Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis while Affording a Fluorescent Conjugate

Abstract

Molecules that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) were conceived of by structure-based design principles and were chemically synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ~ 1400 times faster than the hydrolysis reaction outside the thyroxine binding site. X-ray crystallography confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. A few of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. A few of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore.

Global profiling of lysine reactivity and ligandability in the human proteome

“…4a and Supplementary Table 2). The hyper-reactive lysine K35 in the hormone-binding protein transthyretin TTR, for instance, which has previously been shown to be modified selectively in human plasma by activated (thio)ester and sulfonyl fluoride ligands 19,28 , was preferentially targeted by the dinitrophenyl ester fragment 31 over fragments that showed much greater proteome-wide reactivity (e.g., 29 and 30 ) (Fig. 4a and Supplementary Fig.…”

“…Success of such a program, however, may depend on identifying alternative amine-reactive chemotypes, as the activated esters tested herein are likely too prone to enzymatic and non-enzymatic hydrolysis for development into cellular or in vivo probes. Alternative amine-reactive electrophiles, such as sulfonyl fluorides 28,29 or the N , N ′-diacyl-pyrazolecarboxamidines explored herein may offer more suitable starting points for optimization of lysine-targeting covalent ligands that are more suitable for cell biological studies. Alternative electrophiles, when used as broad profiling probes, may also provide access to additional lysine residues in the proteome, although the chemoselectivity of such probes could present a challenge.…”

“…Individual lysine residues within functional protein pockets are also susceptible to modification by electrophilic small molecules, including natural products, such as Wortmannin 18 , which targets a lysine in the active sites of PI3K kinases, activated esters that react with a lysine in transthyretin (TTR) 19 , and boronic acid carbonyl antagonists of the apoptosis regulatory protein MCL-1 13 . Additional electrophiles that have been shown to react with proteinaceous lysine residues include dichlorotriazines 20,21 , imidoesters 22 , 2-acetyl- or 2-formyl-benzeneboronic acids 13,23 , isothiocyanates 24,25 , pyrazolecarboxamidines 26,27 , sulfonyl fluorides 28,29 , and vinyl sulfonamides 30 .…”

A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis elegans

“…For example, the majority of fluorogenic probes published to date target enzymes 4,7,14,18–24 . It would be ideal if fluorogenic probes could be routinely developed for non-enzyme proteins and other macromolecules 8,13,15,16,25–30 . Moreover, a better understanding of the physicochemical underpinnings of fluorogenicity would enable more efficient design 18,31,32 .…”

“…Encouraged by the development of TTR-selective fluorogenic probes for the aforementioned uses, as well as for additional applications 15,16,25,35 , we explored the design of additional environment-sensitive, cell-permeable fluorogenic TTR probes wherein the little studied aryl fluorosulfate group (ArOSO 2 F) is incorporated 45 .…”