RESUMEN
sp2-Iminosugar glycolipids (sp2-IGLs) represent a consolidated family of glycoconjugate mimetics encompassing a monosaccharide-like glycone moiety with a pseudoamide-type nitrogen replacing the endocyclic oxygen atom of carbohydrates and an axially-oriented lipid chain anchored at the pseudoanomeric position. The combination of these structural features makes them promising candidates for the treatment of a variety of conditions, spanning from cancer and inflammatory disorders to parasite infections. The exacerbated anomeric effect associated to the putative sp2-hybridized N-atom imparts chemical and enzymatic stability to sp2-IGLs and warrants total α-anomeric stereoselectivity in the key glycoconjugation step. A variety of O-, N-, C- and S-pseudoglycosides, differing in glycone configurational patterns and lipid nature, have been previously prepared and evaluated. Here we expand the chemical space of sp2-IGLs by reporting the synthesis of α-d-gluco-configured analogs with a bicyclic (5N,6O-oxomethylidene)nojirimycin (ONJ) core incorporating selenium at the glycosidic position. Structure-activity relationship studies in three different scenarios, namely cancer, Leishmaniasis and inflammation, convey that the therapeutic potential of the sp2-IGLs is highly dependent, not only on the length of the lipid chain (linear aliphatic C12 vs. C8), but also on the nature of the glycosidic atom (nitrogen vs. sulfur vs. selenium). The ensemble of results highlights the α-dodecylseleno-ONJ-glycoside as a promising multitarget drug candidate.
Asunto(s)
Antiinflamatorios/uso terapéutico , Antineoplásicos/uso terapéutico , Antiprotozoarios/uso terapéutico , Glucolípidos/uso terapéutico , Neoplasias/tratamiento farmacológico , Compuestos de Organoselenio/uso terapéutico , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Glucolípidos/síntesis química , Glucolípidos/química , Humanos , Inflamación/tratamiento farmacológico , Leishmaniasis/tratamiento farmacológico , Compuestos de Organoselenio/síntesis química , Compuestos de Organoselenio/químicaRESUMEN
The P4 family of P-type ATPases (P4-ATPases) plays an important role in maintaining phospholipid asymmetry in eukaryotic cell membranes. Leishmania miltefosine transporter (LMT) is a plasma membrane (PM) P4-ATPase that catalyses translocation into the parasite of the leishmanicidal drug miltefosine as well as phosphatidylcholine and phosphatidylethanolamine analogues. In the present study, we analysed the role, in LMT, of a series of highly conserved amino acids previously undescribed in the N-terminal region of P4-ATPases. Seven residues were identified and, according to an LMT structural model, five were located in the cytosolic N-terminal tail (Asn58, Ile60, Lys64, Tyr65 and Phe70) and the other two (Pro72 and Phe79) in the first transmembrane segment (TM1). Alanine-scanning mutagenesis analysis showed that N58A, Y65A and F79A mutations caused a considerable reduction in the LMT translocase activity. These mutations did not affect protein expression levels. We generated additional mutations in these three residues to assess the influence of the conservation degree on LMT translocase activity. Some of these mutations reduced expression levels without affecting the interaction between LMT and its CDC50 subunit, LRos3. Conserved and non-conserved mutations in the invariant residue Asn58 drastically reduced the translocase activity. Consequently, Asn58 may be necessary to achieve optimal catalytic LMT activity as previously described for the potentially equivalent Asn39 of the sarco/endoplasmic reticulum Ca2+-ATPase isoform 1a (SERCA1a). Additionally, conservation of a hydrophobic residue at position 79 is crucial for LMT stability.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Adenosina Trifosfatasas/genética , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Células Cultivadas , Secuencia Conservada/genética , Leishmania donovani , Leishmania infantum , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas/genética , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
The Leishmania LABCG2 transporter has a key role in the redox metabolism of these protozoan parasites. Recently, the involvement of LABCG2 in virulence, autophagy and oxidative stress has been described. Null mutant parasites for LABCG2 present an increase in the intracellular levels of glutathione (GSH) and trypanothione [T(SH)2]. On the other hand, parasites overexpressing LABCG2 transporter export non-protein thiols to the extracellular medium. To explore if LABCG2 may mediate an active transport of non-protein thiols, the effect of these molecules on ATPase activity of LABCG2 as well as the ability of LABCG2 to transport them was determined using a baculovirus-Sf9 insect cell system. Our results indicate that all thiols tested [GSH, T(SH)2] as well as their oxidized forms GSSG and TS2 (trypanothione disulfide) stimulate LABCG2-ATPase basal activity. We have measured the transport of [3H]-GSH in inside-out Sf9 cell membrane vesicles expressing LABCG2-GFP (green fluorescence protein), finding that LABCG2 was able to mediate a rapid and concentration-dependent uptake of [3H]-GSH in the presence of ATP. Finally, we have analyzed the ability of different thiol species to compete for this uptake, T(SH)2 and TS2 being the best competitors. The IC50 value for [3H]-GSH uptake in the presence of increasing concentrations of T(SH)2 was less than 100â µM, highlighting the affinity of this thiol for LABCG2. These results provide the first direct evidence that LABCG2 is an ABC transporter of reduced and oxidized non-protein thiols in Leishmania, suggesting that this transporter can play a role in the redox metabolism and related processes in this protozoan parasite.