Highly Water-Soluble Orotic Acid Nanocrystals Produced by High-Energy Milling.

Orotic acid (OA), a heterocyclic compound also known as vitamin B13, has shown potent antimalarial and cardiac protection activities; however, its limited water solubility has posed a barrier to its use in therapeutic approaches. Aiming to overcome this drawback, OA freeze-dried nanocrystal formulations (FA, FB, and FC) were developed by using the high-energy milling method. Polysorbate 80 (FA) and povacoat® (FC) were used alone and combined (FB) as stabilizers. Nanocrystals were fully characterized by dynamic light scattering, laser diffraction, transmission electron microscopy, thermal analysis (thermogravimetry and derivative thermogravimetry, and differential scanning calorimetry), and X-ray powder diffraction revealing an acceptable polydispersity index, changes in the crystalline state with hydrate formation and z-average of 100-200 nm, a remarkable 200-time reduction compared to the OA raw material (44.3 µm). Furthermore, saturation solubility study showed an improvement of 13 times higher than the micronized powder. In addition, cytotoxicity assay revealed mild toxicity for the FB and FC formulations prepared with povacoat®. OA nanocrystal platform can deliver innovative products allowing untapped the versatile potential of this drug substance candidate.

The mechanistic study of Leishmania major dihydro-orotate dehydrogenase based on steady- and pre-steady-state kinetic analysis.

Leishmania major dihydro-orotate dehydrogenase (DHODHLm) has been considered as a potential therapeutic target against leishmaniasis. DHODHLm, a member of class 1A DHODH, oxidizes dihydro-orotate (DHO) to orotate (ORO) during pyrimidine biosynthesis using fumarate (FUM) as the oxidizing substrate. In the present study, the chemistry of reduction and reoxidation of the flavin mononucleotide (FMN) cofactor in DHODHLm was examined by steady- and pre-steady state kinetics under both aerobic and anaerobic environments. Our results provide for the first time the experimental evidence of co-operative behaviour in class 1A DHODH regulated by DHO binding and reveal that the initial reductive flavin half-reaction follows a mechanism with two steps. The first step is consistent with FMN reduction and shows a hyperbolic dependence on the DHO concentration with a limiting rate (kred) of 110±6 s(-1) and a K(DHO) d of 180±27 µM. Dissociation of the reduced flavin-ORO complex corresponds to the second step, with a limiting rate of 6 s(-1). In the oxidative half-reaction, the oxygen-sensitive reoxidation of the reduced FMN cofactor of DHODHLm by FUM exhibited a hyperbolic saturation profile dependent on FUM concentration allowing estimation of K(FUM) d and the limiting rate (kreox) of 258±53 µM and 35±2 s(-1), respectively. Comparison between steady- and pre-steady-state parameters together with studies of interaction for DHODHLm with both ORO and succinate (SUC), suggests that ORO release is the rate-limiting step in overall catalysis. Our results provide evidence of mechanistic differences between class 1A and class 2 individual half-reactions to be exploited for the development of selective inhibitors.

Insights into the mechanism of oxidation of dihydroorotate to orotate catalysed by human class 2 dihydroorotate dehydrogenase: a QM/MM free energy study.

The dihydroorotate dehydrogenase (DHOD) enzyme catalyzes the unique redox reaction in the de novo pyrimidine biosynthesis pathway. In this reaction, the oxidation of dihydroorotate (DHO) to orotate (OA) and reduction of the flavin mononucleotide (FMN) cofactor is catalysed by DHOD. The class 2 DHOD, to which the human enzyme belongs, was experimentally shown to follow a stepwise mechanism but the data did not allow the determination of the order of bond-breaking in a stepwise oxidation of DHO. The goal of this study is to understand the reaction mechanism at the molecular level of class 2 DHOD, which may aid in the design of inhibitors that selectively impact the activity of only certain members of the enzyme family. In this paper, the catalytic mechanism of oxidation of DHO to OA in human DHOD was studied using a hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) approach and Molecular Dynamics (MD) simulations. The free energy barriers calculated reveal that the mechanism in human DHOD occurs via a stepwise reaction pathway. In the first step, a proton is abstracted from the C5 of DHO to the deprotonated Ser215 side chain. Whereas, in the second step, the transfer of the hydride or hydride equivalent from the C6 of DHO to the N5 of FMN, where free energy barrier calculated by the DFT/MM level is 10.84 kcal mol(-1). Finally, a residual decomposition analysis was carried out in order to elucidate the influence of the catalytic region residues during DHO oxidation.

Computational study of the mechanism of half-reactions in class 1A dihydroorotate dehydrogenase from Trypanosoma cruzi.

Chagas' disease is considered to be a health problem affecting millions of people in Latin America. This disease is caused by the parasite Trypanosoma cruzi. Recently dihydroorotate dehydrogenase class 1A from Trypanosoma cruzi (TcDHODA) was shown to be essential for the survival and growth of T. cruzi and proposed as a drug target against Chagas' disease. This enzyme catalyzes the oxidation of (S)-dihydroorotate to orotate, with a proposed catalytic cycle consisting of two half-reactions. In the first half-reaction dihydroorotate is oxidized to orotate, with the consequent reduction of the flavin mononucleotide cofactor. In the second half-reaction fumarate is reduced to succinate. The first oxidation half-reaction may occur via a concerted or a stepwise mechanism. Herein, the catalytic mechanism of TcDHODA has been studied using hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) Molecular Dynamics (MD) simulations. The free energy profiles derived from the bidimensional potential of mean force reveal more details for two half-reaction processes.

Crystal structure of dihydroorotate dehydrogenase from Leishmania major.

Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases.

Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii.

The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60µM for l-dihydroorotate, and a K(m)=29µM for decylubiquinone (Q(D)). Quinones lacking or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The IC(50)s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91µM, 96µM, and 60µM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite's mitochondrion, revealing this organelle as the site of orotate production in T. gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs.

Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension.

Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.