity of PLD and NADPH oxidase, but this stimulation is inhibited by pretreatment with bosentan (ET-1 receptor antagonist), FIPI (PLD inhibitor), apocynin (NADPH oxidase inhibitor), and EGCG and ECG (catechins having a galloyl group), but not EGC and EC (catechins devoid of a galloyl group). Herein, using molecular docking analyses based on our biochemical studies, we determined the probable mechanism by which the catechins containing a galloyl group inhibit the stimulation of PLD activity induced by ET-1. The ET-1-induced stimulation of PLD activity was inhibited by SecinH3 (inhibitor of cytohesin). Arf6 and cytohesin-1 are associated in the cell membrane, which is not inhibited by the catechins during ET-1 treatment of the cells. However, EGCG and ECG inhibited the binding of GTPγS with Arf6, even in the presence of cytohesin-1. The molecular docking analyses revealed that the catechins containing a galloyl group (EGCG and ECG) with cytohesin-1-Arf6GDP, but not the catechins without a galloyl group (EGC and EC), prevent GDP-GTP exchange in Arf6, which seems to be an important mechanism for inhibiting the activation of PLD induced by ET-1, and subsequently increases the activity of NADPH oxidase.

Dynamics and modulation studies of human voltage gated Kv1.5 channel.

The voltage gated Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) and play critical role in repolarization of action potential duration. It is the most rapidly activated channel and has very little or no inactivated states. In human cardiac cells, these channels are expressed more extensively in atrial myocytes than ventricle. From the evidences of its localization and functions, Kv1.5 has been declared a selective drug target for the treatment of atrial fibrillation (AF). In this present study, we have tried to identify the rapidly activating property of Kv1.5 and studied its mode of inhibition using molecular modeling, docking, and simulation techniques. Channel in open conformation is found to be stabilized quickly within the dipalmitoylphosphatidylcholine membrane, whereas most of the secondary structure elements were lost in closed state conformation. The obvious reason behind its ultra-rapid property is possibly due to the amino acid alteration in S4-S5 linker; the replacement of Lysine by Glutamine and vice versa. The popular published drugs as well as newly identified lead molecules were able to inhibit the Kv1.5 in a very similar pattern, mainly through the nonpolar interactions, and formed sable complexes. V512 is found as the main contributor for the interaction along with the other important residues such as V505, I508, A509, V512, P513, and V516. Furthermore, two screened novel compounds show surprisingly better inhibitory potency and can be considered for the future perspective of antiarrhythmic survey.

Probing binding mechanism of interleukin-6 and olokizumab: in silico design of potential lead antibodies for autoimmune and inflammatory diseases.

Computer-aided antibody engineering has been successful in the design of new biologics for disease diagnosis and therapeutic interventions. Interleukin-6 (IL-6), a well-recognized drug target for various autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis, was investigated in silico to design potential lead antibodies. Here, crystal structure of IL-6 along with monoclonal antibody olokizumab was explored to predict antigen-antibody (Ag - Ab)-interacting residues using DiscoTope, Paratome, and PyMOL. Tyr56, Tyr103 in heavy chain and Gly30, Ile31 in light chain of olokizumab were mutated with residues Ser, Thr, Tyr, Trp, and Phe. A set of 899 mutant macromolecules were designed, and binding affinity of these macromolecules to IL-6 was evaluated through Ag - Ab docking (ZDOCK, ClusPro, and Rosetta server), binding free-energy calculations using Molecular Mechanics/Poisson Boltzman Surface Area (MM/PBSA) method, and interaction energy estimation. In comparison to olokizumab, eight newly designed theoretical antibodies demonstrated better result in all assessments. Therefore, these newly designed macromolecules were proposed as potential lead antibodies to serve as a therapeutics option for IL-6-mediated diseases.

Molecular dynamics of Kv1.3 ion channel and structural basis of its inhibition by scorpion toxin-OSK1 derivatives.

Kv1.3 is one of the widely distributed Shaker type voltage gated potassium channel which performs the outward flow of K(+) ions in excitable cells. In immunological synapse, Kv1.3 plays a pivotal role in antigen dependent activation and proliferation of lymphocytes along with the KCa3.1. The up-regulation of Kv1.3 leads to several T-cell-mediated autoimmune diseases, hence considered as an attractive pharmacological drug target. Here, we have employed molecular modeling, docking and simulation techniques to examine the dynamical properties of Kv1.3 in both open and closed state conformation embedded in DPPC membrane as well as its modes of inhibition against the popularly known scorpion venom OSK1 and its three mutant analogues. The Kv1.3 in open conformation took comparatively more time to get stabilized than the closed state. Both conformations ascertain their stability and the transition between closed to active states is more consistent with the paddle model of channel gating. The binding modes of channel-toxin complexes are well established by identifying strongly interacting amino acids lining at their polar surfaces. Our findings suggest that, two mutant derivatives OSK1-K16,D20 & OSK1-P12,K16,D20 have increased inhibitory potency against Kv1.3. We also pointed out some particular residues responsible for binding of OSK1 with Kv1.3 over other Shaker-type ion channels. We believe that the insights came from Kv1.3-OSK1 interaction will be valuable in pharmacological studies for strategic development of both potent and selective therapeutic drugs against T-cell-mediated autoimmune diseases.

Differential Interactions of Cytochrome P450 3A5 and 3A4 with Chemotherapeutic Agent-Vincristine: A Comparative Molecular Dynamics Study.

The chemotherapeutic agent vincristine, used for treatment of acute lymphoblastic leukemia is metabolized preferentially by polymorphic cytochrome P450 3A5 (CYP3A5) with higher clearance rate than cytochrome P450 3A4 (CYP3A4). As a result, CYP3A5 expressers have a reduced amount of vincristine-induced peripheral neuropathy than non-expressers. We modeled the structure of CYP3A5 and its interaction with vincristine, compared with CYP3A4-vincristine complex using molecular docking and simulation studies. This relative study helped us to understand the molecular mechanisms behind the interaction at the atomic level through interaction energy, binding free energy, hydrogen bond and solvent accessible surface area analysis - giving an insight into the binding mode and the main residues involved in this particular interaction. Our results show that the interacting groups get closer in CYP3A5-vincristine complex due to different orientation of vincristine. This leads to higher binding affinity of vincristine towards CYP3A5 compared to CYP3A4 and explains the preferential metabolism of vincristine by CYP3A5. We believe that, the results of the current study will be helpful for future studies on structure-based drug design in this area.

Conformational dynamics of shaker-type Kv1.1 ion channel in open, closed, and two mutated states.

The dynamic properties of shaker-type Kv1.1 ion channel in its open, closed, & two mutated (E325D & V408A) states embedded in DPPC membrane have been investigated using all-atom force field-based MD simulation. Here, we represent the detailed channel stability, gating environment of charge-carrying residues, salt bridge interaction among the voltage-sensing domains (VSDs), movement of S4 helix, and ion conduction of pore. At positive potential, the S4 helix undergoes lateral fluctuations in accordance with their gating motions found in every model. During transition from closed to active state conformation, charged residues of S4 move "up" across the membrane with an average tilt angle difference of 24°, which is more consistent with the paddle model of channel gating. The E325D mutation at C-terminal end of S4-S5 helical linker leads the channel to a rapid activated state by pushing the gating charge residues upward beside the VSDs resulting in more prominent tilt of S4. Similarly in V408A mutant model, disruption of hydrophobic gate at S6 C-terminal end takes place, which causes the violation of channel-active conformation by bringing the C-terminal end of S4 to its corresponding resting state. The ion permeation is observed only in open-state conformation.