Isolation and characterization of an iridoid, Arbortristoside-C from Nyctanthes arbor-tristis Linn., a potential drug candidate for diabetes targeting α-glucosidase.

Many parts of the plant Nyctanthes arbor-tristis Linn. are widely investigated for their biological properties. Purified Arbortristosides from seeds are reported as anticancer, anti-leishmania, anti-inflammatory, anti-allergic, immunomodulatory and antiviral. The present study elaborates on the isolation, structural and functional characterization of Arbortristoside-C and its inhibition properties against alpha-glucosidase, an important target for diabetes mellitus. Arbortristoside-C is purified from seeds of N. arbor-tristis by extraction using polar fractionation and chromatographic techniques. Arbortristoside-C has been characterized using Ultra Violet (UV), Mass (MS), Infra-Red (IR) and Nuclear Magnetic Resonance (NMR). Inhibition kinetics and Isothermal Titration Calorimetry (ITC) were used for activity and binding characteristics of acarbose and Arbortristoside-C using in-house purified α-glucosidase from Bos taurus. Modeling, docking and structural comparison with acarbose bound structure revealing the similar binding characteristics of Arbortristoside-C which include interaction with catalytic acid/base Aspartic acid residue. Cytotoxicity assay revealed that 100 µg/ml is the maximum toxic-free concentration of Arbortristoside-C. The purified Arbortristoside-C showed inhibition against mammalian α-glucosidase, suggesting its potential to treat Diabetes mellitus.

Structure-based drug designing towards the identification of potential anti-viral for COVID-19 by targeting endoribonuclease NSP15.

The world is facing health and economic havoc due to the Corona Virus Disease-2019 (COVID-19) pandemic. Given the number of affected people and the mortality rate, the virus is undoubtedly a serious threat to humanity. By analogy with earlier reports about Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome (MERS-CoV) - viruses, the novel Coronavirus' replication mechanism is likely well understood. The structure of an endoribonuclease (NSP15) of SARS-CoV-2 was reported recently. This enzyme is expected to play a crucial role in replication. In this work, attempts were made to identify inhibitors of this enzyme. To achieve the goal, high throughput in silico screening and molecular docking procedures were performed. From an Enamine database of a billion compounds, 3978 compounds with potential antiviral activity were selected for screening and induced fit docking that funneled down to eight compounds with good docking score and docking energy. Detailed analysis of non-covalent interactions at the active site and the apparent match of the molecule with the shape of the binding pocket were assessed. All the compounds show significant interactions for tight binding. Since all the compounds are synthetic with favorable drug-like properties, these may be considered for immediate optimization and downstream applications.

Crystallographic and calorimetric analysis on Pleurotus ostreatus lectin and its sugar complexes - promiscuous binding driven by geometry.

Carbohydrate recognition is established as a property of lectins and implicated in many functions including immunity and defense against pathogens. Many lectins are characterized and proposed for various applications owing to the above said recognition. The crystal structure of a lectin from Pleurotus ostreatus has been determined and shown to be calcium dependent. The overall structure is a tandem repeat of two ß-jelly roll domains, a new fold for lectins. The calcium dependence of sugar binding is analyzed in-detail through isothermal titration calorimetry. The serendipitous observation of malonate and glycerol, the intentional N-Acetyl-D-galactosamine, D-Galactose and L-Rhamnose binding to Pleurotus ostreatus lectin by Ca2+ coordination revealed that the binding site is promiscuous. Among these sugars, Rhamnose binding found to be thermodynamically most favourable. In all these structures, a vicinal diol motif, one at axial and the other at equatorial positions could be established as a specific requirement for binding. Interestingly, when compared with other calcium mediated lectin structures; this geometric requirement is found conserved. This observation could lead to the conclusion that lectins are not 'molecule specific' but 'geometry specific' so that any molecule not necessarily a sugar may be recognized by this lectin if the geometry exists.

Structural insights on starch hydrolysis by plant ß-amylase and its evolutionary relationship with bacterial enzymes.

The conversion of starch to maltose is catalysed in plants by ß-amylase. The enzymatic mechanism has been well-characterized for the soybean and barley enzymes, which utilise a glutamic acid-glutamate pair. In the present study, we present a surprise observation of maltotetraose at the active site, the presence of which elucidates the clear role of Thr344 as a conformational "switch" between substrate binding and product release during hydrolysis. This observation is confirmed by the selection of maltotetraose by the crystallized enzyme although that carbohydrate was present in only trace amounts. The conformation of the residues in the substrate-binding site changed upon substrate binding, leading to the movement of threonine, glutamic acid, and the loop conformation, elucidating a missing link in the existing mechanism. By aligning our substrate-free and maltotetraose-bound structures with other existing structures, the sequence of events from substrate binding to hydrolysis can be visualized. Apart from this, the evolutionary relationship among ß-amylases of bacterial and amyloplastic origin could be established. The presence of a sugar-binding domain in the bacterial enzyme and its absence in the plant counterpart could be attributed to a carbohydrate-rich environment. Interestingly, cladogram analysis indicates the presence of N-terminal additions in some plant ß-amylases. Based on sequence similarity, we postulate that the role of such additions is important for the regulation of enzymatic activity, particularly under stress conditions.

