RESUMO
CONTEXT: Malaria remains a significant global health challenge with emerging resistance to current treatments. Plasmodium falciparum glutathione reductase (PfGR) plays a critical role in the defense mechanisms of malaria parasites against oxidative stress. In this study, we investigate the potential of targeting PfGR with conventional antimalarials and dual drugs combining aminoquinoline derivatives with GR inhibitors, which reveal promising interactions between PfGR and studied drugs. The naphthoquinone Atovaquone demonstrated particularly high affinity and potential dual-mode binding with the enzyme active site and cavity. Furthermore, dual drugs exhibit enhanced binding affinity, suggesting their efficacy in inhibiting PfGR, where the aliphatic ester bond (linker) is essential for effective binding with the enzyme's active site. Overall, this research provides important insights into the interactions between antimalarial agents and PfGR and encourages further exploration of its role in the mechanisms of action of antimalarials, including dual drugs, to enhance antiparasitic efficacy. METHODS: The drugs were tested as PfGR potential inhibitors via molecular docking on AutoDock 4, which was performed based on the preoptimized structures in HF/3-21G-PCM level of theory on ORCA 5. Drug-receptor systems with the most promising binding affinities were then studied with a molecular dynamic's simulation on AMBER 16. The molecular dynamics simulations were performed with a 100 ns NPT ensemble employing GAFF2 forcefield in the temperature of 310 K, integration time step of 2 fs, and non-bond cutoff distance of 6.0 Å.
Assuntos
Antimaláricos , Glutationa Redutase , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Plasmodium falciparum , Antimaláricos/química , Antimaláricos/farmacologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/química , Glutationa Redutase/metabolismo , Ligação Proteica , Domínio Catalítico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , HumanosRESUMO
Falcipain-2 (FP-2) is a Plasmodium falciparum hemoglobinase widely targeted in the search for antimalarials. FP-2 can be allosterically modulated by various noncompetitive inhibitors that have been serendipitously identified. Moreover, the crystal structures of two inhibitors bound to an allosteric site, termed site 6, of the homolog enzyme human cathepsin K (hCatK) suggest that the equivalent region in FP-2 might play a similar role. Here, we conduct the rational identification of FP-2 inhibitors through virtual screenings (VS) of compounds into several pocket-like conformations of site 6, sampled during molecular dynamics (MD) simulations of the free enzyme. Two noncompetitive inhibitors, ZINC03225317 and ZINC72290660, were confirmed using in vitro enzymatic assays and their poses into site 6 led to calculated binding free energies matching the experimental ones. Our results provide strong evidence about the allosteric inhibition of FP-2 through binding of small molecules to site 6, thus opening the way toward the discovery of new inhibitors against this enzyme.
Assuntos
Antimaláricos/farmacologia , Simulação por Computador , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Sítio Alostérico , Antimaláricos/química , Inibidores de Cisteína Proteinase/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Plasmodium falciparum/enzimologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Loop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. To improve malaria diagnosis, new colorimetric LAMP tests were developed using multicopy targets for Plasmodium vivax and Plasmodium falciparum detection. METHODS: The cytochrome oxidase I (COX1) mitochondrial gene and the non-coding sequence Pvr47 for P. vivax, and the sub-telomeric sequence of erythrocyte membrane protein 1 (EMP1) and the non-coding sequence Pfr364 for P. falciparum were targeted to design new LAMP primers. The limit of detection (LOD) of each colorimetric LAMP was established and assessed with DNA extracted by mini spin column kit and the Boil & Spin method from 28 microscopy infections, 101 malaria submicroscopic infections detected by real-time PCR only, and 183 negatives infections by both microscopy and PCR. RESULTS: The LODs for the colorimetric LAMPs were estimated between 2.4 to 3.7 parasites/µL of whole blood. For P. vivax detection, the colorimetric LAMP using the COX1 target showed a better performance than the Pvr47 target, whereas the Pfr364 target was the most specific for P. falciparum detection. All microscopic infections of P. vivax were detected by PvCOX1-LAMP using the mini spin column kit DNA extraction method and 81% (17/21) were detected using Boil & Spin sample preparation. Moreover, all microscopic infections of P. falciparum were detected by Pfr364-LAMP using both sample preparation methods. In total, PvCOX1-LAMP and Pfr364-LAMP detected 80.2% (81 samples) of the submicroscopic infections using the DNA extraction method by mini spin column kit, while 36.6% (37 samples) were detected using the Boil & Spin sample preparation method. CONCLUSION: The colorimetric LAMPs with multicopy targets using the COX1 target for P. vivax and the Pfr364 for P. falciparum have a high potential to improve POC malaria diagnosis detecting a greater number of submicroscopic Plasmodium infections.
