Discovery and characterization of a small-molecule enteropeptidase inhibitor, SCO-792

Abstract

Enteropeptidase, localized into the duodenum brush border, is a key enzyme catalyzing the conversion of pancreatic trypsinogen proenzyme to active trypsin, thereby regulating protein digestion and energy homeostasis. We report the discovery and pharmacological profiles of SCO-792, a novel inhibitor of enteropeptidase. A screen employing fluorescence resonance energy transfer was performed to identify enteropeptidase inhibitors. Inhibitory profiles were determined by in vitro assays. To evaluate the in vivo inhibitory effect on protein digestion, an oral protein challenge test was performed in rats. Our screen identified a series of enteropeptidase inhibitors, and compound optimization resulted in identification of SCO-792, which inhibited enteropeptidase activity in vitro, with IC ₅₀ values of 4.6 and 5.4 nmol/L in rats and humans, respectively. In vitro inhibition of enteropeptidase by SCO-792 was potentiated by increased incubation time, and the calculated K_inact/K_I was 82 000/mol/L s. An in vitro dissociation assay showed that SCO-792 had a dissociation half-life of almost 14 hour, with a calculated k_off rate of 0.047/hour, which suggested that SCO-792 is a reversible enteropeptidase inhibitor. In normal rats, a ≤4 hour prior oral dose of SCO-792 effectively inhibited plasma elevation of branched-chain amino acids in an oral protein challenge test, which indicated that SCO-792 effectively inhibited protein digestion in vivo. In conclusion, our new screen system identified SCO-792 as a potent and reversible inhibitor against enteropeptidase. SCO-792 slowly dissociated from enteropeptidase in vitro and inhibited protein digestion in vivo. Further study using SCO-792 could reveal the effects of inhibiting enteropeptidase on biological actions.

Keywords: FRET; SCO‐792; covalent; enteropeptidase.

Figure 1

Time course and determination of K _m value of enteropeptidase for various substrates. Enteropeptidase and various concentrations of GDDDDK‐βNA (A), Cy5‐substrate (C), and 5FAM‐substrate (E) were incubated in enteropeptidase assay buffer in a 384‐well plate at room temperature, and the fluorescence was measured. The final concentrations of enteropeptidase were 10 U/mL for GDDDDK‐βNA, 31.3 mU/mL for Cy5 substrate, and 8 mU/mL for 5FAM substrate. Data are presented as mean ± SE values (n = 4). Initial velocities from (A, C, E) are plotted in (B, D, F) as functions of substrate concentration. The data were analyzed using the Michaelis–Menten equation to give K _m values

Figure 2

Time‐dependent inhibition of enteropeptidase by the hit compound T‐0046812. (A) Chemical structure of T‐0046812. (B) Percent inhibition is plotted as a function of inhibitor concentration at 6 and 120 minutes of incubation. (C) Enzyme progress curves were obtained at eight different inhibitor concentrations. (D) Data from (C) were fit to Equation to determine observed rate constants (K _obs). Replotting of K _obs according to Equation yielded K _inact/K_I. The estimated K _inact/K_I was 5300 (mol/L s)

Figure 3

Time‐dependent inhibition of enteropeptidase by SCO‐792. (A) Chemical structure of SCO‐792. (B) Percent inhibition is plotted as a function of inhibitor concentration at 10, 30, 60, and 120 minutes of incubation. (C) Enzyme progress curves were obtained at 16 different inhibitor concentrations. (D) Data from (C) were fit to Equation to determine observed rate constants (K _obs). Replotting of K _obs according to Equation yielded K _inact/K_I. The estimated K _inact/K_I was 82 000 (/mol/L s)

Figure 4

Determination of dissociation half‐life and predicted molecular mechanism of SCO‐792. (A) Enteropeptidase was preincubated for 120 min with and without SCO‐792 and diluted rapidly with substrate solution. Recovery of enteropeptidase activity was detected kinetically. SCO‐792 exhibits very slow reversibility with an estimated t _1/2 value of approximately 14 hours. (B) Predicted mechanism of the inhibitory and reversible activity of SCO‐792

Figure 5

Plasma concentration of SCO‐792 after oral (A) or intravenous (B) administration in rats. SCO‐792 was administrated at the dose of 10 mg/kg orally and 0.2 mg/kg intravenously. Values are presented as the mean ± SD (n = 3). Calculated pharmacokinetic parameters are shown in Table 2

Figure 6

Effect of a single oral dose of SCO‐792 on plasma BCAA levels after an oral protein challenge in rats. Plasma BCAA levels at 1 (A), 2 (B), 4 (C), and 6 hours (D) after oral SCO‐792 dosing in an oral protein challenge test. SCO‐792 effectively suppressed BCAA elevation in this study. Values are presented as the mean ± SD (n = 4). ^# P ≤ 0.025 vs vehicle by one‐tailed Williams’ test