Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Emma J. Shanks; Han B. Ong; David A. Robinson; Stephen Thompson; Natasha Sienkiewicz; Alan H. Fairlamb; Julie A. Frearson

doi:10.1016/j.ab.2009.09.003

Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Emma J. Shanks, Han B. Ong, David A. Robinson, Stephen Thompson, Natasha Sienkiewicz, Alan H. Fairlamb, Julie A. Frearson

Research output: Contribution to journal › Article › peer-review

25 Citations (Scopus)

Abstract

Activity of the pterin- and folate-salvaging enzymes pteridine reductase I (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS. (C) 2009 Elsevier Inc. All rights reserved.

Original language	English
Pages (from-to)	194-203
Number of pages	10
Journal	Analytical Biochemistry
Volume	396
Issue number	2
DOIs	https://doi.org/10.1016/j.ab.2009.09.003
Publication status	Published - 15 Jan 2010

Keywords

Drug discovery
Screening
Pteridine reductase
Dihydrofolate reductase
PARASITE LEISHMANIA-MAJOR
BINDING INHIBITION CONSTANTS
THYMIDYLATE SYNTHASE
TRYPANOSOMA-BRUCEI
ENZYME-INHIBITORS
METHOTREXATE
METABOLISM
PTR1
GENE
REPLACEMENT

Access to Document

10.1016/j.ab.2009.09.003

Aref#d: 19815. Wellcome Trust Centre for Drug Discovery (Strategic Award)
Fairlamb, A. (Investigator), Ferguson, M. (Investigator) & Frearson, J. (Investigator)
1/01/08 → 31/12/12
Project: Research
Aref#d: 18185. Characterization and validation of drug targets in the Kinetoplastida (Principal Research Fellowship/Programme Grant)
Fairlamb, A. (Investigator)
1/10/06 → 30/09/17
Project: Research

Cite this

@article{f4163cde651e4046ac297775c7385334,

title = "Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase",

abstract = "Activity of the pterin- and folate-salvaging enzymes pteridine reductase I (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS. (C) 2009 Elsevier Inc. All rights reserved.",

keywords = "Drug discovery, Screening, Pteridine reductase, Dihydrofolate reductase, PARASITE LEISHMANIA-MAJOR, BINDING INHIBITION CONSTANTS, THYMIDYLATE SYNTHASE, TRYPANOSOMA-BRUCEI, ENZYME-INHIBITORS, METHOTREXATE, METABOLISM, PTR1, GENE, REPLACEMENT",

author = "Shanks, {Emma J.} and Ong, {Han B.} and Robinson, {David A.} and Stephen Thompson and Natasha Sienkiewicz and Fairlamb, {Alan H.} and Frearson, {Julie A.}",

year = "2010",

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doi = "10.1016/j.ab.2009.09.003",

language = "English",

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journal = "Analytical Biochemistry",

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T1 - Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

AU - Shanks, Emma J.

AU - Ong, Han B.

AU - Robinson, David A.

AU - Thompson, Stephen

AU - Sienkiewicz, Natasha

AU - Fairlamb, Alan H.

AU - Frearson, Julie A.

PY - 2010/1/15

Y1 - 2010/1/15

N2 - Activity of the pterin- and folate-salvaging enzymes pteridine reductase I (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS. (C) 2009 Elsevier Inc. All rights reserved.

AB - Activity of the pterin- and folate-salvaging enzymes pteridine reductase I (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS. (C) 2009 Elsevier Inc. All rights reserved.

KW - Drug discovery

KW - Screening

KW - Pteridine reductase

KW - Dihydrofolate reductase

KW - PARASITE LEISHMANIA-MAJOR

KW - BINDING INHIBITION CONSTANTS

KW - THYMIDYLATE SYNTHASE

KW - TRYPANOSOMA-BRUCEI

KW - ENZYME-INHIBITORS

KW - METHOTREXATE

KW - METABOLISM

KW - PTR1

KW - GENE

KW - REPLACEMENT

U2 - 10.1016/j.ab.2009.09.003

DO - 10.1016/j.ab.2009.09.003

M3 - Article

SN - 0003-2697

VL - 396

SP - 194

EP - 203

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 2

ER -

Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Abstract

Keywords

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Projects

Aref#d: 19815. Wellcome Trust Centre for Drug Discovery (Strategic Award)

Aref#d: 18185. Characterization and validation of drug targets in the Kinetoplastida (Principal Research Fellowship/Programme Grant)

Cite this