Combinatorial Library of Peptide Isosters Based on Diels−Alder Reactions:  Identification of Novel Inhibitors against a Recombinant Cysteine Protease fromLeishmania mexicana

Abstract

A combinatorial split-and-mix library of peptide isosters based on a Diels-Alder reaction was synthesized as a "one-bead-two-compounds" library and encoded by ladder synthesis for facile analysis by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In the "one-bead-two-compounds" library approach, each bead contains a library member as a putative protease inhibitor along with a fluorescence-quenched substrate for the protease. When the library was screened with CPB2.8 DeltaCTE, a recombinant cysteine protease from L. mexicana, several beads containing compounds with inhibitory activity could be selected from the library and analyzed by MALDI-TOF MS for structure elucidation. Two types of inhibitors were revealed. One novel class of inhibitors had the bicyclic Diels-Alder product isosteric element incorporated internally in a peptide, while the other type was an N-terminal alpha,beta-unsaturated ketone Michael acceptor used as starting material for the Diels-Alder reaction. Selected hit sequences and constructed consensus sequences based on the observed frequencies of amino acids in different subsites were resynthesized and assayed in solution for inhibitor activity and were shown to have IC(50) values in the high nanomolar to low micromolar range.

CC Bond‐Forming Reactions

“…In-situ generation of dienes: Highly reactive dienes such as silyl-enolates, were obtained upon silylation of support-bound a,b-unsaturated ketones (301,302) [308,309]. The nucleophilicity of some of the silyl-enolates may lead to the formation of Michael adducts with electron-poor dienophiles.…”

Biotechnology to improve health in developing countries: a review

“…Two new classes of drugs against leishmaniasis were discovered using a combinatorial process that produced over 150,000 different compounds (Graven et al 2001). Leishmaniasis is a potentially fatal disease that is estimated to affect 12 million people around the globe.…”

Total Chemical Synthesis, Folding, and Assay of a Small Protein on a Water‐Compatible Solid Support

“…These highly pure proteins could be assayed for inhibitory activity towards a variety of enzymes by using strategies such as the one-bead-two-compound fluorescence-inhibitor assay. [19][20][21][22] We have demonstrated the synthesis and folding of a small trypsin inhibitor, EETI-II, in rapid fashion and high yield by solid-phase chemical ligation on a PEG-based solid support. We have also shown the ability to perform enzyme assays with the resin-immobilized inhibitor, as well as recovery of both the enzyme and inhibitor from the resin.…”