Tetrahydro-1H,5H-pyrazolo[1,2-a]pyrazole-1-carboxylates as inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

The reactions between 5-substituted pyrazolidine-3-ones, aldehydes, and methyl methacrylate provided tetrahydropyrazolo[1,2-a]pyrazole-1-carboxylates as mixtures of syn- and anti-diastereomers. Testing for inhibition of dihydroorotate dehydrogenase of Plasmodium falciparum (PfDHODH) revealed high activity of some anti-isomers of the methyl esters, while the corresponding carboxylic acids and carboxamides were not active. The most active representative, methyl (1S*,3S*,5R*)-1,5-dimethyl-7-oxo-3-phenyltetrahydro-1H,5H-pyrazolo[1,2-a]pyrazole-1-carboxylate (IC₅₀ = 2.9 ± 0.3 μM), also exhibited very high selectivity of the parasite enzyme vs. the human enzyme, PfDHODH/HsDHODH > 350. According to the molecular docking score, this high activity is explainable by synergic interactions of the methyl, phenyl and the CO₂Me substituent with the hydrophobic pockets in the active site of the enzyme. The carboxylic acid and carboxamides derived from this compound did not inhibit PfDHODH.     

Isolation of mitochondria from Plasmodium falciparum showing dihydroorotate dependent respiration.

Using N₂ cavitation, we established a protocol to prepare the active mitochondria from Plasmodium falciparum showing a higher succinate dehydrogenase activity than previously reported and a dihydroorotate-dependent respiration. The fact that fumarate partially inhibited the dihydroorotate dependent respiration suggests that complex II (succinate–ubiquinone reductase/quinol–fumarate reductase) in the erythrocytic stage cells of P. falciparum functions as a quinol–fumarate reductase.     

Molecular docking analysis of Plasmodium falciparum dihydroorotate dehydrogenase towards the design of effective inhibitors

Malaria remains a global public health burden with significant mortality and morbidity. Despite the several approved drugs available for its management, the parasite has developed resistance to virtually all known antimalarial drugs. The development of a new drug that can combat resistant to Artemisinin based Combination Therapies (ACTs) for malaria is imperative. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH), a flavin-dependent mitochondrial enzyme is vital in the parasite''s pyrimidine biosynthesis is a well-known drug target. Therefore, it is of interest to document the MOLECULAR DOCKING analysis (using Maestro, Schrodinger) data of DIHYDROOROTATE DEHYDROGENASE PfDHODH from P. falciparum towards the design of effective inhibitors. The molecular docking features of 10 compounds with reference to chloroquine with PfDHODH are documented in this report for further consideration.     

微生物来源的二氢乳清酸脱氢酶抑制剂F01WB-1315A，B

摘要：目的 从微生物次生代谢产物中筛选免疫相关疾病治疗药物重要靶点—－二氢乳清酸脱氢酶的抑制剂。方法 利用自建的快速、高效的二氢乳清酸脱氢酶抑制剂的高通量筛选方法,从4560株真菌菌株中筛选阳性菌株。阳性菌株的发酵产物进行分离纯化获得活性化合物,再通过对活性化合物的紫外、质谱、核磁等理化数据的分析进行结构鉴定。结果 筛选分离得到2个活性化合物F01WB-1315A和F01WB-1315B。F01WB-1315A对二氢乳清酸脱氢酶有强的抑制活性,IC50=0.07 μg/mL,于20 μg/mL浓度下对体外     

The first de novo designed inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

The de novo molecular design program SPROUT has been applied to the X-ray crystal structures of Plasmodium and human dihydroorotate dehydrogenase, respectively. The resulting design templates were used to prepare a series of molecules which, in keeping with predictions, showed useful levels of species-selective enzyme inhibition.     

