Experimental Active-Site Mapping by Fragments: Hot Spots Remote from the Catalytic Center of Endothiapepsin

Successful optimization of a given
lead scaffold requires thorough
binding-site mapping of the target protein particular in regions remote
from the catalytic center where high conservation across protein families
is given. We screened a 361-entry fragment library for binding to
the aspartic protease endothiapepsin by crystallography. This enzyme
is frequently used as a surrogate for the design of renin and β-secretase
inhibitors. A hit rate of 20% was achieved, providing 71 crystal structures.
Here, we discuss 45 binding poses of fragments accommodated in pockets
remote from the catalytic dyad. Three major hot spots are discovered
in remote binding areas: Asp81, Asp119, and Phe291. Compared to the
dyad binders, bulkier fragments occupy these regions. Many of the
discovered fragments suggest an optimization concept on how to grow
them into larger ligands occupying adjacent binding pockets that will
possibly endow them with the desired selectivity for one given member
of a protein family.