Irreversible Small Molecule Inhibitor Characterization


The majority of small molecule inhibitor assays tested with label-free real-time biosensor technologies are reversible interactions, characterized by second order kinetic rate models. However, a significant fraction of therapeutic enzyme inhibitors on the market function through covalent modification of the target. Irreversible enzyme inhibitors function through a two-step binding model which is characterized by the equation in Figure 1.

Conventional SPR-based fragment screening workflow

Figure 1: Rate equations for irreversible inhibition, where E is enzyme, I is inhibitor, E•I is binding encounter complex, E-I is adduct or covalent complex, kon is association rate constant, koff is dissociation rate constant, kinact is the reaction rate constant of the covalent inactivation.

Biochemical plate-based assays are often used to characterize these irreversible inhibitors through the lead optimization process but modeling the time dependence of kinact can prove to be difficult. ForteBio's Pioneer FE is an SPR platform that can be used with regenerable Streptavidin Sensors to reversibly capture protein targets and quantify the efficiency of covalent inhibitors binding to target. The Pioneer FE system's irreversible inhibitor applications method has the key benefits of:

  • Determining the compound’s commitment factor (Cc), an important efficiency metric for irreversible inhibitors
  • Simultaneously determining the association rate constant (kon), important for structure-activity relationship (SAR) of small molecule inhibitors
  • Improved target activity over similar methods in the field
  • No requirement for specialized streptavidin reagents
  • A reproducible capture yield for many test cycles

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