Peptide Mapping, In Silico and In Vivo Analysis of Allergenic Sorghum Profilin Peptides.

BACKGROUND AND OBJECTIVES: Nearly 20-30% of the world's population suffers from allergic rhinitis, among them 15% are progressing to asthma conditions. Sorghum bicolor profilin (Sorb PF), one of the panallergens, was identified, but the allergen specificity is not yet characterized. MATERIALS AND METHODS: To map the antigenic determinants responsible for IgE binding, the present study is focused on in silico modeling, simulation of Sorb PF and docking of the Sorb PF peptides (PF1-6) against IgG and IgE, followed by in vivo evaluation of the peptides for its allergenicity in mice. RESULTS: Peptide PF3 and PF4 displayed high docking G-scores (-9.05) against IgE only. The mice sensitized with PF3 peptide showed increased levels of IL5, IL12, TNF-alpha, and GMCSF when compared to other peptides and controls, signifying a strong, Th2-based response. Concurrently, the Th1 pathway was inhibited by low levels of cytokine IL2, IFN-γ, and IL-10 justifying the role of PF3 in allergenic IgE response. CONCLUSIONS: Based on the results of overlapping peptides PF3 and PF4, the N-terminal part of the PF3 peptide (TGQALVI) plays a crucial role in allergenic response of Sorghum profilin.

Palmitoylethanolamide (PEA) Inhibits SARS-CoV-2 Entry by Interacting with S Protein and ACE-2 Receptor.

Lipids play a crucial role in the entry and egress of viruses, regardless of whether they are naked or enveloped. Recent evidence shows that lipid involvement in viral infection goes much further. During replication, many viruses rearrange internal lipid membranes to create niches where they replicate and assemble. Because of the close connection between lipids and inflammation, the derangement of lipid metabolism also results in the production of inflammatory stimuli. Due to its pivotal function in the viral life cycle, lipid metabolism has become an area of intense research to understand how viruses seize lipids and to design antiviral drugs targeting lipid pathways. Palmitoylethanolamide (PEA) is a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that also counteracts SARS-CoV-2 entry and its replication. Our work highlights for the first time the antiviral potency of PEA against SARS-CoV-2, exerting its activity by two different mechanisms. First, its binding to the SARS-CoV-2 S protein causes a drop in viral infection of ~70%. We show that this activity is specific for SARS-CoV-2, as it does not prevent infection by VSV or HSV-2, other enveloped viruses that use different glycoproteins and entry receptors to mediate their entry. Second, we show that in infected Huh-7 cells, treatment with PEA dismantles lipid droplets, preventing the usage of these vesicular bodies by SARS-CoV-2 as a source of energy and protection against innate cellular defenses. This is not surprising since PEA activates PPAR-α, a transcription factor that, once activated, generates a cascade of events that leads to the disruption of fatty acid droplets, thereby bringing about lipid droplet degradation through ß-oxidation. In conclusion, the present work demonstrates a novel mechanism of action for PEA as a direct and indirect antiviral agent against SARS-CoV-2. This evidence reinforces the notion that treatment with this compound might significantly impact the course of COVID-19. Indeed, considering that the protective effects of PEA in COVID-19 are the current objectives of two clinical trials (NCT04619706 and NCT04568876) and given the relative lack of toxicity of PEA in humans, further preclinical and clinical tests will be needed to fully consider PEA as a promising adjuvant therapy in the current COVID-19 pandemic or against emerging RNA viruses that share the same route of replication as coronaviruses.

Structural and simulation analysis of hotspot residues interactions of SARS-CoV 2 with human ACE2 receptor.

The novel corona virus disease 2019 (SARS-CoV 2) pandemic outbreak was alarming. The binding of SARS-CoV (CoV) spike protein (S-Protein) Receptor Binding Domain (RBD) to Angiotensin converting enzyme 2 (ACE2) receptor initiates the entry of corona virus into the host cells leading to the infection. However, considering the mutations reported in the SARS-CoV 2 (nCoV), the structural changes and the binding interactions of the S-protein RBD of nCoV were not clear. The present study was designed to elucidate the structural changes, hot spot binding residues and their interactions between the nCoV S-protein RBD and ACE2 receptor through computational approaches. Based on the sequence alignment, a total of 58 residues were found mutated in nCoV S-protein RBD. These mutations led to the structural changes in the nCoV S-protein RBD 3d structure with 4 helices, 10 sheets and intermittent loops. The nCoV RBD was found binding to ACE2 receptor with 11 hydrogen bonds and 1 salt bridge. The major hot spot amino acids involved in the binding identified by interaction analysis after simulations includes Glu 35, Tyr 83, Asp 38, Lys 31, Glu 37, His 34 amino acid residues of ACE2 receptor and Gln 493, Gln 498, Asn 487, Tyr 505 and Lys 417 residues in nCoV S-protein RBD. Based on the hydrogen bonding, RMSD and RMSF, total and potential energies, the nCoV was found binding to ACE2 receptor with higher stability and rigidity. Concluding, the hotspots information will be useful in designing blockers for the nCoV spike protein RBD. [Formula: see text]Communicated by Ramaswamy H. Sarma.

