Kinetic target-guided synthesis represents an efficient hit-identification strategy, in which the protein assembles its own inhibitors from a pool of building blocks via an irreversible reaction. Herein, we pioneered an in situ Ugi reaction for the identification of novel inhibitors of a model enzyme and binders for an important drug target, namely, the aspartic protease endothiapepsin and the bacterial ß-sliding clamp DnaN, respectively. Highly sensitive mass-spectrometry methods enabled monitoring of the protein-templated reaction of four reaction partners, which occurred in a background-free manner for endothiapepsin or with a clear amplification of two binders in the presence of DnaN. The Ugi products show low micromolar activity on endothiapepsin or moderate affinity for DnaN. We succeeded in expanding the portfolio of chemical reactions and biological targets and demonstrated the efficiency and sensitivity of this approach, which can find application on any drug target.
KTGS Ugi-4CR Supporting Information