Maria Cristina Nonato

Isolation and biochemical, functional and structural characterization of a novel l-amino acid oxidase from Lachesis muta snake venom

The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K(m) of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 μg of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC₅₀: 22.7 μg/mL) and on MCF-7 cell line (breast adenocarcinoma, IC₅₀:1.41 μg/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC₅₀: 2.22 μg/mL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions.

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.

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 microM and V(max)=4.1+/-0.7 microMs(-1)) and fumarate (K(M)=120+/-9 microM and V(max)=6.71+/-0.15 microMs(-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.

Discovery of Antimalarial Azetidine-2-carbonitriles That Inhibit P. falciparum Dihydroorotate Dehydrogenase

Dihydroorotate dehydrogenase (DHODH) is an enzyme necessary for pyrimidine biosynthesis in protozoan parasites of the genus Plasmodium, the causative agents of malaria. We recently reported the identification of novel compounds derived from diversity-oriented synthesis with activity in multiple stages of the malaria parasite life cycle. Here, we report the optimization of a potent series of antimalarial inhibitors consisting of azetidine-2-carbonitriles, which we had previously shown to target P. falciparum DHODH in a biochemical assay. Optimized compound BRD9185 (27) has in vitro activity against multidrug-resistant blood-stage parasites (EC50 = 0.016 μM) and is curative after just three doses in a P. berghei mouse model. BRD9185 has a long half-life (15 h) and low clearance in mice and represents a new structural class of DHODH inhibitors with potential as antimalarial drugs.

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.

3-Epiabruslactone A, a New Triterpene Lactone Isolated from Austroplenckia populnea

A new lactonic triterpene isolated from the heartwood of Austroplenckia populnea (Celastraceae) was characterized as 3a-hydroxyolean-12-en-29,22a-olide (the g-lactone of the 3a,22a-dihydroxyolean-12-en-29a-oic acid), the 3-epimer of the abruslactone A, on the basis of its spectral data, chemical transformations, and single crystal X-ray analysis.

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 3.4 Å resolution.

Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication.

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

Recombinant production, crystallization and crystal structure determination of dihydroorotate dehydrogenase from Leishmania (Viannia) braziliensis

The enzyme dihydroorotate dehydrogenase (DHODH) is a flavoenzyme that catalyses the oxidation of dihydroorotate to orotate in the de novo pyrimidine-biosynthesis pathway. In this study, a reproducible protocol for the heterologous expression of active dihydroorotate dehydrogenase from Leishmania (Viannia) braziliensis (LbDHODH) was developed and its crystal structure was determined at 2.12 Å resolution. L. (V.) braziliensis is the species responsible for the mucosal form of leishmaniasis, a neglected disease for which no cure or effective therapy is available. Analyses of sequence, structural and kinetic features classify LbDHODH as a member of the class 1A DHODHs and reveal a very high degree of structural conservation with the previously reported structures of orthologous trypanosomatid enzymes. The relevance of nucleotide-biosynthetic pathways for cell metabolism together with structural and functional differences from the respective host enzyme suggests that inhibition of LbDHODH could be exploited for antileishmanicidal drug development. The present work provides the framework for further integrated in vitro, in silico and in vivo studies as a new tool to evaluate DHODH as a drug target against trypanosomatid-related diseases.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of recombinant human fumarase

Human fumarase (HsFH) is a well-known citric acid cycle enzyme and is therefore a key component in energy metabolism. Genetic studies on human patients have shown that polymorphisms in the fumarase gene are responsible for diseases such as hereditary leiomyomatosis and renal cell cancer. As a first step in unravelling the molecular basis of the mechanism of fumarase deficiency in genetic disorders, the HsFH gene was cloned in pET-28a, heterologously expressed in Escherichia coli, purified by nickel-affinity chromatography and crystallized using the vapour-diffusion technique. X-ray diffraction experiments were performed at a synchrotron source and the structure was solved at 2.1 Å resolution by molecular replacement.