Rational Design of Thermodynamic and Kinetic Binding Profiles by Optimizing Surface Water Networks Coating Protein Bound Ligands

A previously studied congeneric series of thermolysin inhibitors addressing the solvent-accessible S₂′ pocket with different hydrophobic substituents showed modulations of the surface water layers coating the protein-bound inhibitors. Increasing stabilization of water molecules resulted in an enthalpically more favorable binding signature, overall enhancing affinity. Based on this observation, we optimized the series by designing tailored P₂′ substituents to improve and further stabilize the surface water network. MD simulations were applied to predict the putative water pattern around the bound ligands. Subsequently, the inhibitors were synthesized and characterized by high-resolution crystallography, microcalorimetry, and surface plasmon resonance. One of the designed inhibitors established the most pronounced water network of all inhibitors tested so far, composed of several fused water polygons, and showed 50-fold affinity enhancement with respect to the original methylated parent ligand. Notably, the inhibitor forming the most perfect water network also showed significantly prolonged residence time compared to the other tested inhibitors

Six Biophysical Screening Methods Miss a Large Proportion of Crystallographically Discovered Fragment Hits A Case Study

Fragment-based lead discovery (FBLD) has become a pillar in drug development. Typical applications of this method comprise at least two biophysical screens as prefilter and a follow-up crystallographic experiment on a subset of fragments. Clearly, structural information is pivotal in FBLD, but a key question is whether such a screening cascade strategy will retrieve the majority of fragment-bound structures. We therefore set out to screen 361 fragments for binding to endothiapepsin, a representative of the challenging group of aspartic proteases, employing six screening techniques and crystallography in parallel. Crystallography resulted in the very high number of 71 structures. Yet alarmingly, 44% of these hits were not detected by any biophysical screening approach. Moreover, any screening cascade, building on the results from two or more screening methods, would have failed to predict at least 73% of these hits. We thus conclude that, at least in the present case, the frequently applied biophysical prescreening filters deteriorate the number of possible X-ray hits while only the immediate use of crystallography enables exhaustive retrieval of a maximum of fragment structures, which represent a rich source guiding hit-to-lead-to-drug evolution

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 surrogate for the design of renin and ß-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