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Leishmania major/metabolismo , Proteínas Protozoarias/metabolismo , Espermidina/análogos & derivados , Transportadoras de Casetes de Unión a ATP/genética , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Transporte Biológico Activo , Membrana Celular/química , Membrana Celular/metabolismo , Clonación Molecular , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Leishmania major/genética , Oxidación-Reducción , Estrés Oxidativo , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Sf9 , Espermidina/metabolismo , SpodopteraRESUMEN
The unique stereoelectronic properties of sp2-iminosugars enable their participation in glycosylation reactions, thereby behaving as true carbohydrate chemical mimics. Among sp2-iminosugar conjugates, the sp2-iminosugar glycolipids (sp2-IGLs) have shown a variety of interesting pharmacological properties ranging from glycosidase inhibition to antiproliferative, antiparasitic, and anti-inflammatory activities. Developing strategies compatible with molecular diversity-oriented strategies for structure-activity relationship studies was therefore highly wanted. Here we show that a reaction sequence consisting in stereoselective C-allylation followed by thiol-ene "click" coupling provides a very convenient access to α-C-glycoside sp2-IGLs. Both the glycone moiety and the aglycone tail can be modified by using sp2-iminosugar precursors with different configurational profiles (d-gluco or d-galacto in this work) and varied thiols, as well as by oxidation of the sulfide adducts (to the corresponding sulfones in this work). A series of derivatives was prepared in this manner and their glycosidase inhibitory, antiproliferative and antileishmanial activities were evaluated in different settings. The results confirm that the inhibition of glycosidases, particularly α-glucosidase, and the antitumor/leishmanicidal activities are unrelated. The data are also consistent with the two later activities arising from the ability of the sp2-IGLs to interfere in the immune system response in a cell line and cell context dependent manner.
Asunto(s)
Química Clic , Glucolípidos/síntesis química , Glucolípidos/farmacología , Glicósidos/química , Iminoazúcares/química , Compuestos de Sulfhidrilo/química , Antiprotozoarios/síntesis química , Antiprotozoarios/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucolípidos/química , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/química , Humanos , Pruebas de Sensibilidad ParasitariaRESUMEN
We have identified and characterized ABCI3 as a new mitochondrial ABC transporter from Leishmania major Localization studies using confocal microscopy, a surface biotinylation assay, and trypsin digestion after digitonin permeabilization suggested that ABCI3 presents a dual localization in both mitochondria and the plasma membrane. From studies using parasites with a single knockout of ABCI3 (ABCI3+/-), we provide evidence that ABCI3 is directly involved in susceptibility to the trivalent form of antimony (SbIII) and metal ions. Attempts to obtain parasites with a double knockout of ABCI3 were unsuccessful, suggesting that ABCI3 could be an essential gene in L. majorABCI3+/- promastigotes were 5-fold more resistant to SbIII than the wild type, while ABCI3+/- amastigotes were approximately 2-fold more resistant to pentavalent antimony (SbV). This resistance phenotype was associated with decreased SbIII accumulation due to decreased SbIII uptake. ABCI3+/- parasites presented higher ATP levels and generated less mitochondrial superoxide after SbIII incubation. Finally, we observed that ABCI3+/- parasites showed a slightly higher infection capacity than wild-type and add-back ABCI3+/-::3×FABCI3 parasites; however, after 72 h the number of ABCI3+/- intracellular parasites per macrophage increased significantly. Our results show that ABCI3 is responsible for SbIII transport inside mitochondria, where it contributes to enhancement of the general toxic effects caused by SbIII To our knowledge, ABCI3 is the first ABC transporter which is involved in susceptibility toward antimony, conferring SbIII resistance to parasites when it is partially deleted.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Antimonio/metabolismo , Antiprotozoarios/farmacología , Leishmania major , Animales , Antimonio/farmacología , Antiprotozoarios/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Células Cultivadas , Resistencia a Medicamentos/genética , Técnicas de Inactivación de Genes , Leishmania major/efectos de los fármacos , Leishmania major/genética , Leishmania major/metabolismo , Macrófagos/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Mitocondrias/metabolismo , Pruebas de Sensibilidad Parasitaria , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
