Stilbene Vinyl Sulfonamides as Fluorogenic Sensors of and Traceless Covalent Kinetic Stabilizers of Transthyretin that Prevent Amyloidogenesis

摘要

Small molecules that react selectively with a specific non-enzyme drug-target protein in a complex biological environment without displacement of a leaving group (tracelessly) are rare and highly desirable. Herein, we describe the development of a family of fluorogenic stilbene-based vinyl amides and sulfonamides that covalently modify transthyretin (TTR) tracelessly. These small molecules bind selectively to TTR in complex biological environments and then undergo a rapid and chemoselective 1,4-Michael addition with the pKa-perturbed Lys-15 ε-amino group of TTR. Replacing the vinyl amide in >2 with the more reactive vinyl sulfonamide in >4 hastens conjugation kinetics. X-ray co-crystallography verifies formation of the secondary amine bond mediating conjugation in the case of >2 and >4 and confirms the expected orientation of the stilbene within the TTR binding sites. Vinyl amide >2 and sulfonamide >4 potently inhibit TTR dissociation and amyloid fibril formation in vitro. The TTR binding selectivity, modification yield, and reaction chemoselectivity of vinyl sulfonamide >4 is good enough in human plasma to serve as a starting point for medicinal chemistry efforts. Moreover, vinyl sulfonamide >4 is fluorogenic, i.e. it exhibits minimal background fluorescence in complex biological environments, remains dark upon binding to TTR, and only becomes fluorescent upon reaction with TTR. The fluorogenicity of >4 was utilized to accurately quantify the native TTR concentration in E. coli lysate using a fluorescence plate reader.