RESUMEN
VT-1129 is a novel fungal enzyme-specific Cyp51 inhibitor with potent cryptococcal activity. Because of its long half-life (>6 days in mice) and our desire to quickly reach potent efficacy, we evaluated a VT-1129 loading dose-maintenance dose strategy against cryptococcal meningitis. VT-1129 plasma and brain pharmacokinetics were first studied in healthy mice, and these data were used to model loading dose-maintenance dose regimens to generate different steady-state concentrations. Mice were inoculated intracranially with Cryptococcus neoformans, and oral treatment began 1 day later. Treatment consisted of placebo or one of three VT-1129 loading dose-maintenance dose regimens, i.e., loading dose of 1, 3, or 30 mg/kg on day 1, followed by once-daily maintenance doses of 0.15, 0.5, or 5 mg/kg, respectively. In the fungal burden arm, therapy continued for 14 days and brains were collected on day 15 for fungal burden assessments. In the survival arm, treatment continued for 10 days, after which mice were monitored without therapy until day 30. VT-1129 plasma and brain concentrations were also measured. All VT-1129 doses significantly improved survival and reduced fungal burdens, compared to placebo. VT-1129 plasma and brain levels correlated with fungal burden reductions (R2 = 0.72 and R2 = 0.67, respectively), with a plasma concentration of 1 µg/ml yielding a reduction of â¼5 log10 CFU/g. With the highest loading dose-maintenance dose regimen, fungal burdens were undetectable in one-half of the mice in the fungal burden arm and in one-fourth of the mice in the survival arm, 20 days after the final dose. These data support a loading dose-maintenance dose strategy for quickly reaching highly efficacious VT-1129 concentrations for treating cryptococcal meningitis.
Asunto(s)
Antifúngicos/farmacología , Meningitis Criptocócica/tratamiento farmacológico , Piridinas/farmacología , Tetrazoles/farmacología , Animales , Encéfalo/microbiología , Cryptococcus neoformans/efectos de los fármacos , Masculino , Meningitis Criptocócica/microbiología , Ratones , Ratones Endogámicos ICR , Pruebas de Sensibilidad Microbiana/métodosRESUMEN
Cryptococcal meningitis is a significant cause of morbidity and mortality in immunocompromised patients. VT-1129 is a novel fungus-specific Cyp51 inhibitor with potent in vitro activity against Cryptococcus species. Our objective was to evaluate the in vivo efficacy of VT-1129 against cryptococcal meningitis. Mice were inoculated intracranially with Cryptococcus neoformans Oral treatment with VT-1129, fluconazole, or placebo began 1 day later and continued for either 7 or 14 days, and brains and plasma were collected on day 8 or 15, 1 day after therapy ended, and the fungal burden was assessed. In the survival study, treatment continued until day 10 or day 28, after which mice were monitored off therapy until day 30 or day 60, respectively, to assess survival. The fungal burden was also assessed in the survival arm. VT-1129 plasma and brain concentrations were also measured. VT-1129 reached a significant maximal survival benefit (100%) at a dose of 20 mg/kg of body weight once daily. VT-1129 at doses of ≥0.3 mg/kg/day and each dose of fluconazole significantly reduced the brain tissue fungal burden compared to that in the control after both 7 and 14 days of dosing. The fungal burden was also undetectable in most mice treated with a dose of ≥3 mg/kg/day, even ≥20 days after dosing had stopped, in the survival arm. In contrast, rebounds in fungal burden were observed with fluconazole. These results are consistent with the VT-1129 concentrations, which remained elevated long after dosing had stopped. These data demonstrate the potential utility of VT-1129 to have a marked impact in the treatment of cryptococcal meningitis.
