Biosensor-based kinetic and thermodynamic characterization of opioids interaction with human μ-opioid receptor.

Development of opioid analgesics with minimal side effects requires substantial knowledge on structure–kinetic and –thermodynamic relationship of opioid-receptor interactions. Here, combined kinetics and thermodynamics of opioid agonist binding to human μ-opioid receptor (h-μOR) was investigated using real-time label-free surface plasmon resonance (SPR)–based method. The N-terminal end truncated and C-terminal 6His-tagged h-μOR was constructed and expressed in E. coli. Receptor was purified, detergent-solubilized and characterized by circular dichroism. The uniform immobilization of h-μOR on Ni-NTA chips was achieved using hybrid capture-coupling approach followed by reconstitution in lipid bilayer. Thermodynamic equilibrium affinities of opioids were in narrow nanomolar range and in near quantitative agreement with their Ki values. However, they did not correlate with their in vitro EC50 values, indicating that they might not have thermodynamic selectivity. Contrary, on and off rates exhibited much larger dispersion and well correlated with EC50 values, indicating that opioids might exhibit kinetic-selectivity towards their target. Temperature-dependent SPR assays provided access to rate and equilibrium thermodynamic data, which demonstrated binding of morphine and naloxone to μOR was exothermic and essentially enthalpy driven. This work suggests that kinetic-based structure-activity of opioids in drug design and incorporation into the pharmacokinetics-pharmacodynamics predictions may have more value than thermodynamic equilibrium constants alone.