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A high-throughput screening assay for pyruvate carboxylase.
Brittney N Wyatt1, Leggy A Arnold2, Martin St Maurice1
1Department of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA.
Analytical Biochemistry
|April 16, 2018
Summary
A new assay was developed to detect small molecule regulators of pyruvate carboxylase (PC), an enzyme crucial for metabolism. This method aids in discovering potential drug candidates for various organisms.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- Pyruvate carboxylase (PC) is a key metabolic enzyme converting pyruvate to oxaloacetate (OAA).
- Small molecule regulators of PC are needed for metabolic studies and potential therapeutic development.
- Currently, no specific and potent small molecule regulators for PC are available.
Purpose of the Study:
- To develop a novel, high-throughput screening (HTS) assay for identifying small molecule effectors of pyruvate carboxylase (PC).
- To validate the assay's suitability for screening compound libraries against PC from different microbial species.
Main Methods:
- A fixed-time colorimetric assay was developed utilizing the reaction of OAA with Fast Violet B (FVB).
- The assay measures the formation of a colored adduct with maximum absorbance at 530 nm.
- Assay performance was validated through plate uniformity testing and a pilot screen of 1280 compounds.
Main Results:
- The developed assay is reproducible, sensitive, and responsive to known PC effectors.
- The assay demonstrated effectiveness with PC enzymes from Rhizobium etli, Staphylococcus aureus, and Listeria monocytogenes.
- Pilot screening indicated the assay's suitability for HTS of small molecule libraries.
Conclusions:
- A novel and validated fixed-time assay enables the screening of small molecules for pyruvate carboxylase (PC) regulation.
- This assay is suitable for high-throughput screening, facilitating the discovery of novel PC inhibitors or activators.
- The developed method supports research into the metabolic roles of PC and the development of new pharmacological agents.
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