Screening of caspase-3 inhibitors from natural molecule database using e-pharmacophore and docking studies.

Caspase a protease family member, have a vital role in cell death and inflammation process. Caspase-3, an effector caspase controls the regulation of apoptosis and has an anti apoptotic function. The mechanical significance of restoring apoptosis signaling to selectively target malignant cells is utilized to develop strong therapeutic strategies by the caspase family of mortality - induction molecules. Caspase-3 has currently no clear role in treatment for tumor progression and tumor sensitivity. The present study was aimed to screen caspase for potential inhibitors using computer aided docking methodologies. For this, zinc natural molecule database molecules were screened using e-pharmacophore and ADME protocols along with docking studies. Docking analysis selected two molecules, namely ZINC13341044 and ZINC13507846 with G-scores -5.27 and -6.19 respectively. These two potential hits are predicted as caspase inhibitors based on the results and can be further processed for in vitro validation.

Specific panallergen peptide of Sorghum Polcalcin showing IgE response identified based on in silico and in vivo peptide mapping.

In India, Sorghum plant allergenicity was reported to be approximately 54.9%. Sorghum bicolor Polcalcin (Sorb PC) was identified as the panallergen but the specificity of this allergen is yet to be characterized. The present study was aimed to characterize the antigenic determinants of Sorb PC that are responsible for eliciting the IgE response. In silico modeling, simulation studies and docking of Sorb PC peptides (PC1-11) against IgG and IgE followed by in vivo evaluation was adopted. Peptide docking studies revealed PC 6 with highest G-score -12.85 against IgE followed by PC-11, 5, 1 and 7 (-10.91) peptides. The mice sensitized with PC7 peptide showed interleukin (IL) 4 (IL-4), IL-5, IL-12, TNF-α and GMCSF levels increased when compared with other peptides and controls, signifying a strong T helper type 2 (Th2)-based response. In tandem, the T helper type 1 (Th1) pathway was inhibited by low levels of cytokine IL-2, interferon γ (IFN-γ) and increased IL-10 levels justifying the role of PC7 in allergic IgE response. Considering the above data of overlapping peptides of PC6 and PC7, N-terminal part of the PC7 peptide (DEVQRMM) is found to play a crucial role in Sorghum Polcalcin allergenic response.

Pharmacophore Directed Screening of Agonistic Natural Molecules Showing Affinity to 5HT2C Receptor.

Obesity prevalence continues to be a foremost health concern across the globe leading to the development of major health risk conditions like type II diabetes, hyperlipidemia, hypertension and even cancers. Because of the deprived drug-based management system, there is an urgent need for the development of new drugs aiming at satiety and appetite control targets. Among the reported satiety signaling targets, 5HT2C receptor plays a crucial role in decreasing appetite and has become a promising target for the development of anti-obesity drugs. Lorcaserin, a 5HT2C receptor agonist and the only drug available in the market, was designed based on the receptor mechanism of action. Due to limited drug options available and considering the adverse drug effects of Lorcaserin, the development of new drugs which are highly specific toward the 5HT2C target and with lesser side effects is essential. The present study is majorly focused on developing new 5HT2C agonists through computational approaches like screening, docking, and simulation using Phase, QikProp, Glide and Desmond applications of the Schrodinger suite. Screening protocols resulted in eight best hit molecules with affinity for the receptor and among them, five hits displayed binding affinity toward the conserved residue Asp 134 of the receptor. The stability of the five molecules in complex with the 5HT2C receptor was studied through molecular dynamic simulations. Three molecules, ZINC32123870, ZINC40312983 and ZINC32124535, maintained stable interactions with the Asp 134 residue throughout the 50 ns simulation run time. Further, due to the high sequence similarity seen among the receptors of 5HT2 family, the three potential hits were cross validated against other subtypes 5HT2A and 5HT2B of the 5HT2 family to determine the specificity of the molecules against the target. Among the three hits, ZINC32124535 was identified as the best potential hit based on the hydrogen bond interaction percentage with Asp residue [5HT2A (Asp 155:60%); 5HT2B (Asp155: No interaction); 5HT2C (Asp 134:86%)]. The ZINC32124535 molecule produced one salt bridge and hydrogen bond interactions with Asp 134, alike the known drug Lorcaserin. Based on the results, ZINC32124535 was identified as the best potential hit against the 5HT2C receptor.

In vitro and in silico characterization of angiogenic inhibitors from Sophora interrupta.

Sophora interrupta Bedd, (Fabaceae) is used in Indian folk medicine to treat cancer. Angiogenesis is one of the crucial characteristics of cancer metastasis and is regulated by vascular endothelial growth factor (VEGF). In this study, we examined the antiangiogenic properties of the root ethyl acetate extract of Sophora interrupta by various methods. In vitro antioxidant activity (100-600 µg/ml) of S. interrupta ethyl acetate (SEA) extract was evaluated by DPPH and ABTS, anti-inflammatory activity (50, 100 and 150 µg/ml) by estimating nitric oxide (NO) levels, anti-angiogenic activity (200 and 500 µg/ml) was validated by chorio allantoic membrane (CAM) assay and in silico molecular dynamic (MD) simulations analyses (25 ns) were performed to identify the anti-angiogenic compounds extracted from root extract. The antioxidative activity of SEA extract at IC50 (200 ± 0.6 µg/mL) is equal to that of ascorbic acid at IC50 (50 ± 0.6 µg/mL), and the anti-inflammatory activity of SEA extract at IC50 (150 ± 0.2 µg/mL) was inhibited significantly by nitric oxide (NO) production. The SEA extract significantly reduced the sprouting of new blood vessels at ID50 500 ± 0.13 µg/mL in the CAM assay. Gas chromatography-mass spectrometry analysis of the SEA extract detected 34 secondary metabolites, of which 6a,12a-dihydro-6H-(1,3)dioxolo(5,6)benzofuro(3,2-c)chromen-3-ol (maackiain) and funiculosin formed strong hydrogen bond interactions with Lys 920, Thr 916 and Cys 919 (2H), as well as Glu 917 of VEGFR2, and these interactions were similar to those of the anti-angiogenic compound axitinib. Significant findings in all the assays performed indicate that SEA extract has potential anti-angiogenic compounds that may interfere with VEGF-induced cancer malignancy.

Ketorolac salt is a newly discovered DDX3 inhibitor to treat oral cancer.

DDX3 belongs to DEAD box RNA helicase family and is involved in the progression of several types of cancer. In this work, we employed a High Throughput Virtual screening approach to identify bioactive compounds against DDX3 from ZINC natural database. Ketorolac salt was selected based on its binding free energy less than or equals to -5 Kcal/mol with reference to existing synthetic DDX3 inhibitors and strong hydrogen bond interactions as similar to crystallized DDX3 protein (2I4I). The anti-cancer activity of Ketorolac salt against DDX3 was tested using oral squamous cell carcinoma (OSCC) cell lines. This compound significantly down regulated the expression of DDX3 in human OSCC line (H357) and the half maximal growth inhibitory concentration (IC50) of Ketorolac salt in H357 cell line is 2.6 µM. Ketorolac salt also inhibited the ATP hydrolysis by directly interacting with DDX3. More importantly, we observed decreased number of neoplastic tongue lesions and reduced lesion severity in Ketorolac salt treated groups in a carcinogen induced tongue tumor mouse model. Taken together, our result demonstrates that Ketorolac salt is a newly discovered bioactive compound against DDX3 and this compound can be used as an ideal drug candidate to treat DDX3 associated oral cancer.

High-throughput virtual screening with e-pharmacophore and molecular simulations study in the designing of pancreatic lipase inhibitors.

