Thermodynamic, Kinetic and Crystallographic Investigations of Benzenesulfonamides as Ligands of Human Carbonic Anhydrase II
The data presented here show that different geometries of para-alkyl substituents of a benzenesulfonamide scaffold can have a significant influence on the thermodynamic and kinetic binding parameters and suggest, that the fine-tuning of the ligand geometry to match the geometry of the active site le...
|PDF Full Text
No Tags, Be the first to tag this record!
|The data presented here show that different geometries of para-alkyl substituents of a benzenesulfonamide scaffold can have a significant influence on the thermodynamic and kinetic binding parameters and suggest, that the fine-tuning of the ligand geometry to match the geometry of the active site leads to a reduced dissociation rate of the protein-ligand complex and thus to a prolonged binding in the active site. In addition, it was found that the kinetic data extracted by isothermal titration calorimetry correlate with data from experiments with surface plasmon resonance for a subset of compounds, despite a difference of one order of magnitude that could not be explained so far. Furthermore, the investigation of fluorinated benzenesulfonamide ligands revealed complex structural-thermodynamic and structural-kinetic relationships, which could not be fully elucidated herein, but suggest that fluorination of a meta-position of the aromatic part of the benzenesulfonamide backbone favors high association as well as small dissociation rates. In addition, it has been shown that a higher degree of fluorination does not necessarily have an advantage for the thermodynamic or kinetic binding profile or affnity itself. Furthermore, structurally similar ligands with only one para-substituent but different acidities indicate that the association rate of complex formation benefits from increased acidity. A new measurement protocol for microcalorimetric measurements was analyzed for its precision and compared with other possible measurement protocols and showed that it is best suited for the
reliable simultaneous determination of thermodynamic and kinetic data by microcalorimetry. The microcalorimetric investigation of an already known and supposedly very potent Carbonic Anhydrase inhibitor showed an unexpected two-step binding behavior, which, however, excludes a 2 : 1 binding of inhibitor and protein. Time-dependent experiments suggest that the binding process between protein and ligand leads to a thermodynamically and kinetically preferred complex. The investigation of a chiral heterocyclic Carbonic Anhydrase inhibitor could not conclusively clarify, whether the crystallographically apparently non-binding enantiomer also shows inhibitory activity. However, microcalorimetric investigations allow the conclusion that racemization occurs under experimental conditions, which makes the clarification of the original question considerably more difficult. Soaking of Carbonic Anhydrase crystals in solutions of different concentrations of a ligand showed, that the occupancy of a second binding site was too weak to allow modeling of the ligand, which made it impossible to investigate the concentration
dependence of the occupancy in this binding site. Concentration differences, however, resulted in different occupancies of the active site. A crystallographic model based on neutron diffraction data of human Carbonic Anhydrase II in complex with saccharin was not obtained. However, an attempt was made to assess the protonation state of saccharin in the active site of the enzyme by experimental phasing using high-resolution X-ray diffraction data. The continuation of a fragment screening showed the binding of further small molecules in Carbonic Anhydrase crystals. An analysis with the Pan-Dataset Density Analysis program suggests, that electron density maps are strongly dependent on the time of soaking of the crystals.