Activation state-selective kinase inhibitor assay based on ion mobility-mass spectrometry

Abstract

The discovery of activation state dependent kinase inhibitors, which bind specifically to the inactive conformation of the protein, is considered to be a promising pathway to improved cancer treatments. Identifying such inhibitors is challenging, however, because they can have Kd values similar to molecules known to inhibit kinase function by interacting with the active form. Further, while inhibitor induced changes within the kinase tertiary structure are significant, few technologies are able to correctly assign inhibitor binding modes in a high-throughput fashion based exclusively on protein-inhibitor complex formation and changes in local protein structure. We have developed a new assay, using ion mobility-mass spectrometry, capable of both rapidly detecting inhibitor binding and classifying the resultant kinase binding modes. Here, we demonstrate the ability of our approach to classify a broad set of kinase inhibitors, using micrograms of protein, without the need for protein modification or tagging.

Figure 1

Schematic describing the basic steps involved in the CIU assay to determine kinase inhibitor binding modes. Protein ligand complexes are ionized by nESI in a range of charge states (A), a single charge state is selected for activation in a quadrupole mass filter (B). Following selection, collisional activation with argon (varying the amount of accelerating voltage) is used to initiate three different processes: charge stripping, inhibitor dissociation, and gas-phase protein unfolding (C). IM drift time is monitored and recorded over a broad range of collision voltages to create a complete CIU fingerprint, which creates a contour plot of the intensity of ion populations as a function of these two parameters (D).

Figure 2

CIU fingerprints for indicated 11⁺ Abl-inhibitor complex ions (A). Dashed areas are between 40 and 44 volts of acceleration voltage, and are color-coded to correspond with the known binding mode of the inhibitor (as previously). Average IM drift time data derived from integrating the dashed-box regions from A (B). Data from selected types (I and II as indicated) are averaged and displayed, and overlayed below. Average IM drift time data in this region provide the greatest dissimilarity between inhibitor binding types, revealing sufficient detail to classify all of the inhibitors studied in this report.