RESUMO
Atrial fibrillation (AF) is the most common cardiac arrhythmia, and a significant risk factor for ischemic stroke and heart failure. Marketed anti-arrhythmic drugs can restore sinus rhythm, but with limited efficacy and significant toxicities, including potential to induce ventricular arrhythmia. Atrial-selective ion channel drugs are expected to restore and maintain sinus rhythm without risk of ventricular arrhythmia. One such atrial-selective channel target is GIRK1/4 (G-protein regulated inwardly rectifying potassium channel 1/4). Here we describe 14b, a potent GIRK1/4 inhibitor developed to cardiovert AF to sinus rhythm while minimizing central nervous system exposure - an issue with preceding GIRK1/4 clinical candidates.
Assuntos
Fibrilação Atrial , Humanos , Fibrilação Atrial/tratamento farmacológico , Cardioversão Elétrica , Átrios do Coração , EncéfaloRESUMO
Synthetic modification of cyclosporin A at P3-P4 positions led to the discovery of NIM258, a next generation cyclophilin inhibitor with excellent anti-hepatitis C virus potency, with decreased transporter inhibition, and pharmacokinetics suitable for coadministration with other drugs. Herein is disclosed the evolution of the synthetic strategy to from the original medicinal chemistry route, designed for late diversification, to a convergent and robust development synthesis. The chiral centers in the P4 fragment were constructed by an asymmetric chelated Claisen rearrangement in the presence of quinidine as the chiral ligand. Identification of advanced crystalline intermediates enabled practical supply of key intermediates. Finally, macrocyclization was carried out at 10% weight concentration by a general and unconventional "slow release" concept.
Assuntos
Antivirais/química , Ciclosporina/química , Hepacivirus/fisiologia , Antivirais/síntese química , Antivirais/farmacologia , Ciclização , Ciclosporina/síntese química , Ciclosporina/farmacologia , Dipeptídeos/síntese química , Dipeptídeos/química , Desenho de Fármacos , Quinidina/química , Estereoisomerismo , Replicação Viral/efeitos dos fármacosRESUMO
The continuing emergence of antibacterial resistance reduces the effectiveness of antibiotics and drives an ongoing search for effective replacements. Screening compound libraries for antibacterial activity in standard growth media has been extensively explored and may be showing diminishing returns. Inhibition of bacterial targets that are selectively important under in vivo (infection) conditions and, therefore, would be missed by conventional in vitro screens might be an alternative. Surrogate host models of infection, however, are often not suitable for high-throughput screens. Here, we adapted a medium-throughput Tetrahymena pyriformis surrogate host model that was successfully used to identify inhibitors of a hyperviscous Klebsiella pneumoniae strain to a high-throughput format and screened circa 1.2 million compounds. The screen was robust and identified confirmed hits from different chemical classes with potent inhibition of K. pneumoniae growth in the presence of T. pyriformis that lacked any appreciable direct antibacterial activity. Several of these appeared to inhibit capsule/mucoidy, which are key virulence factors in hypervirulent K. pneumoniae. A weakly antibacterial inhibitor of LpxC (essential for the synthesis of the lipid A moiety of lipopolysaccharides) also appeared to be more active in the presence of T. pyriformis, which is consistent with the role of LPS in virulence as well as viability in K. pneumoniae.
