| Assay Method Information | |
| | Coupled Diaphorase Assay |
| Description: | The inhibitory properties of the compounds were investigated using a coupled enzyme assay that links the lactate dehydrogenase (LDH) reaction to the production of fluorescent resorutin by diaphorase.Human lactate dehydrogenases (LDH) catalyze the reversible interconversion between pyruvate and lactate. LDH is capable of catalyzing both the forward (pyruvate to lactate) and the reverse (lactate to pyruvate) reaction, using either NADH or NAD+ as a cofactor. The reaction proceeds in either direction dependent on various factors, such as substrate availability, the presence of necessary cofactors, temperature and pH. Different isoforms (LDH A, B, and C) of the enzyme favor different reaction directions LDHA prefers the conversion from pyruvate to lactate, whereas LDHB preferentially oxidizes lactate to pyruvate.The coupled assay relies on the oxidation of NAD+ to NADH throughout the conversion of lactate to pyruvate by LDH (isoforms A, B and C). The produced NADH serves as cofactor in the diaphorase reaction, which reduces non-fluorescent resazurin to fluorescent resorufin. Therefore, the assay indirectly monitors the rate of pyruvate production. Although the consumption of NADH can be directly monitored due to the intrinsic fluorescence of the molecule (excitation: 340 nm, emission: 460 nm) there are problems linked to the direct readout method. It has been shown that many compounds in chemical libraries interfere with the assay due to fluorescent properties similar to NADH. Shifting the assay to longer wavelengths by coupling the LDH reaction to the conversion of resazurin to fluorescent resorufin by diaphorase reduces this compound interference. The assay direction was thus chosen to provide a robust and reliable assay.Applying the LDHA reaction in the preferred direction for the conversion of pyruvate to lactate under oxidation of NADH to NAD+ would necessitate running the LDHA reaction to about 80% completion and adding the diaphorase assay reagents afterwards in order to avoid enzyme competition for NADH. As a result, such a method would be expected to be more prone to errors, since too high conversion rates will lead to extenuation of the IC50 values obtained (Davis et al., ASSAY and Drug Dev. Tech. 14 (3): 175-179, 2016). When not running the assay in the preferred direction for LDHA, more conservative IC50 values would be expected to be obtained compared to earlier published results for other LDHA inhibitor compounds. Therefore, actual IC50 values could thus be expected to be lower.For the determination of IC50 values a coupled diaphorase assay was adopted from Bembenek et al. (A Fluorescence-Based Coupling Reaction for Monitoring the Activity of Recombinant Human NAD Synthetase. ASSAY and Drug Development Technologies, 2005. 3(5): 533-541). Compounds were tested in duplicates using 2-fold, 3-fold or 4-fold serial dilutions including 11 individual concentrations, starting from 5000 μM to 30 μM. A no-substrate control representing 100% inhibition or oxamate-inhibition controls (28.7 mM final oxamate concentration in assay) and a control containing the complete substrate solution as well as DMSO representing the fully uninhibited reaction were added. Oxamate is a well characterized inhibitor of LDH that inhibits LDH enzyme activity in the mM range in vitro with high specificity (Papacostantinou el al., J. Biol. Chem. 236: 278-284, 1961). The controls allowed for the calculation of the percentage inhibition for each data point. The assay buffer consisted of 50 mM HEPES pH 7.4, 5 mM MgCl2 and 0.05% pluronic acid F-127. Enzyme solution leading to final concentrations of 4-7 nM LDHA or 6 nM LDHB, as well as 0.2 U/ml diaphorase in the reaction well was dispensed into 384-well plates (Greiner bio-one) using a CyBi -SELMA robotic pipettor. Compound dilutions and the enzyme were incubated for at least 20 min at room temperature. Thereafter, the substrate solution was added. |
| Affinity data for this assay | |
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