Identification and characterization of small molecule inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

Plasmodium falciparum causes the most deadly form of malaria and accounts for over one million deaths annually. The malaria parasite is unable to salvage pyrimidines and relies on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHOD), a mitochondrially localized flavoenzyme, catalyzes the rate-limiting step of this pathway and is therefore an attractive antimalarial chemotherapeutic target. Using a target-based high throughput screen, we have identified a series of potent, species-specific inhibitors of P. falciparum DHOD (pfDHOD) that are also efficacious against three cultured strains (3D7, HB3, and Dd2) of P. falciparum. The primary antimalarial mechanism of action of these compounds was confirmed to be inhibition of pfDHOD through a secondary assay with transgenic malaria parasites, and the structural basis for enzyme inhibition was explored through in silico structure-based docking and site-directed mutagenesis. Compound-mediated cytotoxicity was not observed with human dermal fibroblasts or renal epithelial cells. These data validate pfDHOD as an antimalarial drug target and provide chemical scaffolds with which to begin medicinal chemistry efforts.     

High-throughput screening for potent and selective inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC(50) values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC(50) against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.     

Detergent-dependent kinetics of truncated Plasmodium falciparum dihydroorotate dehydrogenase

The survival of the malaria parasite Plasmodium falciparum is dependent upon the de novo biosynthesis of pyrimidines. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the fourth step in this pathway in an FMN-dependent reaction. The full-length enzyme is associated with the inner mitochondrial membrane, where ubiquinone (CoQ) serves as the terminal electron acceptor. The lipophilic nature of the co-substrate suggests that electron transfer to CoQ occurs at the two-dimensional lipid-solution interface. Here we show that PfDHODH associates with liposomes even in the absence of the N-terminal transmembrane-spanning domain. The association of a series of ubiquinone substrates with detergent micelles was studied by isothermal titration calorimetry, and the data reveal that CoQ analogs with long decyl (CoQ(D)) or geranyl (CoQ(2)) tails partition into detergent micelles, whereas that with a short prenyl tail (CoQ(1)) remains in solution. PfDHODH-catalyzed reduction of CoQ(D) and CoQ(2), but not CoQ(1), is stimulated as detergent concentrations (Tween 80 or Triton X-100) are increased up to their critical micelle concentrations, beyond which activity declines. Steady-state kinetic data acquired for the reaction with CoQ(D) and CoQ(2) in substrate-detergent mixed micelles fit well to a surface dilution kinetic model. In contrast, the data for CoQ(1) as a substrate were well described by solution steady-state kinetics. Our results suggest that the partitioning of lipophilic ubiquinone analogues into detergent micelles needs to be an important consideration in the kinetic analysis of enzymes that utilize these substrates.     

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.     

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.     

Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain

Trypanosoma cruzi is the etiological agent of Chagas’ disease, a pathogenesis that affects millions of people in Latin America. Here, we report the crystal structure of dihydroorotate dehydrogenase (DHODH) from T. cruzi strain Y solved at 2.2 Å resolution. DHODH is a flavin mononucleotide containing enzyme, which catalyses the oxidation of l-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Genetic studies have shown that DHODH is essential for T. cruzi survival, validating the idea that this enzyme can be considered an attractive target for the development of antichagasic drugs. In our work, a detailed analysis of T. cruzi DHODH crystal structure has allowed us to suggest potential sites to be further exploited for the design of highly specific inhibitors through the technology of structure-based drug design.     

Analysis of flavin oxidation and electron-transfer inhibition in Plasmodium falciparum dihydroorotate dehydrogenase

Plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) is a flavin-dependent mitochondrial enzyme that provides the only route to pyrimidine biosynthesis in the parasite. Clinically significant inhibitors of human DHODH (e.g., A77 1726) bind to a pocket on the opposite face of the flavin cofactor from dihydroorotate (DHO). This pocket demonstrates considerable sequence variability, which has allowed species-specific inhibitors of the malarial enzyme to be identified. Ubiquinone (CoQ), the physiological oxidant in the reaction, has been postulated to bind this site despite a lack of structural evidence. To more clearly define the residues involved in CoQ binding and catalysis, we undertook site-directed mutagenesis of seven residues in the structurally defined A77 1726 binding site, which we term the species-selective inhibitor site. Mutation of several of these residues (H185, F188, and F227) to Ala substantially decreased the affinity of pfDHODH-specific inhibitors (40-240-fold). In contrast, only a modest increase in the Kmapp for CoQ was observed, although mutation of Y528 in particular caused a substantial reduction in kcat (40-100-fold decrease). Pre-steady-state kinetic analysis by single wavelength stopped-flow spectroscopy showed that the mutations had no effect on the rate of the DHO-dependent reductive half-reaction, but most reduced the rate of the CoQ-dependent flavin oxidation step (3-20-fold decrease), while not significantly altering the Kdox for CoQ. As with the mutants, inhibitors that bind this site block the CoQ-dependent oxidative half-reaction without affecting the DHO-dependent step. These results identify residues involved in inhibitor binding and electron transfer to CoQ. Importantly, the data provide compelling evidence that the binding sites for CoQ and species-selective site inhibitors do not overlap, and they suggest instead that inhibitors act either by blocking the electron path between flavin and CoQ or by stabilizing a conformation that excludes CoQ binding.     

Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry

Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) catalyzes the oxidation of l-dihydroorotate to orotate with concomitant reduction of fumarate to succinate in the de novo pyrimidine biosynthetic pathway. Based on the important need to characterize catalytic mechanism of TcDHODH, we have tailored a protocol to measure TcDHODH kinetic parameters based on isothermal titration calorimetry. Enzymatic assays lead to Michaelis-Menten curves that enable the Michaelis constant (K_M) and maximum velocity (V_max) for both of the TcDHODH substrates: dihydroorotate (K_M = 8.6 ± 2.6 μM and V_max = 4.1 ± 0.7 μM s^-1) and fumarate (K_M = 120 ± 9 μM and V_max = 6.71 ± 0.15 μM s^-1). TcDHODH activity was investigated using dimethyl sulfoxide (10%, v/v) and Triton X-100 (0.5%, v/v), which seem to facilitate the substrate binding process with a small decrease in K_M. Arrhenius plot analysis allowed the determination of thermodynamic parameters of activation for substrates and gave some insights into the enzyme mechanism. Activation entropy was the main contributor to the Gibbs free energy in the formation of the transition state. A factor that might contribute to the unfavorable entropy is the hindered access of substrates to the TcDHODH active site where a loop at its entrance regulates the open-close channel for substrate access.     

In-Silico molecular docking and simulation studies on novel chalcone and flavone hybrid derivatives with 1, 2, 3-triazole linkage as vital inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

The structural motifs of chalcones, flavones, and triazoles with varied substitutions have been studied for the antimalarial activity. In this study, 25 novel derivatives of chalcone and flavone hybrid derivatives with 1, 2, 3-triazole linkage are docked with Plasmodium falciparum dihydroorotate dehydrogenase to establish their inhibitory activity against Plasmodium falciparum. The best binding conformation of the ligands at the catalytic site of dihydroorotate dehydrogenase are selected to characterize the best bound ligand using the best consensus score and the number of hydrogen bond interactions. The ligand namely (2E)-3-(4-{[1-(3-chloro-4-fluorophenyl)-1H-1, 2, 3-triazol-4-yl]methoxy}-3-methoxyphenyl-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one, is one the among the five best docked ligands, which interacts with the protein through nine hydrogen bonds and with a consensus score of five. To refine and confirm the docking study results, the stability of complexes is verified using Molecular Dynamics Simulations, Molecular Mechanics /Poisson–Boltzmann Surface Area free binding energy analysis, and per residue contribution for the binding energy. The study implies that the best docked Plasmodium falciparum dihydroorotate dehydrogenase–ligand complex is having high negative binding energy, most stable, compact, and rigid with nine hydrogen bonds. The study provides insight for the optimization of chalcone and flavone hybrids with 1, 2, 3-triazole linkage as potent inhibitors.     

Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei

The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite.     

Leishmania mexicana: Conversion of dihydroorotate to orotate in amastigotes and promastigotes

The specific activity of dihydroorotate dehydrogenase, catalysing the conversion of l-5,6-dihydroorotate (l-DHO) to orotate, in Leishmania mexicana mexicana was found to be 22.1 ± 3.5 nmole/hr/mg protein in the amastigote, and 28.7 ± 4.6 nmole/hr/mg protein in the promastigote. The enzyme was found to be soluble and to require molecular O₂ for activity in both forms of the parasite. Oxygen utilisation was not mediated through the mitochondrial cytochrome-containing respiratory chain, and phenazine methosulphate and ferricyanide could be used as electron acceptors by the enzyme in both aerobic and anaerobic conditions. The enzyme from both amastigote and promastigote had a pH optimum of 7.0, and the K_m values for l-DHO were 11.8 ± 4.9 and 2.3 ± 0.4 μM, respectively. The pyrimidine analogs 5-methylorotate (K_i = 8.8 μM) and 5-aminoorotate (K_i = 57 μM) were shown to be competitive inhibitors of the promastigote enzyme, as was the reaction product orotate (K_i = 10 μM).     

Isolation of mitochondria from Plasmodium falciparum showing dihydroorotate dependent respiration

Using N₂ cavitation, we established a protocol to prepare the active mitochondria from Plasmodium falciparum showing a higher succinate dehydrogenase activity than previously reported and a dihydroorotate-dependent respiration. The fact that fumarate partially inhibited the dihydroorotate dependent respiration suggests that complex II (succinate–ubiquinone reductase/quinol–fumarate reductase) in the erythrocytic stage cells of P. falciparum functions as a quinol–fumarate reductase.     

Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain

The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP. The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis. The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone. These inhibitors have shown promising results as anti-proliferative, immunosuppressive, and antiparasitic agents. A unique feature of the class 2 DHODs is their N-terminal extension, which folds into a separate domain comprising two alpha-helices. This domain serves as the binding site for the two inhibitors and the respiratory quinones acting as the second substrate for the class 2 DHODs. The orientation of the first N-terminal helix is very different in the two complexes of rat DHOD (DHODR). Binding of atovaquone causes a 12 A movement of the first residue in the first alpha-helix. Based on the information from the two structures of DHODR, a model for binding of the quinone and the residues important for the interactions could be defined. His 56 and Arg 136, which are fully conserved in all class 2 DHODs, seem to play a key role in the interaction with the electron acceptor. The differences between the membrane-bound rat DHOD and membrane-associated class 2 DHODs exemplified by the Escherichia coli DHOD has been investigated by GRID computations of the hydrophobic probes predicted to interact with the membrane.     

Biochemical and structural characterization of the apicoplast dihydrolipoamide dehydrogenase of Plasmodium falciparum

PDC (pyruvate dehydrogenase complex) is a multi-enzyme complex comprising an E1 (pyruvate decarboxylase), an E2 (dihydrolipomide acetyltransferase) and an E3 (dihydrolipoamide dehydrogenase). PDC catalyses the decarboxylation of pyruvate and forms acetyl-CoA and NADH. In the human malaria parasite Plasmodium falciparum, the single PDC is located exclusively in the apicoplast. Plasmodium PDC is essential for parasite survival in the mosquito vector and for late liver stage development in the human host, suggesting its suitability as a target for intervention strategies against malaria. Here, PfaE3 (P. falciparum apicoplast E3) was recombinantly expressed and characterized. Biochemical parameters were comparable with those determined for E3 from other organisms. A homology model for PfaE3 reveals an extra anti-parallel β-strand at the position where human E3BP (E3-binding protein) interacts with E3; a parasite-specific feature that may be exploitable for drug discovery against PDC. To assess the biological role of Pfae3, it was deleted from P. falciparum and although the mutants are viable, they displayed a highly synchronous growth phenotype during intra-erythrocytic development. The mutants also showed changes in the expression of some mitochondrial and antioxidant proteins suggesting that deletion of Pfae3 impacts on the parasite''s metabolic function with downstream effects on the parasite''s redox homoeostasis and cell cycle.