Mapping of Methyl Epitopes of a Peptide-Drug with Its Receptor by 2D STDD-Methyl TROSY NMR Spectroscopy.

The current trend in the biopharmaceutical market has boosted the development and production of biological drugs with high efficacy and fidelity for receptor binding. While high-resolution structural insights into binding epitopes of the receptor are indispensable for better therapeutic design, it is tedious and costly. In this work, we develop a protocol by integrating two well-known NMR-based solution-state methods. Saturation transfer double-difference with methyl-TROSY (STDD-Methyl TROSY NMR) was used to probe methyl binding epitopes of the ligand in a label-free environment. This study was carried out with Human insulin as a model peptide drug, with the insulin growth factor receptor (IGFR), which is an off-target receptor for insulin. Methyl epitopes identified from STDD-Methyl TROSY NMR spectroscopy were validated through the HADDOCK platform to generate a drug-receptor model. Since this method can be applied at natural abundance, it has the potential to screen a large set of peptide-drug interactions for optimum receptor binding. Thus, we propose STDD-Methyl TROSY NMR spectroscopy as a technique for rapid screening of biologics for the development of optimized biopharmaceutics.

In silico design of multi-epitope-based peptide vaccine against SARS-CoV-2 using its spike protein.

SARS-CoV-2 has been efficient in ensuring that many countries are brought to a standstill. With repercussions ranging from rampant mortality, fear, paranoia, and economic recession, the virus has brought together countries to look at possible therapeutic countermeasures. With prophylactic interventions possibly months away from being particularly effective, a slew of measures and possibilities concerning the design of vaccines are being worked upon. We attempted a structure-based approach utilizing a combination of epitope prediction servers and Molecular dynamic (MD) simulations to develop a multi-epitope-based subunit vaccine that involves the two subunits of the spike glycoprotein of SARS-CoV-2 (S1 and S2) coupled with a substantially effective chimeric adjuvant to create stable vaccine constructs. The designed constructs were evaluated based on their docking with Toll-Like Receptor (TLR) 4. Our findings provide an epitope-based peptide fragment that can be a potential candidate for the development of a vaccine against SARS-CoV-2. Recent experimental studies based on determining immunodominant regions across the spike glycoprotein of SARS-CoV-2 indicate the presence of the predicted epitopes included in this study.Communicated by Ramaswamy H. Sarma.

Combining cysteine scanning with chemical labeling to map protein-protein interactions and infer bound structure in an intrinsically disordered region.

The Mycobacterium tuberculosis genome harbours nine toxin-antitoxin (TA) systems of the mazEF family. These consist of two proteins, a toxin and an antitoxin, encoded in an operon. While the toxin has a conserved fold, the antitoxins are structurally diverse and the toxin binding region is typically intrinsically disordered before binding. We describe high throughput methodology for accurate mapping of interfacial residues and apply it to three MazEF complexes. The method involves screening one partner protein against a panel of chemically masked single cysteine mutants of its interacting partner, displayed on the surface of yeast cells. Such libraries have much lower diversity than those generated by saturation mutagenesis, simplifying library generation and data analysis. Further, because of the steric bulk of the masking reagent, labeling of virtually all exposed epitope residues should result in loss of binding, and buried residues are inaccessible to the labeling reagent. The binding residues are deciphered by probing the loss of binding to the labeled cognate partner by flow cytometry. Using this methodology, we have identified the interfacial residues for MazEF3, MazEF6 and MazEF9 TA systems of M. tuberculosis. In the case of MazEF9, where a crystal structure was available, there was excellent agreement between our predictions and the crystal structure, superior to those with AlphaFold2. We also report detailed biophysical characterization of the MazEF3 and MazEF9 TA systems and measured the relative affinities between cognate and non-cognate toxin-antitoxin partners in order to probe possible cross-talk between these systems.

Evaluation of (Anti)androgenic Activities of Environmental Xenobiotics in Milk Using a Human Liver Cell Line and Androgen Receptor-Based Promoter-Reporter Assay.

The recent reports on milk consumption and its associated risk with hormone related disorders necessitates the evaluation of dairy products for the presence of endocrine disrupting chemicals (EDCs) and ensure the safety of consumers. In view of this, we investigated the possible presence of (anti)androgenic contaminants in raw and commercialized milk samples. For this purpose, a novel HepARE-Luc cell line that stably expresses human androgen receptor (AR) and the androgen responsive luciferase reporter gene was generated and used in the present study. Treatment of this cell line with androgens and corresponding antiandrogen (flutamide) stimulated or inhibited expression of reporter luciferase, respectively. Real time polymerase chain reaction and immunostaining results exhibited transcription response and translocation of AR from the cytoplasm to the nucleus in response to androgen. Observations implied that a cell-based xenobiotic screening assay via AR response can be conducted for assessing the (anti)androgenic ligands present in food chain including milk. Therefore, the cell line was further used to screen the (anti)androgenic activity of a total of 40 milk fat samples procured as raw or commercial milk. Some of the raw and commercial milk fat samples distinctly showed antiandrogenic activities. Subsequently, some commonly used environmental chemicals were also evaluated for their (anti)androgenic activities. Initial observations with molecular docking studies of experimental compounds were performed to assess their interaction with AR ligand binding domain. Furthermore, (anti)androgenic activities of these compounds were confirmed by performing luciferase assay using the HepARE-Luc cell line. None of the test compounds showed androgenic activities rather some of them like Bisphenol A (BPA) and rifamycin showed antiandrogenic activities. In conclusion, our results provide a valuable information about the assessment of (anti)androgenic activities present in milk samples. Overall, it is proposed that a robust cell-based CALUX assay can be used to assess the (anti)androgenic activities present in milk which can be attributed to different environmental chemicals present therein.

Investigation of the interaction of allergens of Glycine max with IgE-antibody for designing of peptidomimetics based anti-allergen.

Allergen induced IgE dependent type I hypersensitivity is the main cause of the allergy, which would be a burden on medical setup in coming years. Allergens of Glycine max have been isolated, and their disease relationships are documented. Therefore, it becomes important to investigate the interaction of different allergens of Glycine max with IgE and also screen suitable therapeutics to prevent this interaction. The amino acid sequences of all allergens of Glycine max and their isoallergens have been taken, and 3D structure of allergens (Gly m 3, Gly m 4, Gly m 5, Gly m 6 and Gly m 8) and their isoallergens were generated using Modeller v9.17. The modeled structures were further validated using PSVS, ProSA, RAMPAGE, and PDBsum. HL domain of Fab region of human IgE (PDBID: 2R56) was generated using UCSFchimera. The HL domain was minimized by Schrodinger software using the OPLS_2005 force field. SiteMap identified epitope binding site of the minimized domain. All the predicted epitopes of different allergens were docked to the binding site of HL domain using the Patchdock server. We have also designed a peptidomimetics based inhibitor targeted at interaction interface of Gly m8 and IgE, using in-silico virtual screening, molecular mechanics, and molecular dynamics simulation studies. These studies identified BDE32166344 ((N-(1-{[1-(1-aminocyclopentanecarbonyl)-3-hydroxypyrrolidin-3-yl]methyl}piperidin-4-yl)acetamide) as a peptidomimetics based lead with binding energy of -72.77 kcal/mol. Therefore, the present study investigates the interaction between different Gly m allergens and IgE antibody and identifies peptidomimetics based lead that might be developed as a suitable therapeutics against allergy caused by allergen of Glycine max.