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.