Evaluation of Rate Constants from Protein-Ligand Interactions with Weak Affinity Chromatography

• Main Author: Jönsson, Daniel
• Format: Others
• Language: English
• Published: Linnéuniversitetet, Institutionen för naturvetenskap, NV 2012
• Subjects: peak profile method; dissociation / association rate constants; weak affinity chromatography; band-broadening; Chemistry; Kemi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-19974

The paradigm of drug discovery have been to find the strongest possible binder to the target by high-throughput screening (HTS) but high affinity interactions are related to low kinetic off rates and thus result in severe side-effects and non-approved drugs. Lead molecules working in a transient manner (KD > µM) will allow for rapid off rates and possibly less side-effects. In this study the peak profile method applied to weak affinity chromatography (WAC) was evaluated as a simple way to provide the kinetics of the interaction and thereby allowing for high-throughput determinations. In the peak profile formula all band-broadening effects except the stationary mass transfer is subtracted which simplifies the calculations for the kinetics of the interaction tremendously. The technique was evaluated by screening of 3 different benzamidines at 3 linear flow-rates using zonal chromatography and human α-thrombin as immobilized target protein. The kinetics of the interaction could unfortunately not be determined. This was possibly due to the flow-rates not being high enough as indicated by a low critical ratio (η < 1). Higher flow-rates would increase the contribution to band-broadening due to kinetic effects but would also require more precise estimation of peak variance.