Targeting folate metabolism in Brugia malayi parasitic model using synthetic chalcone derivatives as probe

Context: Formidable disability burden associated with human lymphatic filariasis has compelled WHO to campaign for its global elimination programme. Diethyl-carbamazine (DEC) is the mainstay for mass drug administration strategy under this programme. However, till date proper rationale of this almost solitary drug is unknown. Moreover, it has no direct action on the parasite. These limitations call for the search of novel therapeutic options. Cardinal importance in cellular proliferation due to direct involvement in DNA synthesis made folate metabolism, a lucrative therapeutic target. Polyphenols might be a feasible candidate for inhibition of key enzyme of folate metabolism, dihydrofolate reductase (DHFR), since it shares common origin with folate from shikimate pathway. Chalcones are one significant subset of such polyphenols. With this perspective, our research question was that whether synthetic chalcone derivatives can be used as probable antifilarial agent against Brugia malayi by targeting parasitic folate pathway. Aims: 1. To validate possible effect of certain chalcone derivatives against B. malayi microfilariae and its therapeutic safety against peripheral blood mononuclear cell (PBMCs). 2. To explore the apoptotic activity of the proposed compounds by AO-EB staining. 3. To validate B. malayi DHFR enzyme as a plausible target of the drug by both in vivo and in silico studies. Setting: Filarial parasitic model in vitro study. Design: Basic experimental study design. Methods: A series of chalcone compounds was synthesized, characterized and then screened against microfilariae of B. malayi to test their therapeutic potential in terms of loss of parasitic motility. Further, best selected compound was used for determination of inhibitory concentration required and also for the possible lethal dose against human PBMCs. Both in silico molecular docking and further in vitro folate reversal studies were carried out to validate the parasitic target. Possible induction of apoptosis was tested by MTT and cytoplasmic cytochrome c ELISA assay with the proposed drug treated parasite along with suitable controls. Statistical Analysis: Suitable statistical methods for comparative analysis were used. Results: Among 17 chalcone derivatives, M1R showed marked antifilarial effect in micro-molar dosage; also wide difference between IC50and LD50ensured therapeutic safety. Molecular docking through bioinformatics approach with the proposed drug against pre-validated DHFR protein structure of B. malayi showed favourable thermodynamic parameters with lower inhibition constant as compared to positive control. Significant reversal of antifilarial effect of drug mediated DHFR inhibition by addition of folate indicated plausible mechanism of competitive inhibition. Further, significant apoptosis recorded by MTT assay and cytoplasmic cytochrome c ELISA in drug treated parasite against untreated control confirmed a presumable outcome of inhibition of folate metabolism by M1R compound. Conclusion: These results lead us to propose that filarial folate metabolism can be targeted through DHFR with consequent induction of apoptosis for effective therapeutic intervention, using a rationale of structural analogy based competitive inhibition of DHFR by chalcone derivatives.