Recently, we reported ethyl nicotinates as antagonists of the P2Y12 receptor, which is an important target in antiplatelet therapies. A potential liability of these compounds was their generally high ...in vivo clearance due to ethyl ester hydrolysis.
Shape and electrostatic similarity matching was used to select five-membered heterocycles to replace the ethyl ester functionality. The 5-methyl and 5-ethyl-oxazole bioisosteres retained the sub-micromolar potency levels of the parent ethyl esters. Many oxazoles showed a higher CYP450 dependent microsomal metabolism than the corresponding ethyl esters. Structure activity relationship investigations supported by ab initio calculations suggested that a correctly positioned alkyl substituent and a strong hydrogen bond acceptor were necessary structural motifs for binding. In rat pharmacokinetics, the low clearance was retained upon replacement of an ethyl ester with a 5-ethyl-oxazole.
The use of shape and electrostatic similarity led to the successful replacement of a metabolically labile ethyl ester functionality with 5-alkyl-oxazole bioisosteres.
The accurate determination of the maximum turnover number and Michaelis constant for membrane enzymes remains challenging. Here, this problem has been solved by observing in parallel the hydrolysis ...of thousands of individual fluorescently labeled immobilized liposomes each processed by a single phospholipase A2 molecule. The release of the reaction product was tracked using total internal reflection fluorescence microscopy. A statistical analysis of the hydrolysis kinetics was shown to provide the Michaelis–Menten parameters with an accuracy better than 20 % without variation of the initial substrate concentration. The combined single‐liposome and single‐enzyme mode of operation made it also possible to unravel a significant nanoscale dependence of these parameters on membrane curvature.
Verdauung für Statistiker: Die Kinetik des Phospholipase‐A2‐Verdaus einzelner Liposome aus farbstoffmodifizierten Lipiden wurde mit Fluoreszenzbildgebung mit totaler interner Reflexion untersucht (siehe Bild). Sowohl kcat als auch Km konnten ermittelt werden, ohne dass die Anfangskonzentration des Phospholipidsubstrats variiert werden musste. Dieser Ansatz mit einzelnen Liposomen und Enzymmolekülen könnte Informationen über Auswirkungen der Membranmorphologie auf die Reaktionskinetik liefern.
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