Asunto(s)
Inhibidores de 14 alfa Desmetilasa/farmacología , Cryptococcus neoformans/efectos de los fármacos , Meningitis Criptocócica/tratamiento farmacológico , Piridinas/farmacología , Esterol 14-Desmetilasa/metabolismo , Tetrazoles/farmacología , Animales , Antifúngicos/farmacología , Criptococosis/tratamiento farmacológico , Fluconazol/farmacología , Ratones , Pruebas de Sensibilidad Microbiana/métodos , Modelos TeóricosRESUMEN
INTRODUCTION: Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis. METHODS: PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting. RESULTS: We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-4) and P=0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis. CONCLUSIONS: Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Microtúbulos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Moduladores de Tubulina/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Docetaxel , Sinergismo Farmacológico , Femenino , Amplificación de Genes , Expresión Génica , Humanos , Pronóstico , Transducción de Señal/efectos de los fármacos , Taxoides/farmacología , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
A series of alpha-sulfone piperidine hydroxamate TACE inhibitors 11a-n bearing a quinolinyl methyl P1' group was prepared, and their activity was compared to analogous alpha- and beta-sulfone piperidine hydroxamates with a butynyloxy P1' group. The quinolinyl methyl P1' group affords increased inhibitory enzyme activity relative to the corresponding butynyloxy P1' analogs in the alpha-sulfone piperidine hydroxamate series, and greater selectivity than the corresponding butynyloxy P1' analogs in the beta-sulfone piperidine hydroxamate series.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Ácidos Hidroxámicos/síntesis química , Piperidinas/química , Inhibidores de Proteasas/síntesis química , Sulfonas/química , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animales , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Ratones , Piperidinas/síntesis química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Sulfonas/síntesis químicaRESUMEN
The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Benzoatos/síntesis química , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Sulfonamidas/síntesis química , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/tratamiento farmacológico , Benzoatos/química , Benzoatos/farmacología , Disponibilidad Biológica , Broncoconstricción/efectos de los fármacos , Calorimetría , Carragenina , Línea Celular , Inhibidores de la Ciclooxigenasa 2/síntesis química , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/farmacología , Edema/inducido químicamente , Edema/tratamiento farmacológico , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inhibidores , Masculino , Ratones , Unión Proteica , Ratas , Ratas Sprague-Dawley , Ovinos , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
Cytosolic phospholipase A(2) alpha (cPLA(2)alpha, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA(2)alpha inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA(2)alpha and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA(2)alpha inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 degrees C and pH 7.4. The compounds tested bound cPLA(2)alpha with a 1:1 stoichiometry, and the dissociation constant K(d) of the inhibitors calculated from the ITC experiments correlated well with the IC(50) values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA(2)alpha was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA(2)alpha. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA(2)alpha was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (DeltaH), entropy (DeltaS), and free energy (DeltaG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics.
Asunto(s)
Citosol/enzimología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Animales , Ácidos Araquidónicos/metabolismo , Ácidos Araquidónicos/farmacología , Benzofenonas/química , Benzofenonas/metabolismo , Benzofenonas/farmacología , Células CHO , Dominio Catalítico , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Fosfolipasas A2 Grupo IV/metabolismo , Humanos , Indoles/química , Indoles/metabolismo , Indoles/farmacología , Organofosfonatos/metabolismo , Organofosfonatos/farmacología , Unión Proteica , Pirrolidinas/química , Pirrolidinas/metabolismo , Pirrolidinas/farmacología , Solubilidad , TermodinámicaRESUMEN
The synthesis and structure-activity relationship of a series of benzenesulfonamide indole inhibitors of cPLA(2)alpha are described. Substitution of the benzenesulfonamide led to analogues with 50-fold improvement in potency versus the unsubstituted benzenesulfonamide lead compound. Rat pharmacokinetics in a minimal formulation was used to prioritize compounds, leading to the discovery of a potent inhibitor of cPLA(2)alpha with oral efficacy in models of rat carrageenan paw edema and Ascaris suum airway challenge in naturally sensitized sheep.
