Practical Evaluation of Time-Dependent Inhibition Kinetics Through Small-Volume Size-Exclusion Chromatography

Abstract

Accurate identification and characterization of the mechanism of time-dependent inhibitors (TDIs) is essential for reporting key kinetic parameters, which can significantly improve the efficiency of early-stage drug discovery campaigns. A practical, quantitative method that is amendable to integration into upstream lead characterization pipelines can provide this critical context, as classical potency determinations alone may be misleading if a time-dependent element of inhibitor binding is unaccounted for. Here we have developed a new method for the determination of compound binding reversibility and the quantitative assessment of reversible inhibitors for guiding optimization of the structure-kinetic relationship of candidate TDI compounds.

Currently, a well-established strategy for determining the mechanism of inhibitor reversibility in a highthroughput, label-free manner involves rapid dilution of a saturated kinase-inhibitor complex to drive compound dissociation. The complex is created by incubating the kinase with a concentration of inhibitor well above an estimated IC50 value. From here, a continuous, kinetic assay format can be leveraged to measure activity recovery of the enzyme over time, through which the relative reversibility of the inhibitor can be visualized. In the case of reversible inhibitors, one can also quantify the residence time τ from this progress curve, further improving characterization. Despite the utility of the assay, however, this rapid dilution strategy – known as a “jump dilution” – faces significant challenges when deployed to characterize highly potent inhibitors. The high concentrations of enzyme in the preincubation step make it difficult to avoid the tight-binding limit of the assay.

Further, the elevated inhibitor concentration in the assay – particularly if adjusted to account for the tight-binding limit – introduces the caveat of compound re-binding during the recovery of activity, necessitating further steps in the protocol. To eliminate for these complications, we demonstrate a novel application of small-volume size-exclusion chromatography (SEC) via desalting columns as a means to facilitate the removal of free compound from the kinase-inhibitor complex, driving dissociation in a manner similar to that of a jump dilution without requiring an elevation of kinase concentration. Following this SEC-facilitated compound clearance, we are able to detect and measure the recovery of active kinase using a continuous assay format. This allows us to directly observe reversibility and quantify off-rates through activity recovery in the progress curve. We demonstrate these quantitative applications through the side-by-side comparisons of well-known EGFR and ABL1 kinase inhibitors with various time-dependent characteristics. Through a combination of SEC-facilitated compound clearance and continuous kinetic monitoring, we are able to capture reversibility behavior and – for reversible inhibitors – determine residence times in agreement with the general literature.

Key Takeaways

Incorporation of separation techniques to power activity-driven assessments of time-dependent inhibition can enable early detection of desirable binding kinetics among early lead candidates. This can be leveraged into an informed, efficient downstream discovery and optimization program while ensuring that correct kinetic metrics are captured to best assess relevant parameters: 

• Separation experiments can be introduced following dose-response experiments that combine progress curve analysis (Figure 2) with detection of IC50 shifts as a function of preincubation prior to instantaneous Vi calculation. 
  
  
• SEC-driven separation can be scaled to achieve the desired throughput, either as individual columns or as multi-column plates, to facilitate large-scale TDI assessment through activity-based readouts. 
  
  
• The tight-binding limitation imposed by elevated [kinase] in the jump dilution protocol can be more easily circumvented, as final [inhibitor] is tied to binding site concentration and can be modulated by adjusting [kinase].