Inhibition properties of free and conjugated leupeptin analogues

Abstract

Leupeptin is a naturally occurring inhibitor of various proteases, in particular serine proteases. Following its discovery, the inhibitory properties of several other peptidyl argininals have been studied. The specificity of leupeptin is most likely due to the Leu-Leu-Argininal sequence, and its C-terminal aldehyde group has been suggested to enhance the binding efficiency and to be essential for function. The terminal aldehyde group makes the structure less vulnerable to carboxypeptidases. Here, we investigated whether the inhibitory function of leupeptin toward serine proteases is retained after oxidation or reduction of the aldehyde group. The oxidized form, which corresponds to the natural precursor, was shown to be superior to the reduced form in terms of inhibitory properties. However, the original leupeptin possessed enhanced inhibitory properties as compared with the oxidized form. Based on these results, new synthetic leupeptin analogues, 6-aminohexanoic acid (Ahx)-Phe-Leu-Arg-COOH and Ahx-Leu-Leu-Arg-COOH, were prepared by solid-phase peptide synthesis using the Fmoc strategy. In these analogues, the N-terminal capping acetyl group was replaced with a 6-aminohexanoyl group to allow conjugation. The structures of the modified leupeptin and the synthetic peptides were confirmed by mass spectrometry. Determination of the inhibitory properties against trypsin (IEC 3.4.21.4, Chymotrypsin IEC 3.4.21.1) revealed that these further modified tripeptides were tight binding inhibitors to their target enzyme, similar to the naturally occurring leupeptin, with K_i values generally in the micromolar range. The Ahx-Phe-Leu-Arg-COOH analogue was selected for conjugation to inorganic oxide nanoparticles and agarose gel beads. All conjugates exhibited inhibitory activity in the same range as for the free peptides.

Keywords: conjugation; inhibition; inhibitor design; leupeptin analogues; tight binding.

Fig. 1

Leupeptin in its natural state (Ac–Leu–Leu–Arg–CHO).

Fig. 2

(A) MS spectra of oxidized leupeptin with a peak at 443 m/z corresponding to the molecular ion [M + H]. (B) MS spectra of reduced leupeptin with a peak at 429 m/z corresponding to the molecular ion [M + H], confirming the identity of both products.

Fig. 3

Tentative comparison of active site binding mode for original, reduced and oxidized leupeptin, respectively.

Fig. 4

The ratio v_i/v ₀ is plotted as a function of the inhibitor peptide Ahx–Phe–Leu–Arg–COOH (A) and Ahx–Leu–Leu–Arg–COOH (B). The conditions for the experiments and calculations were [trypsin] = 0.25 μm, [BAPA] = 1 mm and K _m = 0.82 mm. Error bars represent standard deviation.

Fig. 5

v_i/v ₀ is plotted as a function of the conjugated TiO₂–Ahx–Phe–Leu–Arg–COOH (A), ZnO–Ahx–Phe–Leu–Arg–COOH (B) and WorkBeads™‐Ahx‐Phe‐Leu‐Arg‐COOH (C). The conditions for the experiments and calculations were [trypsin] = 0.25 μm, [BAPA] = 1 mm and K _m = 0.82 mm. Error bars represent standard deviation.

Fig. 6

Relative area increase rate for free peptide 1, conjugated peptide 1 and free particles.

Fig. 7

The oxidation of leupeptin using Tollen’s test.

Fig. 8

The reduction of leupeptin using sodium borohydride.

Fig. 9

Structure of the two tripeptides.

Fig. 10

Conjugate of Ahx–Phe–Leu–Arg–COOH to TiO₂ particles, where R = (CH₂)₃NHC(=O)–Ahx–Phe–Leu–Arg–COOH.

Fig. 11

Conjugate of agarose gel bead and peptide 1. The gel bead in comparison with the peptide is about 40 000 times larger, and the gel bead can conjugate a number of peptides. This figure demonstrates only the conjugation between the bead and the peptide itself, and the components are not in correct scale.