Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface-modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide-co-glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU-NF). The GLU-NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer-by-layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA-GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer-by-layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU-NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU-NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization.

Zika virus is a mosquito-transmitted flavivirus that causes devastating fetal outcomes in the context of maternal infection during pregnancy. An important target for drugs combatting Zika virus pathogenicity is NS2B-NS3 protease, which plays an essential role in hydrolysis and maturation of the flavivirus polyprotein. We identify hydroxychloroquine, a drug that already has approved uses in pregnancy, as a possible inhibitor of NS2B-NS3 protease by using a Food and Drug Administration-approved drug library, molecular docking, and molecular dynamics simulations. Further, to gain insight into its inhibitory potential toward NS2B-NS3 protease, we performed enzyme kinetic studies, which revealed that hydroxychloroquine inhibits protease activity with an inhibition constant (Ki) of 92.34 ± 11.91 μM. Additionally, hydroxychloroquine significantly decreases Zika virus infection in placental cells.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. The outbreak of CHIKV infection has been seen in many tropical and subtropical regions of the biosphere. Current reports evidenced that after outbreaks in 2005–06, the fitness of this virus propagating in Aedes albopictus enhanced due to the epistatic mutational changes in its envelope protein. In our study, we evaluated the prevalence of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) in CHIKV proteome. IDPs/IDPRs are known as members of a ‘Dark Proteome’ that defined as a set of polypeptide segments or whole protein without unique three-dimensional structure within the cellular milieu but with significant biological functions, such as cell cycle regulation, control of signaling pathways, and maintenance of viral proteomes. However, the intrinsically disordered aspects of CHIKV proteome and roles of IDPs/IDPRs in the pathogenic mechanism of this important virus have not been evaluated as of yet. There are no existing reports on the analysis of intrinsic disorder status of CHIKV. To fulfil this goal, we have analyzed the abundance and functionality of IDPs/IDPRs in CHIKV proteins, involved in the replication and maturation. It is likely that these IDPs/IDPRs can serve as novel targets for disorder based drug design.

The NSP1– C terminal structure in complex with ribosome using cryo-EM is available now, and the N-terminal region structure in isolation is also deciphered in literature. However, as a reductionist approach, the conformation of NSP1– C terminal region (NSP1-CTR; amino acids 131–180) has not been studied in isolation. We found that NSP1-CTR conformation is disordered in an aqueous solution. Further, we examined the conformational propensity towards alpha-helical structure using trifluoroethanol, we observed induction of helical structure conformation using CD spectroscopy. Additionally, in SDS, NSP1-CTR shows a conformational change from disordered to ordered, possibly gaining alpha-helix in part. But in the presence of neutral lipid DOPC, a slight change in conformation is observed, which implies the possible role of hydrophobic interaction and electrostatic interaction on the conformational changes of NSP1. Fluorescence-based studies have shown a blue shift and fluorescence quenching in the presence of SDS, TFE, and lipid vesicles. In agreement with these results, fluorescence lifetime and fluorescence anisotropy decay suggest a change in conformational dynamics. The zeta potential studies further validated that the conformational dynamics are primarily because of hydrophobic interaction. These experimental studies were complemented through Molecular Dynamics (MD) simulations, which have shown a good correlation and testifies our experiments. We believe that the intrinsically disordered nature of the NSP1-CTR will have implications for enhanced molecular recognition feature properties of this IDR, which may add disorder to order transition and disorder-based binding promiscuity with its interacting proteins.

Short α-helical sequences of proteins fail to maintain their native conformation when taken out of their protein context. Several covalent constraints have been designed, including the covalent H-bond surrogate (HBS)where a peptide backbone i + 4 → i H-bond is replaced by a covalent surrogateto nucleate α-helix in short sequences (>7 < 15 amino acids). But constraining the shortest sequences (four amino acids) into a single α-helical turn is still a significant challenge. Here, we introduce an HBS model that can be placed in unstructured tetrapeptides without excising any of its residues, and that biases them predominantly into remarkably stable single α-helical turns in varying solvents, pH values, and temperatures. Circular dichroism (CD), Fourier transform infrared (FT-IR) absorption, one-dimensional (1D)-NMR, two-dimensional (2D)-NMR spectral and computational analyses of the HBS-constrained tetrapeptide analogues reveal that (a) the number of sp 2 atoms in the HBS-constrained backbone influences their predominance and rigidity in the α-helical conformation; and (b) residue preferences at the unnatural HBS-constrained positions influence their α-helicities, with Moc[GFA]G-OMe (1a) showing the highest known α-helicity (θ n→π*MRE ∼−25.3 × 10 3 deg cm 2 dmol −1 at 228 nm) for a single α-helical turn. Current findings benefit chemical biological applications desiring predictable access to single α-helical turns in tetrapeptides.

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Given the COVID-19 pandemic, currently, there are many drugs in clinical trials against this virus. Among the excellent drug targets of SARS-CoV-2 are its proteases (Nsp3 and Nsp5) that plays vital role in polyprotein processing giving rise to functional nonstructural proteins, essential for viral replication and survival. Nsp5 (also known as M pro) hydrolyzes replicase polyprotein (1ab) at eleven different sites. For targeting M pro , we have employed drug repurposing approach to identify potential inhibitors of SARS-CoV-2 in a shorter time span. Screening of approved drugs through docking reveals Hyaluronic acid and Acarbose among the top hits which are showing strong interactions with catalytic site residues of M pro. We have also performed docking of drugs Lopinavir, Ribavirin, and Azithromycin on SARS-CoV-2 M pro. Further, binding of these compounds (Hyaluronic acid, Acarbose, and Lopinavir) is validated by extensive molecular dynamics simulation of 500 ns where these drugs show stable binding with M pro. We believe that the high-affinity binding of these compounds will help in designing novel strategies for structure-based drug discovery against SARS-CoV-2.

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