Asunto(s)
Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Fosfolipasas A2 Grupo IV/metabolismo , Indoles/farmacología , Sulfonamidas/química , Administración Oral , Animales , Ascariasis/tratamiento farmacológico , Ascariasis/parasitología , Ascaris suum/fisiología , Calorimetría , Humanos , Indoles/química , Indoles/uso terapéutico , Estructura Molecular , Ratas , Ovinos , Relación Estructura-Actividad , Temperatura , BencenosulfonamidasRESUMEN
The synthesis and structure-activity relationship of a series of indole inhibitors of cytosolic phospholipase A2alpha (cPLA2alpha, type IVA phospholipase) are described. Inhibitors of cPLA2alpha are predicted to be efficacious in treating asthma as well as the signs and symptoms of osteoarthritis, rheumatoid arthritis, and pain. The introduction of a benzyl sulfonamide substituent at C2 was found to impart improved potency of these inhibitors, and the SAR of these sulfonamide analogues is disclosed. Compound 123 (Ecopladib) is a sub-micromolar inhibitor of cPLA2alpha in the GLU micelle and rat whole blood assays. Compound 123 displayed oral efficacy in the rat carrageenan air pouch and rat carrageenan-induced paw edema models.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Benzoatos/síntesis química , Citosol/enzimología , Indoles/síntesis química , Fosfolipasas A/antagonistas & inhibidores , Sulfonamidas/síntesis química , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Antiinflamatorios no Esteroideos/farmacología , Benzoatos/farmacocinética , Benzoatos/farmacología , Carragenina , Edema/inducido químicamente , Edema/tratamiento farmacológico , Fosfolipasas A2 Grupo IV , Humanos , Técnicas In Vitro , Indoles/farmacocinética , Indoles/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologíaRESUMEN
Compound 1 was previously reported to be a potent inhibitor of cPLA(2)alpha in both artificial monomeric substrate and cell-based assays. However, 1 was inactive in whole blood assays previously used to characterize cyclooxygenase and lipoxygenase inhibitors. The IC(50) of 1 increased dramatically with cell number or lipid/detergent concentration. In an attempt to insert an electrophilic ketone between the indole and benzoic acid moieties, we discovered that increasing the distance between the two moieties gave a compound with activity in the GLU (7-hydroxycoumarinyl-gamma-linolenate) micelle assay, which contains lipid and detergent. Extensive structure-activity relationship work around this lead identified a potent pharmacophore for cPLA(2)alpha inhibition. The IC(50)s between the GLU micelle and rat whole blood assays correlated highly. No correlation was found for other parameters, including lipophilicity or acidity of the required acid functionality. Compounds 25, 39, and 94 emerged as potent, selective inhibitors of cPLA(2)alpha and represent well-validated starting points for further optimization.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Indoles/síntesis química , Indoles/farmacología , Fosfolipasas A/antagonistas & inhibidores , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Citosol/enzimología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Fosfolipasas A2 Grupo IV , Humanos , Indoles/química , Masculino , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid, is a natural product antagonist of the hedgehog pathway. In a previous report, a seven-membered D-ring semisynthetic analogue of cyclopamine, IPI-269609 (2), was shown to have greater acid stability and better aqueous solubility compared to cyclopamine. Further modifications of the A-ring system generated three series of analogues with improved potency and/or solubility. Lead compounds from each series were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926 (compound 28), a novel semisynthetic cyclopamine analogue with substantially improved pharmaceutical properties and potency and a favorable pharmacokinetic profile relative to cyclopamine and compound 2. As a result, complete tumor regression was observed in a Hh-dependent medulloblastoma allograft model after daily oral administration of 40 mg/kg of compound 28.
Asunto(s)
Descubrimiento de Drogas , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Transducción de Señal/efectos de los fármacos , Alcaloides de Veratrum/administración & dosificación , Alcaloides de Veratrum/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular , Humanos , Hígado/citología , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Estereoisomerismo , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacocinéticaRESUMEN
Herein is reported the synthesis of a novel class of hedgehog antagonists derived from cyclopamine. The acid sensitive D-ring of cyclopamine was homologated utilizing a sequence of chemoselective cyclopropanation and stereoselective acid-catalyzed rearrangement. Further modification of the A/B-ring homoallylic alcohol to the conjugated ketone led to the discovery of new cyclopamine analogues with improved pharmaceutical properties and in vitro potency (EC 50) ranging from 10 to 1000 nM.