RESUMEN
Single-molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM data set is an essential aspect of the image processing and analysis pipeline. Here, we present a simple method to estimate the localization spread function (LSF) of a static SMLM data set directly from acquired localizations, exploiting the correlated dynamics of individual emitters and properties of the pair autocorrelation function evaluated in both time and space. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labeled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. We show that experimentally obtained images have LSFs that are broader than expected from the localization precision alone, due to additional uncertainty accrued when localizing molecules imaged over time.
Asunto(s)
Microscopía , Imagen Individual de Molécula , ADN/química , Procesamiento de Imagen Asistido por Computador/métodos , Imagen Individual de Molécula/métodosRESUMEN
A series of pyrrolopyridinones was designed and synthesized as constrained analogs of the pyrazole CB-1 antagonist rimonabant. Certain examples exhibited very potent hCB-1 receptor binding affinity and functional antagonism with Ki and Kb values below 10 nM, and with high selectivity for CB-1 over CB-2 (>100-fold). A representative analog was established to cause significant appetite suppression and reduction in body weight gain in industry-standard rat models used to develop new therapeutics for obesity.
Asunto(s)
Fármacos Antiobesidad/síntesis química , Fármacos Antiobesidad/farmacología , Piperidinas/síntesis química , Piperidinas/farmacología , Pirazoles/síntesis química , Pirazoles/farmacología , Piridonas/síntesis química , Pirroles/síntesis química , Receptor Cannabinoide CB1/antagonistas & inhibidores , Animales , Fármacos Antiobesidad/farmacocinética , Peso Corporal/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Ingestión de Alimentos/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Modelos Moleculares , Obesidad/tratamiento farmacológico , Piridonas/farmacocinética , Piridonas/farmacología , Pirroles/farmacocinética , Pirroles/farmacología , Ratas , Ratas Wistar , Ratas Zucker , Rimonabant , Relación Estructura-Actividad , Aumento de Peso/efectos de los fármacosRESUMEN
Several imidazole-based cyclohexyl amides were identified as potent CB-1 antagonists, but they exhibited poor oral exposure in rodents. Incorporation of a hydroxyl moiety on the cyclohexyl ring provided a dramatic improvement in oral exposure, together with a ca. 10-fold decrease in potency. Further optimization provided the imidazole 2-hydroxy-cyclohexyl amide 45, which exhibited hCB-1 K(i)=3.7nM, and caused significant appetite suppression and robust, dose-dependent reduction of body weight gain in industry-standard rat models.