RESUMO
trans-Sialidase from Trypanosoma cruzi (TcTS) has emerged as a potential drug target for treatment of Chagas disease. Here, we report the results of virtual screening for the discovery of novel TcTS inhibitors, which targeted both the sialic acid and sialic acid acceptor sites of this enzyme. A library prepared from the Evotec database of commercially available compounds was screened using the molecular docking program GOLD, following the application of drug-likeness filters. Twenty-three compounds selected from the top-scoring ligands were purchased and assayed using a fluorimetric assay. Novel inhibitor scaffolds, with IC(50) values in the submillimolar range were discovered. The 3-benzothiazol-2-yl-4-phenyl-but-3-enoic acid scaffold was studied in more detail, and TcTS inhibition was confirmed by an alternative sialic acid transfer assay. Attempts to obtain crystal structures of these compounds with TcTS proved unsuccessful but provided evidence of ligand binding at the active site.
Assuntos
Química Farmacêutica/métodos , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Glicoproteínas/antagonistas & inibidores , Neuraminidase/antagonistas & inibidores , Animais , Sítios de Ligação , Domínio Catalítico , Química Farmacêutica/instrumentação , Cristalização , Cristalografia por Raios X/métodos , Inibidores Enzimáticos/química , Glicoproteínas/química , Concentração Inibidora 50 , Cinética , Ligantes , Modelos Químicos , Ácido N-Acetilneuramínico/química , Neuraminidase/química , Trypanosoma cruziRESUMO
A series of novel imidazolyluracil conjugates were rationally designed and synthesised to probe the active site constraints of the angiogenic enzyme, thymidine phosphorylase (TP, E.C. 2.4.2.4). The lead compound in the series, 15d, showed good binding in the active site of human TP with an inhibition in the low muM range. The absence of a methylene bridge between the uracil and the imidazolyl subunits (series 16) decreased potency (up to 3-fold). Modelling suggested that active site residues Arg202, Ser217 and His116 are important for inhibitor binding.
Assuntos
Inibidores Enzimáticos/farmacologia , Timidina Fosforilase/antagonistas & inibidores , Uracila/análogos & derivados , Uracila/farmacologia , Sítios de Ligação , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Relação Estrutura-Atividade , Timidina Fosforilase/metabolismo , Uracila/síntese químicaRESUMO
Thymidine phosphorylase (TP) is an important target enzyme for cancer chemotherapy because it is expressed at high levels in the hypoxic regions of many tumors and inhibitors of TP have been shown in animal model studies to inhibit angiogenesis and metastasis, and to promote tumor cell apoptosis. The 5-halo-6-[(2'-aminoimidazol-1'-yl)methyl]uracils (3, X = Cl, Br) are very potent inhibitors of E. coli and human TP with IC(50) values of approximately 20 nM when the enzyme concentration is approximately 40 nM. Their 4'-aminoimidazol-1'-yl analogues (4, X = Cl, Br) are >350-fold less active with IC(50) values of approximately 7 microM. The 5-unsubstituted analogues (3 and 4, X = H) were both less active than their 5-halo derivatives. Determination of pK(a) values and molecular modeling studies of these compounds in the active site of human TP was used to rationalize their activities. The finding that 3, X = Br has a poor pharmacokinetic (PK) profile in mice, coupled with the desire for tumor selectivity, led us to design prodrugs. The corresponding 2'-nitroimidazol-1'-ylmethyluracils (5, X = Cl, Br) are >1000-fold less active (IC(50) 22-24 microM) than their 2'-amino analogues and are reduced to the 2'-amino inhibitors (3, X = Cl, Br) by xanthine oxidase (XO). As XO is also highly expressed in many tumors, the 2'-nitro prodrugs have the potential to selectively deliver the potent 2'-aminoimidazol-1'-yl TP inhibitors into hypoxic solid tumors.
Assuntos
Antineoplásicos/síntese química , Imidazóis/síntese química , Pró-Fármacos/síntese química , Timidina Fosforilase/antagonistas & inibidores , Uracila/análogos & derivados , Uracila/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Disponibilidade Biológica , Escherichia coli/química , Humanos , Imidazóis/química , Imidazóis/farmacologia , Camundongos , Modelos Moleculares , Nitroimidazóis/síntese química , Nitroimidazóis/química , Nitroimidazóis/farmacologia , Oxirredução , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Uracila/farmacologiaRESUMO
Thymidine phosphorylase (TP) is an angiogenic growth factor and a target for anticancer drug design. Molecular modeling suggested that 2'-aminoimidazolylmethyluracils would be potent inhibitors of TP. The novel 5-halo-2-aminoimidazolylmethyluracils (4b/4c) were very potent inhibitors of E. coli TP (IC50 approximately 20 nM). Contrastingly, the corresponding 2'-nitroimidazolylmethyluracil (as bioreductively activated) prodrugs (3b/3c) were 1000-fold less active (IC50 22-24 microM). This approach may be used to selectively deliver TP inhibitors into hypoxic regions of solid tumors where TP is overexpressed.
Assuntos
Inibidores da Angiogênese/síntese química , Inibidores Enzimáticos/síntese química , Imidazóis/síntese química , Timidina Fosforilase/antagonistas & inibidores , Uracila/análogos & derivados , Uracila/síntese química , Inibidores da Angiogênese/química , Sítios de Ligação , Inibidores Enzimáticos/química , Escherichia coli/química , Humanos , Imidazóis/química , Imidazóis/farmacologia , Modelos Moleculares , Relação Estrutura-Atividade , Timidina Fosforilase/química , Uracila/químicaRESUMO
Benzoic acid and pyridine derivatives inhibit recombinant trans-sialidase from Trypanosoma cruzi with I50 values between 0.4 and 1mM. The best compounds, 4-acetylamino-3-hydroxymethylbenzoic acid and 5-acetylamino-6-aminopyridine-2-carboxylic acid, provide new leads to inhibitors not containing the synthetically complex sialic acid structure. The weak inhibition by such compounds contrasts with their much stronger inhibition of neuraminidase from Influenza virus.
Assuntos
Ácido Benzoico/farmacologia , Inibidores Enzimáticos/farmacologia , Glicoproteínas/antagonistas & inibidores , Neuraminidase/antagonistas & inibidores , Piridinas/farmacologia , Trypanosoma cruzi/enzimologia , Animais , Ácido Benzoico/química , Inibidores Enzimáticos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Piridinas/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Thymidine phosphorylase (TP) is an important target enzyme for cancer chemotherapy but currently available inhibitors lack in vivo potency. Related enzymes also are therapeutic targets. A greater understanding of enzyme structure and mechanism may help in the design of improved drugs and this work assists in that regard. Also important is the correct identification of the ionization states and tautomeric forms of substrates and products when bound to the enzyme and during the course of the reaction. Approximate methods for estimating some deltapK(a)s between aqueous and protein-bound substrates are exemplified for nucleobases and nucleosides. The estimates demonstrate that carbonyl-protonated thymidine and hydroxy tautomers of thymine are not involved in TP's actions. Other estimates indicate that purine nucleoside phosphorylase binds inosine and guanosine as zwitterionic tautomers and that phosphorolysis proceeds through these forms. Extensive molecular modeling based on an X-ray structure of human TP indicates that TP is likely to be mechanistically similar to all other natural members of the class in proceeding through a alpha-oxacarbenium-like transition state or states.
Assuntos
Modelos Biológicos , Timidina Fosforilase/metabolismo , Animais , Sítios de Ligação , Humanos , Cinética , Fósforo/química , Fósforo/metabolismo , Especificidade por Substrato , Timidina Fosforilase/químicaRESUMO
Thymidine phosphorylase (EC 2.4.2.4) catalyses the reversible phosphorolysis of pyrimidine 2'-deoxynucleosides, forming 2-deoxyribose-1-phosphate and pyrimidine. 5-Chloro-6-(2-imino-pyrrolidin-1-yl)methyl-uracil hydrochloride (TPI, 1) and its 5-bromo analogue (2), 6-(2-amino-imidazol-1-yl)methyl-5-bromo-uracil (3) and its 5-chloro analogue (4) act as tight-binding stoichiometric inhibitors of recombinant E. coli thymidine phosphorylase, and thus can be used as the first active-site titrants for it using either thymidine or 5-nitro-2'-deoxyuridine as substrate.