RESUMO
Over the past several decades, the frequency of antibacterial resistance in hospitals, including multidrug resistance (MDR) and its association with serious infectious diseases, has increased at alarming rates. Pseudomonas aeruginosa is a leading cause of nosocomial infections, and resistance to virtually all approved antibacterial agents is emerging in this pathogen. To address the need for new agents to treat MDR P. aeruginosa, we focused on inhibiting the first committed step in the biosynthesis of lipid A, the deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by the enzyme LpxC. We approached this through the design, synthesis, and biological evaluation of novel hydroxamic acid LpxC inhibitors, exemplified by 1, where cytotoxicity against mammalian cell lines was reduced, solubility and plasma-protein binding were improved while retaining potent anti-pseudomonal activity in vitro and in vivo.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Amidoidrolases/química , Animais , Antibacterianos/síntese química , Técnicas de Química Sintética , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Feminino , Células Hep G2/efeitos dos fármacos , Humanos , Células K562/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Relação Estrutura-AtividadeRESUMO
Nonimmunosuppressive cyclophilin inhibitors have demonstrated efficacy for the treatment of hepatitis C infection (HCV). However, alisporivir, cyclosporin A, and most other cyclosporins are potent inhibitors of OATP1B1, MRP2, MDR1, and other important drug transporters. Reduction of the side chain hydrophobicity of the P4 residue preserves cyclophilin binding and antiviral potency while decreasing transporter inhibition. Representative inhibitor 33 (NIM258) is a less potent transporter inhibitor relative to previously described cyclosporins, retains anti-HCV activity in cell culture, and has an acceptable pharmacokinetic profile in rats and dogs. An X-ray structure of 33 bound to rat cyclophilin D is reported.
Assuntos
Antivirais/química , Antivirais/farmacologia , Ciclofilinas/antagonistas & inibidores , Ciclosporinas/farmacologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/farmacocinética , Técnicas de Química Sintética , Cristalografia por Raios X , Peptidil-Prolil Isomerase F , Ciclofilinas/química , Ciclofilinas/metabolismo , Ciclosporina/química , Ciclosporina/farmacologia , Ciclosporinas/química , Cães , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunossupressores/química , Imunossupressores/farmacologia , Transportador 1 de Ânion Orgânico Específico do Fígado , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Ratos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacosRESUMO
5'-S-(2-aminoethyl)-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), 5'-S-(2-acetamidoethyl)-6-N-[(4-substituted)benzyl]-5'-thioadenosine analogues, 5'-S-[2-(6-aminohexanamido)]ethyl-6-N-(4-nitrobenzyl)-5'-thioadenosine (SAHENTA), and related compounds were synthesized by S(N)Ar displacement of fluoride from 6-fluoropurine intermediates with 4-(substituted)benzylamines. Conjugation of the pendant amino groups of SAENTA and SAHENTA with fluorescein-5-yl isothiocyanate (FITC) gave fluorescent probes that bound at nanomolar concentrations specifically to human equilibrative nucleoside transporter 1 (hENT1) produced in recombinant form in model expression systems and in native form in cancer cell lines. Transporter binding effects were studied and the ability of the probes to predict the potential antitumor efficacy of 2'-deoxy-2',2'-difluorocytidine (gemcitabine) was demonstrated.
Assuntos
Adenosina/análogos & derivados , Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/síntese química , Corantes Fluorescentes/síntese química , Tionucleosídeos/síntese química , Adenosina/síntese química , Adenosina/química , Animais , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/química , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Humanos , Camundongos , Oócitos/metabolismo , Ligação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Tionucleosídeos/química , Xenopus , Leveduras/efeitos dos fármacos , Leveduras/metabolismo , GencitabinaRESUMO
To understand the mechanism of cellular resistance to the nucleoside analogue cytarabine (1-beta-D-arabinofuranosylcytosine, AraC), two resistant derivatives of the human leukemic line CCRF-CEM were obtained by stepwise selection in different concentrations of AraC. CEM/4xAraC cells showed low AraC resistance, whereas CEM/20xAraC cells showed high resistance. Both cell lines showed similar patterns of cross-resistance to multiple cytotoxic nucleoside analogues, with the exception that CEM/20xAraC cells remained sensitive to 5-fluorouridine and 2-deoxy-5-fluorouridine. Both cell lines were sensitive to 5-fluorouracil and to a variety of natural product drugs. Although both CEM/4xAraC and CEM/20xAraC cells displayed reduced intracellular accumulation of [(3)H]AraC, only CEM/4xAraC cells showed reduced uptake of [(3)H]uridine, which was used to assess