BACKGROUND: Obesity is a progressive metabolic disorder in the current world population, and is characterized by the excess deposition of fat in the adipose tissue. Pancreatic lipase is one of the key enzymes in the hydrolysis of triglycerides into monoglycerides and free fatty acids, and is thus considered a promising target for the treatment of obesity. The present drugs used for treating obesity do not give satisfactory results, and on prolonged usage result in severe side effects. In view of the drastic increase in the obese population day-to-day, there is a greater need to discover new drugs with lesser side effects. MATERIALS AND METHODS: High-throughput virtual screening combined with e-pharmacophore screening and ADME (absorption, distribution, metabolism, and excretion) and PAINS (pan-assay interference compounds) filters were applied to screen out the ligand molecules from the ZINC natural molecule database. The screened molecules were subjected to Glide XP docking to study the molecular interactions broadly. Further, molecular dynamic simulations were used to validate the stability of the enzyme-ligand complexes. Finally, the molecules with better results were optimized for in vitro testing. RESULTS: The screening protocols identified eight hits from the natural molecule database, which were further filtered through pharmacological filters. The final four hits were subjected to extra precision docking, and the complexes were finally studied with molecular dynamic simulations. The results pointed to the zinc 85893731 molecule as the most stable in the binding pocket, producing consistent H-bond interaction with Ser152 (G=-7.18). The optimized lead molecule exhibited good docking score, better fit, and improved ADME profile. CONCLUSION: The present study specifies zinc 85893731 as a lead molecule with higher binding score and energetically stable complex with pancreatic lipase. This lead molecule, along with its various analogs, can be further tested as a novel inhibitor against pancreatic lipase using in vitro protocols.

Shape based virtual screening and molecular docking towards designing novel pancreatic lipase inhibitors.

Increase in obesity rates and obesity associated health issues became one of the greatest health concerns in the present world population. With alarming increase in obese percentage there is a need to design new drugs related to the obesity targets. Among the various targets linked to obesity, pancreatic lipase was one of the promising targets for obesity treatment. Using the in silico methods like structure based virtual screening, QikProp, docking studies and binding energy calculations three molecules namely zinc85531017, zinc95919096 and zinc33963788 from the natural database were reported as the potential inhibitors for the pancreatic lipase. Among them zinc95919096 presented all the interactions matching to both standard and crystal ligand and hence it can be further proceeded to drug discovery process.

Molecular modeling and analysis of human and plant endo-ß-N-acetyl- glucosaminidases for mutations effects on function.

Endo- ß-N-acetylgucosaminidases (ENGases) are the enzymes that catalyze both hydrolysis and transglycosylation reactions. It is of interest to study ENGases because of their ability to synthesize glycopeptides. Homology models of Human, Arabidopsis thaliana and Sorghum ENGases were developed and their active sites marked based on information available from Arthrobacter protophormiae (PDB ID: 3FHQ) ENGase. Further, these models were docked with the natural substrate GlcNAc-Asn and the inhibitor Man3GlcNAc-thiazoline. The catalytic triad of Asn, Glu and Tyr (N171, E173 and Y205 of bacteria) were found to be conserved across the phyla. The crucial Y299F mutation showing 3 times higher transglycosylation activity than in wild type Endo-A is known. The hydrolytic activity remained unchanged in bacteria, while the transglycosylation activity increased. This Y to F change is found to be naturally evolved and should be attributing higher transglycosylation rates in human and Arabidopsis thaliana ENGases. Ligand interactions Ligplots revealed the interaction of amino acids with hydrophobic side chains and polar uncharged side chain amino acids. Thus, structure based molecular model-ligand interactions provide insights into the catalytic mechanism of ENGases and assist in the rational engineering of ENGases.

Inferences from the ADMET analysis of predicted inhibitors to Follicle Stimulating Hormone in the context of infertility.

Follicle stimulating hormone (FSH) is a glycoprotein secreted by gonadotrophs of the anterior pituitary gland that regulates reproduction in mammals. FSH targets its receptor (FSHR) expressed only on grannulosa cells and induce the maturation of ovarian follicles in females. The levels of both FSH and FSHR rise until the middle of estrus cycle and then falls on level at the time of ovulation. It is associated with stimulated sertoli cell proliferation in testes and supports spermatogenesis in males. The interaction between the polypeptide FSH hormone and its corresponding receptor is highly selective. Therefore, it is of interest to inhibit FSH in the context of infertility. The structure of FSH (PDB ID: 1XWD) is screened using molecular docking techniques against the ZINC database (a database of 2.7 million compounds) with reference to known standard compounds. This exercise identifies compounds with better binding and ADMET (Absorption, Digestion, Metabolism, Excretion and Toxicity) properties compared to known standard compounds. These observations find application for the consideration of such compounds for further validation towards inhibiting the FSH.