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.

Novel, Self-Assembling Dimeric Inhibitors of Human β Tryptase

“…By reversibly joining two small molecules through bio-orthogonal linkers, coferons enable the delivery of larger, more specific, and higher-affinity dimeric molecules. 5,6 The coferon chemistry platform employs bio-orthogonal linker chemistries that allow the intracellular self-assembly of two distinct small molecule monomers, composed of a ligand, a connector, and a linker element, into a large dimeric inhibitor molecule designed to be capable of more potent and selective inhibition. This study confirms that polyvalent binding increases the affinity and specificity of interactions between molecules, 13 where a number of self-assembled heterodimeric tryptase inhibitors demonstrate marked improvements over the activity of their monomeric parts.…”

“…A solution of 40 in MeCN (65 mL) was charged with TFA (10 mL) and stirred at RT for 12 h. The reaction mixture was concentrated in vacuo and purified by reverse-phase HPLC, resulting in a TFA salt that was subsequently converted into the HCl salt by stirring the pure product in 2 N HCl for 30 min under a N 2 atmosphere and lyophilized to dryness, which resulted in 81% yield of 9s. Synthesis of [3][4][5]phenyl]piperidine-1carbonyl]-2,3-dimethylphenyl]phenyl]boronic Acid (10a) and [3][4][5]…”

“…3,4 We have developed reversible, self-assembling linkers to deliver two drug monomers in parts, producing more specific and higheraffinity dimeric drug molecules, termed coferons. 5,6 Each drug monomer contains a ligand that has an affinity for a protein binding site and a small linker moiety capable of reversibly joining with a complementary linker from an adjacent monomer that binds a proximal site. Upon binding to their macromolecular target, the monomers are driven to covalently re-associate, where the protein serves as a template for the assembly of the higher-affinity dimer, and more effectively modulate activity than equivalent nonassembling monomers.…”

“…Upon binding to their macromolecular target, the monomers are driven to covalently re-associate, where the protein serves as a template for the assembly of the higher-affinity dimer, and more effectively modulate activity than equivalent nonassembling monomers. 5,6 Our methods are built on protein-directed dynamic combinatorial chemistry methods 7−11 and incorporate key qualities of multivalency 12,13 and fragment-based drug discovery. 14−17 Weakly active drug fragments assembling covalently in situ have been previously shown to form more active compounds, 18 but our application of reversibly covalent linker chemistries gives rise to opportunities to target proteins within the cellular milieu.…”

“…5,6 We have previously reported the use of hydroxy-ketone homodimeric linkers that reversibly cojoin two identical tryptase ligands. 5 This article builds on that precedent and expands the coferon technology to incorporate linkers with the ability to co-join nonidentical pharmacophores to target adjacent protein sites.…”