RESUMEN
Psoriasis is an incurable cutaneous illness characterized by the presence of well-delimited reddish plaques and silvery-white dry scales. So far, there is a limited understanding of its pathogenesis, though recent discoveries on the immunological, genetic and molecular aspects of this disease have significantly contributed to the identification of new targets and the development of novel drugs. Despite these advances, many patients are still dissatisfied, so to improve patient satisfaction, reliability, and clinical outcomes, the individualization of the treatments for this disease becomes a necessity. This review summarizes recent findings related to psoriasis pathogenesis and describes new small molecules and targets recently identified as promising for treatments. Additionally, the current status, challenges and the future directions for achieving individualized therapy for this disease and the need for more collaborative studies are discussed. The individualization of treatments for psoriasis, rather than a goal, is analyzed as a process where a dynamic integration between the needs and characteristics of the patients, the pharmacological progress, and the clinical decisions takes place.
Asunto(s)
Fármacos Dermatológicos/farmacología , Desarrollo de Medicamentos , Medicina de Precisión/métodos , Psoriasis/tratamiento farmacológico , Canales de Calcio Activados por la Liberación de Calcio/antagonistas & inhibidores , Catepsinas/antagonistas & inhibidores , Catepsinas/genética , Fármacos Dermatológicos/uso terapéutico , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Satisfacción del Paciente , Psoriasis/genética , Psoriasis/inmunología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismoRESUMEN
Cutaneous leishmaniasis (CL) is the most common clinical form of American tegumentary leishmaniasis caused by Leishmania (Viannia) braziliensis. CL is associated with a strong Th1 immune response. This exacerbated inflammatory response is correlated with severity of disease and delays the healing time of the ulcer. The fourth-generation immucillin derivative (DI4G), a potent inhibitor of purine nucleoside phosphorylase, has been proposed as a promising agent in the treatment of diseases associated with T cell activation. Herein, we evaluated the in vitro immunomodulatory activity of DI4G in cells of patients presenting with CL. Peripheral blood mononuclear cells (PBMC) from CL patients were stimulated with soluble leishmania antigen (SLA), in the presence or absence of DI4G, and IFN-γ, TNF, CXCL9, and CXCL10 levels were determined by ELISA. Lymphocyte proliferation in the presence or absence of DI4G was also evaluated, using flow cytometry. DI4G was able to decrease (p < 0.05) IFN-γ production but did not change the TNF, CXCL9, and CXCL10 levels. DI4G decreased (p < 0.05) the lymphoproliferative response mediated by CD8+ T cells, but not that by CD4+ T cells. DI4G is able to attenuate the exaggerated immune response in CL, exhibiting immunomodulatory activity in IFN-γ production and in CD8+ T cell proliferation.
Asunto(s)
Adenina/análogos & derivados , Células Asesinas Naturales/inmunología , Leishmania braziliensis/inmunología , Leishmaniasis Cutánea/tratamiento farmacológico , Leucocitos Mononucleares/inmunología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Pirrolidinas/farmacología , Células TH1/inmunología , Adenina/química , Adenina/farmacología , Adenosina/análogos & derivados , Brasil , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Inmunomodulación , Activación de Linfocitos , Pirrolidinas/químicaRESUMEN
Malaria, a tropical parasitic disease caused by Plasmodium spp., continues to place a heavy social burden, with almost 200 million cases and more than 580,000 deaths per year. Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) can be targeted for antimalarial drug design since its inhibition kills malaria parasites both in vitro and in vivo. Although the currently known inhibitors of PfPNP, immucillins, are orally available and of low toxicity to animals and humans, to the best of our knowledge, none of these compounds has entered clinical trials for the treatment of malaria. Using a pharmacophore-based virtual screening coupled to a consensual molecular docking approach, we identified 59 potential PfPNP inhibitors that are predicted to be orally absorbed in a Caco-2 cell model. Although most of these compounds are predicted to have high plasma protein binding levels, poor water solubility (except for compound 25) and CYP3A4 metabolic stability (except for 4, 7 and 8), four structures (4, 7, 8 and 25) remain as potential leads because of their plausible interaction with a specific hydrophobic pocket of PfPNP, which would confer them higher selectivity for PfPNP over human PNP. Additionally, both predicted Gibbs free energies for binding and molecular dynamics suggest that compound 4 may form a more stable complex with PfPNP than 5[Formula: see text]-methylthio-immucillin-H, a potent and selective inhibitor of PfPNP.
