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Filariasis is a tropical disease caused by the parasitic nematodes  Wuchereria bancrofti  and  Brugia malayi . Known inhibitors of dihydrofolate reductase (DHFR) have been previously shown to kill  Brugia malayi  nematodes and to inhibit  Brugia malayi  DHFR ( Bm DHFR) at nanomolar concentrations. These data suggest that  Bm DHFR is a potential target for the treatment of filariasis. Here, protocols for cloning, expression and purification of  Wuchereria bancrofti  DHFR ( Wb DHFR) were developed. The Uniprot entry J9F199-1 predicts a 172 amino acid protein for  Wb DHFR but alignment of this sequence to the previously described  Bm DHFR shows that this  Wb DHFR sequence lacks a crucial, conserved 13 amino acid loop. The presence of the loop in  Wb DHFR is supported by a noncanonical splicing event and the loop sequence was therefore included in the gene design. Subsequently, the K M  for dihydrofolate (3.7 ± 2 μM),  k  cat  (7.4 ± 0.6 s -1 ), and pH dependence of activity were determined. IC 50  values of methotrexate, trimethoprim, pyrimethamine, raltitrexed, aminopterin, (-)-epicatechin gallate, (-)-epicatechin, and vitexin were measured for  Wb DHFR and  Bm DHFR. Methotrexate and structurally related aminopterin were found to be effective inhibitors of  Wb DHFR, with an K I  of 1.2 ± 0.2 nM and 2.1 ± 0.5 nM, respectively, suggesting that repurposing of known antifolate compound may be an effective strategy to treating filariasis. Most compounds showed similar inhibition profiles toward both enzymes, suggesting that the two enzymes have important similarities in their active site environments and can be targeted with the same compound, once a successful inhibitor is identified.

Expression, purification, and inhibition profile of dihydrofolate reductase from the filarial nematode Wuchereria bancrofti