Application of Native ESI-MS to Characterize Interactions between Compounds Derived from Fragment-Based Discovery...

Native electrospray ionization mass spectrometry (ESI-MS) was applied to analyze the binding of compounds generated during fragment-based drug discovery (FBDD) campaigns against two functionally distinct proteins, the X-linked inhibitor of apoptosis protein (XIAP) and cyclin-dependent kinase 2 (CDK2). Compounds of different molecular weights and a wide range of binding affinities obtained from the hits to leads and lead optimization stages of FBDD campaigns were studied, and their dissociation constants (K_d) were measured by native ESI-MS. We demonstrate that native ESI-MS has the potential to be applied to the stages of an FBDD campaign downstream of primary screening for the detection and quantification of protein-ligand binding. Native ESI-MS was used to derive K_d values for compounds binding to XIAP, and the dissociation of the complex between XIAP and a peptide derived from the second mitochondria-derived activator of caspases (SMAC) protein induced by one of the test compounds was also investigated. Affinities of compounds binding to CDK2 gave K_d values in the low nanomolar to low millimolar range, and K_d values generated by MS and isothermal titration calorimetry (ITC) followed the same trend for both proteins. Practical considerations for the application of native ESI-MS are discussed in detail.