Structure-based design and combinatorial chemistry yield low nanomolar inhibitors of cathepsin D

The identification of potent small molecule ligands to receptors and enzymes is one of the major goals of chemical and biological research. Two powerful new tools that can be used in these efforts are combinatorial chemistry and structure-based design. Here we address how to join these methods in a design protocol that produces libraries of compounds that are directed against specific macromolecular targets. The aspartyl class of proteases, which is involved in numerous biological processes, was chosen to demonstrate this effective procedure.

Using cathepsin D, a prototypical aspartyl protease, a number of low nanomolar inhibitors were rapidly identified. Although cathepsin D is implicated in a number of therapeutically relevant processes, potent nonpeptide inhibitors have not been reported previously. The libraries, synthesized on solid support, displayed nonpeptide functionality about the (hydroxyethyl)amine isostere. The (hydroxyethyl)amine isostere, which targets the aspartyl protease class, is a stable mimetic of the tetrahedral intermediate of amide hydrolysis. Structure-based design, using the crystal structure of cathepsin D complexed with the peptide-based natural product pepstatin, was used to select the building blocks for the library synthesis. The library yielded a 'hit rate' of 6-7% at 1 microM inhibitor concentrations, with the most potent compound having a Ki value of 73 nM. More potent, nonpeptide inhibitors (Ki = 9-15 nM) of cathepsin D were rapidly identified by synthesizing and screening a small second generation library.

The success of these studies clearly demonstrates the power of coupling the complementary methods of combinatorial chemistry and structure-based design. We anticipate that the general approaches described here will be successful for other members of the aspartyl protease class and for many other enzyme classes.