RESUMO
As a result of our continued efforts to pursue Gal-3 inhibitors that could be used to fully evaluate the potential of Gal-3 as a therapeutic target, two novel series of benzothiazole derived monosaccharides as potent (against both human and mouse Gal-3) and orally bioavailable Gal-3 inhibitors, represented by 4 and 5, respectively, were identified. These discoveries were made based on proposals that the benzothiazole sulfur atom could interact with the carbonyl oxygen of G182/G196 in h/mGal-3, and that the anomeric triazole moiety could be modified into an N-methyl carboxamide functionality. The interaction between the benzothiazole sulfur and the carbonyl oxygen of G196 in mGal-3 was confirmed by an X-ray co-crystal structure of early lead 9, providing a rare example of using a S···O binding interaction for drug design. It was found that for both the series, methylation of 3-OH in the monosaccharides caused no loss in h & mGal-3 potencies but significantly improved permeability of the molecules.
Assuntos
Galectina 3 , Monossacarídeos , Animais , Humanos , Camundongos , Benzotiazóis/química , Benzotiazóis/farmacologia , Desenho de Fármacos , Galectina 3/antagonistas & inibidores , Galectinas/antagonistas & inibidores , Monossacarídeos/química , Monossacarídeos/farmacologia , Oxigênio , EnxofreRESUMO
Galectin-3 (Gal-3), a ß-galactoside-binding lectin, has been implicated in a plethora of pathological disorders including fibrosis, inflammation, cancer and metabolic diseases. TD139-a thio-digalactoside inhibitor developed by Galecto Biotech as a potential therapeutic for idiopathic pulmonary fibrosis-is the most advanced small-molecule Gal-3 inhibitor in clinical studies. It binds to human Gal-3 with high affinity but has lower affinity towards mouse and rat homologs, which is also manifested in the differential inhibition of Gal-3 function. Using biophysical methods and high-resolution X-ray co-crystal structures of TD139 and Gal-3 proteins, we demonstrate that a single amino acid change corresponding to A146 in human Gal-3 is sufficient for the observed reduction in the binding affinity of TD139 in rodents. Site-directed mutagenesis of A146V (in human Gal-3) and V160A (in mouse Gal-3) was sufficient to interchange the affinities, mainly by affecting the off rates of the inhibitor binding. In addition, molecular dynamics simulations of both wild-type and mutant structures revealed the sustained favorable noncovalent interactions between the fluorophenyl ring and the active site A146 (human Gal-3 and mouse V160A) that corroborate the finding from biophysical studies. Current findings have ramifications in the context of optimization of drug candidates against Gal-3.
Assuntos
Proteínas Sanguíneas , Galectinas , Tiogalactosídeos , Humanos , Sítios de Ligação/efeitos dos fármacos , Proteínas Sanguíneas/antagonistas & inibidores , Proteínas Sanguíneas/metabolismo , Galectinas/antagonistas & inibidores , Galectinas/metabolismo , Tiogalactosídeos/metabolismo , Tiogalactosídeos/farmacologiaRESUMO
Galectin-3 (Gal-3) is a carbohydrate binding protein that has been implicated in the development and progression of fibrotic diseases. Proof-of-principal animal models have demonstrated that inhibition of Gal-3 is a potentially viable pathway for the treatment of fibrosisâwith small molecule Gal-3 inhibitors advanced into clinical trials. We hereby report the discovery of novel galactose-based monosaccharide Gal-3 inhibitors comprising 2-methyl-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (compound 20) and 4-phenyl-4H-1,2,4-triazole (compound 15). Notably, hindered rotation caused by steric interaction between the 3-thione and ortho-trifluoromethyl group of compounds 20, 21 induced formation of thermodynamically stable atropisomers. Distinct X-ray cocrystal structures of 20 and 21 were obtained, which clearly demonstrated that the configuration of 21 proscribes a key halogen bonding σ-hole interaction of 3-chloro with carbonyl oxygen of Gly182, thereby leading to significant loss in potency. Ultimately, 20 and 15 were evaluated in mouse pharmacokinetic studies, and both compounds exhibited oral exposures suitable for further in vivo assessment.
