A Novel, Nonpeptidic, Orally Active Bivalent Inhibitor of Human β-Tryptase

Abstract

β-Tryptase is released from mast cells upon degranulation in response to allergic and inflammatory stimuli. Human tryptase is a homotetrameric serine protease with 4 identical active sites directed toward a central pore. These active sites present an optimized scenario for the rational design of bivalent inhibitors, which bridge 2 adjacent active sites. Using (3-[1-acylpiperidin-4-yl]phenyl)methanamine as the pharmacophoric core and a disiloxane linker to span 2 active sites we have successfully produced a novel bivalent tryptase inhibitor, compound 1a, with a comparable profile to previously described inhibitors. Pharmacological properties of compound 1a were studied in a range of in vitro enzymic and cellular screening assays, and in vivo xenograft models. This non-peptide inhibitor of tryptase demonstrated superior activity (IC50 at 100 pmol/L tryptase = 1.82 nmol/L) compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and 1a demonstrated good oral bioavailability and efficacy in HMC-1 xenograft models. Furthermore, compound 1a demonstrated extremely slow off rates and high selectivity against-related proteases. This highly potent, orally bioavailable and selective inhibitor of human tryptase will be an invaluable tool in future studies to explore the therapeutic potential of attenuating the activity of this elusive target.

Keywords: Bivalent inhibitor; Disiloxane; Protease inhibitors; Tight-binding; X-ray analysis; β-Tryptase.

Figure 1.

A. Dose-response curves for the silanol bivalent compound, 1a, and its monomeric control, 1b, were compared to responses of the bivalent inhibitor, 2a, and its monomeric analog, 2b. B. IC₅₀s and fold difference between nafamostat, the bivalent and control compounds are indicated for increasing concentrations of tryptase.

Figure 2.

A. Co-crystal structures of 1a confirmed a bivalent mechanism of inhibition, providing insights into the relaxation of the disiloxane angular deformation to 145°. Compound 1a is depicted in stick format and the protein as a surface colored by pKa. B. Curvature was observed with 1a in a continuous fluorimetric enzyme assay at 1nM tryptase, particularly in the vicinity of the IC₅₀, consistent with tight binding. C. Extremely slow off-rates of the bivalent silanol were observed in reversibility studies. Tryptase (100nM) was treated with 1a or monomer control 1b (10μM) and unbound inhibitor was removed by gel filtration spin column. The molar ratio of 1a to tryptase subunits was determined to be 1.3 (± 0.3):1 by LC-MS/MS. The subsequent solutions were diluted to 1nM tryptase and assayed daily for tryptase activity. There was no recovery in activity without removal of excess inhibitor.

Figure 3.

A. Tryptase activity is potently inhibited by treatment of HMC-1 cells with 1a (IC₅₀ = 58nM ± 0.05) and 2a (IC₅₀ = 44nM ± 0.05), followed by washing, and induction of degranulation with a calcium ionophore. Monomeric controls were more than 130 less potent than the bivalent inhibitors. B. Inhibition of mast cell associated tryptase activity in formaldehyde-fixed human lung tissue from asthmatic patients. Fixed sections were incubated with DMSO (C), 100μM 1a (D), or 1b (E) for 1h prior to histochemical staining with a colorimetric tryptase substrate. Staining of mast cells (arrows) was completely inhibited by 1a but not its monomeric analog 1b.

Figure 4.

A. The pharmacokinetics of 1a as its formate salt were determined in freely feeding male C57BL/6 mice following single intravenous (10mg/kg in 0.9% sterile saline) and oral (10 and 100mg/kg in water) doses. At 10mg/kg p.o. the oral bioavailability was 96 %, with peak plasma levels of 653nM (± 265). Only monomeric silanol was detected in all bioanalytical samples. B. Nude mice bearing bilateral HMC1 xenograft tumors were treated orally with 1a (7.8mg/kg as TFA salt in water) or intravenously with nafamostat (10mg/kg i.v.). After 4h, tumors were excised, homogenized and assayed for tryptase activity. 1a significantly reduced tryptase activity (p = 0.0057) by 61% (± 5.8), with mean plasma and tumor tissue levels of 4.1ng/ml (± 1.2) and 5.2ng/mg (± 2.2), respectively.