RESUMO
Pendrin is a transmembrane chloride/anion antiporter that is strongly upregulated in the airways in rhinoviral infection, asthma, cystic fibrosis and chronic rhinosinusitis. Based on its role in the regulation of airway surface liquid depth, pendrin inhibitors have potential indications for treatment of inflammatory airways diseases. Here, a completely regioselective route to tetrahydro-pyrazolopyridine pendrin inhibitors based on 1,3-diketone and substituted hydrazine condensation was been developed. Structure-activity relationships at the tetrahydropyridyl nitrogen were investigated using a focused library, establishing the privileged nature of N-phenyl ureas and improving inhibitor potency by greater than 2-fold.
Assuntos
Pirazóis/farmacologia , Piridinas/farmacologia , Transportadores de Sulfato/antagonistas & inibidores , Animais , Camundongos , Estrutura Molecular , Pirazóis/síntese química , Piridinas/síntese química , Ratos Endogâmicos F344 , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
SLC26A6 (also known as putative anion transporter 1 [PAT1]) is a Cl-/HCO3- exchanger expressed at the luminal membrane of enterocytes where it facilitates intestinal Cl- and fluid absorption. Here, high-throughput screening of 50,000 synthetic small molecules in cells expressing PAT1 and a halide-sensing fluorescent protein identified several classes of inhibitors. The most potent compound, the pyrazolo-pyrido-pyrimidinone PAT1inh-B01, fully inhibited PAT1-mediated anion exchange (IC50 ~350 nM), without inhibition of the related intestinal transporter SLC26A3 (also known as DRA). In closed midjejunal loops in mice, PAT1inh-B01 inhibited fluid absorption by 50%, which increased to >90% when coadministered with DRA inhibitor DRAinh-A270. In ileal loops, PAT1inh-B01 blocked fluid absorption by >80%, whereas DRAinh-A270 was without effect. In colonic loops, PAT1inh-B01 was without effect, whereas DRAinh-A270 completely blocked fluid absorption. In a loperamide constipation model, coadministration of PAT1inh-B01 with DRAinh-A270 increased stool output compared with DRAinh-A270 alone. These results provide functional evidence for complementary and region-specific roles of PAT1 and DRA in intestinal fluid absorption, with PAT1 as the predominant anion exchanger in mouse ileum. We believe that PAT1inh-B01 is a novel tool to study intestinal ion and fluid transport and perhaps a drug candidate for small intestinal hyposecretory disorders such as cystic fibrosis-related meconium ileus and distal intestinal obstruction syndrome.
Assuntos
Antiporters/antagonistas & inibidores , Colo/efeitos dos fármacos , Íleo/efeitos dos fármacos , Absorção Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Transportadores de Sulfato/antagonistas & inibidores , Animais , Antidiarreicos/farmacologia , Antiporters/metabolismo , Colo/metabolismo , Constipação Intestinal/induzido quimicamente , Constipação Intestinal/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Íleo/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Jejuno/metabolismo , Loperamida/farmacologia , Camundongos , Bibliotecas de Moléculas Pequenas , Transportadores de Sulfato/metabolismoRESUMO
Diarrhea is a major cause of global mortality, and outbreaks of secretory diarrhea such as cholera remain an important problem in the developing world. Current treatment of secretory diarrhea primarily involves supportive measures, such as fluid replacement. The calcium-sensing receptor (CaSR) regulates multiple biological activities in response to changes in extracellular Ca2+. The FDA-approved drug cinacalcet is an allosteric activator of CaSR used for treatment of hyperparathyroidism. Here, we found by short-circuit current measurements in human colonic T84 cells that CaSR activation by cinacalcet reduced forskolin-induced Cl- secretion by greater than 80%. Cinacalcet also reduced Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide (VIP). The cinacalcet effect primarily involved indirect inhibition of cystic fibrosis transmembrane conductance regulator-mediated (CFTR-mediated) Cl- secretion following activation of CaSR and downstream phospholipase C and phosphodiesterases. In mice, cinacalcet reduced fluid accumulation by more than 60% in intestinal closed loop models of cholera and traveler's diarrhea. The cinacalcet effect involved both inhibition of CFTR-mediated secretion and stimulation of sodium-hydrogen exchanger 3-mediated absorption. These findings support the therapeutic utility of the safe and commonly used drug cinacalcet in CFTR-dependent secretory diarrheas, including cholera, traveler's diarrhea, and VIPoma.
Assuntos
Cinacalcete/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos adversos , Diarreia/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Receptores de Detecção de Cálcio/uso terapêutico , Animais , Toxinas Bacterianas , Linhagem Celular , Toxina da Cólera , Cinacalcete/metabolismo , Colo/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/metabolismo , Enterotoxinas , Escherichia coli , Proteínas de Escherichia coli , Feminino , Humanos , Hiperparatireoidismo/tratamento farmacológico , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Masculino , CamundongosRESUMO
BACKGROUND: The c.3700A>G mutation, a rare cystic fibrosis (CF)-causing CFTR mutation found mainly in the Middle East, produces full-length transcript encoding a missense mutation (I1234V-CFTR), and a cryptic splice site that deletes 6 amino acids in nucleotide binding domain 2 (I1234del-CFTR). METHODS: FRT cell models expressing I1234V-CFTR and I1234del-CFTR were generated. We also studied an I1234del-CFTR-expressing gene-edited human bronchial (16HBE14o-) cell model, and primary cultures of nasal epithelial cells from a c.3700A>G homozygous subject. To identify improved mutation-specific CFTR modulators, high-throughput screening was done using I1234del-CFTR-expressing FRT cells. Motivated by the in vitro findings, Trikafta was tested in two c.3700A>G homozygous CF subjects. RESULTS: FRT cells expressing full-length I1234V-CFTR had similar function to that of wildtype CFTR. I1234del-CFTR showed reduced activity, with modest activation seen with potentiators VX-770 and GLPG1837, correctors VX-809, VX-661 and VX-445, and low-temperature incubation. Screening identified novel arylsulfonyl-piperazine and spiropiperidine-quinazolinone correctors, which when used in combination with VX-445 increased current ~2-fold compared with the VX-661/VX-445 combination. The combination of VX-770 with arylsulfonamide-pyrrolopyridine, piperidine-pyridoindole or pyrazolo-quinoline potentiators gave 2-4-fold greater current than VX-770 alone. Combination potentiator (co-potentiator) efficacy was also seen in gene-edited I1234del-CFTR-expressing human bronchial epithelial cells. In two CF subjects homozygous for the c.3700A>G mutation, one subject had a 27 mmol/L decrease in sweat chloride and symptomatic improvement on Trikafta, and a second subject showed a small improvement in lung function. CONCLUSIONS: These results support the potential benefit of CFTR modulators, including co-potentiators, for CF caused by the c.3700A>G mutation.