Asunto(s)
Antimaláricos/química , Antimaláricos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Plasmodium falciparum/enzimología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Células CACO-2 , Simulación por Computador , Bases de Datos Farmacéuticas , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Purinas/metabolismoRESUMEN
MTAP is a ubiquitously expressed gene important for adenine and methionine salvage. The gene is located at 9p21, a chromosome region often deleted in breast carcinomas, similar to CDKN2A, a recognized tumor suppressor gene. Several research groups have shown that MTAP acts as a tumor suppressor, and some therapeutic approaches were proposed based on a tumors´ MTAP status. We analyzed MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression in seven breast cancer cell lines and evaluated their promoter methylation patterns to better characterize the contribution of these genes to breast cancer. Cytotoxicity assays with inhibitors of de novo adenine synthesis (5-FU, AZA and MTX) after MTAP gene knockdown showed an increased sensitivity, mainly to 5-FU. MTAP expression was also evaluated in two groups of samples from breast cancer patients, fresh tumors and paired normal breast tissue, and from formalin-fixed paraffin embedded (FFPE) core breast cancer samples diagnosed as Luminal-A tumors and triple negative breast tumors (TNBC). The difference of MTAP expression between fresh tumors and normal tissues was not statistically significant. However, MTAP expression was significantly higher in Luminal-A breast tumors than in TNBC, suggesting the lack of expression in more aggressive breast tumors and the possibility of using the new approaches based on MTAP status in TNBC.
Asunto(s)
Adenocarcinoma Mucinoso/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Purina-Nucleósido Fosforilasa/genética , Neoplasias de la Mama Triple Negativas/genética , Adenocarcinoma Mucinoso/tratamiento farmacológico , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patología , Antineoplásicos/farmacología , Azacitidina/farmacología , Carcinoma Ductal de Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/tratamiento farmacológico , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patología , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metilación de ADN , Receptor alfa de Estrógeno/deficiencia , Receptor alfa de Estrógeno/genética , Femenino , Fluorouracilo/farmacología , Humanos , Metástasis Linfática , Metotrexato/farmacología , Especificidad de Órganos , Regiones Promotoras Genéticas , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor ErbB-2/deficiencia , Receptor ErbB-2/genética , Receptores de Progesterona/deficiencia , Receptores de Progesterona/genética , Transducción de Señal , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening¸ the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme-substrate complex than for the free enzyme, and inhibition constant of 55.1±9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein.
Asunto(s)
Compuestos de Bencilo/farmacología , Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , Xantina Oxidasa/antagonistas & inhibidores , Animales , Compuestos de Bencilo/síntesis química , Compuestos de Bencilo/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Guanina/síntesis química , Guanina/química , Guanina/farmacología , Humanos , Estructura Molecular , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/metabolismo , Schistosoma mansoni/enzimología , Relación Estructura-Actividad , Xantina Oxidasa/metabolismoRESUMEN
The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 µmol L (-1) and 164 ± 13.4 µmol L (-1), respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.
Asunto(s)
Bioensayo/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/química , Schistosoma mansoni/enzimología , Esquistosomicidas/química , Espectrofotometría Ultravioleta/instrumentación , Adsorción , Animales , Diseño de Fármacos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/química , Enzimas Inmovilizadas/química , Diseño de Equipo , Análisis de Falla de Equipo , Purina-Nucleósido Fosforilasa/análisis , Reproducibilidad de los Resultados , Esquistosomicidas/administración & dosificación , Esquistosomicidas/análisis , Sensibilidad y EspecificidadRESUMEN
Purine nucleoside phosphorylase (PNP) is a purine-metabolizing enzyme that catalyzes the reversible phosphorolysis of 6-oxypurine (deoxy)nucleosides to their respective bases and (deoxy)ribose-1-phosphate. It is a key enzyme in the purine salvage pathway of mammalian cells. The present investigation sought to determine whether the PNP transition state analog inhibitor (Immucillin-H) arrests bone loss in two models of induced periodontal disease in rats. Periodontal disease was induced in rats using ligature or LPS injection followed by administration of Immucillin-H for direct analysis of bone loss, histology and TRAP staining. In vitro osteoclast differentiation and activation of T CD4+ cells in the presence of Immucillin-H were carried out for assessment of