Introduction of Intrinsic Kinetics of Protein–Ligand Interactions and Their Implications for Drug Design

Structure–kinetic
relationship analyses and identification
of dominating interactions for optimization of lead compounds should
ideally be based on intrinsic rate constants instead
of the more easily accessible observed kinetic constants,
which also account for binding-linked reactions. The intrinsic rate
constants for sulfonamide inhibitors and pharmacologically relevant
isoforms of carbonic anhydrase were determined by a novel surface
plasmon resonance (SPR) biosensor-based approach, using chemodynamic
analysis of binding-linked pH-dependent effects. The observed association
rates (kaobs) were pH-dependent and correlated with the
fraction of deprotonated inhibitor and protonated zinc-bound water
molecule. The intrinsic association rate constants (kaintr) were
pH independent and higher than kaobs. By contrast, the observed
and intrinsic dissociation rate constants were identical and pH-independent,
demonstrating that the observed association and dissociation mechanisms
are inherently different. A model accounting for the differences between
intrinsic and observed rate constants was developed, useful also for
other interactions with binding-linked protonation reactions.