Inhibitors of CPH1-MAP Kinase Pathway: Ascertaining Potential Ligands as Multi-Target Drug Candidate in Candida albicans

A number of proteins contributing in pathogen’s virulence mechanisms offer a potential target for anticandidal therapeutics. CPH1 and its regulatory proteins Cst20, Hst7, Cek1 (MAPK cascade) administers filamentation and morphogenesis in human pathogenic fungus Candida albicans. These proteins are essential targets for their involvement in the successful establishment of the fungi within the host. In silico drug design using virtual screening, docking and (ADME)/Tox analysis for identification of lead compounds is an economic strategy for the development of potent anticandidal agent. The study divulged five persuasive ligands (2-O-prenyl coumaric acid, 2-nitro-4-methyl-cinnamaldehyde, 3,5-diprenyl-4-coumaric acid, VT1161, T-2307) out of 25, which collectively inhibited Cst20, Hst7, Cek1 in C. albicans. They can hence be used as natural drug leads for designing more effective inhibitors of multiple targets for C. albicans survival and progression of the disease. This study will enhance our understanding of the phenomenon “multiple targeting” and multi-target drug discovery further accelerating efficient broad-spectrum antifungal therapeutics development in near future. This study provides a good platform to eradicate the issue of “target shortage” that might facilitate the discovery of novel drugs in near future because the prolonged use of antibiotics over the years has transformed pathogenic fungus into resistant to many drugs.

     

Influence of Hydroxyl Group Position and Temperature on Thermophysical Properties of Tetraalkylammonium Hydroxide Ionic Liquids with Alcohols

In this work, we have explored the thermophysical properties of tetraalkylammonium hydroxide ionic liquids (ILs) such as tetrapropylammonium hydroxide (TPAH) and tetrabutylammonium hydroxide (TBAH) with isomers of butanol (1-butanol, 2-butanol and 2-methyl-2-propanol) within the temperature range 293.15–313.15 K, with interval of 5 K and over the varied concentration range of ILs. The molecular interactions between ILs and butanol isomers are essential for understanding the function of ILs in related measures and excess functions are sensitive probe for the molecular interactions. Therefore, we calculated the excess molar volume (V^E) and the deviation in isentropic compressibility (Δκ_s) using the experimental values such as densities (ρ) and ultrasonic sound velocities (u) that are measured over the whole compositions range at five different temperatures (293.15, 298.15, 303.15, 308.15 and 313.15 K) and atmospheric pressure. These excess functions were adequately correlated by using the Redlich–Kister polynomial equation. It was observed that for all studied systems, the V^E and Δκ_s values are negative for the whole composition range at 293.15 K. And, the excess function follows the sequence: 2-butanol>1-butanol>2-methyl-2-propanol, which reveals that (primary or secondary or tertiary) position of hydroxyl group influence the magnitude of interactions with ILs. The negative values of excess functions are contributions from the ion-dipole interaction, hydrogen bonding and packing efficiency between the ILs and butanol isomers. Hence, the position of hydroxyl group plays an important role in the interactions with ILs. The hydrogen bonding features between ILs and alcohols were analysed using molecular modelling program by using HyperChem 7.     

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.     

Inhibitory mechanisms and binding site location for serotonin selective reuptake inhibitors on nicotinic acetylcholine receptors

Functional and structural approaches were used to examine the inhibitory mechanisms and binding site location for fluoxetine and paroxetine, two serotonin selective reuptake inhibitors, on nicotinic acetylcholine receptors (AChRs) in different conformational states. The results establish that: (a) fluoxetine and paroxetine inhibit hα1β1γδ AChR-induced Ca²⁺ influx with higher potencies than dizocilpine. The potency of fluoxetine is increased ～10-fold after longer pre-incubation periods, which is in agreement with the enhancement of [³H]cytisine binding to resting but activatable Torpedo AChRs elicited by these antidepressants, (b) fluoxetine and paroxetine inhibit the binding of the phencyclidine analog piperidyl-3,4-³H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine to the desensitized Torpedo AChR with higher affinities compared to the resting AChR, and (c) fluoxetine inhibits [³H]dizocilpine binding to the desensitized AChR, suggesting a mutually exclusive interaction. This is supported by our molecular docking results where neutral dizocilpine and fluoxetine and the conformer of protonated fluoxetine with the highest LUDI score interact with the domain between the valine (position 13′) and leucine (position 9′) rings. Molecular mechanics calculations also evidence electrostatic interactions of protonated fluoxetine at positions 20′, 21′, and 24′. Protonated dizocilpine bridges these two binding domains by interacting with the valine and outer (position 20′) rings. The high proportion of protonated fluoxetine and dizocilpine calculated at physiological pH suggests that the protonated drugs can be attracted to the channel mouth before binding deeper within the AChR ion channel between the leucine and valine rings, a domain shared with phencyclidine, finally blocking ion flux and inducing AChR desensitization.     

