Surface plasmon resonance applications in drug discovery : with an emphasis on small molecule and low affinity systems

Surface plasmon resonance (SPR) technology evolved into a key technology for the
characterization of biomolecular interactions, and is integrated in many stages of the
drug discovery process. Despite recent developments in the area of instrument
sensitivity and data processing, working with small molecules and low affinity
interactions still remains a major challenge. The aim of this thesis was therefore to
evaluate and develop different methods for the accurate and reliable determination of
thermodynamic and kinetic information of such interaction systems.
Through participation in the international ABRF-MIRG’02 study, the instrument used
for this thesis was validated for small molecular analyses. The results obtained for a
small sulfonamide analyte binding to bovine carbonic anhydrase II were very close to
the study average and corresponded well with solution-based methods. Screening
experiments with human serum albumin and a set of known plasma protein binders
further confirmed the effectiveness of SPR for small molecule assays. However, the
albumin assay also revealed some limitations; while neutral and cationic drugs
generated very reproducible KD values, the deviations were usually larger for free
acids. Some compounds like diazepam or L-tryptophan showed a more complex
binding behavior. Most of these atypical signal effects could be attributed to ligand- or
pH-induced structural changes of albumin, which are well described in literature.
Finally, a new immobilization method for human serum albumin was developed by
targeting its single free cysteine residue for a reversible coupling to the sensor chip.
The interaction of monovalent carbohydrates with their protein targets is one of the
most prominent examples of small molecule/low affinity systems. They play an
important role in many biological processes from cellular recognition to infection
diseases. In order to characterize such carbohydrate-protein interactions, a diagnostic
monoclonal antibody (GSLA-2) directed against a carbohydrate epitope was
investigated using a combination of SPR and nuclear magnetic resonance. By screening
the tetrasaccharide antigen sialyl Lewisa and a set of structurally related compounds,
the thermodynamic and kinetic binding properties as well as the recognition pattern
could be successfully described. With a KD in the low micromolar range and fast
kinetic on- (~104 M-1s-1) and off-rates (>0.1 s-1), the interaction correlated very well
with earlier reports about carbohydrate-protein interactions. Truncation of the antibody
to its antigen-binding parts led to a significant increase in binding activity and reduced
non-specific binding.
The human hepatic asialoglycoprotein receptor served as a more complex example of
carbohydrate-binding proteins. This C-type lectin is involved in the clearance of
desialylated glycoproteins from blood and is regarded as an important target for
selective delivery of genes and drugs to the liver. After expression of the carbohydrate
recognition domain in E.coli, the lectin could be successfully purified using a
combination of different chromatographic steps and was immobilized on a SPR sensor
chip. Binding of the physiological glycoprotein ligands asialofetuin and
asialoorosomucoid was characterized by a polyvalent interaction pattern with slow
dissociation rates and sub-nanomolar KD values. In contrast, monovalent sugars like
galactose or N-acetyl