RESUMO
To signal properly, excitable cells must establish and maintain the correct balance of various types of ion channels that increase or decrease membrane excitability. The mechanisms by which this balance is regulated remain largely unknown. Here, we describe a regulatory mechanism uncovered by a Drosophila behavioral mutant, down and out (dao). At elevated temperatures, dao loss-of-function mutants exhibit seizures associated with spontaneous bursts of neural activity. This phenotype closely resembles that of seizure mutations, which impair activity of ether-a-go-go-related gene (Erg)-type potassium channels. Conversely, neural over-expression of wild-type Dao confers dominant temperature-sensitive paralysis with kinetics reminiscent of paralytic sodium-channel mutants. The over-expression phenotype of dao is suppressed in a seizure mutant background, suggesting that Dao acts by an effect on Erg channels. In support of this hypothesis, functional expression of Erg channels in a heterologous system is dependent on the presence of Dao. These results indicate that Dao has an important role in establishing the proper level of neuronal membrane excitability by regulating functional expression of Erg channels.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila/genética , Drosophila/fisiologia , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/fisiologia , Animais , Animais Geneticamente Modificados , Comportamento Animal , Mapeamento Cromossômico , Feminino , Expressão Gênica , Genes de Insetos , Técnicas In Vitro , Mutação , Oócitos/metabolismo , Fenótipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
Galectin-3 (Gal-3), a member of the ß-galactoside-binding protein family, is implicated in a wide variety of human diseases. Identification of Gal-3 inhibitors with the right combination of potency (against both human and mouse Gal-3) and pharmacokinetic properties to fully evaluate the potential of Gal-3 for therapeutic intervention has been a major challenge due to the characteristics of its binding pocket: high hydrophilicity and key structural differences between human Gal-3 and the mouse ortholog. We report the discovery of a novel series of monosaccharide-based, highly potent, and orally bioavailable inhibitors of human and mouse Gal-3. The novel monosaccharide derivatives proved to be selective for Gal-3, the only member of the chimeric type of galectins, over Gal-1 and Gal-9, representative of the prototype and tandem-repeat type of galectins, respectively. The proposed binding mode for the newly identified ligands was confirmed by an X-ray cocrystal structure of a representative analogue bound to Gal-3 protein.
Assuntos
Galectina 3 , Monossacarídeos , Animais , Galectina 3/metabolismo , Galectinas , Humanos , Ligantes , CamundongosRESUMO
Galectin-3 is a member of a family of ß-galactoside-binding proteins. A substantial body of literature reports that galectin-3 plays important roles in cancer, inflammation, and fibrosis. Small-molecule galectin-3 inhibitors, which are generally lactose or galactose-based derivatives, have the potential to be valuable disease-modifying agents. In our efforts to identify novel galectin-3 disaccharide mimics to improve drug-like properties, we found that one of the monosaccharide subunits can be replaced with a suitably functionalized tetrahydropyran ring. Optimization of the structure-activity relationships around the tetrahydropyran-based scaffold led to the discovery of potent galectin-3 inhibitors. Compounds 36, 40, and 45 were selected for further in vivo evaluation. The synthesis, structure-activity relationships, and in vivo evaluation of novel tetrahydropyran-based galectin-3 inhibitors are described.
Assuntos
Dissacarídeos/química , Galectina 3/antagonistas & inibidores , Piranos/química , Animais , Sítios de Ligação , Quimiotaxia/efeitos dos fármacos , Cristalografia por Raios X , Dissacarídeos/síntese química , Dissacarídeos/metabolismo , Dissacarídeos/farmacologia , Galectina 3/metabolismo , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Conformação Molecular , Simulação de Dinâmica Molecular , Permeabilidade/efeitos dos fármacos , Ligação Proteica , Relação Estrutura-Atividade , Triazóis/químicaRESUMO
Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long-QT syndrome (LQTS). Biochemical and functional studies have demonstrated that I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homooligomers of the original hERG 1a isolate. Here, we examine currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We find that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a 2-fold increase in the apparent rates of activation and recovery from inactivation, thus reducing rectification and facilitating current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the 2 channel types. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding 4-fold shift in the IC50. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients that was absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells dramatically reduced 1b protein levels. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr and enhance drug sensitivity, and represent a potential mechanism underlying inherited or acquired LQTS.