Assuntos
Agonistas dos Canais de Cloreto/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Proteínas Mutantes/efeitos dos fármacos , Mutação de Sentido Incorreto , Aminofenóis , Aminopiridinas , Benzodioxóis , Células Cultivadas , Humanos , Indóis , Pirazóis , Piridinas , Pirrolidinas , QuinolonasRESUMO
Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of 'co-potentiators' (combination-potentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC50 down to ~300 nM following initial structure-activity studies. Increased CFTR chloride conductance by up to 8-fold was observed when a co-potentiator (termed 'Class II potentiator') was used with a classical potentiator ('Class I potentiator') such as VX-770 or GLPG1837. To investigate the range of CFTR mutations benefitted by co-potentiators, 14 CF-associated CFTR mutations were studied in transfected cell models. Co-potentiator efficacy was found for CFTR missense, deletion and nonsense mutations in nucleotide binding domain-2 (NBD2), including W1282X, N1303K, c.3700A > G and Q1313X (with corrector for some mutations). In contrast, CFTR mutations G85E, R334W, R347P, V520F, R560T, A561E, M1101K and R1162X showed no co-potentiator activity, even with corrector. Co-potentiator efficacy was confirmed in primary human bronchial epithelial cell cultures generated from a N1303K homozygous CF subject. The Class II potentiators identified here may have clinical benefit for CF caused by mutations in the NBD2 domain of CFTR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Descoberta de Drogas , Sinergismo Farmacológico , Ensaios de Triagem em Larga Escala , Humanos , Mutação , Piperidinas/uso terapêutico , Pirazóis/uso terapêutico , Relação Estrutura-AtividadeRESUMO
SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl- exchange with HCO3-, I-, and thiocyanate (SCN-), with an IC50 of ~0.2 µM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.
Assuntos
Antiporters/farmacologia , Constipação Intestinal/tratamento farmacológico , Transportadores de Sulfato/antagonistas & inibidores , Transportadores de Sulfato/metabolismo , Animais , Antiporters/antagonistas & inibidores , Antiporters/química , Antiporters/genética , Antiporters/metabolismo , Antiportadores de Cloreto-Bicarbonato/farmacologia , Cloretos/metabolismo , Fibrose Cística , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Transporte de Íons , Loperamida/farmacologia , Camundongos , Ratos , Ratos Endogâmicos F344 , Trocador 3 de Sódio-Hidrogênio/farmacologia , Transportadores de Sulfato/genética , Transportadores de Sulfato/farmacologiaRESUMO
BACKGROUND: Vaniprevir with P/R improved SVR rates over P/R alone in treatment-experienced patients with chronic HCV-genotype 1 infection, but treatment failure presents therapeutic challenges. We identified RAVs from non-cirrhotic patients failing to achieve SVR on vaniprevir-containing regimens from a dose/duration-ranging trial of triple-combination therapy. METHODS: Using population analysis, resistance sequencing was performed on all baseline samples and on samples at virologic failure in the vaniprevir arms. Longitudinal clonal analyses were performed on viral isolates from six vaniprevir recipients experiencing breakthrough viremia. RESULTS: Baseline RAVs were detected in two patients subsequently experiencing virologic failure. At virologic failure, the majority of RAVs had substitutions at R155, A156, or D168. Clonal analyses identified novel double/triple variants emerging with continuing vaniprevir dosing. CONCLUSIONS: RAVs were predominantly observed at R155, A156, and/or D168 during virologic failure on vaniprevir/P/R. Double/triple RAVs were identified in patients remaining viremic on triple therapy, suggesting evolution of resistance under selective pressure.
Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Variação Genética , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Indóis/uso terapêutico , Peptídeo Hidrolases , Adolescente , Adulto , Idoso , Substituição de Aminoácidos , Antivirais/administração & dosagem , Antivirais/farmacologia , Ciclopropanos , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C Crônica/virologia , Humanos , Indóis/administração & dosagem , Indóis/farmacologia , Isoindóis , Lactamas Macrocíclicas , Leucina/análogos & derivados , Masculino , Pessoa de Meia-Idade , Peptídeo Hidrolases/genética , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacologia , Polietilenoglicóis/uso terapêutico , Prolina/análogos & derivados , RNA Viral/genética , Ribavirina/administração & dosagem , Ribavirina/farmacologia , Ribavirina/uso terapêutico , Sulfonamidas , Falha de Tratamento , Resultado do Tratamento , Viremia/tratamento farmacológico , Viremia/virologia , Adulto JovemRESUMO
We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7-10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH).