Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
2.
Nature ; 560(7717): 192-197, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30046105

RESUMEN

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.


Asunto(s)
Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Leishmania donovani/efectos de los fármacos , Leishmania donovani/enzimología , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Terapia Molecular Dirigida , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Quinasa 9 Dependiente de la Ciclina/química , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Simulación del Acoplamiento Molecular , Proteoma/efectos de los fármacos , Proteómica , Pirazoles/química , Pirazoles/uso terapéutico , Pirimidinas/química , Pirimidinas/uso terapéutico , Reproducibilidad de los Resultados , Especificidad por Sustrato
3.
Artículo en Inglés | MEDLINE | ID: mdl-31307977

RESUMEN

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening condition that has become a global issue. Current treatment is limited to two medicines that require prolonged dosing and are associated with multiple side effects, which often lead to treatment discontinuation and failure. One way to address these shortcomings is through target-based drug discovery on validated T. cruzi protein targets. One such target is the proteasome, which plays a crucial role in protein degradation and turnover through chymotrypsin-, trypsin-, and caspase-like catalytic activities. In order to initiate a proteasome drug discovery program, we isolated proteasomes from T. cruzi epimastigotes and characterized their activity using a commercially available glow-like luminescence-based assay. We developed a high-throughput biochemical assay for the chymotrypsin-like activity of the T. cruzi proteasome, which was found to be sensitive, specific, and robust but prone to luminescence technology interference. To mitigate this, we also developed a counterscreen assay that identifies potential interferers at the levels of both the luciferase enzyme reporter and the mechanism responsible for a glow-like response. Interestingly, we also found that the peptide substrate for chymotrypsin-like proteasome activity was not specific and was likely partially turned over by other catalytic sites of the protein. Finally, we utilized these biochemical tools to screen 18,098 compounds, exploring diverse drug-like chemical space, which allowed us to identify 39 hits that were active in the primary screening assay and inactive in the counterscreen assay.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Sistema Libre de Células , Luminiscencia , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Reproducibilidad de los Resultados , Trypanosoma cruzi/química
4.
PLoS One ; 19(4): e0300021, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38635818

RESUMEN

Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas' disease, a parasitic infection responsible for significant morbidity and mortality in Latin America. The current treatments have many serious drawbacks and new drugs are urgently required. In the UK, T. cruzi is classified by the Advisory Committee on Dangerous Pathogens (ACDP) as a Hazard Group 3 organism and strict safety practices must be adhered to when handling this pathogen in the laboratory. Validated inactivation techniques are required for safe T. cruzi waste disposal and removal from Containment Level 3 (CL3) facilities for storage, transportation and experimental analysis. Here we assess three T. cruzi. inactivation methods. These include three freeze-thaw cycles, chemical inactivation with Virkon disinfectant, and air drying on Whatman FTA cards (A, B, C, Elute) and on a Mitra microsampling device. After each treatment parasite growth was monitored for 4-6 weeks by microscopic examination. Three freeze-thaw cycles were sufficient to inactivate all T. cruzi CLBrener Luc life cycle stages and Silvio x10/7 A1 large epimastigote cell pellets up to two grams wet weight. Virkon treatment for one hour inactivated T. cruzi Silvio x10/7 subclone A1 and CLBrener Luc both in whole blood and cell culture medium when incubated at a final concentration of 2.5% Virkon, or at ≥1% Virkon when in tenfold excess of sample volume. Air drying also inactivated T. cruzi CLBrener Luc spiked blood when dried on FTA A, B or Elute cards for ≥30 minutes and on a Mitra Microsampler for two hours. However, T. cruzi CLBrener Luc were not inactivated on FTA C cards when dried for up to two hours. These experimentally confirmed conditions provide three validated T. cruzi inactivation methods which can be applied to other related ACDP Hazard Group 2-3 kinetoplastid parasites.


Asunto(s)
Aminopiridinas , Enfermedad de Chagas , Ácidos Sulfúricos , Trypanosoma cruzi , Humanos , Enfermedad de Chagas/parasitología , Peróxidos
5.
ACS Infect Dis ; 6(3): 515-528, 2020 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-31967783

RESUMEN

Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.