In 2014, a published report of the high-throughput screen of>42,000 kinase inhibitors from GlaxoSmithKline against T. brucei identified 797 potent and selective hits. From this rich data set, we selected NEU-0001101 (1) for hit-to-lead optimization. Through our preliminary compound synthesis and SAR studies, we have confirmed the previously reported activity of 1 in a T. brucei cell proliferation assay and have identified alternative groups to replace the pyridyl ring in 1. Pyrazole 24 achieves improvements in both potency and lipophilicity relative to 1, while also showing good in vitro metabolic stability. The SAR developed on 24 provides new directions for further optimization of this novel scaffold for anti-trypanosomal drug discovery.
Asunto(s)
Diseño de Fármacos , Pirazoles/síntesis química , Quinazolinonas/química , Tripanocidas/síntesis química , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Pirazoles/farmacología , Pirazoles/uso terapéutico , Quinazolinonas/síntesis química , Quinazolinonas/farmacología , Quinazolinonas/uso terapéutico , Relación Estructura-Actividad , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/tratamiento farmacológicoRESUMEN
Treatment for leishmaniasis, which is caused by Leishmania protozoan parasites, currently relies on a reduced arsenal of drugs. However, the significant increase in the incidence of drug therapeutic failure and the growing resistance to first-line drugs like antimonials in some areas of Northern India and Nepal limit the control of this parasitic disease. Understanding the molecular mechanisms of resistance in Leishmania is now a matter of urgency to optimize drugs used and to identify novel drug targets to block or reverse resistant mechanisms. Some members of the family of ATP-binding cassette (ABC) transporters in Leishmania have been associated with drug resistance. In this study, we have focused our interest to characterize LABCG2's involvement in drug resistance in Leishmania. Leishmania major parasites overexpressing the ABC protein transporter LABCG2 were generated in order to assess how LABCG2 is involved in drug resistance. Assays of susceptibility to different leishmanicidal agents were carried out. Analysis of the drug resistance profile revealed that Leishmania parasites overexpressing LABCG2 were resistant to antimony, as they demonstrated a reduced accumulation of Sb(III) due to an increase in drug efflux. Additionally, LABCG2 was able to transport thiols in the presence of Sb(III) Biotinylation assays using parasites expressing LABCG2 fused with an N-terminal green fluorescent protein tag revealed that LABCG2 is partially localized in the plasma membrane; this supports data from previous studies which suggested that LABCG2 is localized in intracellular vesicles that fuse with the plasma membrane during exocytosis. In conclusion, Leishmania LABCG2 probably confers antimony resistance by sequestering metal-thiol conjugates within vesicles and through further exocytosis by means of the parasite's flagellar pocket.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antimonio/farmacología , Antiprotozoarios/farmacología , Leishmania major/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Proteínas Protozoarias/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Resistencia a Medicamentos/genética , Leishmania major/genética , Pruebas de Sensibilidad Parasitaria , Proteínas Protozoarias/genéticaRESUMEN
Together with vector control, chemotherapy is an essential tool for the control of visceral leishmaniasis (VL), but its efficacy is jeopardized by growing resistance and treatment failure against first-line drugs. To delay the emergence of resistance, the use of drug combinations of existing antileishmanial agents has been tested systematically in clinical trials for the treatment of visceral leishmaniasis (VL). In vitro, Leishmania donovani promastigotes are able to develop experimental resistance to several combinations of different antileishmanial drugs after 10 weeks of drug pressure. Using an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics approach, we identified metabolic changes in lines that were experimentally resistant to drug combinations and their respective single-resistant lines. This highlighted both collective metabolic changes (found in all combination therapy-resistant [CTR] lines) and specific ones (found in certain CTR lines). We demonstrated that single-resistant and CTR parasite cell lines show distinct metabolic adaptations, which all converge on the same defensive mechanisms that were experimentally validated: protection against drug-induced and external oxidative stress and changes in membrane fluidity. The membrane fluidity changes were accompanied by changes in drug uptake only in the lines that were resistant against drug combinations with antimonials, and surprisingly, drug accumulation was higher in these lines. Together, these results highlight the importance and the central role of protection against oxidative stress in the different resistant lines. Ultimately, these phenotypic changes might interfere with the mode of action of all drugs that are currently used for the treatment of VL and should be taken into account in drug development.
