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Expression, purification, and inhibition profile of dihydrofolate reductase from the filarial nematode Wuchereria bancrofti
- Source :
- PLoS ONE, Vol 13, Iss 5, p e0197173 (2018), PLoS ONE
- Publication Year :
- 2018
- Publisher :
- Public Library of Science (PLoS), 2018.
-
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 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.
- Subjects :
- 0301 basic medicine
Nematoda
lcsh:Medicine
Protein Sequencing
medicine.disease_cause
Brugia malayi
Database and Informatics Methods
chemistry.chemical_compound
Dihydrofolate reductase
Medicine and Health Sciences
Cloning, Molecular
Enzyme Inhibitors
Brugia Malayi
lcsh:Science
Peptide sequence
chemistry.chemical_classification
Multidisciplinary
biology
Organic Compounds
Chemistry
Drugs
Eukaryota
Filariasis
Pyrimethamine
Wuchereria bancrofti
Helminth Infections
Physical Sciences
Antifolate
Sequence Analysis
Wuchereria
Research Article
medicine.drug
Bioinformatics
Research and Analysis Methods
Aminopterin
Gene Expression Regulation, Enzymologic
Antimalarials
03 medical and health sciences
parasitic diseases
Parasitic Diseases
Brugia
medicine
Animals
Amino Acid Sequence
Molecular Biology Techniques
Sequencing Techniques
Molecular Biology
Imidazole
Flavonoids
Pharmacology
030102 biochemistry & molecular biology
Organic Chemistry
lcsh:R
Organisms
Chemical Compounds
Biology and Life Sciences
biology.organism_classification
Invertebrates
Molecular biology
Tetrahydrofolate Dehydrogenase
Methotrexate
030104 developmental biology
Enzyme
biology.protein
lcsh:Q
Sequence Alignment
Subjects
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 13
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- PLoS ONE
- Accession number :
- edsair.doi.dedup.....aa2d3b020a513a1767eb5a22314de81e