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

Abstract

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.

Rapid Discovery of Aspartyl Protease Inhibitors Using an Anchoring Approach

“…Typical warheads for Asp proteases include primary and secondary amines, guanidines, amidines, hydrazides, carboxylic acids, alcohols, imidazoles and pyrazoles . However, it is surprising the absence of a warhead with equal interaction to the two oxygens of an aspartic acid residue.…”

A False‐Positive Screening Hit in Fragment‐Based Lead Discovery: Watch out for the Red Herring

“…EP specifically recognizes the final reaction product 2 through three hydrogen bonds, one of which is mediated by water (Figure A). In particular, the aldehyde oxygen of 2 accepts an H‐bond from Thr222, although this residue usually acts as an acceptor . This phenomenon is part of several structural adaptations that take place upon binding of the ligand.…”

“…In particular, the aldehyde oxygen of 2 accepts an H-bond from Thr222, although this residue usually acts as an acceptor. [11] This phenomenon is part of several structural adaptations that take place upon binding of the ligand. Structure 2 would sterically interfere with the apo protein, which therefore experiences an induced fit as shown in Figure 4B and simultaneously takes up aglycerol molecule from the soaking buffer at the position of the catalytic water.…”

A Proof-of-Concept Fragment Screening of a Hit-Validated 96-Compounds Library against Human Carbonic Anhydrase II

“…Detected fragment hits are subsequently elaborated into lead compounds by growing, merging or linking, which relies upon the potentiation of binding affinity upon combination of weak-affinity contributors [3,4,8]. Based on the considerable knowledge about fragment-based research that our group has amassed over the years [9][10][11][12][13][14][15], a 96-entry fragment library was assembled, to make the utility of fragments available to the broader public [16]. We tested its versatility in screenings against several established proteins, among these the well-established drug target and model protein human carbonic anhydrase II (hCAII).…”