Two Methods, One Goal: Structural Differences between Cocrystallization and Crystal Soaking to Discover Ligand Binding Poses

Abstract

In lead optimization, protein crystallography is an indispensable tool to analyze drug binding. Binding modes and non‐covalent interaction inventories are essential to design follow‐up synthesis candidates. Two protocols are commonly applied to produce protein–ligand complexes: cocrystallization and soaking. Because of its time and cost effectiveness, soaking is the more popular method. Taking eight ligand hinge binders of protein kinase A, we demonstrate that cocrystallization is superior. Particularly for flexible proteins, such as kinases, and larger ligands cocrystallization captures more reliable the correct binding pose and induced protein adaptations. The geometrical discrepancies between soaking and cocrystallization appear smaller for fragment‐sized ligands. For larger flexible ligands that trigger conformational changes of the protein, soaking can be misleading and underestimates the number of possible polar interactions due to inadequate, highly impaired positions of protein amino‐acid side and main chain atoms. Thus, if applicable cocrystallization should be the gold standard to study protein–ligand complexes.

Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

“…In principle, co-crystallization and soaking are two common strategies for collecting diffraction data from protein-drug complex structures [43]. The soaking method involves "soaking" the protein crystal in the drug solution at a determined concentration, whereas co-crystallization mixes the drug and the target protein prior to crystallization.…”

“…Therefore, it was hypothesized that montelukast may also have antiviral activity against SARS-CoV-2 [27]. Studies related to SARS-CoV-2-montelukast interaction are mostly in silico- [43][44][45] and in vitro- [17,42] oriented. Accordingly, we suggest that although montelukast interacts with Mpro, because of the limitations on the soaking experiments and/or used drug concentrations in the current study, we could not receive noticeable electron densities.…”

Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

“…In principle, co-crystallization and soaking are two common strategies for collecting diffraction data from protein-drug complex structures [44]. The soaking method involves 'soaking' the protein crystal in the drug solution at a determined concentration, whereas co-crystallization mixes the drug and the target protein prior to crystallization.…”

“…Therefore, it was hypothesized that montelukast may also have antiviral activity against SARS-CoV-2 [27]. Studies related to SARS-CoV-2-montelukast interaction are mostly in silico - [44,45,46] and in vitro - [17,42] oriented. Accordingly, we suggest that although montelukast interacts with Mpro, it may interfere through an allosteric effect, disrupting its conformation and perturbing the crystal lattice contacts.…”

Large-Scale Ligand Perturbations of the Protein Conformational Landscape Reveal State-Specific Interaction Hotspots

“…Although co-crystallization would imply that lid conformations stabilize crystal packing, soaking would raise concerns that pre-packed crystals restrict lid mobility to accommodate ligands and may explain some of the curious cases observed here. Pose changes of fragments upon soaking vs co-crystallization have been previously observed for Hsp90 and protein kinase A . While the lack of consistent reporting prohibits us from disentangling cause and effect, packing artifacts that distort the protein conformational landscape also impede our ability to rationally explore the landscape’s features during ligand discovery .…”