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  • Several antimalarial drugs in clinical studies target pyrimi

    2020-07-30

    Several antimalarial drugs in clinical studies target pyrimidine nucleotide biosynthesis , because parasites rely on fast and large replication of DNA to infect during liver and blood stages. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of -dihydroorotate (L-DHO) to -orotate in the fourth step in the de novo pyrimidine biosynthetic pathway. It is essential for species survival because, unlike humans, malaria parasites are unable to scavenge preformed pyrimidines . This is the only redox and rate-limiting step in uridine monophosphate (UMP) formation, the precursor to all the other pyrimidines used to synthesize DNA, RNA, and various cofactors . DHODH (DHODH) belongs to family 2, found in gram-negative bacteria and eukaryotes. The enzyme is attached to the inner mitochondrial membrane and contains a tightly bound flavin mononucleotide (FMN) cofactor that is reduced on L-DHO oxidation to -orotate in the first half of the reaction cycle. This cofactor is recycled to its oxidized form in the second half of the reaction, transferring the electrons to the ubiquinone (CoQ) that acts as natural final hiv integrase acceptor, chemically coupling pyrimidine biosynthesis to the respiratory chain . One key function of the parasite mitochondrion is to maintain the mitochondrial electron transport chain (mETC) to regenerate the CoQ required as electron acceptor for DHODH. This is demonstrated by the fact that parasites are very sensitive to mETC inhibitors, but transgenic strains expressing ubiquinone-nondependent DHODH from are resistant to them. These results provide a genetic validation of DHODH as an attractive antimalarial target . Potent inhibitors of the human enzyme, such as lapachol, brequinar, and leflunomide, are poorly active against DHODH. Thus, these data suggest that it should be feasible to exploit active-site differences to identify inhibitors that exhibit a high degree of selectivity toward malarial DHODH . The sequence of the L-DHO binding site is highly conserved, but the sequence of the quinone-binding N-terminal domain is variable . This variability is thought to be responsible for the high degree of species-related preferential inhibition observed among DHODH family 2 members. So, therapeutic agents, both those targeted to rapidly proliferating human cells and those targeted to human pathogens, could be designed to explicitly exploit these differences. 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 compound 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.