Extracellular release of two peptidases dominates generation of the trypanosome quorum-sensing signal

Trypanosomes causing African sleeping sickness use quorum-sensing (QS) to generate transmission-competent stumpy forms in their mammalian hosts. This density-dependent process is signalled by oligopeptides that stimulate the signal transduction pathway leading to stumpy formation. Using mass spectrometry analysis, peptidases released by trypanosomes were identified and, for 12 peptidases, their extracellular delivery was confirmed. Thereafter, the contribution of each peptidase to QS signal production was determined using systematic inducible overexpression in vivo, activity being confirmed to operate through the physiological QS signalling pathway. Gene knockout of the QS-active peptidases identified two enzymes, oligopeptidase B and metallocarboxypeptidase I, that significantly reduced QS when ablated individually. Further, a combinatorial gene knockout of both peptidases confirmed their dominance in the generation of the QS signal, with peptidase release of oligopeptidase B mediated via an unconventional protein secretion pathway. This identifies how the QS signal driving trypanosome virulence and transmission is generated in mammalian hosts.

The Crystal Structure of Nα-p-tosyl-lysyl Chloromethylketone-Bound Oligopeptidase B from Serratia Proteamaculans Revealed a New Type of Inhibitor Binding

A covalent serine protease inhibitor—Na-p-tosyl-lysyl chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues.

First Crystal Structure of Bacterial Oligopeptidase B in an Intermediate State: The Roles of the Hinge Region Modification and Spermine

Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states—closed and open—in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Antileishmanial Potential of Berberine Alkaloids From Berberis glaucocarpa Roots: Molecular Docking Suggests Relevant Leishmania Protein Targets

Leishmaniases are a spectrum of poverty-linked neglected parasitic diseases that are endemic in 88 countries around the globe and affect millions of people every year. Currently available chemotherapeutic options are inadequate due to side effects, high cost, prolonged treatment, and parasite resistance. Thus, there is an existing need to develop new potent and safer leishmanicidal drugs. Considering the folkloric antiulcer and leishmanicidal use of the genus Berberis and its alkaloids, 5 reported alkaloids, namely berberine (1), palmatine (2), columbamine (3), 8-trichloromethyldihydroberberine (4), and jatrorrhizine (5), were isolated from the roots of Berberis glaucocarpa using classical (column and preparative chromatography) and modern isolation techniques (Sephadex LH-20). Their structures were elucidated and established from 1D and 2D spectroscopic data. The isolated alkaloids displayed excellent antileishmanial potential with IC50 values ranging from 1.50 to 2.56 µM: 1 (1.50 ± 0.53 µM), 2 (2.31 ± 0.37 µM), 3 (2.56 ± 0.48 µM), 4 (1.40 ± 0.90 µM), 5 (2.44 ± 1.34 µM). While the IC50 value for the standard drug (Amphotericin-B) was found to be 1.08 ± 0.95 µM. All of the isolated alkaloids displayed excellent antileishmanial potential as well as minimal cytotoxicity against THP-1 monocytic cells. Molecular docking analysis has revealed Leishmania N-myristoyl transferase, methionyl-tRNA synthetase, pteridine reductase 1, oligopeptidase B, tyrosyl-tRNA synthetase, and/or glycerol-3-phosphate dehydrogenase to be potential protein targets for the alkaloids.

DOCKING MOLECULAR E AVALIAÇÃO DA ATIVIDADE ANTILEISHMANIA IN VITRO DE UM COMPLEXO METÁLICO DE RUTÊNIO COM EPIISOPILOTURINA E ÓXIDO NÍTRICO

