Active Site Mapping of an Aspartic Protease by Multiple Fragment Crystal Structures: Versatile Warheads To Address a Catalytic Dyad

J Med Chem. 2016 Nov 10;59(21):9743-9759. doi: 10.1021/acs.jmedchem.6b01195. Epub 2016 Oct 28.


Crystallography is frequently used as follow-up method to validate hits identified by biophysical screening cascades. The capacity of crystallography to directly screen fragment libraries is often underestimated, due to its supposed low-throughput and need for high-quality crystals. We applied crystallographic fragment screening to map the protein-binding site of the aspartic protease endothiapepsin by individual soaking experiments. Here, we report on 41 fragments binding to the catalytic dyad and adjacent specificity pockets. The analysis identifies already known warheads but also reveals hydrazide, pyrazole, or carboxylic acid fragments as novel functional groups binding to the dyad. A remarkable swapping of the S1 and S1' pocket between structurally related fragments is explained by either steric demand, required displacement of a well-bound water molecule, or changes of trigonal-planar to tetrahedral geometry of an oxygen functional group in a side chain. Some warheads simultaneously occupying both S1 and S1' are promising starting points for fragment-growing strategies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspartic Acid Proteases / chemistry*
  • Aspartic Acid Proteases / metabolism*
  • Biocatalysis*
  • Calorimetry
  • Carboxylic Acids / chemistry
  • Carboxylic Acids / metabolism*
  • Catalytic Domain
  • Crystallography, X-Ray
  • Hydrazines / chemistry
  • Hydrazines / metabolism*
  • Models, Molecular
  • Pyrazoles / chemistry
  • Pyrazoles / metabolism*


  • Carboxylic Acids
  • Hydrazines
  • Pyrazoles
  • pyrazole
  • Aspartic Acid Proteases