Evaluation of a Two-Stage Screening Procedure in the Combinatorial Search for Serine Protease-Like Activity

Abstract

A series of peptidosteroid derivatives containing two independent peptide chains in which Ser and His are incorporated were synthesized by solid-phase peptide synthesis. The activity of the different compounds in the hydrolysis of the activated substrate NF31 was assessed in a stepwise fashion. First, the different resin-bound derivatives 6a-l and 6x-z were individually assayed for serine esterification in the absence of water. The use of a colored substrate allowed for a visual identification of the most active compounds. Through the inclusion of control substances, the involvement of histidine in the mechanism for serine acylation was shown. Second, the hydrolysis and methanolysis of the different acylated derivatives 8a-l and 8x were evaluated using UV spectroscopy, again indicating the involvement of histidine. The feasibility of applying the above procedures in a combinatorial context was proven via the screening of artificial libraries, created by mixing the different resin-bound peptidosteroid compounds. In this respect, the use of a photocleavable linker allowed for the unambiguous structural characterization of the selected members via application of single-bead electrospray tandem mass spectrometry.

Design and automated generation of artificial estrogen receptor as potential endocrine disruptor chemical binders

“…15,16 The typical A-B cis connection with appendages originating from the C-3 and C-12 positions imposes a rigid and parallel orientation upon connection of peptide chains; this in contrast to the flexible nature of our previous tripodal design. Combining the more preorganized nature of the chains with the intrinsic hydrophobic cavity presented by the cholic acid should allow for the development of artificial receptors with more potent EDC binding properties.…”

“…16. IR spectra were recorded on a PerkineElmers 1000 equipped with HATR and reported in wavenumber (cm À1 ).…”

“…Although the cholic acid template has been used in a variety of designs, the reported and earlier developed synthetic methodology for construction of compound collections was centered around the manual generation of either single compound 17 or mix-split libraries. 15,16 As for generation of parallel libraries, using a standard automated peptide synthesis apparatus, the orthogonality scheme of 1 presents some serious drawbacks.…”

Evaluation of non-covalent interaction between Seryl-Histidine dipeptide and cyclophilin A using NMR and molecular modeling

“…It could not only cleave DNA and carboxyl esters by a hydrolysis mechanism [7,8], but also cleave bovine serum albumen (BSA) [8][9][10] and green fluorescence protein (GFP) [11] in a wide range of pH and temperature. Therefore, as a protease mimics, Ser-His is attracting increasingly more attention for its special biological function [12,13]. Elucidation of molecular mechanisms by which the mini-protease Ser-His functions, especially the molecular mechanism of Ser-His cleaving proteins, will be helpful to understand the repeated selection in the evolution of diverse groups of enzymes.…”

Synthesis of Functionalised Nucleosides for Incorporation into Nucleic Acid-Based Serine Protease Mimics

“…In a first approach, we developed non-peptidic organic molecules containing an array of functional groups in a suitable geometry for possible hydrolytic activity [22]. In a second combinatorial approach, a dipodal scaffold based on a cholic acid template was synthesized and two independent peptide chains were attached, each containing one residue of the catalytic triad [23,24]. In addition, a model based on a tripodal scaffold, with a rigid structure possessing three independent functionalised peptide chains, was also synthesized [25].…”