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

Andrew M. Tobias, Dea Toska, Keith Lange, Tyler Eck, Rohit Bhat, Cheryl A. Janson, David Rotella, Ueli Gubler, Nina Goodey

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Article numbere0197173
JournalPLoS ONE
Volume13
Issue number5
DOIs
StatePublished - 1 May 2018

Fingerprint

Wuchereria bancrofti
Aminopterin
dihydrofolate reductase
Folic Acid Antagonists
Tetrahydrofolate Dehydrogenase
Brugia malayi
Methotrexate
Purification
Nematoda
Amino Acids
Pyrimethamine
Trimethoprim
Catechin
Cloning
filariasis
Filariasis
Enzymes
aminopterin
Genes
methotrexate

Cite this

Tobias, Andrew M. ; Toska, Dea ; Lange, Keith ; Eck, Tyler ; Bhat, Rohit ; Janson, Cheryl A. ; Rotella, David ; Gubler, Ueli ; Goodey, Nina. / Expression, purification, and inhibition profile of dihydrofolate reductase from the filarial nematode Wuchereria bancrofti. In: PLoS ONE. 2018 ; Vol. 13, No. 5.
@article{083c4fadad61483f84fcf42ad2c8beaf,
title = "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 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.",
author = "Tobias, {Andrew M.} and Dea Toska and Keith Lange and Tyler Eck and Rohit Bhat and Janson, {Cheryl A.} and David Rotella and Ueli Gubler and Nina Goodey",
year = "2018",
month = "5",
day = "1",
doi = "10.1371/journal.pone.0197173",
language = "English",
volume = "13",
journal = "PloS one",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "5",

}

Expression, purification, and inhibition profile of dihydrofolate reductase from the filarial nematode Wuchereria bancrofti. / Tobias, Andrew M.; Toska, Dea; Lange, Keith; Eck, Tyler; Bhat, Rohit; Janson, Cheryl A.; Rotella, David; Gubler, Ueli; Goodey, Nina.

In: PLoS ONE, Vol. 13, No. 5, e0197173, 01.05.2018.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Expression, purification, and inhibition profile of dihydrofolate reductase from the filarial nematode Wuchereria bancrofti

AU - Tobias, Andrew M.

AU - Toska, Dea

AU - Lange, Keith

AU - Eck, Tyler

AU - Bhat, Rohit

AU - Janson, Cheryl A.

AU - Rotella, David

AU - Gubler, Ueli

AU - Goodey, Nina

PY - 2018/5/1

Y1 - 2018/5/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85047468968&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0197173

DO - 10.1371/journal.pone.0197173

M3 - Article

VL - 13

JO - PloS one

JF - PloS one

SN - 1932-6203

IS - 5

M1 - e0197173

ER -