RESUMEN
Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH(3) and CO(2), neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Helicobacter pylori/química , Protones , Urea/metabolismo , Secuencias de Aminoácidos , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Modelos Moleculares , Multimerización de Proteína , Estructura Secundaria de Proteína , Homología Estructural de ProteínaRESUMEN
Chagas' disease, the leading cause of heart failure in Latin America, is caused by the kinetoplastid protozoan Trypanosoma cruzi. The sterols of T. cruzi resemble those of fungi, both in composition and in biosynthesis. Azole inhibitors of sterol 14alpha-demethylase (CYP51) successfully treat fungal infections in humans, and efforts to adapt the success of antifungal azoles posaconazole and ravuconazole as second-use agents for Chagas' disease are under way. However, to address concerns about the use of azoles for Chagas' disease, including drug resistance and cost, the rational design of nonazole CYP51 inhibitors can provide promising alternative drug chemotypes. We report the curative effect of the nonazole CYP51 inhibitor LP10 in an acute mouse model of T. cruzi infection. Mice treated with an oral dose of 40 mg LP10/kg of body weight twice a day (BID) for 30 days, initiated 24 h postinfection, showed no signs of acute disease and had histologically normal tissues after 6 months. A very stringent test of cure showed that 4/5 mice had negative PCR results for T. cruzi, and parasites were amplified by hemoculture in only two treated mice. These results compare favorably with those reported for posaconazole. Electron microscopy and gas chromatography-mass spectrometry (GC-MS) analysis of sterol composition confirmed that treatment with LP10 blocked the 14alpha-demethylation step and induced breakdown of parasite cell membranes, culminating in severe ultrastructural and morphological alterations and death of the clinically relevant amastigote stage of the parasite.
Asunto(s)
Aminopiridinas/farmacología , Antiprotozoarios/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Aminopiridinas/administración & dosificación , Aminopiridinas/química , Animales , Antiprotozoarios/administración & dosificación , Antiprotozoarios/química , Dominio Catalítico , Enfermedad de Chagas/parasitología , Sistema Enzimático del Citocromo P-450/química , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Femenino , Humanos , Indoles/administración & dosificación , Indoles/química , Ratones , Ratones Endogámicos C3H , Microscopía Electrónica de Transmisión , Modelos Moleculares , Proteínas Protozoarias/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Esteroles/biosíntesis , Trypanosoma cruzi/ultraestructuraRESUMEN
CYP102A1 is a highly active, water-soluble, bacterial monooxygenase enzyme that contains both substrate-binding heme and diflavin reductase subunits, both in a single polypeptide. Recently we developed a procedure which uses the known structure of the substrate-bound heme domain of CYP102A1 and its sequence homology with a cytochrome P450 of unknown structure, both of which react with a common substrate but produce different products, to create recombinant enzymes which have substrate selectivity different from that of CYP102A1, and produce the product of the enzyme of unknown structure. Insect CYP4C7, a terpene hydroxylase from the cockroach, was chosen as the cytochrome P450 of unknown structure, and farnesol was chosen as the substrate. CYP102A1 oxidizes farnesol to three products (2,3-epoxyfarnesol, 10,11-epoxyfarnesol, and 9-hydroxyfarnesol), whereas CYP4C7 produces 12-hydroxyfarnesol as the major product. In earlier work it was found that the chimera C(78-82,F87L) showed a change in substrate selectivity from fatty acids to farnesol, and was approximately sixfold more active than wild-type CYP102A1 (Chen et al. in J Biol Inorg Chem 13:813-824, 2008), but neither it nor any other earlier chimera produced 12-hydroxyfarnesol. In this work we added amino acid residues 327-332, to create six new full-length, functional chimeric proteins. Four of these, the most active of which was C(78-82,F87L,328-330), produce 12-hydroxyfarnesol as the major product, with approximately twofold increase in turnover number as compared with wild-type CYP102A1 toward farnesol. Methylfarnesoate was metabolized to 12-hydroxymethylfarnesoate (70%) and 10,11-epoxymethylfarnesoate (juvenile hormone III) (30%). The latter is metabolized to 65% 12-hydroxy-10,11-epoxymethylfarnesoate and 35% 15-hydroxy-10,11-epoxymethylfarnesoate. Substitution of residues 328-330, APA, by VPL was crucial to accomplishing this change in product.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cucarachas/enzimología , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Proteínas Mutantes Quiméricas/metabolismo , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/genética , Familia 4 del Citocromo P450 , Farnesol/análogos & derivados , Farnesol/química , Farnesol/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas de Insectos/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , NADPH-Ferrihemoproteína Reductasa/genética , Especificidad por SustratoRESUMEN
The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reactivation of mutant p53 by small molecules is an exciting potential cancer therapy. Although several compounds restore wild-type function to mutant p53, their binding sites and mechanisms of action are elusive. Here computational methods identify a transiently open binding pocket between loop L1 and sheet S3 of the p53 core domain. Mutation of residue Cys124, located at the centre of the pocket, abolishes p53 reactivation of mutant R175H by PRIMA-1, a known reactivation compound. Ensemble-based virtual screening against this newly revealed pocket selects stictic acid as a potential p53 reactivation compound. In human osteosarcoma cells, stictic acid exhibits dose-dependent reactivation of p21 expression for mutant R175H more strongly than does PRIMA-1. These results indicate the L1/S3 pocket as a target for pharmaceutical reactivation of p53 mutants.