nucleoside transport activities. Genes encoding proteins known to be involved in nucleoside transport, efflux, and metabolism were analyzed for the presence of mutations in the two cell lines. In CEM/4xAraC cells, independent mutations were identified at each allele of human equilibrative nucleoside transporter 1 (hENT1; SLC29A1), one corresponding to a single-nucleotide change in exon 4, the other being a complex intronic mutation disrupting splicing of exon 13. In contrast to CEM/20xAraC cells, CEM/4xAraC cells did not bind the hENT1/SLC29A1 ligand nitrobenzylmercaptopurine ribonucleoside and lacked detectable hENT1/SLC29A1 protein. In CEM/20xAraC cells, independent intronic mutations impairing splicing of exons 2 and 3 were found at each allele of the deoxycytidine kinase gene. These studies point to at least two distinct mechanisms of AraC resistance in leukemic cells.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Citarabina/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Antimetabólitos Antineoplásicos/farmacocinética , Citarabina/farmacocinética , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Transportador Equilibrativo 1 de Nucleosídeo/biossíntese , Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
Equilibrative nucleoside transporters (ENTs) are important for the metabolic salvage of nucleosides and the cellular uptake of antineoplastic and antiviral nucleoside analogs. Human equilibrative nucleoside transporter 1 (hENT1) is inhibited by nanomolar concentrations of structurally diverse compounds, including dipyridamole, dilazep, nitrobenzylmercaptopurine ribonucleoside (NBMPR), draflazine, and soluflazine. Random mutagenesis and screening by functional complementation for inhibitor-resistant mutants in yeast revealed mutations at Phe-334 and Asn-338. Both residues are predicted to lie in transmembrane segment 8 (TM 8), which contains residues that are highly conserved in the ENT family. F334Y displayed increased V(max) values that were attributed to increased rates of catalytic turnover, and N338Q and N338C displayed altered membrane distributions that appeared to be because of protein folding defects. Mutations of Phe-334 or Asn-338 impaired interactions with dilazep and dipyridamole, whereas mutations of Asn-338 impaired interactions with draflazine and soluflazine. A helical wheel projection of TM 8 predicted that Phe-334 and Asn-338 lie in close proximity to other highly conserved and/or hydrophilic residues, suggesting that they form part of a structurally important region that influences interactions with inhibitors, protein folding, and rates of conformational change during the transport cycle.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Compostos Heterocíclicos/farmacologia , Conformação Proteica , Dobramento de Proteína , Adenosina/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Dilazep/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/química , Transportador Equilibrativo 1 de Nucleosídeo/genética , Teste de Complementação Genética , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese , Mutação , Nucleosídeos/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Tioinosina/farmacologiaRESUMO
To better understand nucleoside transport processes and intracellular fates of nucleosides, we have developed a pair of fluorescent nucleoside analogues, FuPmR and dFuPmR, that differ only in the sugar moiety (ribofuranosyl versus 2'-deoxy, respectively), for real-time analysis of nucleoside transport into living cells by confocal microscopy. The binding and transportability of the two compounds were assessed for five recombinant human nucleoside transporters (hENT1/2, hCNT1/2/3) produced in Saccharomyces cerevisiae and/or oocytes of Xenopus laevis. The ribosyl derivative (FuPmR) was used to demonstrate proof of principle in live cell imaging studies in 11 cultured human cancer cell lines with different hENT1 activities. The autofluorescence emitted from FuPmR enabled direct visualization of its movement from the extracellular medium into the intracellular compartment of live cells, and this process was blocked by inhibitors of hENT1 (nitrobenzylmercaptopurine ribonucleoside, dipyridamole, and dilazep). Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake: a fast first stage that represented the initial uptake rate (i.e., transport rate) followed by a slow long-lasting second stage. The accumulation of FuPmR and/or its metabolites in nuclei and mitochondria was also visualized by live cell imaging. Measurements of fluorescence intensity increases in nuclei and mitochondria revealed rate-limited processes of permeant translocation across intracellular membranes, demonstrating for the first time the intracellular distribution of nucleosides and/or nucleoside metabolites in living cells. The use of autofluorescent nucleosides in time-lapse confocal microscopy is a novel strategy to quantitatively study membrane transport of nucleosides and their metabolites that will provide new knowledge of nucleoside biology.