In silico studies of urease inhibitors to explore ligand-enzyme interactions

In continuation of our previous study on the urease inhibition by a number of chalcones, 2,3-dihydro-1,5-benzothiazepines and 2,3,4,5-tetrahydro-1,5-benzothiazepines, FlexX docking has been exploited to get a deeper insight into the mechanism of their inhibitory action. A comparison of the IC₅₀ values of the active compounds reveals that, of the three classes of compounds studied, 2,3-dihydro-1,5-benzothiazepines were the most potent urease inhibitors. An in silico examination of these compounds showed that the activity is related to the interaction of ligand with the nickel metallocentre, its interaction with two amino acid residues, Asp224 and Cys322, in addition to the orientation of rings A and B in the catalytic core of the enzyme. The most active compound 2,3-dihydro-1,5-benzothiazepine (4) anchor tightly through a network of interactions with Ni701 and Ni702. This includes a number of hydrogen bonds and hydrophobic contacts with the amino acid residues in its vicinity. For their reduced analogs, the difference in the activity of different diastereomers has been observed to be configuration-dependent. This may be ascribed mainly to the difference in the orientation of ring B of the two stereoisomers and the extent of their interaction with Asp224 and Cys322 present in the catalytic core of the enzyme.     

Association mapping for soilborne pathogen resistance in synthetic hexaploid wheat

Soilborne pathogens such as cereal cyst nematode (CCN; Heterodera avenae) and root lesion nematode (Pratylenchus neglectus; PN) cause substantial yield losses in the major cereal-growing regions of the world. Incorporating resistance into wheat cultivars and breeding lines is considered the most cost-effective control measure for reducing nematode populations. To identify loci with molecular markers linked to genes conferring resistance to these pathogens, we employed a genome-wide association approach in which 332 synthetic hexaploid wheat lines previously screened for resistance to CCN and PN were genotyped with 660 Diversity Arrays Technology (DArT) markers. Two sequence-tagged site markers reportedly linked to genes known to confer resistance to CCN were also included in the analysis. Using the mixed linear model corrected for population structure and familial relatedness (Q+K matrices), we were able to confirm previously reported quantitative trait loci (QTL) for resistance to CCN and PN in bi-parental crosses. In addition, we identified other significant markers located in chromosome regions where no CCN and PN resistance genes have been reported. Seventeen DArT marker loci were found to be significantly associated with CCN and twelve to PN resistance. The novel QTL on chromosomes 1D, 4D, 5B, 5D and 7D for resistance to CCN and 4A, 5B and 7B for resistance to PN are suggested to represent new sources of genes which could be deployed in further wheat improvement against these two important root diseases of wheat.     

Design,Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors

Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their K_i and IC₅₀ values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC₅₀ values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).     

Role of N-Cadherin cis and trans Interfaces in?the Dynamics of Adherens Junctions in Living Cells

Cadherins, Ca²⁺-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca²⁺-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca²⁺-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca²⁺-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins.     

CD160Ig Fusion Protein Targets a Novel Costimulatory Pathway and Prolongs Allograft Survival

CD160 is a cell surface molecule expressed by most NK cells and approximately 50% of CD8⁺ cytotoxic T lymphocytes. Engagement of CD160 by MHC class-I directly triggers a costimulatory signal to TCR-induced proliferation, cytokine production and cytotoxic effector functions. The role of CD160 in alloimmunity is unknown. Using a newly generated CD160 fusion protein (CD160Ig) we examined the role of the novel costimulatory molecule CD160 in mediating CD4⁺ or CD8⁺ T cell driven allograft rejection. CD160Ig inhibits alloreactive CD8⁺ T cell proliferation and IFN-γ production in vitro, in particular in the absence of CD28 costimulation. Consequently CD160Ig prolongs fully mismatched cardiac allograft survival in CD4^−/−, CD28^−/− knockout and CTLA4Ig treated WT recipients, but not in WT or CD8^−/− knockout recipients. The prolonged cardiac allograft survival is associated with reduced alloreactive CD8⁺ T cell proliferation, effector/memory responses and alloreactive IFN-γ production. Thus, CD160 signaling is particularly important in CD28-independent effector/memory CD8⁺ alloreactive T cell activation in vivo and therefore may serve as a novel target for prevention of allograft rejection.     

In vitro selection and field responses of somaclonal variant plants of rice cv PR113 for drought tolerance

Drought is the major environmental stress that limits rice productivity worldwide. In vitro somaclonal variation using different selection agents has been used for crop improvement. Here, rice plants of cv PR113 were selected in vitro on 30, 50 and 70 g L^-1 polyethylene glycol 6,000 (PEG). Callus growth, proliferation, calli volume (first and second culture) and plantlet regeneration (third culture) were found to be decreased upto a certain level to acquire tolerance to PEG-induced drought. From the field data, 30 g L^-1 PEG lines showed higher vegetative growth (plant height, tiller number, leaf number, shoot weight and root growth) as compared with 50 g L^-1 PEG selected somaclone lines under limited irrigation. The yield parameters-panicle length, panicle weight, grains per panicle, 1,000-grain weight, grain yield per plant, harvest index and grain straw ratio were also higher in 30 g L^-1 PEG lines as compared with 50 g L^-1 PEG lines. The results, therefore indicate that 30 g L^-1 PEG selected somaclone lines were more suited than 50 g L^-1 PEG selected somaclone lines under stress as compared with WT. The finding suggests that rice cv PR113 somaclones generated on PEG are found to be drought tolerant under field condition with better yield.     