Assuntos
Substituição de Aminoácidos , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Mutação de Sentido Incorreto , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Antiarrítmicos/farmacologia , Linhagem Celular , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Cinética , Modelos Biológicos , Piperidinas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , PiridinasRESUMO
BACKGROUND AND PURPOSE: Activators of Kv 11.1 (hERG) channels have potential utility in the treatment of acquired and congenital long QT (LQT) syndrome. Here, we describe a new hERG channel activator, 5-(((1H-indazol-5-yl)oxy)methyl)-N-(4-(trifluoromethoxy)phenyl)pyrimidin-2-amine (ITP-2), with a chemical structure distinct from previously reported compounds. EXPERIMENTAL APPROACH: Conventional electrophysiological methods were used to assess the effects of ITP-2 on hERG1a and hERG1a/1b channels expressed heterologously in HEK-293 cells. KEY RESULTS: ITP-2 selectively increased test pulse currents (EC50 1.0 µM) and decreased tail currents. ITP-2 activated hERG1a homomeric channels primarily by causing large depolarizing shifts in the midpoint of voltage-dependent inactivation and hyperpolarizing shifts in the voltage-dependence of activation. In addition, ITP-2 slowed rates of inactivation and made recovery from inactivation faster. hERG1a/1b heteromeric channels showed reduced sensitivity to ITP-2 and their inactivation properties were differentially modulated. Effects on midpoint of voltage-dependent inactivation and rates of inactivation were less pronounced for hERG1a/1b channels. Effects on voltage-dependent activation and activation kinetics were not different from hERG1a channels. Interestingly, hERG1b channels were inhibited by ITP-2. Inactivation-impairing mutations abolished activation by ITP-2 and led to inhibition of hERG channels. ITP-2 exerted agonistic effect from extracellular side of the membrane and could activate one of the arrhythmia-associated trafficking-deficient LQT2 mutants. CONCLUSIONS AND IMPLICATIONS: ITP-2 may serve as another novel lead molecule for designing robust activators of hERG channels. hERG1a/1b gating kinetics were differentially modulated by ITP-2 leading to altered sensitivity. ITP-2 is capable of activating an LQT2 mutant and may be potentially useful in the development of LQT2 therapeutics.
Assuntos
Canal de Potássio ERG1/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Células Cultivadas , Relação Dose-Resposta a Droga , Canal de Potássio ERG1/metabolismo , Células HEK293 , Humanos , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
We have recently disclosed 5-phenyl-N-(pyridin-2-ylmethyl)-2-(pyrimidin-5-yl)quinazolin-4-amine 1 as a potent IKur current blocker with selectivity versus hERG, Na and Ca channels, and an acceptable preclinical PK profile. Upon further characterization in vivo, compound 1 demonstrated an unacceptable level of brain penetration. In an effort to reduce the level of brain penetration while maintaining the overall profile, SAR was developed at the C2' position for a series of close analogues by employing hydrogen bond donors. As a result, 5-[5-phenyl-4-(pyridin-2-ylmethylamino)quinazolin-2-yl]pyridine-3-sulfonamide (25) was identified as the lead compound in this series. Compound 25 showed robust effects in rabbit and canine pharmacodynamic models and an acceptable cross-species pharmacokinetic profile and was advanced as the clinical candidate. Further optimization of 25 to mitigate pH-dependent absorption resulted in identification of the corresponding phosphoramide prodrug (29) with an improved solubility and pharmacokinetic profile.
Assuntos
Fibrilação Atrial/tratamento farmacológico , Bloqueadores dos Canais de Potássio/uso terapêutico , Quinazolinas/uso terapêutico , Bloqueadores dos Canais de Sódio/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Cães , Espectrometria de Massas , Bloqueadores dos Canais de Potássio/farmacologia , Espectroscopia de Prótons por Ressonância Magnética , Quinazolinas/química , Quinazolinas/farmacologia , Coelhos , Bloqueadores dos Canais de Sódio/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
A new series of phenylquinazoline inhibitors of Kv 1.5 is disclosed. The series was optimized for Kv 1.5 potency, selectivity versus hERG, pharmacokinetic exposure, and pharmacodynamic potency. 5-Phenyl-N-(pyridin-2-ylmethyl)-2-(pyrimidin-5-yl)quinazolin-4-amine (13k) was identified as a potent and ion channel selective inhibitor with robust efficacy in the preclinical rat ventricular effective refractory period (VERP) model and the rabbit atrial effective refractory period (AERP) model.
RESUMO
Defects in the trafficking of subunits encoded by the human ether-à-go-go-related gene (hERG1) can lead to catastrophic arrhythmias and sudden cardiac death due to a reduction in I(Kr)-mediated repolarization. Native I(Kr) channels are composed of two alpha subunits, hERG 1a and 1b. In heterologous expression systems, hERG 1b subunits efficiently produce current only in heteromeric combination with hERG 1a. We used Western blot analysis and electrophysiological recordings in HEK-293 cells and Xenopus oocytes to monitor hERG 1b maturation in the secretory pathway and to determine the factors regulating surface expression of hERG 1b subunits. We found that 1b subunits expressed alone were largely retained in the endoplasmic reticulum (ER), thus accounting for the poor functional expression of homomeric 1b currents. Association with hERG 1a facilitated 1b ER export and surface expression. We show that hERG 1b subunits fail to mature because of an "RXR" ER retention signal specific to the 1b N terminus of the human sequence and not conserved in other species. Mutating the RXR facilitated maturation and functional expression of homomeric hERG 1b channels in a charge-dependent manner. Co-expression of the 1b RXR mutants with hERG 1a did not further enhance 1b maturation, suggesting that hERG 1a promotes 1b trafficking by overcoming the RXR-mediated retention. Thus, selective trafficking mechanisms regulate subunit composition of surface hERG channels.