Crystal structure of dihydrofolate reductase from the filarial nematode W. bancrofti in complex with NADPH and folate

Author summaryLymphatic filariasis is a disease commonly characterized by urogenital swelling, or scrotal hydrocele and lymphedema, which affects nearly 50 million people worldwide as of 2018. It is caused by parasitic worms transmitted through mosquitos. Dihydrofolate reductase (DHFR) is a ubiquitous enzyme involved in folate metabolism and provides building blocks for DNA synthesis. DHFR is an established pharmacological target in the treatment of cancer and infectious diseases through the use of DHFR specific inhibitors known as antifolates. Using recombinant DHFR from the parasitic worm W. bancrofti (Wb), we have recently shown that known antifolate compounds (i.e. methotrexate and others) potently inhibit this enzyme. However, the absence of structural information for WbDHFR has limited the investigation of in-depth structure-function relationships. We now report the first crystal structure of WbDHFR. Using in silico approaches, a series of known antifolates was docked into the active site of the enzyme to gain insights into their interactions with WbDHFR. Such data will facilitate the rational design of inhibitors specific for filarial DHFRs. The availability of such inhibitors will be crucial to establish DHFR as a valid target for the treatment of filariasis and will also help determine whether existing antifolates may be repurposed for treatment. 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 angstrom 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.