Asunto(s)
Antiparasitarios/farmacología , Resistencia a Medicamentos/efectos de los fármacos , Leishmania donovani/efectos de los fármacos , Adaptación Fisiológica , Animales , Membrana Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , ADN Protozoario/genética , Combinación de Medicamentos , Resistencia a Medicamentos/genética , Leishmania donovani/genética , Leishmania donovani/metabolismo , Espectrometría de Masas , Fluidez de la Membrana/efectos de los fármacos , Metabolómica , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills different T. brucei spp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca(2+), and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significant in vitro activity against T. brucei.
Asunto(s)
Aminoquinolinas/farmacología , Antimaláricos/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Administración Oral , Aminoquinolinas/metabolismo , Antimaláricos/metabolismo , Humanos , Lisosomas/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Leishmaniasis is the protozoan disease second in importance for human health, superseded only by malaria; however, the options for chemotherapeutic treatment are increasingly limited due to drug resistance and toxicity. Under this perspective, a quest for new chemical compounds is urgently needed. An N-substituted 2-aminoalkan-1-ol scaffold has been shown to be a versatile scaffold for antiparasitic activity. Knowledge about its mechanism of action is still rather limited. In this work, we endeavored to define the leishmanicidal profile of such ß-amino alkanol derivatives using a set of 15 N-mono- and disubstituted surrogates, tested on Leishmania donovani promastigotes and intracellular amastigotes. The best compound (compound 5), 2-ethylaminododecan-1-ol, had a 50% effective concentration (EC50) of 0.3 µM and a selectivity index of 72 for infected THP-1 cells and was selected for further elucidation of its leishmanicidal mechanism. It induced fast depletion of intracellular ATP content in promastigotes in the absence of vital dye intracellular entry, ruling out plasma membrane permeabilization as its origin. Confocal and transmission electron microscopy analyses showed that compound 5 induced severe mitochondrial swelling and vesiculation. Polarographic analysis using an oxygen electrode demonstrated that complex II of the respiratory chain (succinate reductase) was strongly inhibited by compound 5, identifying this complex as one of the primary targets. Furthermore, for other ß-amino alkanols whose structures differed subtly from that of compound 5, plasma membrane permeabilization or interference with membrane traffic was also observed. In all, N-substituted ß-amino alkanols were shown as appealing leishmanicidal candidates deserving further exploration.
Asunto(s)
Antiprotozoarios/química , Antiprotozoarios/farmacología , Leishmania donovani/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Humanos , Leishmania donovani/ultraestructura , Estructura Molecular , Consumo de Oxígeno/efectos de los fármacosRESUMEN
Multidrug resistance (MDR) caused by the overexpression of ABC drug transporters is a major obstacle in clinical cancer chemotherapy and underlines the urgent need for the development of new, potent, and safe reversal agents. Toward this goal, reported herein are the structure elucidation and biological activity of nine new (1-9) and four known (10-13) dihydro-ß-agarofuran sesquiterpenes, isolated from the leaves of Celastrus vulcanicola, as reversers of MDR mediated by human P-glycoprotein expression. The structures of these compounds were elucidated by extensive NMR spectroscopic and mass spectrometric analysis, and their absolute configurations were determined by circular dichroism studies, chemical correlations (1a, 8a, and 8b), and biogenetic means. Four compounds from this series were discovered as potent chemosensitizers for MDR1-G185 NIH-3T3 murine cells (3, 4, 6, and 7), showing higher efficacies than the classical P-glycoprotein inhibitor verapamil, a first-generation chemosensitizer, when reversing resistance to daunomycin and vinblastine at the lowest concentration tested of 1 µM.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Celastrus/química , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Dicroismo Circular , Cristalografía por Rayos X , Daunorrubicina/farmacología , Resistencia a Múltiples Medicamentos , El Salvador , Humanos , Ratones , Conformación Molecular , Estructura Molecular , Células 3T3 NIH , Resonancia Magnética Nuclear Biomolecular , Hojas de la Planta/química , Sesquiterpenos/química , Vinblastina/farmacologíaRESUMEN