. A leishmaniose é uma doença infecciosa crônica, ocasionada em humanos pela picada de um mosquito infectado classificado como flebotomíneo. Após a infecção, os parasitas metacíclicos de Leishmania, irão interagir principalmente com células fagocitárias que irão infectar e atingir os tecidos e órgãos, a depender da espécie de Leishmania e da interação imune e genética do hospedeiro. A L. major possui características únicas dentro do seu gênero, que a diferencia da T. brucei e T. cruzi metabolicamente. Além de terem características próprias dentro do seu genoma, também apresentam organelas particulares tais como os glicossomos, que funcionam como reservatórios de enzimas glicolíticas e cinetoplasto. A Química Quântica Computacional disponibiliza de dados algoritmos que nos permite localizar e decifrar estado de transição em transformações químicas nos fornecendo informações relevantes sobre interações moleculares de um potencial biológico, onde os resultados quânticos são de 95% de confiança. O presente trabalho tem como objetivo realizar testes de docking molecular e avaliação da atividade antileishmania in vitro de um complexo metálico de rutênio com epiisopiloturina e óxido nítrico (Epiruno2). As estruturas das proteínas em 3D dos alvos de L. major foram obtidas no banco de dados Protein Data Bank (PDB) com os códigos 1e7w (Pteridine reductase), 5nzg (UDP-glucose Pyrophosphorylase), 5g20 (Glycyl Peptide N-tetradecanoyltransferase), 5c7p (Nucleoside diphosphate kinase), 1ezr (Nucleoside hydrolase), 1lml (Leishmanolysin), 4ef8 (Dihydroorotate Dehydrogenase), 5l42 (Pteridine reductase 1), 3ogz (UDP-sugar pyrophosphorylase), 3tue (Tryparedoxin peroxidase) e 2xe4 (Oligopeptidase B).

Análise e caracterização de um promissor alvo terapêutico identificado em Leishmania spp

A Leishmaniose é uma doença negligenciada causada por protozoários do gênero Leishmania spp., o qual atinge cerca de 1,6 milhão indivíduos a cada ano sendo que 500 mil se apresentam na forma visceral. No Brasil ocorrem cerca de 30.000 novos casos a cada ano. Além disso, o país é responsável por 90% dos casos notificados de Leishmaniose Visceral, sendo essa forma mais grave da doença. Aliado a esses fatos, o tratamento vigente se mostra ineficaz, contribuindo para o estabelecimento de cepas resistentes. Atualmente, o tratamento apresenta diversos efeitos colaterais e danos permanentes à saúde dos pacientes, tal fato têm contribuído para a procura de novos fármacos contra a leishmaniose. A enzima oligopeptidase B (OPB) tem sido estudada como possível alvo terapêutico no desenvolvimento de agentes antiparasitários. Desta forma, o objetivo desse trabalho é construir o modelo tridimensional da enzima Oligopeptidase B de diferentes espécies de Leishmania spp. e compara-las entre si. Para tal, foi utilizado o método de modelagem comparativa. Nesse método foi feita a construção dos modelos das espécies L. brasiliensis, L. donovani, L. infantum, L. mexicana e L. panamensis, utilizando o programa MODELLER. Uma vez com os modelos prontos foram realizados o processo de validação dos mesmos e subsequentemente caracterizados, o qual foi possível constatar um grau de semelhança promissor entre os modelos. Por fim, tais modelos foram submetidos ao método de análise por modos normais, os quais obtiveram um padrão de movimento semelhante, com isso foi possível constatar um movimento em uma região específica de uma alfa-hélice, levando consequentemente a tríade da enzima se mostrar exposta, podendo ser indicativo de um mecanismo de ação. Por fim, espera-se utilizar os modelos construídos para auxiliar no desenvolvimento de uma nova terapia promissora para o tratamento da leishmaniose.

Analysis and characterization of a promising therapeutic target identified in Leishmania spp

Leishmaniasis is a neglected disease caused by protozoa of the genus Leishmania spp., which affects about 1.6 million individuals each year and 500,000 present themselves in the visceral form. In Brazil there are about 30,000 new cases each year. In addition, the country is responsible for 90% of reported cases of Visceral Leishmaniasis, and this is more severe form of the disease. Allied to these facts, the current treatment is ineffective, contributing to the establishment of resistant strains. Currently, the treatment has several side effects and permanent damage to the health of patients, this fact has contributed to the search for new drugs against leishmaniasis. The enzyme oligopeptidase B (OPB) has been studied as a possible therapeutic target in the development of antiparasitic agents. Thus, the objective of this work is to construct the three-dimensional model of the enzyme Oligopeptidase B of different species of Leishmania spp. and compare them to each other. For this purpose, the comparative modeling method was used. In this method, the models of the species L. brasiliensis, L. donovani, L. infantum, L. mexicana and L. panamensis were constructed using the MODELLER program. Once the models were ready, the validation process was carried out and subsequently characterized, which was possible to verify a promising degree of similarity between the models. Finally, these models were submitted to the method of analysis by normal modes, which obtained a similar movement pattern, thus it was possible to verify a movement in a specific region of an alpha-helix, consequently leading to the triad of the enzyme being exposed, which may be indicative of a mechanism of action. Finally, it is expected to use the models built to assist in the development of a promising new therapy for the treatment of leishmaniasis.