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 (BmDHFR) at nanomolar concentrations. These data suggest that BmDHFR is a potential target for the treatment of filariasis. Here, protocols for cloning, expression and purification of Wuchereria bancrofti DHFR (WbDHFR) were developed. The Uniprot entry J9F199-1 predicts a 172 amino acid protein for WbDHFR but alignment of this sequence to the previously described BmDHFR shows that this WbDHFR sequence lacks a crucial, conserved 13 amino acid loop. The presence of the loop in WbDHFR is supported by a noncanonical splicing event and the loop sequence was therefore included in the gene design. Subsequently, the KM for dihydrofolate (3.7 ± 2 μM), kcat (7.4 ± 0.6 s-1), and pH dependence of activity were determined. IC50 values of methotrexate, trimethoprim, pyrimethamine, raltitrexed, aminopterin, (-)-epicatechin gallate, (-)-epicatechin, and vitexin were measured for WbDHFR and BmDHFR. Methotrexate and structurally related aminopterin were found to be effective inhibitors of WbDHFR, with an KI 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.

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Lymphatic Filariasis, commonly regarded as Elephantiasis, is a vastly untreated parasitic disease primarily caused by the worms Wuchereria bancrofti and Brugia malayi which affects ~63 million people in 73 countries. Current drug treatments, such as diethylcarbamazine and ivermectin, are effective drug treatments when the host is initially infected; however, upon onset of limb swelling, these treatments fail to eradicate the parasites. Dihydrofolate Reductase (DHFR), a vital and ubiquitous enzyme that aids in DNA synthesis and folate metabolism, is a well‐established drug target and may result in a treatment option for many suffering from the disease. Selective inhibition of DHFR in both organisms may lead to progress in combating the disease and therefore aiding those who are infected. Currently, the lack of three‐dimensional understanding of both enzymes poses as an obstacle for selective inhibition. Crystallization and X‐Ray diffraction of these enzymes would elucidate their respective binding sites which will provide an understanding of how these enzymes function and thus, providing insight as to how to introduce inhibition. Once crystal structures of both enzymes are obtained with and without ligand bound, rational drug design can begin. Currently, both DHFR enzymes are being expressed and purified by Dr. Goodey's research lab and several inhibition studies have already been carried about; however, crystallization procedures have recently begun to ultimately provide a detailed model as to how the enzymes function within these organisms. In the future and as a result of this project, kinetic studies can then subsequently be carried out to test possible selective inhibitors.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lymphatic filariasis is a debilitating illness with an estimated 50 million cases as of 2018. The majority of cases are caused by the parasitic worm W. bancrofti and additional cases by the worms B. malayi and B. timori. Dihydrofolate reductase (DHFR) is an established target in the treatment of cancer, bacterial, and protozoal infections and may be a potential target for drugs targeting parasitic worm infections, including filariasis. Recent studies have shown that known antifolate compounds, including methotrexate, inhibit the activity of W. bancrofti DHFR (WbDHFR). However, the absence of structural information for filarial DHFRs has limited the study of more in-depth structure-function relationships. We report the structure of WbDHFR complexed with NADPH and folate using X-ray diffraction data measured to 2.47 Å resolution. The structure of WbDHFR reveals the usual DHFR fold and is currently only the second nematode DHFR structure in the Protein Data Bank. The equilibrium dissociation constants for NADPH (90 ± 29 nM) and folate (23 ± 4 nM) were determined by equilibrium titrations. The interactions of known antifolates with WbDHFR were analyzed using molecular docking programs and molecular dynamics simulations. Antifolates with a hydrophobic core and extended linker formed favorable interactions with WbDHFR. These combined data should now facilitate the rational design of filarial DHFR inhibitors, which in turn can be used to determine whether DHFR is a viable drug target for filariasis and whether existing antifolates may be repurposed for its treatment.