DHODH activity has been traditionally
DHODH activity has been traditionally measured with the standard colorimetric assay that monitors 2,6-dichloroindophenol (DCIP) reduction as absorbance decrease at 600 nm . This assay has permitted the identification of several families of DHODH inhibitors in a successful high-throughput screening (HTS) campaign of a chemical library containing 220,000 compounds in 384-well plates and 50 μl final volume . The optimization of the initial hits resulted in the identification of DMS265 . This molecule is a potent, first in class inhibitor of DHODH with an in vivo potency similar to chloroquine, and has been recently progressed to clinical studies in phase 2 . This fact has renewed the interest in finding new DHODH inhibitors, and GlaxoSmithKline (GSK) has accomplished the screen of a 77 8 collection of 1.5 million in HTS format. To achieve this task, we started by trying to miniaturize the colorimetric assay to 10 μl in a 1536-well format, but we found it to be not robust enough. Colorimetric assay conditions include the presence of detergent to solubilize the quinone substrate, glycerol to stabilize the enzyme, and sodium dodecyl sulfate (SDS) to stop the reaction. Preliminary trials were unsuccessful because buffer components made the mixing steps hard, resulting in nonreproducible dispensations, and formation of bubbles disturbed the absorbance reading in 1536-well plates. Thus, we explored the possibility of developing a fluorescence assay to make it more amenable to ultra-high-throughput mode.
Here we report the development of a new fluorescence intensity (FLINT), signal increase, high-throughput assay that measures the oxidation of L-DHO to -orotate by DHODH. In this reaction, the reducing equivalents from the oxidation of the L-DHO are transferred to the FMN, yielding the reduced enzyme form that, in the second-half reaction, becomes reoxidized by an electron acceptor. In the physiological environment, the final acceptor, and the second substrate of the reaction, is the respiratory ubiquinone, CoQ. In our assay, the CoQ has been replaced by resazurin, a redox-sensitive fluorogenic dye that changes from a blue nonfluorescent state to a pink highly fluorescent state on reduction to resorufin (). In the standard colorimetric assay, the ubiquinone is present in the reaction, and electrons are transferred from CoQ to the final artificial acceptor DCIP. It is reported the DCIP acts as alternative substrate to CoQ and the observed activity is the sum of the reduction of both the quinone and the dye . Under typical assay conditions, and depending on the quinone substrate, 10–30% of the observed activity is due to the direct reduction of DCIP by DHODH. The presence of two substrates, one of them silent, complicates inhibition experiments and leads to errors if both substrates are not explicitly considered in analysis .
Alternative assay formats have been used to simplify data interpretation in the mechanism of inhibition studies such as colorimetric quantification of -orotate, ferricyanide, and FMN fluorescence transitions , . In the FLINT assay described here, the quinone is removed and substituted by resazurin because the combined addition of both substrates, silent quinone and resazurin, decreased resorufin production and sensitivity to inhibition. Doing so, the assay increases its sensitivity to find inhibitors that compete for the quinone binding site. We report the kinetic parameters and optimal assay conditions to test and human () DHODH (DHODH) activities in a FLINT ultra-high-throughput format that allows the detection of inhibitors.
Materials and methods
Results and discussion
Conclusions We have developed a very sensitive fluorescence assay that can be used to assess the activity of flavoenzymes belonging to the dihydroorotate dehydrogenase family 2. The objective of our work was to develop an ultra-high-throughput assay to test the GSK compound collection against the PfDHODH to find new chemical entities to treat malaria. The development of this new assay allowed very fast HTS with no failed plates, highlighting the robustness of the assay. It also enabled fixing both substrate concentrations around their apparent KM values, thereby making the assay more sensitive to inhibitors with different mechanisms of action. This fluorometric assay uses resazurin as the single electron acceptor, avoiding the presence of a silent alternative substrate such as CoQD as in the standard colorimetric assay, which may mask the effect of some inhibitors. Data correlation of the inhibitors tested in both the colorimetric and fluorometric assays was excellent. All the DHODH inhibitors found with the colorimetric assay displayed equal or higher potency compared with the fluorometric assay, keeping potency ranking. The fact that this assay format was also valid to assess the activity of the human enzyme opens the scope of this original objective because this enzyme is the target for several drugs developed to treat cancer, transplant rejection, psoriasis, and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis .