RESUMO
Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2-BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.
Assuntos
Antineoplásicos/síntese química , Compostos Bicíclicos com Pontes/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Mitocondriais/química , Neoplasias/tratamento farmacológico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/toxicidade , Proteínas Reguladoras de Apoptose , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/toxicidade , Linhagem Celular Tumoral , Simulação por Computador , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
We investigated the specificity of glycosyltransferases toward donor substrates in two complementary directions. First we prepared simple N-acetyl-alpha-D-glucosamine 1-diphosphates: methyl-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-diphosphate, benzyl-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-diphosphate, 4-phenylbutyl-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-diphosphate, by the coupling of the corresponding activated alkyl phosphates with N-acetyl-alpha-D-glucosamine 1-phosphate. These diphosphates as well as 2-acetamido-2-deoxy-alpha-D-glucopyranose 1-diphosphate, tested as donors of N-acetylglucosamine in a reaction catalyzed by Neisseria meningitidis N-acetylglucosaminyltransferase (LgtA), proved to be devoid of activity. Evaluated as inhibitors, only 2-acetamido-2-deoxy-alpha-D-glucopyranose 1-diphosphate showed some inhibitory activity with an IC50 value of 7 mM. In the second approach, we prepared sugar nucleotide mimics having the diphosphate bridge replaced by the oxycarbonylaminosulfonyl linker. The surrogate of GDP-Fuc was synthesized as a 9:1 alpha/beta anomeric mixture, in 40% yield, starting from chlorosulfonyl isocyanate, perbenzylated l-fucopyranose, and a guanosine derivative, protected on the exocyclic amine and secondary hydroxyl functions of ribose. Then two deprotection steps, hydrogenolysis and enzymatic hydrolysis catalyzed by penicillin G amidase afforded the target molecule to be tested as fucose donor with recombinant human alpha-(1-->3/4)-fucosyltransferase (FucT-III). Tested as a 4:1 alpha/beta anomeric mixture, both in the absence and in the presence of cationic cofactors, this new guanosine fucose conjugate proved to be ineffective. Its inhibitory activity toward FucT-III evaluated through a competition fluorescence assay was very poor (IC50 value of 20 mM). The surrogate of UDP-GlcNAc that was already known as its protected acetylated derivative, tested as N-acetylglucosamine donor with LgtA in the presence of Mn(2+) turned out not to be active either.
Assuntos
Glicosiltransferases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicosiltransferases/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Açúcares de Nucleosídeo Difosfato/síntese química , Açúcares de Nucleosídeo Difosfato/química , Açúcares de Nucleosídeo Difosfato/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
An efficient and convergent method for the synthesis of mannose-1-phosphate prodrugs is described as a potential therapy for congenital disorders of glycosylation-Ia (CDG-Ia). The key feature of the proposed approach is the silver assisted nucleophilic substitution of 2,3,4,6-tetra-O-protected-alpha-d-mannopyranosyl bromides with various silver phosphate salts to afford mono, di, and tri-mannopyranosyl phosphates. A preliminary biological evaluation of the synthesized phosphate prodrugs has been carried out.
Assuntos
Defeitos Congênitos da Glicosilação/tratamento farmacológico , Manosefosfatos/química , Organofosfatos/química , Pró-Fármacos/síntese química , Sequência de Carboidratos , Humanos , Dados de Sequência Molecular , Organofosfatos/síntese química , Organofosfatos/uso terapêutico , Pró-Fármacos/química , Pró-Fármacos/uso terapêuticoRESUMO
New unnatural sugar nucleotides, UDP-Fuc and CDP-Fuc were synthesized from fucose-beta-1-phosphate and nucleotide monophosphates activated as morpholidates. Furthermore, a nucleotide analogue was prepared by phosphorylation of 1-(beta-D-ribofuranosyl)cyanuric acid, itself obtained as a protected derivative by condensation of the persilylated derivative of cyanuric acid with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in 74% yield. This phosphate activated according to the same procedure was condensed with fucose-beta-1-phosphate, affording a new sugar nucleotide conjugate (NDP-Fuc) which was evaluated together with UDP-Fuc, CDP-Fuc and ADP-Fuc, as fucose donors in alpha-(1-->4/3)-fucosyltransferase (FucT-III) catalyzed reaction. Fucose transfer could be observed with each of the donors and kinetic parameters were determined using a fluorescent acceptor substrate. Efficiency of the four analogues towards FucT-III was in the following order: UDP-Fuc=ADP-Fuc>NDP-Fuc>CDP-Fuc. According to the same strategy ADP-GlcNAc was prepared from AMP-morpholidate and N-acetylglucosamine-alpha-1-phosphate; tested as a glucosaminyl donor towards Neisseria meningitidis N-acetylglucosaminyl transferase (LgtA), ADP-GlcNAc was recognized with 0.1% efficiency as compared with UDP-GlcNAc, the natural donor substrate.
