Modulating furin activity with designed mini‐PDX peptides: Synthesis and in vitro kinetic evaluation

Abstract

A peptide was designed from reactive site loop structure of a1 Antitrypsin Portland known as a1 PDX as a novel mini-PDX inhibitor of furin. The sequence was derived from (367-394) that contains the crucial furin cleavage motif RIPR 382 . A P3 mutant replacing Ile 380 by Leu was prepared as a first model peptide. A Cys residue was inserted at each terminal of the peptide for purpose of cyclisation which was accomplished by air or iodine-induced oxidation. This mini-PDX peptide both cyclic and acyclic form inhibited in vitro furin activity (IC 50 in nM) when measured against either substrates Boc-RVRRßMCA or QVEGF-C [Abz-QVHSIIRRßSLP-Y(NO 2 )-A-CONH 2 , Abz = 2-amino benzoic acid and Y(NO 2 ) = 3-nitro tyrosine], latter being derived from vascular endothelial growth factor-C (VEGF-C) processing site. The geometrically constrained structure mimicking PDX reactive loop is crucial for enzyme inhibition. Our study further revealed that both mini-PDX peptides inactivate furin in a slow tight binding manner, with disulfidebridged cyclic form being slightly more potent. Unlike PDX, these peptides inhibit furin via a different mechanistic pathway. The study provides an alternate strategy for development of efficient peptide-based inhibitors of Proprotein Convertases including furin.

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

“…To date, most reported inhibitors against the proprotein convertase furin have been either proteins (Dahlen et al, 1998;Dufour et al, 2001;Komiyama et al, 2003;Richer et al, 2004) or peptides/peptide derivatives (Cameron et al, 2000a;Villemure et al, 2003;Basak and Lotfipour, 2005). Nonprotein, nonpeptide convertase inhibitors reported thus far are the natural products of the andrographalide family and their succinoyl ester derivatives (Basak et al, 1999); certain metal complexes (Podsiadlo et al, 2004); dicoumarol and its derivatives (Komiyama et al, 2009); and the bicyclic guanidine and pyrrolidine bis-piperazine derivatives we previously identified as PC2 inhibitors (Kowalska et al, 2009).…”

Proteolytic truncation of human transthyretin linked to amyloidosis is mediated by a trypsin like enzyme: In vitro demonstration using model peptides

“…Peaks were collected and lyophilized twice to a white fluffy powder. It was then subjected to analysis by mass spectrometry for their identifications as described in [39].…”

“…SELDI-tof mass spectrum was performed on re-usable clean gold plate chips with 2 μl of sample and 2 μl of CHCA or DHB matrix solution as described earlier [38,39]. Each mass spectrum was calibrated against pure commercial ACTH (1-17) peptide (MW 2,093.71 Da), purchased from Sigma-Aldrich Company) both as an internal and external standard.…”

“…Typically 5 μl of freshly prepared trypsin solution (0.1 mg/ml in water) was incubated with peptidyl fluorogenic substrate in an aqueous buffer consisting of 25 mM Tris + 25 mM Mes, pH 7.4 and the fluorescence released at various times was measured with a fluorometer instrument with excitation and emission wavelengths fixed at 370 and 460 nm respectively. The experiment was conducted in a 96-microtitre well plate (black color, 50 μl volume capacity) using fluorescence spectrophotometer (Molecular Devices Co, USA) [38,39].…”

“…The release of fluorescence was monitored by a spectrofluorometer (Spectramax, Gemini XS) at excitation and emission wavelengths of 360 nm and 470 nm respectively. The K i (inhibition constant) was derived from Cornish-Bowden plot using the protocol published earlier [39,40].…”