Protonation states serve as an essential molecular recognition motif for biological processes. Their correct consideration is key to successful drug design campaigns, since chemoinformatic tools usually deal with default protonation states of ligands and proteins and miss atypical protonation states. The protonation pattern for the Endothiapepsin/PepstatinA (EP/pepA) complex is investigated using different dry lab and wet lab techniques. ITC experiments revealed an uptake of more than one mole of protons upon pepA binding to EP. Since these experiments were performed at physiological conditions (and not at pH=4.6 at which a large variety of crystal structures is available), a novel crystal structure at pH=7.6 was determined. This crystal structure showed that only modest structural changes occur upon increasing the pH value. This lead to computational studies Poisson‐Boltzmann calculations and constant pH MD simulation to reveal the exact location of the protonation event. Both computational studies could reveal a significant pKa shift resulting in non‐default protonation state and that the catalytic dyad is responsible for the uptake of protons. This study shows that assessing protonation states for two separate systems (protein and ligand) might result in the incorrect assignment of protonation states and hence incorrect calculation of binding energy.

Fragment screening directly on protein crystals has been applied using AnalytiCon’s collection of intermediates that have been utilized to generate libraries of larger synthetic natural product-like molecules. The fragments with well-balanced physicochemical properties show an impressively high hit rate for a screen using the aspartic protease endothiapepsin. The subsequent validation and expansion of the discovered fragment hits benefits from AnalytiCon’s comprehensive library design. Since the screened fragments are intermediates that share a common core with larger and closely related analogs with modulated substitution patterns, they allow for the retrieval of off-the-shelf follow-up compounds, which enable the development of design strategies for fragment optimization. A promising bicyclic core scaffold found in several fragment hits could be validated by selecting a set of enlarged follow-up compounds. Due to unexpected changes in binding mode and no significant improvement in ligand efficiency, this series was quickly deemed unsuitable and therefore discontinued. The structures of follow-up compounds of two other fragments helped to evaluate a putative fusion of two overlapping fragment hits. A design concept on how to fuse the two fragments could be proposed and helps to plan a suitable substitution pattern and promising central bridging element.

1 Protonation states serve as an essential molecular recognition motif for biological processes. Their correct consideration is key to successful drug design campaigns, since chemoinformatic tools usually deal with default protonation states of ligands and proteins and miss atypical protonation states. The protonation pattern for the Endothiapepsin/PepstatinA (EP/pepA) complex is investigated using different dry lab and wet lab techniques. ITC experiments revealed an uptake of more than one mole of protons upon pepA binding to EP. Since these experiments were performed at physiological conditions (and not at pH=4 at which a large variety of crystal structures is available), a novel crystal structure at pH=7.6 was determined. This crystal structure showed that only modest structural changes occur upon increasing the pH value. This lead to computational studies to reveal the exact location of the protonation event. Both computational studies could reveal a significant pKa shift resulting in non-default protonation state and that the catalytic dyad is responsible for the uptake of protons. This study shows that assessing protonation states for two separate systems (protein and ligand) might result in the incorrect assignment of protonation states and hence incorrect calculation of binding energy.

In this study, we utilized a high-performance soaking system of protein kinase A (PKA) to perform a crystallographic screening of a natural product-like fragment library. We resolved 36 fragment-bound structures, corresponding to a hit rate of 41%. Nine fragments bound within the ATP site, nine peripherally, and 18 interacted with both the ATP and peripheral sites. One fragment binds to the same site as the approved allosteric kinase inhibitor asciminib, while another induces an unexpected conformational change. Systematic database mining revealed that both the fragments and their natural product parents have not been previously associated with PKA or kinase activity. A scaffold/chemotype analysis further underscored their novelty. Cheminformatics analyses confirmed that these fragments occupy a distinct chemical space, enriched in saturation, spatial complexity and molecular three-dimensional character compared to kinase binders from reference data sets. These properties have previously been linked to increased selectivity, reduced CYP450 inhibition, and higher overall clinical success rates.

In this study we utilized a high-performance soaking system of protein kinase A (PKA) to perform a crystallographic screening of a natural product-like fragment library. We resolved 36 fragment-bound structures, corresponding to a hit rate of 41%. Nine fragments bound within the ATP site, nine peripherally, and 18 interacted with both the ATP and peripheral sites. One fragment binds to the same site as the approved allosteric kinase inhibitor asciminib, while another induces an unexpected conformational change. Systematic database mining revealed that both the fragments and their natural product parents have not been previously associated with PKA or kinase activity. A scaffold/chemotype analysis further underscored their novelty. Cheminformatics analyses confirmed that these fragments occupy a distinct chemical space, enriched in saturation, spatial complexity and molecular three-dimensional character compared to kinase binders from reference datasets. These properties have previously been linked to increased selectivity, reduced CYP450 inhibition, and higher overall clinical success rates. Abstract Figure: