Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: Deconvolution strategy

Abstract

Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X1-X2-hinge-X3-X4-NH2 (capped) and X1-hinge-X2-X3 (uncapped), where X1 through X4 are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.

The Strange Case of the Botulinum Neurotoxin: Using Chemistry and Biology to Modulate the Most Deadly Poison

“…[97] The linker was chosen to alter the conformation of the amino acid backbone in efforts to enhance BoNT recognition. Further efforts extended the repertoire of amino acids to 12 (giving 12 4 different peptides for study) [98] and used positional scanning synthetic combinatorial library (PS-SCL) technology [99] with the aim of improving the deconvolution strategy to identify the optimal peptide.…”

“…LC/B inhibition of over 80 % was observed with peptide libraries of the formula Ac-X 1 C 2 -linker-X 3 C 4 -NH 2 and C 1 -linker-X 2 F 3 . [99] Several years ago, Schmidt et al recognized the potential for developing BoNT/A inhibitors by exploiting the metalloprotease (LC) properties of the BoNT. [100] Since sulfhydryl groups readily complex zinc, it was reasoned that substitution of key amino acid residues with Cys would provide molecules that inhibit the enzymatic activity of BoNTs.…”

Der seltsame Fall des Botulinum‐Neurotoxins: chemische und biologische Modulierung des tödlichsten aller Gifte

“…LC/BInhibierungen von über 80 % wurden mit Peptiden vom Typ Ac-X 1 C 2 -Linker-X 3 C 4 -NH 2 und C 1 -Linker-X 2 F 3 erzielt. [99] Vor einigen Jahren erkannten Schmidt und Mitarbeiter, dass die spezifischen Eigenschaften der Metalloproteasedomäne (LC) zur Entwicklung von BoNT/A-Inhibitoren genutzt werden können.…”

Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A

“…The activated light chain cleaves the substrate into two products of 11 and 6 amino acids. The substrate has been used in vitro to detect peptidase activity with the activated light chain of BoNT/A and used as a tool to design and discover novel inhibitors in combinatorial peptide libraries [6][7][8]. Previously HPLC was the method of analysis used to measure the concentrations of substrate and products in reaction mixtures [6,8].…”

“…The substrate has been used in vitro to detect peptidase activity with the activated light chain of BoNT/A and used as a tool to design and discover novel inhibitors in combinatorial peptide libraries [6][7][8]. Previously HPLC was the method of analysis used to measure the concentrations of substrate and products in reaction mixtures [6,8]. Several limitations of HPLC such as the length of time for analysis (approximately 1 h per sample) and the inability to completely resolve and easily quantitate substrate and products in reaction mixtures, especially in the presence of combinatorial peptide libraries, impeded the search for inhibitor compounds.…”