Cdc50 (cell-cycle control protein 50) is a family of conserved eukaryotic proteins that interact with P4-ATPases (phospholipid translocases). Cdc50 association is essential for the endoplasmic reticulum export of P4-ATPases and proper translocase activity. In the present study, we analysed the role of Leishmania infantum LiRos3, the Cdc50 subunit of the P4-ATPase MLF (miltefosine) transporter [LiMT (L. infantum MLF transporter)], on trafficking and complex functionality using site-directed mutagenesis and domain substitution. We identified 22 invariant residues in the Cdc50 proteins from L. infantum, human and yeast. Seven of these residues are found in the extracellular domain of LiRos3, the conservation of which is critical for ensuring that LiMT arrives at the plasma membrane. The substitution of other invariant residues affects complex trafficking to a lesser extent. Furthermore, invariant residues located in the N-terminal cytosolic domain play a role in the transport activity. Partial N-glycosylation of LiRos3 reduces MLF transport and total N-deglycosylation completely inhibits LiMT trafficking to the plasma membrane. One of the N-glycosylation residues is invariant along the Cdc50 family. The transmembrane and exoplasmic domains are not interchangeable with the other two L. infantum Cdc50 proteins to maintain LiMT interaction. Taken together, these findings indicate that both invariant and N-glycosylated residues of LiRos3 are implicated in LiMT trafficking and transport activity.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Secuencia Conservada/fisiología , Evolución Molecular , Proteínas de Transporte de Membrana/fisiología , Proteínas Protozoarias/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Espacio Extracelular/química , Glicosilación , Humanos , Leishmania infantum , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Conejos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The antileishmanial activity of a series of bis-pyridinium derivatives that are analogues of pentamidine have been investigated, and all compounds assayed were found to display activity against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania major, with 50% effective concentrations (EC50s) lower than 1 µM in most cases. The majority of compounds showed similar behavior in both Leishmania species, being slightly more active against L. major amastigotes. However, compound VGP-106 {1,1'-(biphenyl-4,4'-diylmethylene)bis[4-(4-bromo-N-methylanilino)pyridinium] dibromide} exhibited significantly higher activity against L. donovani amastigotes (EC50, 0.86 ± 0.46 µM) with a lower toxicity in THP-1 cells (EC50, 206.54 ± 9.89 µM). As such, VGP-106 was chosen as a representative compound to further elucidate the mode of action of this family of inhibitors in promastigote forms of L. donovani. We have determined that uptake of VGP-106 in Leishmania is a temperature-independent process, suggesting that the compound crosses the parasite membrane by diffusion. Transmission electron microscopy analysis showed a severe mitochondrial swelling in parasites treated with compound VGP-106, which induces hyperpolarization of the mitochondrial membrane potential and a significant decrease of intracellular free ATP levels due to the inhibition of ATP synthesis. Additionally, we have confirmed that VGP-106 induces mitochondrial ROS production and an increase in intracellular Ca(2+) levels. All these molecular events can activate the apoptotic process in Leishmania; however, propidium iodide assays gave no indication of DNA fragmentation. These results underline the potency of compound VGP-106, which may represent a new avenue for the development of novel antileishmanial compounds.
Asunto(s)
Leishmania donovani/efectos de los fármacos , Leishmania major/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Dilatación Mitocondrial/efectos de los fármacos , Pentamidina/farmacología , Adenosina Trifosfato/biosíntesis , Antiprotozoarios/farmacología , Transporte Biológico , Calcio/metabolismo , Línea Celular , Colina Quinasa/antagonistas & inhibidores , Humanos , Macrófagos/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria , Pentamidina/análogos & derivados , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVES: To identify reversal agents for the Leishmania ABCI4 transporter that confers resistance to antimony. METHODS: Selective ABCI4 inhibitors among a series of 15 flavonoid and trolox derivatives or analogues were investigated by evaluating their ability to reverse antimony resistance in Leishmania parasites overexpressing ABCI4. Among the compounds screened, N-ethyltrolox carboxamide (compound D2) produced the highest reversal activity. In order to optimize the activity of D2, we synthesized a series of 10 derivatives by condensation of various amines with trolox. RESULTS: Analysis of antimony resistance reversal activity showed that N-propyltrolox carboxamide (compound D4) was the most potent ABCI4 inhibitor, with reversal activity being maintained in the intracellular amastigote stage. In addition, trolox derivatives significantly reverted the resistance to zinc protoporphyrin. The mechanism of action of these active derivatives was found to be related to significant reversion of Sb(III) and zinc protoporphyrin accumulation and to a decrease in drug efflux. CONCLUSIONS: Our findings suggest that trolox derivatives D2 and D4 could be considered to be specific reversal agents targeting the Leishmania ABCI4 transporter. The structure-activity relationship obtained in the present study highlights the importance of the size and length of the alkyl substituent linked to trolox. Furthermore, the structural data obtained provide valuable information for the further development of new, even more specific and potent Leishmania ABCI4 reversal agents.
