In silico exploration and in vitro validation of the filarial thioredoxin reductase inhibitory activity of Scytonemin and its derivatives.

Lymphatic filariasis (LF) caused by the vector borne parasitic nematode Wuchereria bancrofti is of major concern of the World Health Organization (WHO). Lack of potential drug candidates worsens the situation. Presently available drugs are promising in killing the microfilaria (mf) but are not effective as adulticidal therapeutics. Previous studies have revealed that routine administration of the available drugs (albendazole, ivermectin and albendazole) sometime is associated with severe adverse effects (SAEs) in co-infection state. Therefore, potential and safe therapeutics are still required. Earlier studies on filarial thioredoxin reductase (TrxR) have shown that successful inhibition of it can lead to apoptotic death of the parasites. TrxR in filarial parasites plays a significant role in disease progression and pathogenesis, hence efficient non-reversible inhibition of TrxR can be a good strategy to treat LF. In this research, inhibitory potential of Scytonemin, a cyanobacterial metabolite on filarial TrxR was evaluated via different in silico methods and validated through in vitro experiments. Parasite death upon exposure to Scytonemin can be correlated with the TrxR inhibiting capacity of the compound. Therefore, this cyanobacterial-derived compound may possibly be used further as novel and safe therapeutic candidate against filarial infection.Communicated by Ramaswamy H. Sarma.

Management of Black Root Disease-Causing Fungus Fusarium solani CRP1 by Endophytic Bacillus siamensis CNE6 through Its Metabolites and Activation of Plant Defense Genes.

Black root rot disease of Cicer arietinum L. is accountable for substantial loss in chickpea production worldwide. Endophytic Bacillus siamensis CNE6 has previously shown multifaceted plant growth-promoting, broad-spectrum antifungal, and chickpea plant-colonizing potential. In the present study, the strain Bacillus siamensis CNE6 was used for controlling black root rot disease caused by Fusarium solani CRP1 in chickpea. CNE6 showed strong antagonistic potential against CRP1 both in vivo and in vitro. Scanning electron microscopic studies indicated cellular deformation of CRP1 due to production of ß-glucanase, protease, and other secondary metabolites. A total of five compounds were detected from the cell-free supernatant (CFS) of the ethyl acetate (EA) fraction of CNE6 through gas chromatography-mass spectrometry analysis. A confocal microscopic study demonstrated strong inhibition of biofilm formation of the pathogen CRP1 by the EA fraction of CFS of CNE6. Molecular docking analysis revealed that one compound, (2E)-6-methoxy-2-[(4-methoxyphenyl)methylidene]-2,3-dihydro-1-benzofuran-3-one, may inhibit the activity of lanosterol 14-alpha demethylase, which is involved in ergosterol biosynthesis and beta-tubulin assembling. In vivo experiments also showed the efficacy of CNE6 for increasing chickpea growth as well as upregulation of four defense genes (CHI1, PAMP, PR2B, and TF1082) upon pathogenic challenge. Thus, our results strongly suggest a positive role for CNE6 as a prospective biocontrol agent for combating Fusarium solani in chickpea. IMPORTANCE The present work was undertaken to explore an effective biocontrol agent against the destructive black root rot disease of chickpea. We have used an efficient bacterial endophyte, CNE6, which can colonize in the chickpea root system, produce secondary metabolites and enzymes to degrade pathogenic cellular integrity, inhibit pathogenic establishment by rupturing biofilm formation, and induce host immunity upon treatment. Interaction of the bacterial metabolite was also observed with lanosterol 14-alpha demethylase, which is an important component in fungal membrane functioning. Being an endophyte, Bacillus siamensis CNE6 fulfills a suitable criterion as a biocontrol agent to control black root rot disease in chickpea and has huge prospects for use commercially.

Filarial thioredoxin reductase exerts anti-inflammatory effects upon lipopolysaccharide induced inflammation in macrophages.

Lymphatic filariasis and its associated health hazards have taken enormous tolls especially in the tropical and sub-tropical countries round the globe. Our present work contemplates the immunomodulatory role of filarial Thioredoxin reductase (TrxR) for the survival of the parasite inside the human host. For this, the protein TrxR was purified from the filarial parasite Setaria cervi and further substantiated through specific anti-TrxR antibody raised in mice. Both commercially available anti-TrxR antibody and laboratory raised antibody produced a single band with a molecular mass of ~80 kDa on western blot. The protein is optimally active at pH 7.0 and at temperature 37 °C. This protein contains both alpha helix and beta pleated sheet with selenocysteine at its active site. The Km was found to be 2.75 ± 0.49 mM. TrxR was found to downregulate lipopolysaccharide (LPS)-induced inflammation in macrophages due to inhibition of TLR4-NF-κB pathway. The result was further supported by the downregulation of inflammasome pathway and activation of alternatively activated macrophages upon TrxR treatment. Hence this study projects insights into the importance of filarial TrxR in host-parasite interface as well as it illustrates novel therapeutic strategy towards anti-filarial drug development.

