Back to Search
Start Over
Label-free high-throughput assays to screen and characterize novel lactate dehydrogenase inhibitors
- Source :
- Analytical biochemistry. 441(2)
- Publication Year :
- 2013
-
Abstract
- Catalytic turnover of pyruvate to lactate by lactate dehydrogenase (LDH) is critical in maintaining an intracellular nicotinamide adenine dinucleotide (NAD⁺) pool for continuous fueling of the glycolytic pathway. In this article, we describe two label-free high-throughput assays (a kinetic assay detecting the intrinsic reduced nicotinamide adenine dinucleotide (NADH) fluorescence and a mass spectrometric assay monitoring the conversion of pyruvate to lactate) that were designed to effectively identify LDH inhibitors, characterize their different mechanisms of action, and minimize potential false positives from a small molecule compound library screen. Using a fluorescence kinetic image-based reader capable of detecting NADH fluorescence in the ultra-high-throughput screening (uHTS) work flow, the enzyme activity was measured as the rate of NADH conversion to NAD⁺. Interference with NADH fluorescence by library compounds was readily identified during the primary screen. The mass spectrometric assay quantitated the lactate and pyruvate levels simultaneously. The multiple reaction monitoring mass spectrometric method accurately detected each of the two small organic acid molecules in the reaction mixture. With robust Z' scores of more than 0.7, these two high-throughput assays for LDH are both label free and complementary to each other in the HTS workflow by monitoring the activities of the compounds on each half of the LDH redox reaction.
- Subjects :
- Biophysics
Nicotinamide adenine dinucleotide
Biochemistry
Redox
Mass Spectrometry
chemistry.chemical_compound
Lactate dehydrogenase
Neoplasms
Pyruvic Acid
Humans
Glycolysis
Lactic Acid
Enzyme Inhibitors
Molecular Biology
Enzyme Assays
chemistry.chemical_classification
biology
L-Lactate Dehydrogenase
Selected reaction monitoring
Cell Biology
NAD
Small molecule
Enzyme assay
High-Throughput Screening Assays
Spectrometry, Fluorescence
chemistry
biology.protein
Organic acid
Subjects
Details
- ISSN :
- 10960309
- Volume :
- 441
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Analytical biochemistry
- Accession number :
- edsair.doi.dedup.....cf1c70def5a6733b746084a32c2b016d