Asunto(s)
Antiparasitarios/farmacología , Citocromos b/antagonistas & inhibidores , Descubrimiento de Drogas , Leishmania donovani/efectos de los fármacos , Leishmania donovani/genética , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Antiparasitarios/química , Antiparasitarios/aislamiento & purificación , Enfermedad de Chagas/tratamiento farmacológico , Citocromos b/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Leishmaniasis Visceral/tratamiento farmacológico
6.
PLoS Negl Trop Dis ; 10(9): e0004977, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27606593

RESUMEN

Trypanosoma vivax is one of the causative agents of Animal African Trypanosomosis in cattle, which is endemic in sub-Saharan Africa and transmitted primarily by the bite of the tsetse fly vector. The parasite can also be mechanically transmitted, and this has allowed its spread to South America. Diagnostics are limited for this parasite and in farm settings diagnosis is mainly symptom-based. We set out to identify, using a proteomic approach, candidate diagnostic antigens to develop into an easy to use pen-side lateral flow test device. Two related members the invariant surface glycoprotein family, TvY486_0045500 and TvY486_0019690, were selected. Segments of these antigens, lacking N-terminal signal peptides and C-terminal transmembrane domains, were expressed in E. coli. Both were developed into ELISA tests and one of them, TvY486_0045500, was developed into a lateral flow test prototype. The tests were all evaluated blind with 113 randomised serum samples, taken from 37 calves before and after infection with T. vivax or T. congolense. The TvY486_0045500 and TvY486_0019690 ELISA tests gave identical sensitivity and specificity values for T. vivax infection of 94.5% (95% CI, 86.5% to 98.5%) and 88.0% (95% CI, 75.7% to 95.5%), respectively, and the TvY486_0045500 lateral flow test prototype a sensitivity and specificity of 92.0% (95% CI, 83.4% to 97.0%) and 89.8% (95% CI, 77.8% to 96.6%), respectively. These data suggest that recombinant TvY486_0045500 shows promise for the development of a pen-side lateral flow test for the diagnosis of T. vivax animal African trypanosomosis.


Asunto(s)
Antígenos de Protozoos/inmunología , Enfermedades de los Bovinos/diagnóstico , Bovinos/parasitología , Pruebas Inmunológicas/métodos , Trypanosoma vivax/inmunología , Tripanosomiasis Africana/veterinaria , Animales , Anticuerpos Antiprotozoarios/sangre , Enfermedades de los Bovinos/parasitología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Proteómica , Sensibilidad y Especificidad , Trypanosoma congolense/inmunología , Tripanosomiasis Africana/diagnóstico
7.
PLoS Negl Trop Dis ; 8(7): e2976, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25033401

RESUMEN

BACKGROUND: The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT. METHODOLOGY/PRINCIPLE FINDINGS: Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank. CONCLUSION/SIGNIFICANCE: In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use.


Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Juego de Reactivos para Diagnóstico/parasitología , Tripanosomiasis Africana/diagnóstico , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunología , Estudios de Cohortes , Humanos
8.
PLoS Negl Trop Dis ; 8(6): e2936, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24922510

RESUMEN

Animal African Trypanosomosis (AAT) presents a severe problem for agricultural development in sub-Saharan Africa. It is caused by several trypanosome species and current means of diagnosis are expensive and impractical for field use. Our aim was to discover antigens for the detection of antibodies to Trypanosoma congolense, one of the main causative agents of AAT. We took a proteomic approach to identify potential immunodiagnostic parasite protein antigens. One hundred and thirteen proteins were identified which were selectively recognized by infected cattle sera. These were assessed for likelihood of recombinant protein expression in E. coli and fifteen were successfully expressed and assessed for their immunodiagnostic potential by ELISA using pooled pre- and post-infection cattle sera. Three proteins, members of the invariant surface glycoprotein (ISG) family, performed favorably and were then assessed using individual cattle sera. One antigen, Tc38630, evaluated blind with 77 randomized cattle sera in an ELISA assay gave sensitivity and specificity performances of 87.2% and 97.4%, respectively. Cattle immunoreactivity to this antigen diminished significantly following drug-cure, a feature helpful for monitoring the efficacy of drug treatment.


Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/análisis , Proteómica/métodos , Trypanosoma congolense/aislamiento & purificación , África del Sur del Sahara , Animales , Bovinos , Enfermedades de los Bovinos/diagnóstico , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Expresión Génica , Ratones Endogámicos BALB C , Distribución Aleatoria , Sensibilidad y Especificidad , Trypanosoma congolense/inmunología , Tripanosomiasis Africana/diagnóstico , Tripanosomiasis Africana/veterinaria
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA