Abstract
The facile synthesis and detailed investigation of a class of highly potent protease inhibitors based on 1,4-naphthoquinones with a dipeptidic recognition motif (HN-l-Phe-l-Leu-OR) in the 2-position and an electron-withdrawing group (EWG) in the 3-position is presented. One of the compound representatives, namely the acid with EWG = CN and with R = H proved to be a highly potent rhodesain inhibitor with nanomolar affinity. The respective benzyl ester (R = Bn) was found to be hydrolyzed by the target enzyme itself yielding the free acid. Detailed kinetic and mass spectrometry studies revealed a reversible covalent binding mode. Theoretical calculations with different density functionals (DFT) as well as wavefunction-based approaches were performed to elucidate the mode of action.
Highlights
Acting drugs have traditionally been considered as problematic due to their potential to elicit off-target effects
The potential inhibitors were synthesized by an addition/elimination reaction on the naphthoquinone unit carrying two potential leaving groups (Scheme 1)
To further characterize the interaction between rhodesain and the two benzyl ester-based inhibitors 1 and 2, we performed a liquid-chromatography–mass spectrometric (LC–massspectra spectra (MS)) analysis of rhodesain that had been incubated with the compounds
Summary
Acting drugs have traditionally been considered as problematic due to their potential to elicit off-target effects. Since we were interested in the inhibition properties of the two warheads, we used the same recognition unit, the dipeptide sequence H2 N-l-Phe-l-Leu-OBn and attached the 1,4-naphthoquinone unit to its N-terminus This dipeptide sequence was chosen since it was previously shown to be an appropriate recognition unit for cathepsin-like cysteine proteases [29,30]. We previously observed that rhodesain is able to hydrolyze compounds with the dipeptidic H2 N-l-Phe-l-Leu-OBn recognition unit [30] In agreement with these findings, we observed the protease-mediated hydrolysis of the benzyl ester moieties of the naphthoquinone-modified inhibitors to yield the free acids (see Sections 2.4 and 2.6). To evaluate which theoretical approaches are sufficiently reliable to predict the inhibition mechanisms and potencies of such groups, a benchmark study employing various wave functions as well as density-based approaches were performed
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