RESUMEN
BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 µM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 µM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.
Asunto(s)
Enfermedad de Chagas , Tripanocidas , Trypanosoma cruzi , Humanos , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/farmacología , Leucil Aminopeptidasa/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Descubrimiento de Drogas , Antiparasitarios/uso terapéutico , Tripanocidas/uso terapéuticoRESUMEN
Parasite M17 leucine aminopeptidases (LAPs) have been associated with critical roles in different key functions such as the nutrition, migration, and invasion of the natural host. Native or recombinant LAP used as a vaccine antigen has proved effective to elicit protection against Fasciola hepatica infection in sheep, pointing to potential vaccine candidates against fascioliasis in ruminant species. Previously, the FhLAP1, abundantly secreted in vitro by the mature adult parasite was used as a vaccine antigen obtaining promising protection results against F. hepatica challenge in small ruminants. Here, we report the biochemical characterization of a second recombinant LAP (FhLAP2) associated with the juvenile stage of F. hepatica. FhLAP2 showed aminopeptidase activity using different synthetic substrates, including leucine, arginine, and methionine and was increased in the presence of Mn+ 2 and Mg+ 2. The activity was inhibited by bestatin, 1,10-phenanthroline, and EDTA, specific inhibitors of aminopeptidase and/or metalloproteases. Finally, the recombinant FhLAP2 functional form was tested in combination with Freund's incomplete adjuvant in an immunization trial in mice followed by an experimental challenge with F. hepatica metacercariae. The immunization with FhLAP2/FIA resulted in a significant reduction of parasite recovery compared to control groups. The immunized group elicited total specific IgG and subclasses IgG1 and IgG2 antibody responses. This study highlights the potential of a new candidate vaccine formulation with potential applications in natural ruminant hosts, especially those targeting the juvenile stage.
Asunto(s)
Fasciola hepatica , Fascioliasis , Enfermedades de las Ovejas , Vacunas , Ovinos , Ratones , Animales , Fascioliasis/prevención & control , Fascioliasis/veterinaria , Leucil Aminopeptidasa/química , Leucina , Anticuerpos Antihelmínticos , Enfermedades de las Ovejas/parasitologíaRESUMEN
Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.
Asunto(s)
Aminopeptidasas , Leucil Aminopeptidasa , Humanos , Aminopeptidasas/química , Aminopeptidasas/metabolismo , Leucil Aminopeptidasa/química , Péptidos/química , Antígenos CD13RESUMEN
The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appKM = 74 µM, appkcat = 4.4 s-1). The optimal temperature is 50 °C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a Ki value of 881 nM. The enzyme is a good target for inhibitor identification.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Leucil Aminopeptidasa/biosíntesis , Proteínas Protozoarias/biosíntesis , Trypanosoma cruzi/enzimología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/microbiología , Concentración de Iones de Hidrógeno , Cinética , Leucina/análogos & derivados , Leucina/química , Leucil Aminopeptidasa/antagonistas & inhibidores , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/aislamiento & purificación , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , TemperaturaRESUMEN
Leucine aminopeptidase from Vibrio proteolyticus is a broad specificity N-terminal aminopeptidase that is widely used in pharmaceutical processes where the removal of N-terminal residues in recombinant proteins is required. We previously reported the expression of a heterologous construction of the mature protein fused to a 6-histidine tag that presents a reasonable refolding rate for its use at industrial level. Here, we investigate this recombinant leucine aminopeptidase (rLAP) to explain the gain of activity observed when incubated at 37 °C after its production. Unfolding transitions of rLAP as a function of urea concentration were monitored by circular dichroism (CD) and fluorescence (FL) spectroscopy exhibiting single transitions by both techniques. Free energy change for unfolding measured by CD and FL spectroscopy are 2.8 ± 0.4 and 3.7 ± 0.4 kcal mol(-1), respectively. Thermal stability conformation of rLAP is 2.6 ± 0.1 and 6.1 kcal mol(-1) for CD and Nano-Differential Scanning Calorimetry (Nano-DSC), respectively. Enzyme activity was assessed with L-leucine-p-nitroanilide (L-pNA) as substrate. The catalytic efficiency was 3.87 ± 0.10 min(-1) µM(-1) at 37 °C and pH 8.0. Kinetic and conformation studies show differences between the enzyme native and rLAP; however rLAP is selective and specific to remove N-terminal groups from amino acids.
Asunto(s)
Leucil Aminopeptidasa/química , Proteínas Recombinantes/química , Activación Enzimática , Estabilidad de Enzimas , Cinética , Leucil Aminopeptidasa/metabolismo , Conformación Proteica , Replegamiento Proteico/efectos de los fármacos , Desplegamiento Proteico/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Termodinámica , Urea/farmacología , Vibrio/enzimologíaRESUMEN
BACKGROUND: Pathogens depend on peptidase activities to accomplish many physiological processes, including interaction with their hosts, highlighting parasitic peptidases as potential drug targets. In this study, a major leucyl aminopeptidolytic activity was identified in Trypanosoma cruzi, the aetiological agent of Chagas disease. RESULTS: The enzyme was isolated from epimastigote forms of the parasite by a two-step chromatographic procedure and associated with a single 330-kDa homohexameric protein as determined by sedimentation velocity and light scattering experiments. Peptide mass fingerprinting identified the enzyme as the predicted T. cruzi aminopeptidase EAN97960. Molecular and enzymatic analysis indicated that this leucyl aminopeptidase of T. cruzi (LAPTc) belongs to the peptidase family M17 or leucyl aminopeptidase family. LAPTc has a strong dependence on neutral pH, is mesophilic and retains its oligomeric form up to 80°C. Conversely, its recombinant form is thermophilic and requires alkaline pH. CONCLUSIONS: LAPTc is a 330-kDa homohexameric metalloaminopeptidase expressed by all T. cruzi forms and mediates the major parasite leucyl aminopeptidolytic activity. Since biosynthetic pathways for essential amino acids, including leucine, are lacking in T. cruzi, LAPTc could have a function in nutritional supply.
Asunto(s)
Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/metabolismo , Multimerización de Proteína , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Citoplasma/metabolismo , Descubrimiento de Drogas , Hidrólisis , Leucil Aminopeptidasa/clasificación , Leucil Aminopeptidasa/aislamiento & purificación , Datos de Secuencia Molecular , Filogenia , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Trypanosoma cruzi/citología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
A new leucyl aminopeptidase activity has been identified in the fission yeast Schizosaccharomyces pombe. The enzyme, which has been purified and named leucyl aminopeptidase yspII (LAP yspII), had a molecular mass of 320 and 54 kDa by gel filtration and SDS/PAGE, respectively, suggesting a homohexameric structure. The enzyme cleaved synthetic aminoacyl-4-nitroanilides at an optimum of pH 8.5, and preferred leucine and methionine as N-terminal amino acids. A clear dependence on Mn2+ concentration for activity was found, and an apparent association constant of 0.33 mM was calculated for the metal ion. Bestatin behaved as a competitive inhibitor of LAP yspII (K(i) = 0.14 microM), while chelating agents such as chloroquine, EDTA and 1,10-phenanthroline also reduced enzyme activity. A MALDI-MS analysis, followed by sequencing of two of the resulting peptides, showed that LAP yspII undoubtedly corresponds to the putative aminopeptidase C13A11.05 identified in the S. pombe genome project. The protein exhibited nearly 40% sequence identity to fungal and mammalian aminopeptidases belonging to the M17 family of metallopeptidases. Catalytic residues (Lys292 and Arg366), as well as those involved in coordination with the cocatalytic metal ions (Lys280, Asp285, Asp303, Asp362 and Glu364) and those forming the hydrophobic pocket for substrate binding (Met300, Asn360, Ala363, Thr390, Leu391, Ala483 and Met486), were perfectly conserved among all known aminopeptidases. The S. pombe enzyme is predicted to be formed two clearly distinguished domains with a well conserved C-terminal catalytic domain showing a characteristic topology of eight beta-sheets surrounded by alpha-helical segments in the form of a saddle.
Asunto(s)
Citosol/enzimología , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/metabolismo , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Cationes Bivalentes/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Leucina/análogos & derivados , Leucina/farmacología , Leucil Aminopeptidasa/antagonistas & inhibidores , Leucil Aminopeptidasa/genética , Leucil Aminopeptidasa/aislamiento & purificación , Manganeso/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Anopheles nuneztovari is considered an important vector of human malaria in several localities in Venezuela and Colombia. Its status as a vector of human malaria is still unresolved in areas of the Brazilian Amazon, in spite of have been found infected with Plasmodium sp.. For a better understanding of the genetic differentiation of populations of A. nuneztovari, electrophoretic analysis using 11 enzymes was performed on four populations from Brazil and two from Colombia. The results showed a strong differentiation for two loci: alpha-glycerophosphate dehydrogenase (alpha-Gpd) and malate dehydrogenase (Mdh) from 16 loci analyzed. Diagnostic loci were not detected. The populations of A. nuneztovari from the Brazilian Amazon showed little genetic structure and low geographic differentiation, based on the F(IS) (0.029), F(ST) (0.070), and genetic distance (0.001-0.032) values. The results of the isozyme analysis do not coincide with the indication of two lineages in the Amazon Basin by analysis of mitochondrial DNA, suggesting that this evolutionary event is recent. The mean F(ST) value (0.324) suggests that there is considerable genetic divergence among populations from the Brazilian Amazon and Colombia. The genetic distance among populations from the Brazilian Amazon and Colombia is ranges from 0.047 to 0.148, with the highest values between the Brazilian Amazon and Sitronela (SIT) (0.125-0.148). These results are consistent with those observed among members of anopheline species complexes. It is suggested that geographic isolation has reduced the gene flow, resulting in the genetic divergence of the SIT population. Dendrogram analysis showed three large groups: one Amazonian and two Colombia, indicating some genetic structuring. The present study is important because it attempted to clarify the taxonomic status of A. nuneztovari and provide a better understanding of the role of this mosquito in transmission of human malaria in northern South America.
Asunto(s)
Anopheles/genética , Variación Genética/genética , Insectos Vectores/genética , Malaria/transmisión , Aconitato Hidratasa/química , Aconitato Hidratasa/genética , Animales , Anopheles/clasificación , Anopheles/enzimología , Brasil , Colombia , Electroforesis en Gel de Agar , Electroforesis en Gel de Almidón , Esterasas/química , Esterasas/genética , Femenino , Glucosa-6-Fosfato Isomerasa/química , Glucosa-6-Fosfato Isomerasa/genética , Glicerolfosfato Deshidrogenasa/química , Glicerolfosfato Deshidrogenasa/genética , Humanos , Insectos Vectores/clasificación , Insectos Vectores/enzimología , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/genética , Isoenzimas/química , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/genética , Malato Deshidrogenasa/química , Malato Deshidrogenasa/genética , Fosfogluconato Deshidrogenasa/química , Fosfogluconato Deshidrogenasa/genética , Filogenia , Xantina Deshidrogenasa/química , Xantina Deshidrogenasa/genéticaRESUMEN
An aminopeptidase activity capable of hydrolyzing different aminomethylcoumarin amino acids, but mainly leucine-7-amino-4-methylcoumarin (Leu-NHMc), was detected in deoxycholic soluble extracts from adult Fasciola hepatica. The enzyme (EC 3.4.11.1) was partially purified by gel filtration and EAH-Sepharose affinity chromatography using bestatin as a ligand. Results obtained by gel filtration, direct fluorogenic substrate analysis in polyacrylamide gel, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that in a native form the enzyme might be aggregated as a high molecular weight complex. By affinity chromatography on concanavalin A Sepharose, the enzyme did not bond to the column showing that it lacks mannose residues. The F. hepatica aminopeptidase was characterized as a metalloproteinase based on its activation by Mn2+ and Mg2+, and its inhibition by EDTA, 1,10-phenanthroline, and bestatin. It has an optimal activity at a pH between 8.0 and 8.5. Histochemical localization revealed strong leucine naphthylamidase activity at the cells lining the gut epithelium of the parasite.