RANKL expression, PNP and Cathepsin K activity. Immucillin-H inhibited bone loss induced by ligatures and LPS, leading to a reduced number of infiltrating osteoclasts and inflammatory cells. In vitro assays revealed that Immucillin-H could not directly abrogate differentiation of osteoclast precursor cells, but affected lymphocyte-mediated osteoclastogenesis. On the other hand, incubation of pre-activated T CD4+ with Immucillin-H decreased RANKL secretion with no compromise of cell viability. The PNP transition state analog Immucillin-H arrests bone loss mediated by T CD4+ cells with no direct effect on osteoclasts. PNP inhibitor may have an impact in the treatment of diseases characterized by the presence of pathogens and imbalances of bone metabolism.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Enfermedades Periodontales/prevención & control , Nucleósidos de Purina/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Pirimidinonas/farmacología , Animales , Linfocitos T CD4-Positivos/inmunología , Técnicas de Cocultivo , Activación de Linfocitos , Ratones , Purina-Nucleósido Fosforilasa/metabolismo , Ratas , Ratas WistarRESUMEN
Consumption has been a scourge of mankind since ancient times. This illness has charged a high price to human lives. Many efforts have been made to defeat Mycobacterium tuberculosis (Mt). The M. tuberculosis purine nucleoside phosphorylase (MtPNP) is considered an interesting target to pursuit new potential inhibitors, inasmuch it belongs to the purine salvage pathway and its activity might be involved in the mycobacterial latency process. Here we present the MtPNP crystallographic structure associated with acyclovir and phosphate (MtPNP:ACY:PO(4)) at 2.10 Å resolution. Molecular dynamics simulations were carried out in order to dissect MtPNP:ACY:PO(4) structural features, and the influence of the ligand in the binding pocket stability. Our results revealed that the ligand leads to active site lost of stability, in agreement with experimental results, which demonstrate a considerable inhibitory activity against MtPNP (K(i) = 150 nM). Furthermore, we observed that some residues which are important in the proper ligand's anchor into the human homologous enzyme do not present the same importance to MtPNP. Therewithal, these findings contribute to the search of new specific inhibitors for MtPNP, since peculiarities between the mycobacterial and human enzyme binding sites have been identified, making a structural-based drug design feasible.
Asunto(s)
Aciclovir/farmacología , Mycobacterium tuberculosis/enzimología , Purina-Nucleósido Fosforilasa/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Simulación de Dinámica Molecular , Análisis de Componente Principal , Conformación Proteica , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/química , Espectrometría de FluorescenciaRESUMEN
The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The K(M) value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 ± 29.2 µM and 133 ± 14.9 µM, respectively). A new fourth-generation immucillin derivative (DI4G; IC(50)=40.6 ± 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay.
Asunto(s)
Reactores Biológicos , Descubrimiento de Drogas/métodos , Enzimas Inmovilizadas/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Cromatografía Liquida , Enzimas Inmovilizadas/antagonistas & inhibidores , Enzimas Inmovilizadas/química , Diseño de Equipo , Compuestos Heterocíclicos con 1 Anillo/química , Compuestos Heterocíclicos con 1 Anillo/metabolismo , Humanos , Hipoxantina/análisis , Hipoxantina/metabolismo , Inosina/metabolismo , Cinética , Ligandos , Modelos Lineales , Modelos Químicos , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/química , Análisis de Regresión , Reproducibilidad de los ResultadosRESUMEN
Schistosomiasis is considered the second most important tropical parasitic disease, with severe socioeconomic consequences for millions of people worldwide. Schistosoma mansoni , one of the causative agents of human schistosomiasis, is unable to synthesize purine nucleotides de novo, which makes the enzymes of the purine salvage pathway important targets for antischistosomal drug development. In the present work, we describe the development of a pharmacophore model for ligands of S. mansoni purine nucleoside phosphorylase (SmPNP) as well as a pharmacophore-based virtual screening approach, which resulted in the identification of three thioxothiazolidinones (1-3) with substantial in vitro inhibitory activity against SmPNP. Synthesis, biochemical evaluation, and structure-activity relationship investigations led to the successful development of a small set of thioxothiazolidinone derivatives harboring a novel chemical scaffold as new competitive inhibitors of SmPNP at the low-micromolar range. Seven compounds were identified with IC(50) values below 100 µM. The most potent inhibitors 7, 10, and 17 with IC(50) of 2, 18, and 38 µM, respectively, could represent new potential lead compounds for further development of the therapy of schistosomiasis.
