Our docking program, Fitted, implemented in our computational platform, Forecaster, has been modified to carry out automated virtual screening of covalent inhibitors. With this modified version of the program, virtual screening and further docking-based optimization of a selected hit led to the identification of potential covalent reversible inhibitors of prolyl oligopeptidase activity. After visual inspection, a virtual hit molecule together with four analogues were selected for synthesis and made in one-five chemical steps. Biological evaluations on recombinant POP and FAPα enzymes, cell extracts, and living cells demonstrated high potency and selectivity for POP over FAPα and DPPIV. Three compounds even exhibited high nanomolar inhibitory activities in intact living human cells and acceptable metabolic stability. This small set of molecules also demonstrated that covalent binding and/or geometrical constraints to the ligand/protein complex may lead to an increase in bioactivity.

The convenient and efficient synthesis of two macrocyclic ligands (15- and 18-membered) based on a dipyrido-6,7,8,9-tetrahydrophenazine (dpqc) or 2,2':6',2''-terpyridine (tpy) heterocycle and a DTTA (diethylenetriaminetriacetic acid) skeleton is described. In these ligands the DTTA skeleton contains an additional extracyclic functionality (NH(2) group) suitable for covalent attachment to bioactive molecules. These octa- and nonadentate ligands form very stable and luminescent neutral lanthanide complexes in aqueous solutions at physiological pH. The corresponding Eu(III) and Tb(III) complexes are characterized by a maximum absorption wavelength compatible with nitrogen laser excitation (337 nm) and attractive lifetimes and quantum yields. Further introduction of a maleimide bioconjugatable handle in the Eu(III) complexes was investigated and a valuable luminescence brightness above 1500 dm(3) mol(-1) cm(-1) at 337 nm was obtained with the corresponding Eu(III) tpy-derivative. Finally, these two luminescent chelates were grafted onto thiol residues of a model antibody (Mab GSS11) without loss of their luminescent properties.