RESUMEN
Disruption of protein-protein interactions of essential oligomeric enzymes by small molecules represents a significant challenge. We recently reported some linear and cyclic peptides derived from an α-helical region present in the homodimeric interface of Leishmania infantum trypanothione reductase ( Li-TryR) that showed potent effects on both dimerization and redox activity of this essential enzyme. Here, we describe our first steps toward the design of nonpeptidic small-molecule Li-TryR dimerization disruptors using a proteomimetic approach. The pyrrolopyrimidine and the 5-6-5 imidazole-phenyl-thiazole α-helix-mimetic scaffolds were suitably decorated with substituents that could mimic three key residues (K, Q, and I) of the linear peptide prototype (PKIIQSVGIS-Nle-K-Nle). Extensive optimization of previously described synthetic methodologies was required. A library of 15 compounds bearing different hydrophobic alkyl and aromatic substituents was synthesized. The imidazole-phenyl-thiazole-based analogues outperformed the pyrrolopyrimidine-based derivatives in both inhibiting the enzyme and killing extracellular and intracellular parasites in cell culture. The most active imidazole-phenyl-thiazole compounds 3e and 3f inhibit Li-TryR and prevent growth of the parasites at low micromolar concentrations similar to those required by the peptide prototype. The intrinsic fluorescence of these compounds inside the parasites visually demonstrates their good permeability in comparison with previous peptide-based Li-TryR dimerization disruptors.
Asunto(s)
Imidazoles/farmacología , Leishmania infantum/efectos de los fármacos , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Multimerización de Proteína/efectos de los fármacos , Pirimidinas/farmacología , Pirroles/farmacología , Tiazoles/farmacología , Leishmania infantum/enzimología , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirroles/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Development is a well-defined stage-to-stage process that allows the coordination and maintenance of the structure and function of cells and their progenitors, in a complete organism embedded in an environment that, in turn, will shape cellular responses to external stimuli. Epigenetic mechanisms comprise a group of process that regulate genetic expression without changing the DNA sequence, and they contribute to the necessary plasticity of individuals to face a constantly changing medium. These mechanisms act in conjunction with genetic pools and their correct interactions will be crucial to zygote formation, embryo development, and brain tissue organization. In this work, we will summarize the main findings related to DNA methylation and histone modifications in embryonic stem cells and throughout early development phases. Furthermore, we will critically outline some key observations on how epigenetic mechanisms influence the rest of the developmental process and how long its footprint is extended from fecundation to adulthood.
Asunto(s)
Encéfalo/embriología , Encéfalo/fisiología , Desarrollo Embrionario/genética , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Neurogénesis/genética , Animales , Metilación de ADN , Exposición a Riesgos Ambientales , Histonas/metabolismo , Humanos , Modelos Animales , ARN no Traducido/metabolismo , Estrés FisiológicoRESUMEN
Trypanothione reductase (TryR) is a well-established target in the search for novel antitrypanosomal and antileishmanial agents. We have previously identified linear and lactam-bridged 13-residue peptides derived from an α-helical region making up part of the dimeric interface of Leishmania infantum TryR (Li-TryR) which prevent trypanothione reduction by disrupting enzyme dimerization. We now show that i,i + 4 side-chain cross-linking with an all-hydrocarbon staple stabilizes the helical structure of these peptides and significantly improves their resistance to protease cleavage relative to previous linear and cyclic lactam analogues. Interestingly, replacement of the amide bridge by the hydrocarbon staple at the same cyclization positions generates derivatives (2 and 3) that similarly inhibit oxidoreductase activity of the enzyme but unexpectedly stabilize the TryR homodimer. The most proteolytically stable peptide 2 covalently linked to oligoarginines displayed potent in vitro leishmanicidal activity against L. infantum parasites.
