Stilbene Boronic Acids Form a Covalent Bond with Human Transthyretin and Inhibit Its Aggregation

Abstract

Transthyretin (TTR) is a homotetrameric protein. Its dissociation into monomers leads to the formation of fibrils that underlie human amyloidogenic diseases. The binding of small molecules to the thyroxin-binding sites in TTR stabilizes the homotetramer and attenuates TTR amyloidosis. Herein, we report on boronic acid-substituted stilbenes that limit TTR amyloidosis in vitro. Assays of affinity for TTR and inhibition of its tendency to form fibrils were coupled with X-ray crystallographic analysis of nine TTR·ligand complexes. The ensuing structure–function data led to a symmetrical diboronic acid that forms a boronic ester reversibly with serine 117. This diboronic acid inhibits fibril formation by both wild-type TTR and a common disease-related variant, V30M TTR, as effectively as does tafamidis, a small-molecule drug used to treat TTR-related amyloidosis in the clinic. These findings establish a new modality for covalent inhibition of fibril formation and illuminate a path for future optimization.

Boronic Acid with High Oxidative Stability and Utility in Biological Contexts

“…To investigate BOL in this context, we designed and synthesized stilbene 1. This compound resembles ligands that are known to bind to the tetrameric form of human transthyretin and thereby deter its aggregation into fibrils that underlie human amyloidogenic diseases (53,54). We found that stilbene 1 attenuates the formation of transthyretin fibrils in a concentration-dependent manner (SI Appendix, Table S14).…”

“…Supporting Information. Human transthyretin was produced with recombinant DNA technology in Escherichia coli as described previously(54). Phosphate-buffered saline (PBS) was made with ingredients from Research Products International and contained (in 1.0 L): 0.2 g KCl, 0.2 g KH2PO4, 8 g NaCl, and 2.16 g Na2HPO4•7H2O at pH 7.3.NMR spectra were obtained with an Avance-400, Avance-500, or Avance-600 spectrometer from Bruker.…”

Boronic Acid with High Oxidative Stability and Utility in Biological Contexts

“…Additional restraints were imposed to provide accurate bond distances and angles, as described previously (54). Fig.…”

Covalent Probes for Aggregated Protein Imaging via Michael Addition

“…The unique catalytic environment of enzymes or binding proteins offers vastly diverse covalent reactions, including acylation, 1,4‐Michael addition, alkylation, carbamylation, and boronation etc [8–10] . These covalent reactions allow us to develop covalent probes targeting folded proteins, including fluorescent probes for bio‐imaging, covalent drugs for therapeutic purposes, [11–16] and activity based probes for chemical proteomics [17–24] etc. Besides these naturally existing chemical reactivity, introduction of unnatural amino acids enormously broadens the horizon of chemical reactivity and enriches the bioorthogonal chemistry toolbox for selective bioconjugation and functionalization of folded proteins [25–28] …”