RESUMO
Pathogenic parasites of the Trichomonas genus are causative agents of sexually transmitted diseases affecting millions of individuals worldwide and whose outcome may include stillbirths and enhanced cancer risks and susceptibility to HIV infection. Trichomonas vaginalis relies on imported purine and pyrimidine nucleosides and nucleobases for survival, since it lacks the enzymatic activities necessary for de novo biosynthesis. Here we show that T. vaginalis additionally lacks homologues of the bacterial or mammalian enzymes required for the synthesis of the nicotinamide ring, a crucial component in the redox cofactors NAD+ and NADP. Moreover, we show that a yet fully uncharacterized T. vaginalis protein homologous to bacterial and protozoan nucleoside hydrolases is active as a pyrimidine nucleosidase but shows the highest specificity toward the NAD+ metabolite nicotinamide riboside. Crystal structures of the trichomonal riboside hydrolase in different states reveals novel intermediates along the nucleoside hydrolase-catalyzed hydrolytic reaction, including an unexpected asymmetry in the homotetrameric assembly. The active site structure explains the broad specificity toward different ribosides and offers precise insights for the engineering of specific inhibitors that may simultaneously target different essential pathways in the parasite.
Assuntos
Hidrolases , Parasitos , Trichomonas vaginalis , Animais , Hidrolases/química , Hidrolases/metabolismo , NAD/metabolismo , Niacinamida/metabolismo , Trichomonas vaginalis/enzimologia , Cristalografia por Raios X , Especificidade por Substrato , Estrutura Terciária de Proteína , Modelos Moleculares , Ligação ProteicaRESUMO
Trichomoniasis is caused by the parasitic protozoan Trichomonas vaginalis. The increasing prevalence of strains resistant to the current 5-nitroimidazole treatments creates the need for novel therapies. T. vaginalis cannot synthesize purine and pyrimidine rings and requires salvage pathway enzymes to obtain them from host nucleosides. The uridine nucleoside ribohydrolase was screened using an 19F NMR-based activity assay against a 2000-compound fragment diversity library. Several series of inhibitors were identified including scaffolds based on acetamides, cyclic ureas or ureas, pyridines, and pyrrolidines. A number of potent singleton compounds were identified, as well. Eighteen compounds with IC50 values of 20 µM or lower were identified, including some with ligand efficiency values of 0.5 or greater. Detergent and jump-dilution counter screens validated all scaffold classes as target-specific, reversible inhibitors. Identified scaffolds differ substantially from 5-nitroimidazoles. Medicinal chemistry using the structure-activity relationship emerging from the fragment hits is being pursued to discover nanomolar inhibitors.
RESUMO
Trichomoniasis is caused by the parasitic protozoan Trichomonas vaginalis and is the most prevalent, nonviral sexually transmitted disease. The parasite has shown increasing resistance to the current 5-nitroimidazole therapies indicating the need for new therapies with different mechanisms. T. vaginalis is an obligate parasite that scavenges nucleosides from host cells and then uses salvage pathway enzymes to obtain the nucleobases. The adenosine/guanosine preferring nucleoside ribohydrolase was screened against a 2000-compound diversity fragment library using a 1H NMR-based activity assay. Three classes of inhibitors with more than five representatives were identified: bis-aryl phenols, amino bicyclic pyrimidines, and aryl acetamides. Among the active fragments were 10 compounds with ligand efficiency values greater than 0.5, including five with IC50 values <10 µM. Jump-dilution and detergent counter screens validated reversible, target-specific activity. The data reveals an emerging SAR that is guiding our medicinal chemistry efforts aimed at discovering compounds with nanomolar potency.
Assuntos
Antiprotozoários/química , Inibidores Enzimáticos/química , N-Glicosil Hidrolases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Trichomonas vaginalis/enzimologia , Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Ligantes , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/química , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/genéticaRESUMO
Trichomonas vaginalis infects approximately 300 million people worldwide annually. Infected individuals have a higher susceptibility to more serious conditions such as cervical and prostate cancer. The parasite has developed increasing resistance to current drug therapies, with an estimated 5% of clinical cases resulting from resistant strains, creating the need for new therapeutic strategies with novel mechanisms of action. Nucleoside salvage pathway enzymes represent novel drug targets as these pathways are essential for the parasite's survival. The guanosine/adenosine/cytidine nucleoside hydrolase (GACNH) may be particularly important as its expression is upregulated under glucose-limiting conditions mimicking those that occur during infection establishment. GACNH was screened against the NIH Clinical Collection to explore its druggability. Seven compounds were identified with IC50 values <20 µM. Extensive overlap was found between inhibitors of GACNH and the adenosine/guanosine nucleoside hydrolase (AGNH), but no overlap was found with inhibitors of the uridine nucleoside hydrolase. The guanosine analog ribavirin was the only compound found to be specific for GACNH. Compounds that inhibit both AGNH and GACNH purine salvage pathway enzymes may prove critical given the role that GACNH appears to play in the early stages of infection.
Assuntos
N-Glicosil Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/enzimologia , Adenosina/análogos & derivados , Adenosina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Concentração Inibidora 50 , N-Glicosil Hidrolases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Quercetina/análogos & derivados , Quercetina/química , Quercetina/metabolismo , Ribavirina/química , Ribavirina/metabolismo , Relação Estrutura-AtividadeRESUMO
Nucleoside salvage pathway enzymes used by Trichomonas vaginalis are distinct from the pathway involved in activation of existing 5-nitroimidazole drugs. They thus represent excellent targets for developing novel, mechanism-based antitrichomonal agents. The purine-specific adenosine/guanosine preferring ribohydrolase (AGNH) was screened against the NIH Clinical Collection to assess its druggability. Eight compounds, including five flavonoids, were identified with IC50 values ⩽10 µM and confirmed in counter screens run in the presence of detergent. The inhibitors are structurally distinct from inhibitors of the pyrimidine-specific uridine ribohydrolase (UNH) thus indicating that AGNH is a distinct, druggable target from UNH.
Assuntos
Antitricômonas/química , Flavonoides/química , N-Glicosil Hidrolases/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosina/química , Ensaios de Triagem em Larga Escala , Quercetina/análogos & derivados , Quercetina/química , Bibliotecas de Moléculas Pequenas/química , Estereoisomerismo , Trichomonas vaginalisRESUMO
Trichomonas vaginalis continues to be a major health problem with drug-resistant strains increasing in prevalence. Novel antitrichomonal agents that are mechanistically distinct from current therapies are needed. The NIH Clinical Compound Collection was screened to find inhibitors of the uridine ribohydrolase enzyme required by the parasite to scavenge uracil for its growth. The proton-pump inhibitors omeprazole, pantoprazole, and rabeprazole were identified as inhibitors of this enzyme, with IC50 values ranging from 0.3 to 14.5 µM. This suggests a molecular mechanism for the in vitro antitrichomonal activity of these proton-pump inhibitors, and may provide important insights toward structure-based drug design.
Assuntos
2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , N-Glicosil Hidrolases/antagonistas & inibidores , Omeprazol/farmacologia , Inibidores da Bomba de Prótons/farmacologia , Rabeprazol/farmacologia , Trichomonas vaginalis/enzimologia , 2-Piridinilmetilsulfinilbenzimidazóis/síntese química , 2-Piridinilmetilsulfinilbenzimidazóis/química , Relação Dose-Resposta a Droga , Estrutura Molecular , N-Glicosil Hidrolases/metabolismo , Omeprazol/síntese química , Omeprazol/química , Pantoprazol , Inibidores da Bomba de Prótons/síntese química , Inibidores da Bomba de Prótons/química , Rabeprazol/síntese química , Rabeprazol/química , Relação Estrutura-AtividadeRESUMO
Trypanosomes are purine-auxotrophic parasites that depend upon nucleoside hydrolase (NH) activity to salvage nitrogenous bases necessary for nucleic acid and cofactor synthesis. Nonspecific and purine-specific NHs have been widely studied, yet little is known about the 6-oxopurine-specific isozymes, although they are thought to play a primary role in the catabolism of exogenously derived nucleosides. Here, we report the first functional and structural characterization of the inosine-guanosine-specific NH from Trypanosoma brucei brucei. The enzyme shows near diffusion-limited efficiency coupled with a clear specificity for 6-oxopurine nucleosides achieved through a catalytic selection of these substrates. Pre-steady-state kinetic analysis reveals ordered product release, and a rate-limiting structural rearrangement that is associated with the release of the product, ribose. The crystal structure of this trypanosomal NH determined to 2.5 Å resolution reveals distinctive features compared to those of both purine- and pyrimidine-specific isozymes in the framework of the conserved and versatile NH fold. Nanomolar iminoribitol-based inhibitors identified in this study represent important lead compounds for the development of novel therapeutic strategies against trypanosomal diseases.