Target-Directed Self-Assembly of Homodimeric Drugs Against β-Tryptase

Abstract

Tryptase, a serine protease released from mast cells, is implicated in many allergic and inflammatory disorders. Human tryptase is a donut-shaped tetramer with the active sites facing inward forming a central pore. Bivalent ligands spanning two active sites potently inhibit this configuration, but these large compounds have poor drug-like properties. To overcome some of these challenges, we developed self-assembling molecules, called coferons, which deliver a larger compound in two parts. Using a pharmacophoric core and reversibly binding linkers to span two active sites, we have successfully produced three novel homodimeric tryptase inhibitors. Upon binding to tryptase, compounds reassembled into flexible homodimers, with significant improvements in IC₅₀ (0.19 ± 0.08 μM) over controls (5.50 ± 0.09 μM), and demonstrate good activity in mast cell lines. These studies provide validation for this innovative technology that is especially well-suited for the delivery of dimeric drugs to modulate intracellular macromolecular targets.

Figure 1

Designs for target-directed self-assembling dimeric drugs.(A) [3-(1-Acylpiperidin-4-yl)phenyl]methanamine was employed as the pharmacophoric core for the development of reversible homodimeric inhibitors of tetrameric human β-tryptase. One monomer binds each catalytically active subunit of tryptase, such that each homodimeric coferon inhibits an adjacent pair of tryptase subunits. (B) Examples of homodimerizing connector plus linker moieties explored in the current manuscript.

Figure 2

Tryptase promotes the assembly of α-hydroxyketo-based coferon dimers resulting in improved potency. (A) α-Hydroxyketo coferons 1a, 2a, and 3a display significant in vitro and cellular potency improvements (ratios) over their nondimerizable racemic vicinal diol analogs (1b, 2b, and 3b, respectively). (B) Cocrystal structure of 3a at 2.3 Å resolution with human β-tryptase indicates that pharmacophore binding sites in adjacent subunits are bridged by a dimeric spiroketal assembly. The (2R,4S)-4-hydroxy-2-(1-hydroxy-1-methyl-ethyl)-1,3-dioxolane diastereomer in left-to-right and right-to-left configuration (depicted displayed on protein surface colored by electrostatic character) were best suited in fitting the bridging density. (C) Contiguous electron density was also observed in the cocrystal structure of 2a (1.65 Å resolution), consistent with occupancy by dimeric spiroketal assemblies. While an R,S-diastereomer, analogous to that of 3a, contributes to the observed 1σ density (depicted), no single spiroketal configuration could account for all aspects (Table S1).