Dataset Information

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

ABSTRACT: 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 ?M), 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.

SUBMITTER: Tobias AM

PROVIDER: S-EPMC5963757 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Tobias Andrew M AM Toska Dea D Lange Keith K Eck Tyler T Bhat Rohit R Janson Cheryl A CA Rotella David P DP Gubler Ueli U Goodey Nina M NM

PloS one 20180522 5

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 en ...[more]

PMID: 29787565

Similar Datasets

Project description:Wuchereria bancrofti (Wb) is the most widely distributed of the three nematodes known to cause lymphatic filariasis (LF), the other two being Brugia malayi and Brugia timori. Current tools available to monitor LF are limited to diagnostic tests targeting DNA repeats, filarial antigens, and anti-filarial antibodies. While these tools are useful for detection and surveillance, elimination programs have yet to take full advantage of molecular typing for inferring infection history, strain fingerprinting, and evolution. To date, molecular typing approaches have included whole mitochondrial genomes, genotyping, targeted sequencing, and random amplified polymorphic DNA (RAPDs). These studies have revealed much about Wb biology. For example, in one study in Papua New Guinea researchers identified 5 major strains that were widespread and many minor strains some of which exhibit geographic stratification. Genome data, while rare, has been utilized to reconstruct evolutionary relationships among taxa of the Onchocercidae (the clade of filarial nematodes) and identify gene synteny. Their phylogeny reveals that speciation from the common ancestor of both B. malayi and Wb occurred around 5-6 millions years ago with shared ancestry to other filarial nematodes as recent as 15 million years ago. These discoveries hold promise for gene discovery and identifying drug targets in species that are more amenable to in vivo experiments. Continued technological developments in whole genome sequencing and data analysis will likely replace many other forms of molecular typing, multiplying the amount of data available on population structure, genetic diversity, and phylogenetics. Once widely available, the addition of population genetic data from genomic studies should hasten the elimination of LF parasites like Wb. Infectious disease control programs have benefited greatly from population genetics data and recently from population genomics data. However, while there is currently a surplus of data for diseases like malaria and HIV, there is a scarcity of this data for filarial nematodes. With the falling cost of genome sequencing, research on filarial nematodes could benefit from the addition of population genetics statistics and phylogenetics especially in dealing with elimination programs. A comprehensive review focusing on population genetics of filarial nematode does not yet exist. Here our goal is to provide a current overview of the molecular epidemiology of W. bancrofti (Wb) the primary causative agent of LF. We begin by reviewing studies utilizing molecular typing techniques with specific focus on genomic and population datasets. Next, we used whole mitochondrial genome data to construct a phylogeny and examine the evolutionary history of the Onchocercidae. Then, we provide a perspective to aid in understanding how population genetic techniques translate to modern epidemiology. Finally, we introduce the concept of genomic epidemiology and provide some examples that will aid in future studies of Wb.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets