Characterization of Citrusnobilis Peel Methanolic Extract for Antioxidant, Antimicrobial, and Anti-Inflammatory Activity.

Currently, the potential utilization of fruits and vegetable waste as a source of micronutrients and antioxidants has increased. The present study, therefore, aimed to determine the antimicrobial and anti-inflammatory activities of Citrus nobilis peel extract. A modified solvent evaporation technique was employed for peel extract preparation. For effective utilization of the natural product, quantitative analysis of phenolic compounds was carried out using liquid chromatography and mass spectroscopy technique. Phenolic and flavonoids were present in high amounts, while ß-carotene and lycopene were present in vestigial amounts. The antimicrobial efficiency of peel extract was evaluated against four bacterial strains including Staphylococcus aureus (MTCC 3160), Klebsiella pneumoniae (MTCC 3384), Pseudomonas aeruginosa (MTCC 2295), and Salmonella typhimurium (MTCC 1254), and one fungal strain Candida albicans (MTCC 183), and zone of inhibition was comparable to the positive control streptomycin and amphotericin B, respectively. The extract of Citrus nobilis peels showed effective anti-inflammatory activity during human red blood cell membrane stabilization (HRBC) and albumin denaturation assay. The extracts also exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity ranging from 53.46 to 81.13%. Therefore, the obtained results suggest that Citrus nobilis peel could be used as an excellent source of polyphenols and transformed into value-added products.

Crystal structure of chikungunya virus nsP2 cysteine protease reveals a putative flexible loop blocking its active site.

Chikungunya virus (CHIKV), a mosquito-borne pathogenic alphavirus is a growing public health threat. No vaccines or antiviral drug is currently available in the market for chikungunya treatment. nsP2pro, the viral cysteine protease, carries out an essential function of nonstructural polyprotein processing and forms four nonstructural proteins (nsPs) that makes the replication complex, hence constitute a promising drug target. In this study, crystal structure of nsP2pro has been determined at 2.59 Å, which reveals that the protein consists of two subdomains: an N-terminal protease subdomain and a C-terminal methyltransferase subdomain. Structural comparison of CHIKV nsP2pro with structures of other alphavirus nsP2 advances that the substrate binding cleft is present at the interface of two subdomains. Additionally, structure insights revealed that access to the active site and substrate binding cleft is blocked by a flexible interdomain loop in CHIKV nsP2pro. This loop contains His548, the catalytic residue, and Trp549 and Asn547, the residues predicted to bind substrate. Interestingly, mutation of Asn547 leads to three-fold increase in Km confirming that Asn547 plays important role in substrate binding and recognition. This study presents the detailed molecular analysis and signifies the substrate specificity residues of CHIKV nsP2pro, which will be beneficial for structure-based drug design and optimization of CHIKV protease inhibitors.

Chikungunya virus inhibition by peptidomimetic inhibitors targeting virus-specific cysteine protease.

Chikungunya virus (CHIKV), a mosquito-borne pathogenic virus that reemerged and caused epidemic in the Indian Ocean island of La Réunion, is a potential public health threat. Currently there is no antiviral drug or vaccine commercially available for the treatment of chikungunya fever, which necessitates the urge for an effective antiviral therapy for chikungunya treatment. In the present study, a FRET based protease assay was used to analyze the proteolytic activity of chikungunya nsP2 protease (CHIKV nsP2pro) - an essential viral enzyme, with fluorogenic substrate peptide. This protease assay was used to assess the inhibitory activity of Pep-I (MMsINC® database ID MMs03131094) and Pep-II (MMsINC® database ID MMs03927237), peptidomimetic compounds identified in a previous study by our group. Both compounds inhibited CHIKV nsP2pro with half maximal inhibition concentration (IC50) values of ∼34 µM and ∼42 µM, respectively. Kinetic studies showed that the inhibition constant (Ki) value is 33.34 ±â€¯2.53 µM for Pep-I and 45.89 ±â€¯4.38 µM for Pep-II. Additionally, these two compounds significantly inhibited CHIKV replication in BHK-21 cells at concentrations much lower than their cytotoxic concentrations. Intriguingly, these compounds did not show inhibitory effect on Sindbis virus. This suggests that Pep-I and Pep-II compounds identified as CHIKV nsP2 substrate peptidomimetics, specifically inhibit CHIKV replication.

Crystal structure of pentapeptide-independent chemotaxis receptor methyltransferase (CheR) reveals idiosyncratic structural determinants for receptor recognition.

Chemotactic methyltransferase, CheR catalyse methylation of specific glutamate residues in the cytoplasmic domain of methyl-accepting chemotactic protein receptors (MCPRs). The methylation of MCPRs is essential for the chemical sensing and chemotactic bacterial mobility towards favorable chemicals or away from unfavorable ones. In this study, crystal structure of B. subtilis CheR (BsCheR) in complex with S-adenosyl-l-homocysteine (SAH) has been determined to 1.8Å resolution. This is the first report of crystal structure belonging to the pentapeptide-independent CheR (PICheR) class. Till date, only one crystal structure of CheR from S. typhimurium (StCheR) belonging to pentapeptide-dependent CheR (PDCheR) class is available. Structural analysis of BsCheR reveals a helix-X-helix motif (HXH) with Asp53 as the linker residue in the N-terminal domain. The key structural features of the PDCheR ß-subdomain involved in the formation of a tight complex with the pentapeptide binding motif in MCPRs were found to be absent in the structure of BsCheR. Additionally, isothermal titration calorimetry (ITC) experiments were performed to investigate S-adenosyl-(l)-methionine (SAM) binding affinity and KD was determined to be 0.32mM. The structure of BsCheR reveals that mostly residues of the large C-terminal domain contribute to SAH binding, with contributions of few residues from the linker region and the N-terminal domain. Structural investigations and sequence analysis carried out in this study provide critical insights into the distinct receptor recognition mechanism of the PDCheR and PICheR methyltransferase classes.