Structures of endothiapepsin–fragment complexes from crystallographic fragment screening using a novel, diverse and affordable 96-compound fragment library

Abstract

Crystallographic screening of the binding of small organic compounds (termed fragments) to proteins is increasingly important for medicinal chemistry-oriented drug discovery. To enable such experiments in a widespread manner, an affordable 96-compound library has been assembled for fragment screening in both academia and industry. The library is selected from already existing protein-ligand structures and is characterized by a broad ligand diversity, including buffer ingredients, carbohydrates, nucleotides, amino acids, peptide-like fragments and various drug-like organic compounds. When applied to the model protease endothiapepsin in a crystallographic screening experiment, a hit rate of nearly 10% was obtained. In comparison to other fragment libraries and considering that no pre-screening was performed, this hit rate is remarkably high. This demonstrates the general suitability of the selected compounds for an initial fragment-screening campaign. The library composition, experimental considerations and time requirements for a complete crystallographic fragment-screening campaign are discussed as well as the nine fully refined obtained endothiapepsin-fragment structures. While most of the fragments bind close to the catalytic centre of endothiapepsin in poses that have been observed previously, two fragments address new sites on the protein surface. ITC measurements show that the fragments bind to endothiapepsin with millimolar affinity.

Protein X-ray Crystallography and Drug Discovery

“…The efforts can therefore be concentrated on the refinement of the few actual complexes. In a fragment-screening project, a hit rate of 5-10% is expected [11,[52][53][54]. This corresponds to 50 to 100 structures that should be fully refined for a 1000-fragment library.…”

FragMAX: the fragment-screening platform at the MAX IV Laboratory

“…Several fragment libraries are available to users, Frag-MAXlib, F2XEntry (Wollenhaupt et al, 2020) and Xtal Frag Screen (Huschmann et al, 2016), and academic users have free access to them. The platform is flexible and can easily adapt to external fragment libraries.…”

“…To encourage users who are new to crystallographic fragment screening, as well as to provide experienced users with additional screening options, FragMAX offers an XFS-focused fragment library. FragMAXlib contains a diverse set of 96 molecules, reflecting a common format for entry-level libraries (Huschmann et al, 2016). The library is useful both for basic research and for validation of the ligandability of a target, supporting downstream FBLD activities.…”

Establishing Trypanosoma cruzi farnesyl pyrophosphate synthase as a viable target for biosensor driven fragment‐based lead discovery

“…Ninety fragments were selected from a 96-membered library designed to have compounds with a large chemical diversity and high solubility, suitable for crystallographic driven lead discovery (Frag Xtal Screen, JENA Bioscience) were used. 26…”