RESUMEN
Previous studies have reported that compounds bearing an arylamide linked to a heterocyclic planar ring have successfully inhibited the hemopexin-like domain (PEX9) of matrix metalloproteinase 9 (MMP9). PEX9 has been suggested to be more selectively targeted than MMP9's catalytic domain in a degrading extracellular matrix under some pathologic conditions, especially in cancer. In this study, we aim to synthesize and evaluate 10 arylamide compounds as MMP9 inhibitors through an enzymatic assay as well as a cellular assay. The mechanism of inhibition for the most active compounds was investigated via molecular dynamics simulation (MD). Molecular docking was performed using AutoDock4.0 with PEX9 as the protein model to predict the binding of the designed compounds. The synthesis was carried out by reacting aniline derivatives with 3-bromopropanoyl chloride using pyridine as the catalyst at room temperature. The MMP9 assay was conducted using the FRET-based MMP9 kits protocol and gelatin zymography assay. The cytotoxicity assay was done using the MTT method, and the MD simulation was performed using AMBER16. Assay on MMP9 demonstrated activities of three compounds (2, 7, and 9) with more than 50% inhibition. Further inhibition on MMP9 expressed by 4T1 showed that two compounds (7 and 9) inhibited its gelatinolytic activity more than 50%. The cytotoxicity assay against 4T1 cells results in the inhibition of the cell growth with an EC50 of 125 µM and 132 µM for 7 and 9, respectively. The MD simulation explained a stable interaction of 7 and 9 in PEX9 at 100 ns with a free energy of binding of -8.03 kcal/mol and -6.41 kcal/mol, respectively. Arylamides have potential effects as selective MMP9 inhibitors in inhibiting breast cancer cell progression.
Asunto(s)
Amidas/farmacología , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Animales , Dominio Catalítico , Chlorocebus aethiops , Diseño de Fármacos , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Células VeroRESUMEN
Hyperactivation of Gq signaling causes cardiac hypertrophy, and ß-adrenergic receptor-mediated Gs signaling is attenuated in hypertrophic cardiomyocytes. Here, we found the increase in a global ubiquitination in hypertrophic mouse heart. The activation of Gq signaling resulted in the ubiquitination of Gαs in neonatal rat cardiomyocytes, reduced Gαs expression, and suppressed cAMP response to ß-adrenergic receptor stimulation. Ectopic expression of Gαq induced a similar suppression, which is due to the degradation of Gαs by a ubiquitin-proteasome pathway. Co-expression of Ric-8B, a positive regulator of Gαs, effectively canceled the Gαq-induced ubiquitination of Gαs and recovered the cAMP accumulation. In vitro, Gαq competes for the binding of Gαs to Ric-8B. These data show a new role of Ric-8B in the crosstalk of two distinct G protein signaling pathways, which are possibly involved in a part of mechanisms of chronic heart failure.