Determination of Affinity and Residence Time of Potent Drug-Target Complexes by Label-free Biosensing

J Med Chem. 2018 Jun 28;61(12):5154-5161. doi: 10.1021/acs.jmedchem.7b01829. Epub 2018 Jun 7.

Abstract

Prolonged drug-target occupancy has become increasingly important in lead optimization, and biophysical assays that measure residence time are in high demand. Here we report a practical label-free assay methodology that provides kinetic and affinity measurements suitable for most target classes without long preincubations and over comparatively short sample contact times. The method, referred to as a "chaser" assay, has been applied to three sets of unrelated kinase/inhibitor panels in order to measure the residence times, where correlation with observed efficacy was suspected. A lower throughput chaser assay measured a residence time of 3.6 days ±3.4% (95% CI) and provided single digit pM sensitivity. A higher throughput chaser methodology enabled a maximum capacity of 108 compounds in duplicate/day with an upper residence time limit of 9 h given an assay dissociation time of 34 min.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azo Compounds / chemistry
  • Biosensing Techniques / instrumentation
  • Biosensing Techniques / methods*
  • Biotin / metabolism
  • Drug Evaluation, Preclinical / instrumentation
  • Drug Evaluation, Preclinical / methods*
  • High-Throughput Screening Assays / instrumentation
  • High-Throughput Screening Assays / methods
  • Models, Theoretical
  • Molecular Probes / chemistry
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinase Inhibitors / metabolism
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinases / chemistry
  • Protein Kinases / metabolism
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology
  • Streptavidin / metabolism
  • Structure-Activity Relationship
  • Time Factors

Substances

  • Azo Compounds
  • Molecular Probes
  • Protein Kinase Inhibitors
  • Small Molecule Libraries
  • HABA
  • Biotin
  • Streptavidin
  • Protein Kinases