RESUMO
ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited efficacy. Here, we report the in vitro study of Abcb11 missense variants identified in PFIC2 patients and their functional rescue using cystic fibrosis transmembrane conductance regulator potentiators. Three ABCB11 disease-causing variations identified in PFIC2 patients (i.e., A257V, T463I and G562D) were reproduced in a plasmid encoding an Abcb11-green fluorescent protein. After transfection, the expression and localization of the variants were studied in HepG2 cells. Taurocholate transport activity and the effect of potentiators were studied in Madin-Darby canine kidney (MDCK) clones coexpressing Abcb11 and the sodium taurocholate cotransporting polypeptide (Ntcp/Slc10A1). As predicted using three-dimensional structure analysis, the three variants were expressed at the canalicular membrane but showed a defective function. Ivacaftor, GLP1837, SBC040 and SBC219 potentiators increased the bile acid transport of A257V and T463I and to a lesser extent, of G562D Abcb11 missense variants. In addition, a synergic effect was observed when ivacaftor was combined with SBC040 or SBC219. Such potentiators could represent new pharmacological approaches for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the function of the transporter.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Regulador de Condutância Transmembrana em Fibrose Cística , Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminofenóis , Animais , Colestase Intra-Hepática , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Cães , Proteínas de Fluorescência Verde/metabolismo , Quinolonas , Ácido Taurocólico/farmacologiaRESUMO
The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary center at C2' or C3' is described. The construction of this all-carbon stereogenic center involves the use of an intramoleculer photoredox-catalyzed reaction. The nucleoside analogues (NA) hydroxyl functional group at C2' was generated by diastereoselective epoxidation. In addition, highly enantioselective and diastereoselective Mukaiyama aldol reactions, diastereoselective N-glycosylations and regioselective triphosphorylation reactions were employed to synthesize the novel NTPs. Two of these compounds are inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the causal virus of COVID-19.
Assuntos
Antivirais/farmacologia , Carbono/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Nucleotídeos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/enzimologia , Antivirais/síntese química , Antivirais/química , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Nucleotídeos/síntese química , Nucleotídeos/química , SARS-CoV-2/efeitos dos fármacos , EstereoisomerismoRESUMO
Protein misfolding is involved in a large number of diseases, among which cystic fibrosis. Complex intra- and inter-domain folding defects associated with mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, among which p.Phe508del (F508del), have recently become a therapeutical target. Clinically approved correctors such as VX-809, VX-661, and VX-445, rescue mutant protein. However, their binding sites and mechanisms of action are still incompletely understood. Blind docking onto the 3D structures of both the first membrane-spanning domain (MSD1) and the first nucleotide-binding domain (NBD1), followed by molecular dynamics simulations, revealed the presence of two potential VX-809 corrector binding sites which, when mutated, abrogated rescue. Network of amino acids in the lasso helix 2 and the intracellular loops ICL1 and ICL4 allosterically coupled MSD1 and NBD1. Corrector VX-445 also occupied two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. Binding of both correctors on MSD1 enhanced the allostery between MSD1 and NBD1, hence the increased efficacy of the corrector combination. These correctors improve both intra-domain folding by stabilizing fragile protein-lipid interfaces and inter-domain assembly via distant allosteric couplings. These results provide novel mechanistic insights into the rescue of misfolded proteins by small molecules.
Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Fibrose Cística/tratamento farmacológico , Mutação , Dobramento de Proteína/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Pirrolidinas/farmacologia , Sítios de Ligação , Agonistas dos Canais de Cloreto/farmacologia , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Quimioterapia Combinada , Células HEK293 , Humanos , Domínios Proteicos , Estrutura Terciária de ProteínaRESUMO
Radical thiol-ene coupling was exploited for the first time to prepare imino-disaccharides and multivalent iminosugars starting from sugar thiols and iminosugar alkenes or iminosugar thiols and tetra-allylated calixarene, respectively.