A novel guaiane sesquiterpene derivative, guai-2-en-10α-ol, from Ulva fasciata Delile inhibits EGFR/PI3K/Akt signaling and induces cytotoxicity in triple-negative breast cancer cells.

A novel guaiane sesquiterpene derivative, guai-2-en-10α-ol, from Ulva fasciata Delile exhibits antimicrobial property. U. fasciata extract was reported to exhibit cytotoxicity against cancer. In the present study, we have studied the anticancer potential of the compound, guai-2-en-10α-ol, from U. fasciata. The compound showed selective cytotoxicity toward triple-negative breast cancer (TNBC) cell line (MDA MB-231) in a dose-dependent manner. In treated cells, the apoptotic hallmarks such as formation of apoptotic bodies, cell shrinkage, and nuclear condensation were observed. Many small molecules affect the function of cellular signaling pathways. As EGFR/PI3K/Akt pathway proteins are frequently altered in TNBC, we have studied the gene expression of key proteins of this pathway. The semiquantitative PCR results demonstrated the down-regulated expression of PDPK1 (positive regulator) and Akt (key activator) as well as up-regulated expression of PTEN (negative regulator), which suggested the interaction of guai-2-en-10α-ol with upstream protein. Further investigation showed the down-regulation of both PI3K and EGFR. As EGFR is the most upstream protein of the pathway, its protein level expression was investigated. Western blotting analysis confirmed the down-regulation of p-EGFR expression and activation of apoptosis upon compound treatment. Cell cycle analysis also evidenced the G1 phase arrest, which can be due to the inhibition of cell survival pathway. Computational studies showed the interaction of guai-2-en-10α-ol with Asp855 residue of EGFR kinase domain in active conformation. All these results demonstrate the anticancer potential of guai-2-en-10α-ol through EGFR/PI3K/Akt pathway.

Crystal structure determination and analysis of 11S coconut allergen: Cocosin.

Allergy is an abnormal immune response against an innocuous target. Food allergy is an adverse reaction caused by common foods most well-known being those involving peanuts. Apart from mono sensitized food allergy, cross-reactivity with other food allergens is also commonly observed. To understand the phenomenon of cross-reactivity related to immune response, three dimensional structures of the allergens and their antigenic epitopes has to be analysed in detail. The X-ray crystal structure of Cocosin, a common 11S food allergen from coconut, has been determined at 2.2Å resolution using molecular replacement technique. The monomer of 52kDa is composed of two ß-jelly roll domains, one with acidic and the other with basic character. The structure shows hexameric association with two trimers facing each other. Though the overall structure of Cocosin is similar to other 11S allergens, the occurrence of experimentally determined epitopes of the peanut allergen Ara h 3 at flexible as well as variable regions could be the reason for the clinically reported result of cross-reactivity that the peanut allergic patients are not sensitized with coconut allergen.

Structural analysis of ß-prism lectin from Colocasia esculenta (L.) S chott.

The Mannose-binding ß-Prism Colocasia esculenta lectin (ß-PCL) was purified from tubers using ion exchange chromatography. The purified ß-PCL appeared as a single band of ∼12kDa on SDS-PAGE. ß-PCL crystallizes in trigonal space group P3121 and diffracted to a resolution of 2.1Å. The structure was solved using Molecular replacement using Crocus vernus lectin (PDB: 3MEZ) as a model. From the final refined model to an R-factor of 16.5% and an Rfree of 20.4%, it has been observed that the biological unit consists of two ß-Prism domains augmented through C-terminals swap over to form one of faces for each domain. Cα superposition of individual domains of ß-PCL with individual domains of other related structures and superposition of whole protein structures were carried out. The higher RMS deviation for the superposition of whole structures suggest that ß-prism domains assume different orientation in each structure.

In vitro studies on α-glucosidase inhibition, antioxidant and free radical scavenging activities of Hedyotis biflora L.

Aim of this study was to evaluate the in vitro α-glucosidase inhibition and antioxidant activity of hexane, ethyl acetate and methanol extracts of Hedyotis biflora L. (Rubiaceae). In in vitro α-glucosidase inhibition and antioxidant activity, the methanol extract showed potent effect compared to hexane and ethyl acetate extracts. The methanol extract of H. biflora (HBMe) showed 50% α-glucosidase inhibition at the concentration of 480.20 ± 2.37 µg/ml. The total phenolic content of HBMe was 206.81 ± 1.11 mg of catechol equivalents/g extract. HBMe showed great scavenging activity on 2,2-diphenyl-picrylhydrazyl (DPPH) (IC(50) 520.21 ± 1.02 µg/ml), hydroxyl (IC(50) 510.21 ± 1.51 µg/ml), nitric oxide (IC(50) 690.20 ± 2.13 µg/ml) and superoxide (IC(50) 510.31 ± 1.45 µg/ml) radicals, as well as high reducing power. HBMe also showed a strong suppressive effect on lipid peroxidation. Using the ß-carotene method, the scavenging values of HBMe was significantly lower than BHT, and metal chelating ability of HBMe also showed a strong inhibition effect when compared to the reference standard. The active compound ursolic acid from HBMe was identified using various spectroscopical studies. The results obtained in this study clearly indicate that HBMe has a significant potential to use as a natural α-glucosidase inhibition, antioxidant agent.