Asunto(s)
Antiprotozoarios/química , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Péptidos/farmacología , Estabilidad de Medicamentos , Hidrocarburos/química , Leishmania infantum/efectos de los fármacos , Péptidos/química , Conformación Proteica en Hélice alfa , Proteolisis , Proteínas Protozoarias/antagonistas & inhibidoresRESUMEN
The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/ß3-peptide foldamers of different length (from 9-mers to 13-mers) and different αâß substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/ß3-peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,ß-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/ß-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Leishmania infantum/enzimología , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Péptidos de Penetración Celular/química , ProteolisisRESUMEN
A series of 9-mer and 13-mer amide-bridged cyclic peptides derived from the linear prototype Ac-PKIIQSVGIS-Nle-K-Nle-NH2 (Toro et al. ChemBioChem2013) has been designed and synthesized by introduction of the lactam between amino acid side chains that are separated by one helical turn (i, i+4). All of these compounds were tested in vitro as both dimerization and enzyme inhibitors of Leishmania infantum trypanothione reductase (Li-TryR). Three of the 13-mer cyclic peptide derivatives (3, 4 and 6) inhibited the oxidoreductase activity of Li-TryR in the low micromolar range and they also disrupted enzyme dimerization. Cyclic analogues 3 and 4 were more resistant to proteases than was the linear prototype. Furthermore, the most potent TryR inhibitors in the linear and cyclic series displayed potent in vitro activity against Leishmania infantum upon conjugation with cationic cell-penetrating peptides. To date, these conjugated peptides can be considered the first example of TryR dimerization inhibitors that are active in cell culture.
Asunto(s)
Antiprotozoarios/farmacología , Inhibidores Enzimáticos/farmacología , Leishmania infantum/efectos de los fármacos , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Péptidos/farmacología , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dimerización , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Leishmania infantum/citología , Leishmania infantum/metabolismo , Simulación de Dinámica Molecular , Estructura Molecular , NADH NADPH Oxidorreductasas/metabolismo , Péptidos/síntesis química , Péptidos/química , Relación Estructura-ActividadRESUMEN
EndoG, a member of the DNA/RNA non-specific ßßα-metal family of nucleases, has been demonstrated to be present in many organisms, including Trypanosomatids. This nuclease participates in the apoptotic program in these parasites by migrating from the mitochondrion to the nucleus, where it takes part in the degradation of genomic DNA that characterizes this process. We now demonstrate that Leishmania infantum EndoG (LiEndoG) is an endo-exonuclease that has a preferential 5' exonuclease activity on linear DNA. Regardless of its role during apoptotic cell death, this enzyme seems to be necessary during normal development of the parasites as indicated by the reduced growth rates observed in LiEndoG hemi-knockouts and their poor infectivity in differentiated THP-1 cells. The pro-life role of this protein is also corroborated by the higher survival rates of parasites that over-express this protein after treatment with the LiEndoG inhibitor Lei49. Taken together, our results demonstrate that this enzyme plays essential roles in both survival and death of Leishmania parasites.
Asunto(s)
Apoptosis/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Exonucleasas/metabolismo , Leishmania infantum/metabolismo , Mitocondrias/metabolismo , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Exonucleasas/genética , Leishmania infantum/genética , Mitocondrias/genéticaRESUMEN
Diselenide and sulfonamide derivatives have recently attracted considerable interest as leishmanicidal agents in drug discovery. In this study, a novel series of sixteen hybrid selenosulfonamides has been synthesized and screened for their in vitro activity against Leishmania infantum intracellular amastigotes and THP-1 cells. These assays revealed that most of the compounds exhibited antileishmanial activity in the low micromolar range and led us to identify three lead compounds (derivatives 2, 7 and 14) with IC50 values ranging from 0.83 to 1.47 µM and selectivity indexes (SI) over 17, much higher than those observed for the reference drugs miltefosine and edelfosine. When evaluated against intracellular amastigotes, hybrid compound 7 emerged as the most active compound (IC50 = 2.8 µM), showing higher activity and much less toxicity against THP-1 cells than edelfosine. These compounds could potentially serve as templates for future drug-optimization and drug-development efforts for their use as therapeutic agents in developing countries.