Matheus P. Pinheiro

Novel insights for dihydroorotate dehydrogenase class 1A inhibitors discovery.

The enzyme dihydroorotate dehydrogenase (DHODH) has been suggested as a promising target for the design of trypanocidal agents. We report here the discovery of novel inhibitors of Trypanosoma cruzi DHODH identified by a combination of virtual screening and ITC methods. Monitoring of the enzymatic reaction in the presence of selected ligands together with structural information obtained from X-ray crystallography analysis have allowed the identification and validation of a novel site of interaction (S2 site). This has provided important structural insights for the rational design of T. cruzi and Leishmania major DHODH inhibitors. The most potent compound (1) in the investigated series inhibits TcDHODH enzyme with Kiapp value of 19.28 μM and possesses a ligand efficiency of 0.54 kcal mol(-1) per non-H atom. The compounds described in this work are promising hits for further development.

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.

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 2.2A 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.

Target sites for the design of anti-trypanosomatid drugs based on the structure of dihydroorotate dehydrogenase.

Trypanosomatids consist of a large group of flagellated parasitic protozoa, including parasites from the genera Leishmania and Trypanosoma, responsible for causing infections in millions of humans worldwide and for which currently no appropriate therapy is available. The significance of pyrimidines in cellular metabolism makes their de novo and salvage pathways ideal druggable targets for pharmacological intervention and open an opportunity for pharmaceutical innovation. In the current review, we discuss the merits in targeting the enzyme dihydroorotate dehydrogenase (DHODH), a flavin-dependent enzyme that catalyzes the fourth and only redox step in pyrimidine de novo biosynthesis, as a strategy for the development of efficient therapeutic strategies for trypanosomatid-related diseases.We also describe the advances and perspectives from the structural biology point of view in order to unravel the structure-function relationship of trypanosomatid DHODHs, and to identify and validate target sites for drug development.

ThermoFMN - A Thermofluor Assay Developed for Ligand-Screening as an Alternative Strategy for Drug Discovery

A tecnica de Termofluor constitui uma importante ferramenta na identificacao de moleculas prototipos a farmacos. No presente trabalho, foi desenvolvido um metodo alternativo para a tecnica de Termofluor, chamado ThermoFMN, que explora o grupo prostetico flavina mononucleotideo (FMN) como sonda fluorescente. A validacao do metodo foi feita atraves do monitoramento da fluorescencia do FMN para diferentes alvos macromoleculares na presenca de uma biblioteca aleatoria de ligantes. Alem disso, farmacos com eficacia comprovada tiveram seus perfis de inibicao seletiva avaliado. Alem de demonstrar que o rendimento quântico do FMN fornece intensidade adequada para deteccao, nossos resultados revelam que o metodo de ThermoFMN utiliza-se de baixas concentracoes de proteina e e compativel com uma vasta quantidade de tampoes e aditivos quimicos. A metodologia apresentada nesse trabalho propoe uma estrategia alternativa na busca por ligantes para proteinas dependentes de FMN, como uma importante ferramenta no desenvolvimento de novas terapias contra doencas negligenciadas. Thermofluor has become a well-known and widely practiced methodology for screening of ligands that enhance stability and solubility of proteins, and also a powerful tool for hit identification in early drug discovery. In the present work, we developed an alternative Thermoflour method, named ThermoFMN, which explores the endogenous prosthetic group flavin mononucleotide (FMN) of flavoproteins as the fluorescent probe. Validation of ThermoFMN method was achieved by monitoring fluorescence signal of FMN of several drug targets in the presence of an unbiased library of ligands. In addition, drugs with known efficacy had their selective inhibition profile evaluated. Besides demonstrating that FMN signal provides sufficient fluorescence intensity for detection, our results revealed that ThermoFMN assay requires low concentration of protein samples and is compatible with a wide range of chemical reagents. The methodology presented here proposes an alternative strategy in the search for ligands of FMN-binding drug targets, therefore an important tool for the development of new therapies against neglected diseases.

