Since 2006, our well-experienced team has been delivering molecular profiles, such as affinity and kinetic characterisation, as well as mechanistic evaluation insights for a wide range of different proteins, drug target types and purposes.
The efficacy of inhibiting a target is dependent on the rate of target inactivation and the residence time of the ligand. Therefore, two different ligands with identical affinity can differ significantly in clinical efficacy due to differences in kinetics.
Gaining control over the association and dissociation rate of your ligand can provide a competitive advantage. In addition, the discovery of ligands with unexpected kinetics can provide an inventive step, making your compounds patentable.
The analysis of protein-ligand interactions is performed by SPR biosensor-based experiments. This entails the immobilisation of the target protein onto a sensor chip, followed by injection of a concentration series of reference and test compounds.
Analysis of the response with respect to injection time allows for the assignment of the interaction mechanism and the determination of the corresponding rate constants as well as dissociation constants.
Examples of kinetic traces of a kinase inhibitor binding via a simple direct fit mechanism (left). Kinetic data can be shown in kon/koff plots (middle). Compounds 1 and 2 are equipotent in terms of affinity but show differences in kinetics. By increasing on-rate for 2 or by decreasing off-rate for 1, the potency of 3 could be reached. Scheme to the right shows a more complex mechanistic mechanism (selected fit) illustrated by structures of HIV-1 RT.
Mechanism and complexity of the interaction
Superior determination of KD values
Kinetic constants (kon, koff and other parameters for more complex mechanisms)
Comparative data for competitor compounds
SPR biosensor-based technology has several advantages for antibody characterisation over conventional methods (such as enzyme immunoassays and radioimmunassay). No labelling is required, sample consumption is low, the method is fast and the output is information rich. Flexible experimental design can provide information about such diverse parameters as interaction model, affinity, rate constants, temperature- and pH dependency as well as epitope specificity.
In epitope mapping, epitope specificity is determined by testing the ability of pairs of monoclonal antibodies (mAbs) to bind simultaneously to an antigen. Typically, a first mAb is immobilised onto the chip surface and then utilised to capture an antigen of interest. A second mAb is subsequently flowed over the antigen surface. If the second mAb is directed towards an epitope distinct from that of the first mAb (non-competitive), it will bind, whereas if it is directed towards an epitope closely related to the epitope of the first mAb it will interfere with binding (competitive).
A) Immobilised mAbs on a chip surface with captured antigen and two types of ”secondary” mAbs. Green mAbs are non-competitive binders whereas red mAbs are competitive.
B) A reactivity matrix showing the binding ability of pairs of mAbs to an antigen along with epitope pattern definitions.
C) A diagram representing the relationship between epitopes. Overlapping epitopes are competitive, non-overlapping epitopes are non-competitive.
Kinetic constants (kon, koff)
Epitope reactivity matrix showing the binding ability of pairs of Abs to an antigen
Temperature and pH dependency of the interactions
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