First evidence of ethylene production by Fusarium mangiferae associated with mango malformation

Malformation is arguably the most crucial disease of mango (Mangifera indica L.) at present. It is receiving great attention not only because of its widespread and destructive nature but also because of its etiology and control is not absolutely understood. Recently, Fusarium mangiferae is found to be associated with mango malformation disease. There are indications that stress ethylene production could be involved in the disease. Here we have shown the first direct evidence of production of ethylene in pure culture of F. mangiferae obtained from mango. The study also revealed that all the isolates dissected from mango acquire morphological features of F. mangiferae showing most similarity to the features of species with accepted standard features. The isolates of F. mangiferae from mango were observed to produce ethylene in significant amounts, ranging from 9.28–13.66 n mol/g dry wt/day. The findings presented here suggest that F. mangiferae could contribute to the malformation of mango by producing ethylene and probably stimulating stress ethylene production in malformed tissue of mango. Ethylene might be produced through 2-oxoglutarate-dependent oxygenase-type ethylene-forming-enzyme (EFE) pathway in Fusarium sp, which needs to be investigated.     

Focus: Allergic Diseases and Type II Immunity: Mast Cells as Regulators of Adaptive Immune Responses in Food Allergy

Mast cells are a critical first line of defense against endogenous and environmental threats. Their participation in innate immunity is well characterized; activation of toll like receptors as well as receptors for complement, adenosine, and a host of other ligands leads to mast cell release of preformed mediators contained within granules along with newly synthesized arachidonic acid metabolites, cytokines, and chemokines. These confer protective effects including the induction of mucus secretion, smooth muscle contraction, and activation of common itch and pain sensations, all of which act to promote expulsion of noxious agents. While their innate immune role as sentinel cells is well established, recent research has brought into focus their separate but also critical function in adaptive immunity particularly in the setting of IgE mediated food allergies. Crosslinking of FcεR1, the high affinity receptor for IgE, when bound to IgE and antigen, triggers the release of the same factors and elicits the same physiologic responses that occur after activation by innate stimuli. Though IgE-activated mast cells are best known for their role in acute allergic reactions, including the most severe manifestation, anaphylaxis, accumulating evidence has suggested an immunoregulatory effect in T cell-mediated immunity, modulating the balance between type 2 immunity and tolerance. In this review, we outline how mast cells act as adjuvants for food antigen driven Th2 cell responses, while curtailing Treg function.     

Role of iNOS in osteoarthritis: Pathological and therapeutic aspects

Accumulating evidence suggests that inflammation has a key role in the pathogenesis of osteoarthritis (OA). Nitric oxide (NO) has been established as one of the major inflammatory mediators in OA and drives many pathological changes during the development and progression of OA. Excessive production of NO in chondrocytes promotes cartilage destruction and cellular injury. The synthesis of NO in chondrocytes is catalyzed by inducible NO synthase (iNOS), which is thereby an attractive therapeutic target for the treatment of OA. A number of direct and indirect iNOS inhibitors, bioactive compounds, and plant-derived small molecules have been shown to exhibit chondroprotective effects by suppressing the expression of iNOS. Many of these iNOS inhibitors hold promise for the development of new, disease-modifying therapies for OA; however, attempts to demonstrate their success in clinical trials are not yet successful. Many plant extracts and plant-derived small molecules have also shown promise in animal models of OA, though further studies are needed in human clinical trials to confirm their therapeutic potential. In this review, we discuss the role of iNOS in OA pathology and the effects of various iNOS inhibitors in OA.     

Probing the intermolecular interactions into serum albumin and anthraquinone systems: a spectroscopic and docking approach

Intermolecular interaction study of human serum albumin (HSA) with two anthraquinones i.e. danthron and quinizarin has been performed through fluorescence, UV-vis and CD spectroscopy along with docking analysis. The titration of drugs into HSA solution brought about the quenching of fluorescence emission by way of complex formation. The binding constants were found to be 1.51 × 10⁴ L mol^?1 and 1.70 × 10⁴ L mol^?1 at λ_exc = 280 nm while at λ_exc = 295 nm, the values of binding constants were 1.81 × 10⁴ L mol^?1 and 1.90 × 10⁴ L mol^?1 which hinted toward binding of both the drugs in the vicinity of subdomain IIA. Different temperature study revealed the presence of static quenching mechanism. Moreover, more effective quenching of the fluorescence emission was observed at λ_exc = 295 nm which also suggested that both the drug molecule bind nearer to Trp-214. Thermodynamic parameters showed that hydrophobic interaction was the major force behind the binding of drugs. The UV-vis spectroscopy testified the formation of complex in both the systems and primary quenching mechanism as static one. The changes in secondary structure and α-helicity in both the systems were observed by circular dichroism spectroscopy. Furthermore, molecular docking analysis predicted the probable binding site of drugs in subdomain IIA of HSA molecule. The types of amino acid residues surrounding the drug molecule advocated that van der Waals forces, hydrophobic forces and electrostatic forces played a vital role in the stabilization of drug-protein complex formed.