Mycosporine-alanine, an oxo-mycosporine, protect Hassallia byssoidea from high UV and solar irradiation on the stone monument of Konark.

Mycosporine-like amino acids (MAAs) are small natural molecules having potent UV-absorbing and antioxidant properties. Hassallia byssoidea is one dominant cyanobacterium found all over the Konark stone monument, a UNESCO World Heritage site. We characterized mycosporine-alanine for the first time from H. byssoidea and studied its biosynthetic pathway from the whole genome data. We found D-alanyl-D-alanine carboxypeptidase, which might convert mycosporine-glycine to mycosporine-alanine by replacing glycine with alanine or by separate methylation, the mycosporine-glycine is converted to mycosporine-alanine. Our in vitro UV-B exposure experiment and exposure of H. byssoidea to natural sunlight show an increase in biosynthesis of mycosporine-alanine with 12 h of UV-B irradiation and high natural sunlight. We also found mycosporine-alanine to have good free radical scavenging activity with an IC50 value of 1.98 mg/ml. Our results show due to the presence of mycosporine-alanine H. byssoidea probably tolerate the UV and high solar radiation and continue to colonize on the Konark stone monument as a dominant cyanobacterium.

Inhibition of thioredoxin reductase (TrxR) triggers oxidative stress-induced apoptosis in filarial nematode Setaria cervi channelized through ASK-1-p38 mediated caspase activation.

Inhibition of an imperative antioxidant enzyme with subsequent death is a victorious and widely accepted strategy to combat various infectious diseases. Among different antioxidant enzymes, thioredoxin reductase (TrxR) is an exclusive one. Studies have revealed that direct inhibition of TrxR by different classes of chemical moieties promptly results in the death of an organism. Especially the structural as well as biochemical modifications of the enzyme upon inhibition project serious threat towards the subject organism. Herein, an attempt was made to inhibit TrxR of filarial species by administering Auranofin, 1 chloro 2,4 dinitrobenzene (CDNB), Curcumin, and a novel carbamo dithioperoxo(thioate) derivative (4a). Our study has revealed that inhibition of TrxR resulted in the induction of the classical CED pathway of apoptosis along with the intrinsic and extrinsic pathways of apoptosis (Caspase mediated) routed through the ASK-1/p38 axis. Druggability analysis of filarial TrxR for the selected compounds was performed in silico through molecular docking studies. Therefore, this study attempts to decipher the mechanism of apoptosis induction following TrxR inhibition. The safety of those four compounds in terms of dose and toxicity was taken under consideration. Thitherto, the mechanism of TrxR mediated initiation of cell death in filarial parasite has remained undercover, and therefore, it is a maiden report on the characterization of apoptosis induction upon TrxR inhibition which will eventually help in generating effective antifilarial drugs in the future.

Shinorine ameliorates chromium induced toxicity in zebrafish hepatocytes through the facultative activation of Nrf2-Keap1-ARE pathway.

Hexavalent chromium, a heavy metal toxicant, abundantly found in the environment showed hepatotoxic potential in zebrafish liver and instigated the Nrf2-Keap1-ARE pathway as a cellular stress response as reported in our previous studies. In the present study we have evaluated the ameliorating effect of shinorine, a mycosporine like amino acid (MAAs) and a mammalian Keap1 antagonist against chromium induced stress in zebrafish hepatocytes. Shinorine was found to be effective in increasing the cell viability of chromium treated hepatocytes through curtailing the cellular ROS content. Trigonelline, an Nrf2 inhibitor was found to reduce the viability of hepatocyte cultures co-exposed to shinorine and chromium. In other words, trigonelline being an Nrf2 blocker neutralised the alleviating effect of shinorine. This indicated that shinorine mediated cyto-protection in Cr [VI]-intoxicated cells is Nrf2 dependent. Further, qRT-PCR analysis revealed comparatively higher expression of nfe2l2 and nqo1 in shinorine + chromium treated hepatocytes than cells exposed to chromium alone indicating a better functioning of Nrf2-Keap1-Nqo1 axis. To further confirm if shinorine can lead to disruption of Nrf2-Keap1 interaction in zebrafish hepatocytes and render cytoprotection to chromium exposure, our in silico analysis through molecular docking revealed that shinorine could bind to the active amino acid residues of the DGR domain, responsible for Nrf2-Keap1 interaction of all the three Keap1s evaluated. This is the first report about shinorine that ameliorates chromium induced toxicity through acting as an Nrf2-Keap1 interaction disruptor. We additionally carried out in-silico pharmacokinetic and ADMET studies to evaluate druglikeness of shinorine whose promising results indicated its potential to be developed as an ideal therapeutic candidate against toxicant induced pathological conditions.