Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

Abstract

Fragment‐based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit‐identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X‐ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis‐acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240‐fold improvement in potency compared to the parent hits. Subsequent X‐ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit‐identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit‐to‐lead optimization.

Dynamic Combinatorial Chemistry: A New Methodology Comes of Age

“…It is an extremely useful technique, because separate signals for each library member allow for statements regarding their concentration. Disadvantages include the time‐consuming assignment of all signals and due to frequent signal overlapping, the limitation to rather small libraries of generally less than twenty compounds, with one exception by Mondal et al., who work with a 57‐membered library (see Table ) . Since ligands bound to the target cannot be reliably detected, the dissociation of target–ligand complexes prior to analysis is a prerequisite (Figure ).…”

“…In the comparative approach, the composition of a library generated in the presence of a target (template library) is compared to a library generated in its absence (blank library) ,–. This approach is often combined with analysis by high‐performance liquid chromatography (HPLC),,––,,,– and generally includes dissociation of the ligand–target complex by either denaturation of the target or displacement of the ligand by a competitor ,,,,,. Subsequently, amplification of compounds can be deduced by comparing template and blank libraries.…”

“…In contrast to imines, acylhydrazones dynamically formed from aldehydes and hydrazides are sufficiently stable for analysis ,,,,. Under acidic conditions, their formation proceeds efficiently and has been successfully applied in tdDCC ,.…”

“…However, because a low pH is often not compatible with protein stability and the formation of acylhydrazones is slow at neutral pH, nucleophilic catalysts activating the aldehyde through an intermediate Schiff base have been applied . Aniline has proven especially useful as a catalyst and has been widely employed ,,. Interestingly, 5‐methoxyanthranilic acid, also known to accelerate acylhydrazone formation, has been linked to erroneous DCC results, indicating that catalysts must be selected with care.…”

“…A library of n thiol building blocks therefore results in ( n 2 + n )/2 products . Obviously, the use of bi‐ or multivalent ligands exhibiting multiple functional moieties amenable to a reversible reaction will lead to a further increase in the number of products and complexity of the libraries ,…”