Inhibition of thioredoxin reductase (TrxR) triggers oxidative stress-induced apoptosis in filarial nematode Setaria cervi channelized through ASK-1-p38 mediated caspase activation
Introduction
Maintenance of the cellular redox homeostasis in any living organism is an inevitable process and significant disruption results in toxic consequences by the formation of the reactive oxygen species (ROS). Highly reactive free radicals readily damage the crucial cellular biomolecules and eventually threaten the survivability of the concerned organism [1]. For maintenance of the redox equilibrium, organisms are equipped with various antioxidant enzymes and non-enzymes. These antioxidants are often targeted as a means of therapeutic strategy against various infectious diseases [[2], [3], [4]]. Here in this article, an important thiol-based antioxidant enzyme, Thioredoxin reductase of the filarial pathogen has been targeted. In a nutshell, Lymphatic filariasis (LF) is a mosquito vector-borne parasitic disease majorly caused by the lymph dwelling nematode parasites of the genus Wuchereria bancrofti, Brugia malayi, and Brugia timori [5]. The disease itself is endowed with imperative societal as well as adverse fiscal impacts in terms of substantial morbidity and serious illnesses [6]. The current treatment regimen is restricted to the administration of diethylcarbamazine citrate, albendazole and ivermectin either alone or in amalgamation [7]. Various studies have revealed that these drugs are only efficacious to the larval stages (microfilaria, Mf) of the filarial parasites with limited adulticidal activities [7,8].
On the other hand, drug resistance has posed a serious obstacle in the treatment of this disease of poverty [9]. From the present treatment scenario, it is quite evident that novel and effective adulticidal therapeutics as well as drug targets are badly needed [[7], [8], [9]]. An interesting feature of these filarial parasites is they are long-lived inside the human lymphatics and are capable of surviving inside a hostile environment [10]. The strong antioxidant systems in the parasites shield them from the unreceptive environment of the host [11]. Therefore, several putative antioxidant enzymes are often targeted for successful therapeutic interventions.
Several important articles have adequately discussed the importance of thiol based TrxR enzyme in the survival of the parasites [3,12]. Available reports on TrxR state that structurally it is a homodimeric flavoenzyme belonging to the pyrimidine nucleotide- disulfide oxidoreductase family and the catalytic activity of it is based on NADPH [4,13]. The detailed biochemical structure of TrxR and the biological activities it exhibits have been described elsewhere [3]. Selective targeting of TrxR by chemical compounds has been shown to inhibit the enzyme activity inside the cellular system resulting in the death of the cell [3,14].
Herein this study, we have evaluated the anti-TrxR potencies of Auranofin, 1 chloro, 2,4 dinitrobenzene (CDNB), Curcumin, and a novel carbamo(dithioperoxo)thioate derivative p-Tolyl piperidine-1-carbo (dithioperoxo) thioate (Compound 4a) and also investigated the underlying mechanism of apoptosis induction on the filarial parasite. Interestingly, the study has revealed the inhibitory action of Auranofin, CDNB, Curcumin, and compound 4a on filarial TrxR that potentially altered the internal redox balance and augmented caspase-mediated apoptosis through TrxR-ASK-1-p38- mediated axis. This pathway of apoptosis induction is a maiden report in filarial biology and likely to be worthy of antifilarial drug development as well.
Section snippets
Chemicals and reagents
Chief purity grade solvents were purchased from Merck, India, and Milli-Q water (Milli-Q Academic with 0.22 μm Millipak R-40) was used. Fetal bovine serum was purchased from Gibco (USA). MTT, NBT, DMSO, EDTA, Potassium dibasic salt, and monobasic salt were procured from Merck (India). Nicotinamide adenine dinucleotide phosphate reduced tetra-sodium salt (NADPH), RPMI-1640, 1-chloro, 2,4 dinitrobenzene (CDNB), 5−5 Dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Himedia (India).
Effect of Auranofin, CDNB, Curcumin and 4a on viability of the parasite
Treatment with Auranofin, CDNB, Curcumin, and 4a caused the death of both Mf and adult parasites. 24 h post-treatment, Auranofin, and CDNB at 12.5 μM dose was potent enough to kill more than 50 % of the Mf (Fig. 1a). Whereas in case of the adult parasites,100 μM and 25 μM of Auranofin and CDNB showed marked inhibition after 12 h of treatment respectively (Fig. 1b). Curcumin at 100 μM dose caused death of both the Mf and adult filarial parasites (**p < 0.001, compared to the control) (Fig. 1
Discussion
Quest for putative drug targets inside the filarial parasite is necessary for the development of effective therapeutic interventions against LF. Amongst various thiol-based antioxidant enzymes present (glutathione reductase and thioredoxin reductase) in the filarial parasites particularly the TrxR have been taken under consideration in this study. Tiwari et al. [17] had already reported that specific inhibition of TrxR resulted in the apoptotic death of the filarial parasites. However, the
Conclusion
En masse, our obtained results demonstrate that inhibition of filarial TrxR results in oxidative stress-mediated caspase activation leading to the death of the parasite. Interestingly, this TrxR mediated apoptosis routed through the ASK-1/p38 axis is not reported hitherto. Therefore, the detailed molecular evidence obtained along with the in-silico docking studies revealed a brand-new mechanism of parasitic death in the filarial parasites at least. In the future, therapeutic molecules can be
Declaration of Competing Interest
The authors report no declarations of interest.
Author contributions
NJ and SPS conceived the idea. NJ and SPS designed the experiments, analyzed the data and wrote the manuscript. AS and JR conducted the in silico study and analyzed the data. NJ, AS, JR and SPS reviewed the final draft of the manuscript. SPS supervised the entire study.
CRediT authorship contribution statement
Nikhilesh Joardar: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review & editing. Animesh Sen: Software, Investigation, Methodology, Validation. Jnanendra Rath: Software, Investigation, Methodology, Validation. Santi P. Sinha Babu: Supervision, Funding acquisition, Project administration, Resources, Writing - review & editing.
Acknowledgement
NJ sincerely acknowledges the Council for Scientific and Industrial Research (CSIR), Govt. of India for providing the Senior Research Fellowship (Grant Number: 09/202/ (0086) 2K19/EMR-I). Sincere thanks to DST-PURSE (SR/PURSE 2/42/G) for the support of chemicals. Sincere thanks to Prof. Anup Kumar Misra, Bose Institute Kolkata for the gift of compound 4a. We acknowledge Dr. Krishna Das Saha CSIR-IICB, Kolkata) for her generous gift of antibodies. We also acknowledge Confocal Microscopy Facility
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