Theoretical and Experimental Study of New Dihydroorotate Dehydrogenase and Tryparedoxin Peroxidase Inhibitors: One More Step in the Study of Leishmaniasis Infection

In this study, the viability of new dihydroorotate dehydrogenase and tryparedoxin peroxidase inhibitors is reported. In vitro antileishmanial activity was evaluated using a Leishmania (V) panamensis strain, and the cytotoxicity of the compounds was assessed using U-937 cells. The in vivo therapeutic response was evaluated in golden hamsters (Mesocricetus auratus) experimentally infected with L. (V) panamensis and treated with a 1% topical formulation of compounds 4a–f. On the other hand, in silico studies considering the synthesized compounds were also carried out. All of the compounds showed promising in vitro activity, with mean EC50 effective concentration values ​​ranging from 3.8 µM to 19.3 µM. Likewise, treatment with compounds 4a–f produced improvement in most of the hamsters and cured some; in particular, those treated with compounds 4b, 4c, 4d, and 4f reacted the best. Molecular dynamics (MD) simulations, computational docking, and MM/GBSA studies indicate the promising bioavailability and absorption characteristics of the studied compounds, which are expected to be orally active. In addition, the studied 2-arylquinolines are absorbable at the blood–brain barrier, but not in the gastrointestinal tract. Finally, ADMET properties suggest that these molecules can be safely used as leishmaniasis inhibitors.

Chalcones identify cTXNPx as a potential antileishmanial drug target

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2’,6’-dihydroxy-4’-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2’,4’,6’- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

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).

Serine Proteases Profiles of Leishmania (Viannia) Braziliensis Clinical Isolates With Distinct Susceptibilities to Antimony

Glucantime® (SbV) is the first-line treatment against leishmaniasis in South America. Its effectiveness has been associated with modulation of the parasite detoxification system that, in turn, is related to serine proteases such as subtilisins. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders - R) and relapse or therapeutic failure (Non-responders - NR) were used. The parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity – measured with z-FR-AMC as substrate and specific inhibitors – and expression of subtilisins and tryparedoxin-peroxidase (TXNPx). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. TLCK inhibited almost 100 % of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70 %, and PMSF showed lower inhibition of specific isolates. Principal component and clustering analysis yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, transcripts of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) were detected in promastigotes and axenic amastigotes from both groups. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.

A Computational Molecular Docking Studies on the Tryparedoxin Peroxidase of Leishmania donovani responsible for visceral leishmaniasis in human

Objective: Hydroperoxide metabolism involving trypanothione, key for the survival of Leishmania, is a validated target for rational drug design. In this study, we aim in silico drug design by targeting tryparedoxin peroxidase (2-Cysperoxiredoxin type) from Leishmania donovani (LdTXNPx) using clioquinol, nelfinavir, and strychnobiflavone as mother compound. Clioquinol, nelfinavir are known for their anti-leishmanial activity and strychnobiflavone showed antileishmanial activity against Leishmania amazonensis and Leishmania infantum amastigotes and promastigotes recently Background: Visceral leishmaniasis, the most lethal form of Leishmaniasis, is caused by Leishmania donovani in the Indian subcontinent and East Africa. Current therapeutics for the disease are associated with a risk of high toxicity and development of drug-resistant strains. Thus, the discovery of potential targets, successful inhibitors and improved drug distribution mechanisms for leishmaniasis diagnosis has become a focus Methods: On this basis, we constructed protein structure using homology modeling, molecular docking of protein with potential drug candidates, interaction analysis and pharmacophore analysis conducted in this study Results: We have revealed two compounds i.e. Nelfinavir mesylate and strychnobiflavone which have desired characteristics in the future drugs for Visceral leishmaniasis Conclusion: Consistently in the future, we will ratify the efficacy of these compounds, essential animal and clinical trials are needed to be performed. We believe that our present study will help to find efficient and effective therapy for treating Visceral leishmaniasis in humans

Serine Proteinases in Leishmania (Viannia) braziliensis Promastigotes Have Distinct Subcellular Distributions and Expression

Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min−1 mg of protein−1 and Suc-AFK-AMC = 252 ± 20 µmol.min−1 mg of protein−1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min−1 mg of protein−1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min−1 mg of protein−1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.

Tryparedoxin peroxidase-deficiency commits trypanosomes to ferroptosis-type cell death

Tryparedoxin peroxidases, distant relatives of glutathione peroxidase 4 in higher eukaryotes, are responsible for the detoxification of lipid-derived hydroperoxides in African trypanosomes. The lethal phenotype of procyclic Trypanosoma brucei that lack the enzymes fulfils all criteria defining a form of regulated cell death termed ferroptosis. Viability of the parasites is preserved by α-tocopherol, ferrostatin-1, liproxstatin-1 and deferoxamine. Without protecting agent, the cells display, primarily mitochondrial, lipid peroxidation, loss of the mitochondrial membrane potential and ATP depletion. Sensors for mitochondrial oxidants and chelatable iron as well as overexpression of a mitochondrial iron-superoxide dismutase attenuate the cell death. Electron microscopy revealed mitochondrial matrix condensation and enlarged cristae. The peroxidase-deficient parasites are subject to lethal iron-induced lipid peroxidation that probably originates at the inner mitochondrial membrane. Taken together, ferroptosis is an ancient cell death program that can occur at individual subcellular membranes and is counterbalanced by evolutionary distant thiol peroxidases.