Assuntos
Alcenos/química , Dissacarídeos/síntese química , Imino Açúcares/síntese química , Compostos de Sulfidrila/química , Alcenos/efeitos da radiação , Calixarenos/química , Calixarenos/efeitos da radiação , Radicais Livres/química , Compostos de Sulfidrila/efeitos da radiação , Raios UltravioletaRESUMO
Recent evidence shows that combination of correctors and potentiators, such as the drug ivacaftor (VX-770), can significantly restore the functional expression of mutated Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an anion channel which is mutated in cystic fibrosis (CF). The success of these combinatorial therapies highlights the necessity of identifying a broad panel of specific binding mode modulators, occupying several distinct binding sites at structural level. Here, we identified two small molecules, SBC040 and SBC219, which are two efficient cAMP-independent potentiators, acting at low concentration of forskolin with EC50 close to 1 µM and in a synergic way with the drug VX-770 on several CFTR mutants of classes II and III. Molecular dynamics simulations suggested potential SBC binding sites at the vicinity of ATP-binding sites, distinct from those currently proposed for VX-770, outlining SBC molecules as members of a new family of potentiators.
Assuntos
Benzamidas/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Purinas/farmacologia , Aminofenóis/farmacologia , Benzamidas/síntese química , Benzamidas/metabolismo , Sítios de Ligação , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Sinergismo Farmacológico , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Purinas/síntese química , Purinas/metabolismo , Quinolonas/farmacologiaRESUMO
2-aminothiophene derivatives (2AT) in which the thiophene ring is fused with a cycloalkyl or a N-acylated piperidine ring by positions 5 and 6 and carrying a 3-carbethoxy group were synthesized and their bacterial growth and enzyme inhibitory effects against efflux proteins of Staphylococcus aureus leading to resistance to fluoroquinolones and erythromycin (ERY) were investigated. Compounds that most effectively decreases the minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) were assayed for their dose and time effects on the accumulation and efflux of ethidium bromide (EtBr) in the SA-1 strain. None of the compounds displayed antibacterial activity however, three derivatives carrying 2-amino, 2-aminoacetyl and 2-aminotrifluoroacetyl group enhanced the activity of CIP and ERY by 8- and 16-fold, respectively, and were able to restore the sensitivity of resistant strains, acting as typical efflux pump inhibitors (EPIs). The 2-aminoacetyl and 2-aminotrifluoroacetyl derivatives and two other piperidinyl 2-aminotrifluoroacetyl derivatives increased EtBr accumulation in a dose- and time-dependent manner, and one of them was also able to inhibit the EtBr efflux. Taken together, these results represent an important advance in the development of new EPIs, and demonstrate that 2AT represent a good scaffold for developing new antibiotic adjuvants.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Staphylococcus aureus/efeitos dos fármacos , Tiofenos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Tiofenos/síntese química , Tiofenos/químicaRESUMO
A one-pot glycosylation and cyclization procedure is described for the synthesis of 6-chloropurine ribonucleosides from chloropyrimidines. From such a procedure and modification of the obtained chloropurine ribonucleosides, many drug candidates or molecular tools for biological study designed from their similarity to naturally occurring nucleosides could be obtained. The synthesis begins by preparation of several amidinoaminochloropyrimidines as precursors for the one-pot procedure. Then, by adding trimethylsilyl trifluoromethanesulfonate (TMSOTf) to a mixture of a pyrimidine and 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D-ribose, different 6-chloropurine ribonucleosides are obtained. This methodology allows the straightforward introduction of an alkyl substituent at position 8 of purine ribonucleosides, which then can be functionalized at positions 2 and 6. © 2018 by John Wiley & Sons, Inc.
Assuntos
Purinas/química , Pirimidinas/química , Ribonucleosídeos/síntese química , Ribonucleosídeos/químicaRESUMO
Highly substituted purines were synthesized in good to high yields through a one-pot straightforward metal-free scalable method, using the Traube synthesis adapted to Vilsmeier-type reagents. From 5-amino-4-chloropyrimidines, new 9-aryl-substituted chloropurines and intermediates for peptide nucleic acid synthesis were prepared. Variant procedures allowing a rapid synthesis of ribonucleosides and 7-benzylpurine from 5-amidino-6-aminopyrimidines are also reported to illustrate the high potential of this versatile toolbox. This route appears to be particularly interesting in the field of nucleic acids for a direct and rapid access to various new 8-alkylpurine nucleosides.
RESUMO
Multivalent nanoconstructs, extensively used for enhancing the recognition of biomolecular targets, have been recently exploited for enzyme inhibition showing interesting properties such as improvement of inhibitory potency and selectivity. We review herein the recent results highlighting the potential of multivalent nanoconstructs for the inhibition of different enzymes, and the emerging trends in the generation and identification of multivalent clusters as enzyme inhibitors.