The dihydroorotate dehydrogenases: Past and present ☆

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.

Crystallization and Preliminary X-ray Diffraction Analysis of Recombinant Chlorocatechol 1 2-dioxygenase from Pseudomonas Putida

Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6(1)22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4u2005Å resolution.

Cloning, expression, purification, crystallization and preliminary X‐ray diffraction analysis of the N‐terminal carbohydrate‐recognition domain of human galectin‐4

Galectin-4 is a tandem-repeat-type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin-4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate-binding specificities of galectin-4 has been focused on. As an initial step, the N-terminal carbohydrate-recognition domain of human galectin-4 (hGal4-CRD-1) has been successfully crystallized using the vapour-diffusion technique, a complete data set has been collected to 2.2 A resolution and the structure has been solved by the molecular-replacement technique. The crystals belonged to space group P6(1)22, with unit-cell parameters a = b = 71.25, c = 108.66 A. The asymmetric unit contained one molecule of hGal4-CRD-1, with a V(M) value of 2.34 A(3) Da(-1) and a solvent content of 47.51%.

Structural and binding studies of a C-type galactose-binding lectin from Bothrops jararacussu snake venom

ABSTRACT BJcuL is a snake venom galactoside‐binding lectin (SVgalL) isolated from Bothrops jararacussu and is involved in a wide variety of biological activities including triggering of pro‐inflammatory response, disruption of microbial biofilm structure and induction of apoptosis. In the present work, we determined the crystallographic structure of BJcuL, the first holo structure of a SVgalL, and introduced the fluorescence‐based thermal stability assay (Thermofluor) as a tool for screening and characterization of the binding mechanism of SVgalL ligands. BJcuL structure revealed the existence of a porous and flexible decameric arrangement composed of disulfide‐linked dimers related by a five‐fold symmetry. Each monomer contains the canonical carbohydrate recognition domain, a calcium ion required for BJcuL lectinic activity and a sodium ion required for protein stabilization. BJcuL thermostability was found to be induced by calcium ion and galactoside sugars which exhibit hyperbolic saturation profiles dependent on ligand concentration. Serendipitously, the gentamicin group of aminoglycoside antibiotics (gAGAs) was also identified as BJcuL ligands. On contrast, gAGAs exhibited a sigmoidal saturation profile compatible with a cooperative mechanism of binding. Thermofluor, hemagglutination inhibition assay and molecular docking strategies were used to identify a distinct binding site in BJcuL localized at the dimeric interface near the fully conserved intermolecular Cys86‐Cys86 disulfide bond. The hybrid approach used in the present work provided novel insights into structural behavior and functional diversification of SVgaLs. HIGHLIGHTSBJcuL is a snake venom galactoside‐binding lectin isolated from Bothrops jararacussu.The structure of holo BJcuL was determined by X‐ray crystallography.BJcuL structure revealed the existence of a porous and flexible decameric arrangement.A novel BJcuL binding site for the gentamicin group of antibiotics was predicted.Insights into structural behavior and functional diversification of BjcuL were provided.

N-terminal phosphorylation of glutaminase C decreases its enzymatic activity and cancer cell migration

The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function. Here, we identified by mass spectrometry a phosphorylated serine at the GLS N-terminal domain (at position 95) and investigated its role on regulating GAC activity. The ectopic expression of the phosphomimetic mutant (GAC.S95D) in breast cancer cells, compared to wild-type GAC (GAC.WT), led to decreased glutaminase activity, glutamine uptake, glutamate release and intracellular glutamate levels, without changing GAC sub-cellular localization. Interestingly, cells expressing the GAC.S95D mutant, compared to GAC.WT, presented decreased migration and vimentin level, an epithelial-to-mesenchymal transition marker. These results reveal that GAC is post-translationally regulated by phosphorylation, which affects cellular glutamine metabolism and glutaminase-related cell phenotype.