Characterization of interactions

Surface plasmon resonance (SPR) bio-sensor instruments provide label-free analysis applicable to a wide range of biomolecular interactions. Drug discovery projects use SPR to measure interactions between an immobilized protein and soluble ligands. In particular this includes fragment based drug discovery, ligand screening, compound characterization, and benchmarking against competitor compounds.

A protein target is immobilized on sensor chips. The chips themselves comprise gold plated glass onto which carboxymethylated dextran is coupled to present a biocompatible hydrophilic environment with low nonspecific binding. Amine coupling to lysine is the most commonly used method of attaching proteins to the chips. In certain cases coupling via thiols or aldehydes, or tagged proteins may be preferred.


Detection relies upon the refractive index (RI) change for a given change of mass concentration in the surface layer. For proteins and peptides this is more or less constant. The analytes flow continuously over the protein on the sensor surface and therefore the interaction of the binding partners is monitored in “real time”. The plot of change in RI, Response Units (RU) versus time is referred to as a sesorgram from which kinetic data is derived.


 

NovAliX instruments include the high sensitivity high precision Biacore™ 3000 and T200 machines (Biomolecular Interaction Analysis =BIA) that are capable of detecting the binding of small molecules to the protein target.

Whilst other primary screening methods often deliver hits based upon affinity, the ultimate goal of clinical efficacy depends upon many more factors. The kinetics of association and dissociation are particularly important and are conveniently measured by SPR. NovAliX provides accurate characterization data enabling the candidate drug selection projects of our clients to move forward on a sound scientific basis.

The medium throughput of 100-1,000 interactions per day per system containing 4 channels is sufficient to validate and to characterize screening hits.

Measurements that can be performed on the Biacore™:

  • Specificity
  • Detailed kinetics, kon, koff
  • Affinity, Kd

The one caveat is that is that the functionality of some proteins does not survive the immobilization process.  The effort required to investigate alternative immobilization conditions or to produce stable protein constructs have then to be weighed against simply switching to an alternative technique.

A Typical Biacore™ sensorgram monitoring binding events in real time

R&DTechnologies_SPR_ChannelBased_Systems

 

 

References
T.Neumann et al. SPR Based Fragment Screening: Advantages and Applications, Current Topics in Medicinal Chemistry, 2007, 7, 1630-1642