Differential responses to cadmium induced oxidative stress in marine macroalga Ulva lactuca (Ulvales, Chlorophyta)

This study describes various biochemical processes involved in the mitigation of cadmium toxicity in green alga Ulva lactuca. The plants when exposed to 0.4 mM CdCl₂ for 4 days showed twofold increase in lipoperoxides and H₂O₂ content that collectively decreased the growth and photosynthetic pigments by almost 30% over the control. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione peroxidase (GPX) enhanced by twofold to threefold and that of catalase (CAT) diminished. Further, the isoforms of these enzymes, namely, Mn-SOD (~85 kDa), GR (~180 kDa) and GPX (~50 kDa) responded specifically to Cd²⁺ exposure. Moreover, the contents of reduced glutathione (3.01 fold) and ascorbate (1.85 fold) also increased substantially. Lipoxygenase (LOX) activity increased by two fold coupled with the induction of two new isoforms upon Cd²⁺ exposure. Among the polyunsaturated fatty acids, although n ? 3 PUFAs and n ? 6 PUFAs (18:3n ? 6 and C18:2n ? 6) showed relatively higher contents than control, the latter ones showed threefold increase indicating their prominence in controlling the cadmium stress. Both free and bound soluble putrescine increased noticeably without any change in spermidine. In contrast, spermine content reduced to half over control. Among the macronutrients analysed in exposed thalli, the decreased K content was accompanied by higher Na and Mn with no appreciable change in Ca, Mg, Fe and Zn. Induction of antioxidant enzymes and LOX isoforms together with storage of putrescine and n ? 6 PUFAs in cadmium exposed thallus in the present study reveal their potential role in Cd²⁺ induced oxidative stress in U. lactuca.     

Alterations in Electrocardiographic Parameters after Subacute Exposure of Fluoride and Ameliorative Action of Aluminium Sulphate in Goats

Fluorosis or crippling disease is one of the existing environmental challenges for animal and human beings in most parts of the globe. In the present study, sodium fluoride alone and with aluminium sulphate (ameliorative agent) was administered orally daily for 30 days in healthy goats of group 1 and 2, respectively, to access the effect on the electrocardiogram. All waves of Lead I, Lead II, Lead III, aVR, aVL and aVF in electrocardiographs were recorded before and after 30 days exposure of fluoride. A significant (P < 0.05) increase in P–R, Q–T and S–T intervals were observed in goats of group 1 as compared to their pre treatment values. The T wave duration was also significantly (P < 0.05) prolonged and as a result, bradycardia was observed after subacute exposure of fluoride for 30 days in group 1. But, in group 2, no such changes were observed. On the basis of results, it may be concluded that subacute toxicity of fluoride produces significant changes in different waves of electrocardiogram and aluminium sulphate has ameliorative efficacy.     

Synthesis of novel 7-imino-2-thioxo-3,7-dihydro-2H-thiazolo [4,5-d] pyrimidine derivatives as adenosine A2A receptor antagonists

Novel bicyclic thiazolopyrimidine compounds (15–26) were synthesized to develop adenosine A_2A receptor (A_2AR) antagonist for the treatment of Parkinson’s disease (PD). The binding affinity of the compounds (15–26) with A_2AR was evaluated using radioligand binding assay on isolated membranes from stably transfected HEK293 cells. Selectivity of the compounds towards A_2AR was assessed by comparing their binding affinities with A₁ receptors (A₁R). cAMP concentrations were measured from HEK293 cells treated with compounds (15–26) as compared to NECA (A_2AR agonist). The compound (16) possessed strongest A_2AR binding affinity (K_i value = 0.0038 nM) and selectivity (737-fold) versus A₁R. Decrease in A_2AR-coupled release of endogenous cAMP from HEK293 cells treated with compounds (15–26) is evocative of their potential as A_2AR antagonist.     

Genome scale prediction of substrate specificity for acyl adenylate superfamily of enzymes based on active site residue profiles

Background  

Enzymes belonging to acyl:CoA synthetase (ACS) superfamily activate wide variety of substrates and play major role in increasing the structural and functional diversity of various secondary metabolites in microbes and plants. However, due to the large sequence divergence within the superfamily, it is difficult to predict their substrate preference by annotation transfer from the closest homolog. Therefore, a large number of ACS sequences present in public databases lack any functional annotation at the level of substrate specificity. Recently, several examples have been reported where the enzymes showing high sequence similarity to luciferases or coumarate:CoA ligases have been surprisingly found to activate fatty acyl substrates in experimental studies. In this work, we have investigated the relationship between the substrate specificity of ACS and their sequence/structural features, and developed a novel computational protocol for in silico assignment of substrate preference.