Asunto(s)
Inhibidores Enzimáticos/química , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Schistosoma mansoni/enzimología , Secuencia de Aminoácidos , Animales , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Purina-Nucleósido Fosforilasa/química , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Selectivity plays a crucial role in the design of enzyme inhibitors as novel antiparasitic agents, particularly in cases where the target enzyme is also present in the human host. Purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive target for the discovery of potential antischistosomal agents. In the present work, kinetic studies were carried out in order to determine the inhibitory potency, mode of action and enzyme selectivity of a series of inhibitors of SmPNP. In addition, crystallographic studies provided important structural insights for rational inhibitor design, revealing consistent structural differences in the binding mode of the inhibitors in the active sites of the SmPNP and human PNP (HsPNP) structures. The molecular information gathered in this work should be useful for future medicinal chemistry efforts in the design of new inhibitors of SmPNP having increased affinity and selectivity.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Schistosoma mansoni/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Cinética , Datos de Secuencia Molecular , Purina-Nucleósido Fosforilasa/química , Schistosoma mansoni/enzimología , Homología de Secuencia de AminoácidoRESUMEN
A novel inhibitor of Schistosoma PNP was identified using an "in silico" approach allied to enzyme inhibition assays. The compound has a monocyclic structure which has not been previously described for PNP inhibitors. The crystallographic structure of the complex was determined and used to elucidate the binding mode within the active site. Furthermore, the predicted pose was very similar to that determined crystallographically, validating the methodology. The compound Sm_VS1, despite its low molecular weight, possesses an IC(50) of 1.3 microM, surprisingly low when compared with purine analogues. This is presumably due to the formation of eight hydrogen bonds with key residues in the active site E203, N245 and T244. The results of this study highlight the importance of the use of multiple conformations for the target during virtual screening. Indeed the Sm_VS1 compound was only identified after flipping the N245 side chain. It is expected that the structure will be of use in the development of new highly active non-purine based compounds against the Schistosoma enzyme.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas del Helminto/química , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/química , Schistosoma/enzimología , Animales , Dominio Catalítico , Cristalografía por Rayos X , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Unión Proteica , Estructura Terciaria de Proteína , Schistosoma/químicaRESUMEN
This work describes for the first time a model of Purine Nucleoside Phosphorylase from Listeria monocytogenes (LmPNP). We modeled the complexes of LmPNP with ligands in order to determine the structural basis for specificity. Comparative analysis of the model of LmPNP allowed identification of structural features responsible for ligand affinities.
Asunto(s)
Biología Computacional , Listeria monocytogenes/enzimología , Purina-Nucleósido Fosforilasa/química , Secuencia de Aminoácidos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Apoenzimas/antagonistas & inhibidores , Apoenzimas/química , Apoenzimas/metabolismo , Sitios de Unión , Diseño de Fármacos , Humanos , Ligandos , Listeria monocytogenes/efectos de los fármacos , Listeriosis/tratamiento farmacológico , Modelos Moleculares , Estructura Terciaria de Proteína , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/metabolismo , Especificidad por SustratoRESUMEN
This work describes for the first time a structural model of purine nucleoside phosphorylase from Streptococcus agalactiae (SaPNP). PNP catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is a potential target for the development of antibacterial drugs. We modeled the complexes of SaPNP with 15 different ligands in order to determine the structural basis for the specificity of these ligands against SaPNP. The application of a novel empirical scoring function to estimate the affinity of a ligand for a protein was able to identify the ligands with high affinity for PNPs. The analysis of molecular dynamics trajectory for SaPNP indicates that the functionally important motifs have a very stable structure. This new structural model together with a novel empirical scoring function opens the possibility to explorer larger library of compounds in order to identify the new inhibitors for PNPs in virtual screening projects.
Asunto(s)
Simulación por Computador , Purina-Nucleósido Fosforilasa/química , Streptococcus agalactiae/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Desoxirribonucleósidos/química , Guanosina/análogos & derivados , Guanosina/farmacología , Humanos , Enlace de Hidrógeno , Inosina/análogos & derivados , Inosina/farmacología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Nucleósidos de Purina/química , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Alineación de Secuencia , Relación Estructura-Actividad , Factores de TiempoRESUMEN
The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.
Asunto(s)
Inhibidores Enzimáticos/química , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/química , Purinas/química , Bazo/enzimología , Animales , Bovinos , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Conformación Proteica , Purinas/farmacologíaRESUMEN
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and thus drugs that inhibit human PNP activity have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Besides, the purine salvage pathway is the only possible way for apicomplexan parasites to obtain the building blocks for RNA and DNA synthesis, which makes PNP from these parasites an attractive target for drug development against diseases such as malaria. Hence, a number of research groups have made efforts to elucidate the mechanism of action of PNP based on structural and kinetic studies. It is conceivable that the mechanism may be different for PNPs from diverse sources, and influenced by the oligomeric state of the enzyme in solution. Furthermore, distinct transition state structures can make possible the rational design of specific inhibitors for human and apicomplexan enzymes. Here, we review the current status of these research efforts to elucidate the mechanism of PNP-catalyzed chemical reaction, focusing on the mammalian and Plamodium falciparum enzymes, targets for drug development against, respectively, T-Cell- and Apicomplexan parasites-mediated diseases.
