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. 2012 Mar;81(3):440-54.
doi: 10.1124/mol.111.077040. Epub 2011 Dec 14.

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-dideoxystreptamine Derivatives

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Free PMC article

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-dideoxystreptamine Derivatives

Mirella Vivoli et al. Mol Pharmacol. .
Free PMC article

Abstract

The prohormone convertases PC1/3 and PC2 are eukaryotic serine proteases involved in the proteolytic maturation of peptide hormone precursors and are implicated in a variety of pathological conditions, including obesity, diabetes, and neurodegenerative diseases. In this work, we screened 45 compounds obtained by derivatization of a 2,5-dideoxystreptamine scaffold with guanidinyl and aryl substitutions for convertase inhibition. We identified four promising PC1/3 competitive inhibitors and three PC2 inhibitors that exhibited various inhibition mechanisms (competitive, noncompetitive, and mixed), with sub- and low micromolar inhibitory potency against a fluorogenic substrate. Low micromolar concentrations of certain compounds blocked the processing of the physiological substrate proglucagon. The best PC2 inhibitor effectively inhibited glucagon synthesis, a known PC2-mediated process, in a pancreatic cell line; no cytotoxicity was observed. We also identified compounds that were able to stimulate both 87 kDa PC1/3 and PC2 activity, behavior related to the presence of aryl groups on the dideoxystreptamine scaffold. By contrast, inhibitory activity was associated with the presence of guanidinyl groups. Molecular modeling revealed interactions of the PC1/3 inhibitors with the active site that suggest structural modifications to further enhance potency. In support of kinetic data suggesting that PC2 inhibition probably occurs via an allosteric mechanism, we identified several possible allosteric binding sites using computational searches. It is noteworthy that one compound was found to both inhibit PC2 and stimulate PC1/3. Because glucagon acts in functional opposition to insulin in blood glucose homeostasis, blocking glucagon formation and enhancing proinsulin cleavage with a single compound could represent an attractive therapeutic approach in diabetes.

Figures

Scheme 1.
Scheme 1.
Synthesis of compounds 166829 and 166830.
Scheme 2.
Scheme 2.
Synthesis of compounds 166369 and 166646.
Fig. 1.
Fig. 1.
2,5-Dideoxystreptamine derivatives screening against PC1/3 (A) and PC2 (B). The compounds were tested at final concentrations of 10, 25, and 50 μM; in this figure, we show the screening experiment performed at 25 μM for PC1/3 and at 10 μM for PC2. The percentage of inhibition was calculated from the equation (1 − Vi/V0) × 100, where Vi and Vo are the enzyme rates (fluorescence units per minute) in the presence and in the absence of inhibitors, respectively. The percentage of inhibition is expressed as the mean ± S.D. and was determined in triplicate.
Fig. 2.
Fig. 2.
Structures of the most active dideoxystreptamine compounds. The structures of the most potent PC inhibitors; the relative 50% inhibitory concentration (IC50) values, determined using inhibitor concentrations ranging between 0 and 500 μM, are shown under each compound (mean ± SD). A, PC1/3; B, PC2.
Fig. 3.
Fig. 3.
Inhibition kinetics for the most potent PC1/3 inhibitors. Lineweaver-Burk plot shows competitive inhibition for compounds 166811 (A), 166812 (B), 166550 (C), and 166631 (D) against PC1/3. The experiment was performed using 0 (●), 5 (■), 10 (▴), and 50 μM (▾) concentrations of inhibitors, and was carried out in duplicate.
Fig. 4.
Fig. 4.
Lineweaver-Burk plots for the most potent PC2 inhibitors: 166829 (A), 166830 (B), and 166369 (C). Kinetic assays were performed in duplicate using the following concentrations: 0 (●), 5 (■), 10 (▴), or 50 μM (▾).
Fig. 5.
Fig. 5.
Dose-response curves for 166369 against various forms of PC1/3. Assays were performed in triplicate using the compound 166369 in the concentration range 0 to 500 μM against the 87-kDa (●) and 66-kDa (○) forms of PC1/3 (A) and against PC2 (B).
Fig. 6.
Fig. 6.
Structures and dose-response curves of compounds 166691 and 166646 against PC2. The structures show the various aryl groups in different positions on the dideoxystreptamine scaffold (A). For compound 166691, the assay was performed in the concentration range 0 to 500 μM (B, 1), whereas for 166646, the dose-response curve was carried out using concentrations between 0 and 100 μM (B, 2).
Fig. 7.
Fig. 7.
Inhibition of proglucagon processing in vitro- proglucagon gel assay. A, proglucagon (PG) processing by PC1/3 in the presence and absence of the best inhibitors. B, proglucagon processing by PC2 in the presence and absence of the best inhibitors. Note that only early proglucagon cleavages are detected by this method.
Fig. 8.
Fig. 8.
Inhibition of proglucagon processing in vitro glucagon radioimmunoassay. Effect of 2,5-dideoxystreptamine derivatives (166829, 166830, and 166369) and the pyrrolidine bis-piperazine 1435-6 on PC2-mediated cleavage of glucagon from proglucagon, as measured by RIA. The experiment was carried out in duplicate using a highly specific glucagon RIA; inhibitor concentrations used were 0.25, 1, 2.5, 5, 10, and 50 μM. The percentage of glucagon production was calculated from the equation (Ci/C0) × 100, where Ci and Co are the concentrations of glucagon obtained in the presence and absence of inhibitors, respectively.
Fig. 9.
Fig. 9.
Inhibition of glucagon synthesis by the 2,5-dideoxystreptamine derivative 166830 and the pyrrolidine bis-piperazine 1435-6 in α-TC6 cells. A, glucagon RIA. α-TC6 cultures were incubated with compounds 166830 and 1435-6 at a final concentration of 75 μM for 36 h. Cell extracts were then collected for total glucagon determination by RIA. Data represent the mean ± S.D. from quadruplicate wells from a representative experiment. *, significantly less (p < 0.0001) than the values obtained from control cultures incubated with the vehicle, Opti-MEM. B, cytotoxicity assay. In a parallel experiment, the WST-1 assay was used to determine the viability of α-TC6 cells after treatment with the inhibitors used above at the same final concentrations. The experiment was carried out using quadruplicate wells, and the data represent the mean ± S.D.
Fig. 10.
Fig. 10.
Binding poses of inhibitors modeled into the PC1/3 active site. A, molecule 166811 is shown in licorice; only polar hydrogen bonds are displayed. The molecular surface of PC1/3 binding site is colored by electrostatic potential. B, overlay of docking poses obtained for four PC1/3 inhibitors.
Fig. 11.
Fig. 11.
Molecular representation of PC2. Overall view. Spheres represent the locations of binding sites: red, active site; orange, allosteric binding sites; and blue, a key allosteric binding site.
Fig. 12.
Fig. 12.
Binding poses of inhibitors modeled into the PC2 active site and allosteric sites (best inhibitors). The arrow shows the entrance of the S1 pocket. A and B have almost the same orientation, showing the active site where compound 166830 is positioned (A) and the potential allosteric site 1 for the binding of compound 166829 (B), which is on the opposite side of the P4 (S4) subsite. C, binding poses of compound 166369 in allosteric site 3, which approaches the active site from the P4 (S4) subsite.

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