Asunto(s)
Biología Computacional/métodos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Compuestos Aza/farmacología , Sitios de Unión , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Cisteína/genética , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Simulación de Dinámica Molecular , Oxepinas/química , Oxepinas/farmacología , Estabilidad Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Reproducibilidad de los Resultados , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority. METHODOLOGY/PRINCIPAL FINDINGS: The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of â¼104,000 small molecules to 185 hits with estimated nanomolar K(D) values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50) <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50) of 17 nM and was trypanocidal at 40 nM. CONCLUSIONS/SIGNIFICANCE: The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.
Asunto(s)
Antiprotozoarios/química , Antiprotozoarios/aislamiento & purificación , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450 , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Simulación de Dinámica Molecular , Pruebas de Sensibilidad ParasitariaRESUMEN
BACKGROUND: Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14alpha-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding. METHODOLOGY/PRINCIPAL FINDINGS: We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 A and 2.27 A), and from the related pathogen T. brucei (resolutions of 2.7 A and 2.6 A), co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180 degrees depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target. CONCLUSIONS/SIGNIFICANCE: The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for rationally designed anti-Chagasic drugs with improved efficacy and reduced toxicity.
Asunto(s)
Antifúngicos/química , Sistema Enzimático del Citocromo P-450/química , Fluconazol/química , Triazoles/química , Trypanosoma brucei brucei/química , Trypanosoma cruzi/química , Secuencia de Aminoácidos , Antifúngicos/metabolismo , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/metabolismo , Fluconazol/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Triazoles/metabolismoRESUMEN
BACKGROUND: The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas' disease chemotherapy is sterol 14alpha-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols. METHODOLOGY/PRINCIPAL FINDING: In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51(Mt)), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51(Mt). Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51(Tc), demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti-T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site. CONCLUSIONS/SIGNIFICANCE: CYP51(Mt)-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51(Tc).
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Mycobacterium tuberculosis/enzimología , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Trypanosoma cruzi , Animales , Bovinos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Sistema Enzimático del Citocromo P-450 , Inhibidores Enzimáticos/efectos adversos , Humanos , Concentración 50 Inhibidora , Ratones , Mycobacterium tuberculosis/efectos de los fármacos , Pruebas de Sensibilidad Parasitaria , Tripanocidas/efectos adversos , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimologíaRESUMEN
CYP102A1 is a highly active water-soluble bacterial monooxygenase that contains both substrate-binding heme and diflavin reductase subunits, all in a single polypeptide that has been called a "self-sufficient enzyme." Several years ago we developed a procedure called "scanning chimeragenesis," where we focused on residues 73-82 of CYP102A1, which contact approximately 40% of the substrates palmitoleic acid and N-palmitoylglycine [Murataliev et al. (2004) Biochemistry 43:1771-1780]. These residues were replaced with the homologous residues of CYP4C7. In the current work, that study has been expanded to include residue 87. Phenylalanine 87 of wild-type CYP102A1 was replaced with the homologous residue of CYP4C7, leucine, as well as with alanine. The full-sized chimeric proteins C(73-78, F87L), C(73-78, F87A), C(75-80, F87L), C(75-80, F87A), C(78-82, F87L) and C(78-82, F87A) have been purified and characterized. Wild-type CYP102A1 is most active toward fatty acids (both lauric and palmitic acids produce omega-1, omega-2, and omega-3 hydroxylated fatty acids), but it also catalyzes the oxidation of farnesol to three products (2, 3- and 10,11-epoxyfarnesols and 9-hydroxyfarnesol). All of the F87-mutant chimeric proteins show dramatic decreases in activities with the natural CYP102A1 substrates. In contrast, C(78-82, F87A) and C(78-82, F87L) have markedly increased activities with farnesol, with the latter showing a 5.7-fold increase in catalytic activity as compared to wild-type CYP102A1. C(78-82, F87L) produces 10,11-epoxyfarnesol as the single primary metabolite. The results show that chimeragenesis involving only the second half of SRS-1 plus F87 is sufficient to change the substrate selectivity of CYP102A1 from fatty acids to farnesol and to produce a single primary product.