Asunto(s)
Antimonio/farmacología , Antiprotozoarios/aislamiento & purificación , Cromanos/aislamiento & purificación , Evaluación Preclínica de Medicamentos/métodos , Flavonoides/aislamiento & purificación , Leishmania/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Antiprotozoarios/química , Antiprotozoarios/farmacología , Cromanos/química , Cromanos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Flavonoides/química , Flavonoides/farmacología , Relación Estructura-ActividadRESUMEN
Recent efforts in the study of vector-borne parasitic diseases (VBPDs) have emphasized an increased consideration for preventing drug resistance and promoting the environmental safety of drugs, from the beginning of the drug discovery pipeline. The intensive use of the few available antileishmanial drugs has led to the spreading of hyper-resistant Leishmania infantum strains, resulting in a chronic burden of the disease. In the present work, we have investigated the biochemical mechanisms of resistance to antimonials, paromomycin, and miltefosine in three drug-resistant parasitic strains from human clinical isolates, using a whole-cell mass spectrometry proteomics approach. We identified 14 differentially expressed proteins that were validated with their transcripts. Next, we employed functional association networks to identify parasite-specific proteins as potential targets for novel drug discovery studies. We used SeqAPASS analysis to predict susceptibility based on the evolutionary conservation of protein drug targets across species. MATH-domain-containing protein, adenosine triphosphate (ATP)-binding cassette B2, histone H4, calpain-like cysteine peptidase, and trypanothione reductase emerged as top candidates. Overall, this work identifies new biological targets for designing drugs to prevent the development of Leishmania drug resistance, while aligning with One Health principles that emphasize the interconnected health of people, animals, and ecosystems.
RESUMEN
Human African trypanosomiasis is among the World Health Organization's designated neglected tropical diseases. Repurposing strategies are often employed in academic drug discovery programs due to financial limitations, and in this instance, we used human kinase inhibitor chemotypes to identify substituted 4-aminoazaindoles, exemplified by 1. Structure-activity and structure-property relationship analysis, informed by cheminformatics, identified 4s as a potent inhibitor of Trypanosoma brucei growth. While 4s appeared to be fast acting and cidal in the in vitro assays, it failed to cure a murine model of infection. Preliminary efforts to identify the potential mechanism of action of the series pointed to arginine kinase, though, as we demonstrate, this does not appear to be the sole target of our compounds. This comprehensive approach to drug discovery, encompassing cheminformatics, structure-potency and structure-property analysis, and pharmacophore identification, highlights our multipronged efforts to identify novel lead compounds for this deadly disease.
Asunto(s)
Indoles , Tripanocidas , Trypanosoma brucei brucei , Trypanosoma brucei brucei/efectos de los fármacos , Relación Estructura-Actividad , Animales , Tripanocidas/farmacología , Tripanocidas/química , Tripanocidas/síntesis química , Indoles/química , Indoles/farmacología , Indoles/síntesis química , Humanos , Ratones , Tripanosomiasis Africana/tratamiento farmacológico , Compuestos Aza/química , Compuestos Aza/farmacología , Compuestos Aza/síntesis química , Estructura Molecular , FarmacóforoRESUMEN
As the world is facing increasing difficulties to treat leishmaniasis with current therapies, deeper investigation into the molecular mechanisms responsible for both drug resistance and treatment failure (TF) is essential in drug discovery and development. So far, few available drugs cause severe side effects and have developed several resistance mechanisms. Drug resistance and TF parasite strains from clinical isolates may have acquired altered expression of proteins that characterize specific mechanisms leading to therapy inefficacy. This work aims to identify the biochemical pathways of THP-1 human monocytes infected by different Leishmania infantum clinical isolates from patients with either resistance or with TF outcome, using whole cell differential Mass Spectrometry proteomics. We have adopted network enrichment analysis to integrate the transcriptomics and the proteomic results of infected cells studies. Transferrin receptor C (TFRC) and nucleoside diphosphate kinase 3 (NDK3) were discovered as overexpressed proteins in THP-1 cells infected with paromomycin, antimony, and miltefosine resistant L. infantum lines. The overall achievements represent founding concepts to confirm new targets involved in the parasitic drug resistance and TF mechanisms, and to consider in perspective the importance of a dual host-guest pharmacological approach to treat the acute stage of the disease.
