Pluripotentialities of a quenched fluorescent peptide substrate library: enzymatic detection, characterization, and isoenzymes differentiation.

Protease inhibitors represent a major class of drugs, even though a large number of proteases remain unexplored. Consequently, a great interest lies in the identification of highly sensitive substrates useful for both the characterization and the validation of these enzyme targets and for the design of inhibitors as potential therapeutic agents through high-throughput screening (HTS). With this aim, a synthetic substrate library, in which the highly fluorescent (L)-pyrenylalanine residue (Pya) is efficiently quenched by its proximity with the p-nitro-(L)-phenylalanine (Nop) moiety, was designed. The cleavage between Pya and Nop leads to a highly fluorescent metabolite providing the required sensitivity. This library, characterized by a water-soluble primary sequence Ac-SGK-Pya-(X)(n)(-)Nop-GGK-NH(2), X being a mixture of 10 natural amino acids (A, I, L, K, F, W, E, Q, T, P) and n varying from 0 to 3, was validated using enzymes belonging to the four main types of hydrolases: serine-, metallo-, cystein-, and aspartyl-proteases. The selectivity of substrates belonging to this library was evidenced by characterizing specific substrates for the isoenzymes NEP-1 and NEP-2. This library easily synthesized is of great interest for the identification and development of selective and specific substrates for still uncharacterized endoproteases.

Identification of an endothelin-converting enzyme-2-specific fluorigenic substrate and development of an in vitro and ex vivo enzymatic assay.

Endothelin-converting enzyme-2 (ECE-2) is a membrane-bound zinc-dependent metalloprotease that shares a high degree of sequence homology with ECE-1, but displays an acidic pH optimum characteristic of maturing enzymes acting late in the secretory pathway. Although ECE-2, like ECE-1, can cleave the big endothelin intermediate to produce the vasoconstrictive endothelin peptide, its true physiological function remains to be elucidated, a task that is hampered by the lack of specific tools to study and discriminate ECE-2 from ECE-1, i.e. specific substrates and/or specific inhibitors. To fill this gap, we searched for novel ECE-specific peptide substrates. To this end, peptides derived from the big endothelin intermediate were tested using ECE-1 and ECE-2, leading to the identification of an ECE-1-specific substrate. Moreover, screening of our proprietary fluorigenic peptide Fluofast® libraries using ECE-1 and ECE-2 allowed the identification of Ac-SKG-Pya-F-W-Nop-GGK-NH(2) (PL405), as a specific and high affinity ECE-2 substrate. Indeed, ECE-2 cleaved PL405 at the Pya-F amide bond with a specificity constant (k(cat)/K(m)) of 8.1 ± 0.9 × 10(3) M(-1) s(-1). Using this novel substrate, we also characterized the first potent (K(i) = 7.7 ± 0.3 nM) and relatively selective ECE-2 inhibitor and developed a quantitative fluorigenic ECE-2 assay. The assay was used to study the ex vivo ECE-2 activity in wild type and ECE-2 knock-out tissues and was found to truly reflect ECE-2 expression patterns. The PL405 assay is thus the first tool to study ECE-2 inhibition using high throughput screening or for ex vivo ECE-2 quantification.

Detection and quantification of botulinum neurotoxin type a by a novel rapid in vitro fluorimetric assay.

Botulinum neurotoxin type A (BoNT/A), the most poisonous substance known to humans, is a potential bioterrorism agent. The light-chain protein induces a flaccid paralysis through cleavage of the 25-kDa synaptosome-associated protein (SNAP-25), involved in acetylcholine release at the neuromuscular junction. BoNT/A is widely used as a therapeutic agent and to reduce wrinkles. The toxin is used at very low doses, which have to be accurately quantified. With this aim, internally quenched fluorescent substrates containing the fluorophore/repressor pair pyrenylalanine (Pya)/4-nitrophenylalanine (Nop) were developed. Nop and Pya were, respectively, introduced at positions 197 and 200 of the cleavable fragment (amino acids 187 to 203) of SNAP-25 (with norleucine at position 202 [Nle(202)]), which is acetylated at its N terminus and amidated at its C terminus. Cleavage of this peptide occurred between positions 197 and 198, as in SNAP-25, and was easily quantified by the strong fluorescence emission of the metabolite. To increase the assay sensitivity, the peptide sequence of the previous substrate was lengthened to account for exosite binding to BoNT/A. We synthesized the peptide PL50 (SNAP-25-NH(2) acetylated at positions 156 to 203 [Nop(197), Pya(200), Nle(202)]) and its analogue PL51, in which all methionines were replaced by nonoxidizable Nle. Consistent with a large increase in affinity for BoNT/A, PL50 and PL51 exhibit catalytic efficiencies of 2.6 x 10(6) M(-1) s(-1) and 8.85 x 10(6) M(-1) s(-1), respectively, and behave as the best fluorigenic substrates of BoNT/A reported to date. Under optimized assay conditions, they allow simple quantification of as little as 100 and 60 pg of BoNT/A, respectively, within 2 h with a classical fluorimeter. Calibration of the method against the mouse 50% lethal dose assay unequivocally validates the enzymatic assay.