Assuntos
Colorimetria/métodos , Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/análise , Plasmodium falciparum/enzimologia , Plasmodium vivax/enzimologia , Proteínas de Protozoários/análiseRESUMO
In this study, eight natural isocoumarins (1-8) were isolated from a marine-derived Exserohilum sp. fungus. To explore their structure-activity relationship and discover potent antimalarial leads, a small library of 22 new derivatives (1a-1n, 2a, 3a-3c, 4a-4c, and 7a) were semisynthesized by varying the substituents of the aromatic ring and the aliphatic side chains. The natural compound (1) and three semisynthetic derivatives (1d, 1n, and 2a), possessing an all-cis stereochemistry, exhibited strong antiplasmodial activity with IC50 values of 1.1, 0.8, 0.4, and 2.6 µM, respectively. Mechanism studies show that 1n inhibits hemozoin polymerization and decreases the mitochondrial membrane potential but also inhibits P. falciparum DNA gyrase. 1n not only combines different mechanisms of action but also exhibits a high therapeutic index (CC50/IC50 = 675), high selectivity, and a notable drug-like profile.
Assuntos
Antimaláricos/farmacologia , Ascomicetos/química , Isocumarinas/farmacologia , Animais , Antozoários/microbiologia , Antimaláricos/síntese química , Organismos Aquáticos/química , China , Chlorocebus aethiops , DNA Girase , Hemeproteínas , Isocumarinas/síntese química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estrutura Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/farmacologia , Células VeroRESUMO
Melatonin and its indoles derivatives are central in the synchronization of malaria parasites. In this research, we discovered that melatonin is unable to increase the parasitemia in the human malaria Plasmodium falciparum that lacks the kinase PfeIK1. The PfeIK1 knockout strain is a valuable tool in the screening of indol-related compound that blocks the melatonin effect in wild-type (WT) parasite development. The assays were performed by using flow cytometry with simultaneous labeling for mitochondria viability with MitoTracker Deep Red and nucleus staining with SYBR Green. We found that Melatotosil leads to an increase in parasitemia in P. falciparum and blocks melatonin effect in the WT parasite. Using microscopy imaging system, we found that Melatotosil at 500 nM is able to induce cytosolic calcium rise in transgenic PfGCaMP3 parasites. On the contrary, the compound Triptiofen blocks P. falciparum cell cycle with IC50 9.76 µM ± 0.6, inhibits melatonin action, and does not lead to a cytosolic calcium rise in PfGCaMP3 parasites. We also found that the synthetic indol-related compounds arrested parasite cycle for PfeIK1 knockout and (WT) P. falciparum (3D7) in 72 hours culture assays with the IC50 values slighting lower for the WT strain. We concluded that the kinase PfeIK1 is central for melatonin downstream signaling pathways involved in parasite cell cycle progression. More importantly, the indol-related compounds block its cycle as an upstream essential mechanism for parasite survival. Our data clearly show that this class of compounds emerge as an alternative for the problem of resistance with the classical antimalarials.
Assuntos
Antimaláricos/farmacologia , Ciclo Celular , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/antagonistas & inibidores , Antimaláricos/química , Humanos , Malária Falciparum/tratamento farmacológico , Melatonina , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Malaria, caused by protozoa of the genus Plasmodium, is a disease that infects hundreds of millions of people annually, causing an enormous social burden in many developing countries. Since current antimalarial drugs are starting to face resistance by the parasite, the development of new therapeutic options has been prompted. The enzyme Plasmodium falciparum enoyl-ACP reductase (PfENR) has a determinant role in the fatty acid biosynthesis of this parasite and is absent in humans, making it an ideal target for new antimalarial drugs. In this sense, the present study aimed at evaluating the in silico binding affinity of natural and synthetic amides through molecular docking, in addition to their in vitro activity against P. falciparum by means of the SYBR Green Fluorescence Assay. The in vitro results revealed that the natural amide piplartine (1a) presented partial antiplasmodial activity (20.54 µM), whereas its synthetic derivatives (1m-IC50 104.45 µM), (1b, 1g, 1k, and 14f) and the natural amide piperine (18a) were shown to be inactive (IC50 > 200 µM). The in silico physicochemical analyses demonstrated that compounds 1m and 14f violated the Lipinski's rule of five. The in silico analyses showed that 14f presented the best binding affinity (- 13.047 kcal/mol) to PfENR and was also superior to the reference inhibitor triclosan (- 7.806 kcal/mol). In conclusion, we found that the structural modifications in 1a caused a significant decrease in antiplasmodial activity. Therefore, new modifications are encouraged in order to improve the activity observed.
