ABSTRACT
The flavoenzyme dihydroorotate dehydrogenase catalyzes the stereoselective oxidation of (S)-dihydroorotate to orotate in the fourth of the six conserved enzymatic reactions involved in the de novo pyrimidine biosynthetic pathway. Inhibition of pyrimidine metabolism by selectively targeting DHODHs has been exploited in the development of new therapies against cancer, immunological disorders, bacterial and viral infections, and parasitic diseases. Through a chronological narrative, this review summarizes the efforts of the scientific community to achieve our current understanding of structural and biochemical properties of DHODHs. It also attempts to describe the latest advances in medicinal chemistry for therapeutic development based on the selective inhibition of DHODH, including an overview of the experimental techniques used for ligand screening during the process of drug discovery.
Subject(s)
Flavoproteins , Oxidoreductases Acting on CH-CH Group Donors , Animals , Bacterial Infections/drug therapy , Bacterial Infections/enzymology , Dihydroorotate Dehydrogenase , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Flavoproteins/antagonists & inhibitors , Flavoproteins/chemistry , Flavoproteins/metabolism , Humans , Immune System Diseases/drug therapy , Immune System Diseases/enzymology , Neoplasms/drug therapy , Neoplasms/enzymology , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Parasitic Diseases/drug therapy , Parasitic Diseases/enzymology , Pyrimidines/chemistry , Pyrimidines/metabolism , Virus Diseases/drug therapy , Virus Diseases/enzymologyABSTRACT
Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme L-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3â Å resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.64, b = 123.92, c = 105.08â Å, ß = 96.03°. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V(M) of 2.12â Å(3)â Da(-1), corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques.