RESUMEN
Pyridoxal kinase (PdxK, EC 2.7.1.35) is an important enzyme of vitamin B6 salvage pathway which is required for phosphorylation of B6 vitamers. In the present study, pyridoxal kinase (pdxK) gene from Leishmania donovani (LdPdxK) was cloned and a 33â¯kDa protein was expressed and kinetically characterized. Site-directed mutagenesis was performed to determine the functional significance of conserved GXGD motif. Mutation of Thr229 to Ala did not affect the catalytic function of LdPdxK however Gly228, Gly230 and Asp231 were found to be indispensible for enzyme activity. To determine the role of LdPdxK in Leishmania promastigotes, LdPdxK overexpressing parasites were generated by episomal expression of the enzyme. The overexpression studies revealed the role of this enzyme in growth and infection of the parasite. In silico analysis of the human and parasite PdxK structure revealed significant differences in the active site region thus highlighting its potential as an antileishmanial drug target. Homology model of LdPdxK was built and was subjected to molecular dynamics simulations. Based on the above information, a pharmacophore was developed and shape based virtual screening was performed to identify potential and selective inhibitors against this essential enzyme. The current data suggests that LdPdxK could be a promising antileishmanial drug target.
Asunto(s)
Piridoxal Quinasa/química , Piridoxal Quinasa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Activación Enzimática , Expresión Génica , Humanos , Leishmania donovani/clasificación , Leishmania donovani/genética , Leishmania donovani/metabolismo , Redes y Vías Metabólicas , Modelos Moleculares , Mutación , Filogenia , Conformación Proteica , Piridoxal Quinasa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Vitamina B 6/química , Vitamina B 6/metabolismoRESUMEN
In the present study, glycyrrhizic acid (GA) the main component of Glycyrrhiza glabra was evaluated for its efficacy as antileishmanial agent and its mode of action explored. GA inhibits promastigotes and intracellular amastigotes in a dose dependent manner at an IC50 value of 34 ± 3.0 µM and 20 ± 4.2 µM respectively. GA was non-toxic against THP-1 macrophage host cell line. GA was found to inhibit recombinant Leishmania donovani HMG-CoA reductase (LdHMGR) enzyme at the half-maximum inhibitory concentration of 24 ± 4.3 µM indicating the sensitivity and specificity of GA towards the enzyme. However, GA could cause only 30% reduction in HMGR activity when measured in Leishmania promastigotes treated with 34 µM of GA. Interestingly western blot analysis revealed fivefold reduced HMGR expression in GLA treated promastigotes. To further study the mode of action of GA, we used transgenic parasites overexpressing LdHMGR. Results indicated that â¼2 fold resistance was exhibited by LdHMGR overexpressing promastigotes to GA with an IC50 value of 74 µM compared to the wild type parasite. This explained the specific binding of GA to LdHMGR enzyme. There was â¼2 fold depletion in ergosterol levels in wild type promastigotes compared to the HMGR overexpressors. This data was further validated by exogenous supplementation of GA treated cells with ergosterol and 40% reversal of growth inhibition was observed. The results obtained suggested that GA kills the parasite by affecting sterol biosynthetic pathway, especially by inhibiting the L. donovani HMGR and altering ergosterol levels. The finding from the current study shows that GA is a potential antileishmanial chemotherapeutic agent.
Asunto(s)
Antiprotozoarios/farmacología , Ergosterol/metabolismo , Ácido Glicirrínico/farmacología , Leishmania donovani/efectos de los fármacos , Western Blotting , Línea Celular , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Ácido Glicirrínico/química , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Concentración 50 Inhibidora , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/metabolismo , Macrófagos/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Ribose-5-phosphate isomerase B from Leishmania donovani (LdRpiB) is one of the potential drug targets against visceral leishmaniasis. In the present study, we have targeted several conserved amino acids for mutational analysis (i.e. Cys69, His11, His102, His138, Asp45, Tyr46, Pro47 and Glu149) to gain crucial insights into their role in substrate binding, catalysis and conformational stability of the enzyme. All the eight LdRpiB variants were cloned, sequenced, expressed and purified. C69S, H102N, D45N and E149A mutants exhibited complete loss of enzyme activity indicating that they are indispensable for the enzyme activity. Kinetic parameters were altered in case of H138N, H11N and P47A variants; however Y46F exhibited similar kinetic behaviour as wild type. All the mutants except H138N exhibited altered protein structure as determined by CD and fluorescence spectral analysis. This data was supported by the atomic level details of the conformational changes and substrate binding using molecular dynamic simulations. LdRpiB also exhibited activity with D-form of various aldose substrates in the order of D-ribose > D-talose > D-allose > D-arabinose. Our study provides insights for better understanding of substrate enzyme interactions which can rationalize the process of drug design against parasite RpiB.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/genética , Secuencia Conservada , Leishmania donovani/genética , Leishmania donovani/metabolismo , Modelos Moleculares , Mutación , Isomerasas Aldosa-Cetosa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
In the present study, 23 derivatives of 1,3,6-trisubstituted ß-carboline were synthesized and evaluated for cytotoxic potential against four human cancer cells, namely A-549, HeLa, Hep G2 and MCF-7 as well as anti-leishmanial activity against Leishmania donovani (MHOM/80/IN/Dd8) promastigotes. Among the studied compounds, compounds 13c and 13q showed potent cytotoxic activity better than the parent compound 10. For instance, compound 13c was found to be the most cytotoxic with IC50 of 4.72, 3.59, 3.65 and 4.17 µM against A-549, HeLa, Hep G2 and MCF-7 respectively, while for compound 13q, IC50 were 15.47, 5.30, 6.15 and 13.39 µM against the same cancer cells respectively. Further, these two compounds were found to be apoptotic in A-549 and MCF-7 cells when observed using Annexin V/propidium iodide staining under confocal microscope. All the compounds were also tested for anti-leishmanial potential. In which, compounds 13u and 13c were found to show moderate inhibition with IC50 of 23.5±9.0 and 68.0±0.0 µM respectively, while compound 10 was the most active with IC50 of 9.0±2.8 µM, suggesting the modification at C-6 detrimental for anti-leishmanial activity. Interestingly, amongst all, compound 13c was found to be the most active for cytotoxic and moderately active for anti-leishmanial activity which can be further developed as a lead for these disease areas.