Asunto(s)
Antiprotozoarios , Leishmania infantum , Humanos , Antiprotozoarios/farmacología , Proteómica/métodos , Células THP-1 , Resistencia a Medicamentos , Espectrometría de MasasRESUMEN
The AT-rich mitochondrial DNA (kDNA) of trypanosomatid parasites is a target of DNA minor groove binders. We report the synthesis, antiprotozoal screening, and SAR studies of three series of analogues of the known antiprotozoal kDNA binder 2-((4-(4-((4,5-dihydro-1H-imidazol-3-ium-2-yl)amino)benzamido)phenyl)amino)-4,5-dihydro-1H-imidazol-3-ium (1a). Bis(2-aminoimidazolines) (1) and bis(2-aminobenzimidazoles) (2) showed micromolar range activity against Trypanosoma brucei, whereas bisarylimidamides (3) were submicromolar inhibitors of T. brucei, Trypanosoma cruzi, and Leishmania donovani. None of the compounds showed relevant activity against the urogenital, nonkinetoplastid parasite Trichomonas vaginalis. We show that series 1 and 3 bind strongly and selectively to the minor groove of AT DNA, whereas series 2 also binds by intercalation. The measured pKa indicated different ionization states at pH 7.4, which correlated with the DNA binding affinities (ΔTm) for series 2 and 3. Compound 3a, which was active and selective against the three parasites and displayed adequate metabolic stability, is a fine candidate for in vivo studies.
Asunto(s)
Antiprotozoarios , Benzamidas , Leishmania donovani , Parásitos , Trypanosoma brucei brucei , Trypanosoma cruzi , Animales , Antiprotozoarios/química , ADN/metabolismo , ADN de Cinetoplasto/metabolismo , Imidazoles/química , Imidazoles/farmacología , Leishmania donovani/metabolismo , Parásitos/efectos de los fármacos , Parásitos/metabolismo , Benzamidas/química , Benzamidas/farmacologíaRESUMEN
Neglected diseases caused by kinetoplastid parasites are a health burden in tropical and subtropical countries. The need to create safe and effective medicines to improve treatment remains a priority. Microbial natural products are a source of chemical diversity that provides a valuable approach for identifying new drug candidates. We recently reported the discovery and bioassay-guided isolation of a novel family of macrolides with antiplasmodial activity. The novel family of four potent antimalarial macrolides, strasseriolides A-D, was isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. In the present study, we analyze these strasseriolides for activity against kinetoplastid protozoan parasites, namely, Trypanosoma brucei brucei, Leishmania donovani and Trypanosoma cruzi. Compounds exhibited mostly low activities against T. b. brucei, yet notable growth inhibition and selectivity were observed for strasseriolides C and D in the clinically relevant intracellular T. cruzi and L. donovani amastigotes with EC50 values in the low micromolar range. Compound C is fast-acting and active against both intracellular and trypomastigote forms of T. cruzi. While cell cycle defects were not identified, prominent morphological changes were visualized by differential interference contrast microscopy and smaller and rounded parasites were visualized upon exposure to strasseriolide C. Moreover, compound C lowers parasitaemia in vivo in acute models of infection of Chagas disease. Hence, strasseriolide C is a novel natural product active against different forms of T. cruzi in vitro and in vivo. The study provides an avenue for blocking infection of new cells, a strategy that could additionally contribute to avoid treatment failure.
Asunto(s)
Enfermedad de Chagas , Parásitos , Trypanosoma brucei brucei , Trypanosoma cruzi , Animales , Enfermedad de Chagas/tratamiento farmacológico , Macrólidos/farmacologíaRESUMEN
[This corrects the article DOI: 10.3389/fmicb.2023.1149145.].