Assuntos
Amidas/farmacologia , Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Amidas/química , Animais , Chlorocebus aethiops , Simulação por Computador , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Células Hep G2 , Humanos , Malária Falciparum , Simulação de Acoplamento Molecular , Piper nigrum , Plasmodium falciparum/enzimologia , Triclosan/farmacologia , Células VeroRESUMO
Malaria is a tropical disease that kills about half a million people around the world annually. Enzymatic reactions within pyrimidine biosynthesis have been proven to be essential for Plasmodium proliferation. Here we report on the essentiality of the second enzymatic step of the pyrimidine biosynthesis pathway, catalyzed by aspartate transcarbamoylase (ATC). Crystallization experiments using a double mutant ofPlasmodium falciparum ATC (PfATC) revealed the importance of the mutated residues for enzyme catalysis. Subsequently, this mutant was employed in protein interference assays (PIAs), which resulted in inhibition of parasite proliferation when parasites transfected with the double mutant were cultivated in medium lacking an excess of nutrients, including aspartate. Addition of 5 or 10 mg/L of aspartate to the minimal medium restored the parasites' normal growth rate. In vitro and whole-cell assays in the presence of the compound Torin 2 showed inhibition of specific activity and parasite growth, respectively. In silico analyses revealed the potential binding mode of Torin 2 to PfATC. Furthermore, a transgenic ATC-overexpressing cell line exhibited a 10-fold increased tolerance to Torin 2 compared with control cultures. Taken together, our results confirm the antimalarial activity of Torin 2, suggesting PfATC as a target of this drug and a promising target for the development of novel antimalarials.
Assuntos
Antimaláricos , Aspartato Carbamoiltransferase/genética , Naftiridinas/farmacologia , Plasmodium falciparum , Proteínas de Protozoários/genética , Antimaláricos/farmacologia , Ácido Aspártico , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genéticaRESUMO
Falcipain-2 (FP-2) is a Plasmodium falciparum cysteine protease that has been extensively targeted to identify novel antimalarials. Remarkably, previous reports have shown that FP-2 can be allosterically modulated and, for a particular noncompetitive chalcone inhibitor, the existing lines of experimental evidence can guide the prediction of its unknown binding mode to the enzyme in a reliable fashion. In this work, we propose a structure of FP-2 in complex with the aforementioned compound that fulfills all of the experimental data, by employing a combination of molecular modeling tools, such as pocket volume measurements, docking, molecular dynamics (MD) simulations, and free energy calculations. Our results show that the studied inhibitor binds a transient pocket occluded in all of the available FP-2 crystal structures and lying in a region previously characterized as a potential allosteric site in related cysteine proteases. In addition, we detected in silico the occurrence of significant community reorganization in FP-2, increased signal transmission between the allosteric pocket and the active site, and change in loop motions and residue pKa values upon the compound binding, thus providing insight into the uncharacterized allosteric mechanism. Overall, this study yields valuable predictions for the design of novel allosteric inhibitors against FP-2 and other cysteine proteases.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Inibidores Enzimáticos/farmacologia , Plasmodium falciparum/enzimologia , Trypanosoma cruzi/enzimologia , Sítios de Ligação/efeitos dos fármacos , Cisteína Endopeptidases/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , TermodinâmicaRESUMO
Even with access to sufficient nutrients and atmosphere, Plasmodium falciparum can barely be cultured at maximum growth capacity in vitro conditions. Because of this behavior, it has been suggested that P. falciparum has self-regulatory mechanisms in response to density stress. Only recently has this process begun to be acknowledged and characteristics of a programmed cell death been assigned to the parasite at high parasitaemia in vitro cultures. In searching for death signals within the parasite community, we have found that extracellular vesicles (EVs) of P. falciparum from high parasitaemia cultures are able to induce programmed cell death processes in the population. A comparative proteomic analysis of EVs from low (EVL) and high (EVH) parasitaemia cultures was conducted, pointing to lactate dehydrogenase from P. falciparum (PfLDH) as the only parasite protein overexpressed in the later. Although the major function of P. falciparum lactate dehydrogenase (PfLDH) is the conversion of pyruvate to lactate, a key process in the production of energy in most living organisms, we investigated its possible role in the mechanism of parasite density control by intercellular signaling, given that PfLDH had already been listed as a component of extracellular vesicles of P. falciparum. In this study we present evidence of the EV-associated PfLDH regulation of parasite population by inducing apoptosis in highly parasitized cultures.
Assuntos
Apoptose , Vesículas Extracelulares/enzimologia , L-Lactato Desidrogenase/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , HumanosRESUMO
BACKGROUND: Nowadays, malaria is still one of the most important and lethal diseases worldwide, causing 445,000 deaths in a year. Due to the actual treatment resistance, there is an emergency to find new drugs. OBJECTIVE: The aim of this work was to find potential inhibitors of phosphoglycerate mutase 1 from P. falciparum. RESULTS: Through virtual screening of a chemical library of 15,123 small molecules, analyzed by two programs, four potential inhibitors of phosphoglycerate mutase 1 from P. falciparum were found: ZINC64219552, ZINC39095354, ZINC04593310, and ZINC04343691; their binding energies in SP mode were -7.3, -7.41, -7.4, and -7.18 kcal/mol respectively. Molecular dynamic analysis revealed that these molecules interact with residues important for enzyme catalysis and molecule ZINC04343691 provoked the highest structural changes. Physiochemical and toxicological profiles evaluation of these inhibitors with ADME-Tox method suggested that they can be considered as potential drugs. Furthermore, analysis of human PGAM-B suggested that these molecules could be selective for the parasitic enzyme. CONCLUSION: The compounds reported here are the first selective potential inhibitors of phosphoglycerate mutase 1 from P. falciparum, and can serve as a starting point in the search of a new chemotherapy against malaria.