Asunto(s)
Apicomplexa/enzimología , Sistemas de Liberación de Medicamentos/métodos , Infecciones por Protozoos/enzimología , Purina-Nucleósido Fosforilasa/metabolismo , Linfocitos T/enzimología , Animales , Apicomplexa/patogenicidad , Humanos , Infecciones por Protozoos/tratamiento farmacológico , Infecciones por Protozoos/parasitología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Linfocitos T/parasitologíaRESUMEN
Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure- based drug design. This methodology may help in the future development of a new generation of PNP inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/química , Sitios de Unión/efectos de los fármacos , Simulación por Computador , Cristalización/métodos , Cristalografía por Rayos X/métodos , Ligandos , Fosfatos/química , Unión Proteica/efectos de los fármacos , Pliegue de Proteína , Nucleósidos de Purina , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis, and hologram quantitative structure-activity relationship (HQSAR) studies were conducted on a series of 52 training set inhibitors of calf spleen purine nucleoside phosphorylase (PNP). Significant cross-validated correlation coefficients (CoMFA, q(2)=0.68; CoMSIA, q(2)=0.66; and HQSAR, q(2)=0.70) were obtained, indicating the potential of the models for untested compounds. The models were then used to predict the inhibitory potency of 16 test set compounds that were not included in the training set, and the predicted values were in good agreement with the experimental results. The final QSAR models along with the information gathered from 3D contour and 2D contribution maps should be useful for the design of novel inhibitors of PNP having improved potency.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Cinética , Modelos Moleculares , Relación Estructura-Actividad CuantitativaRESUMEN
Despite the availability of effective chemotherapy, schistosomiasis continues to be one of the major parasitic infections to affect the human population worldwide. Currently, little is known of the structural biology of the parasites that are responsible for the disease and few attempts have been made to develop second generation drugs, which may become essential if resistance to those currently available becomes an issue. Here, we describe three crystal structures for the enzyme purine nucleoside phosphorylase (PNP) from Schistosoma mansoni, a component of the purine salvage pathway. PNP is known to be essential for the recovery of purine bases and nucleosides in schistosomes, due to an absence of the enzymes for de novo synthesis, making it a sensitive point in the parasite's metabolism. In all three structures reported here, acetate occupies part of the base-binding site and is directly bound to the conserved glutamic acid at position 203. One of the structures presents the crystallization additive sulfobetaine 195 (NDSB195) occupying simultaneously the ribose and phosphate binding sites, whilst a second presents only phosphate in the latter. The observation of sulfobetaine specifically bound to the protein active site was unexpected and is unique to this structure as far as we are aware. Considerable flexibility is observed in the active site, principally due to variable structural disorder in the regions centered on residues 64 and 260. This conformational plasticity extends to the way in which both NDSB195 and phosphate bind to the individual monomers of the trimeric structure reported here. Differences between the parasite and human enzymes are limited principally to the base-binding site, where the substitution of V245 in the mammalian enzymes by S247 introduces additional hydrogen bonding potential to the site. This is satisfied in the structures described here by a water molecule whose presence is normally observed only in complexes with 6-oxopurines. Residue Y202, which replaces F200 in human PNP, is able to reach over the ribose-binding site to interact with H259 and is predicted to form an additional hydrogen bond with the 5' hydroxyl of nucleoside substrates.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Schistosoma mansoni/efectos de los fármacos , Esquistosomiasis/parasitología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Purina-Nucleósido Fosforilasa/química , Schistosoma mansoni/enzimología , Homología de Secuencia de AminoácidoRESUMEN
Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine-salvage pathway, which allows cells to utilize preformed bases and nucleosides in order to synthesize nucleotides. PNP is specific for purine nucleosides in the beta-configuration and exhibits a strong preference for purines containing a 6-keto group and ribosyl-containing nucleosides relative to the corresponding analogues. PNP was crystallized in complex with ligands and data collection was performed using synchrotron radiation. This work reports the structure of human PNP in complex with guanosine (at 2.80 A resolution), 3'-deoxyguanosine (at 2.86 A resolution) and 8-azaguanine (at 2.85 A resolution). These structures were compared with the PNP-guanine, PNP-inosine and PNP-immucillin-H complexes solved previously.