Enthalpy arrays for enzymatic assays in fragment-based screening

Enthalpy arrays are arrays of nanocalorimeters that enable label-free detection of molecular interactions using small sample volumes and short measurement times. Fragments identified in fragment-based screens (FBS) typically exhibit low binding affinity (0.1 to 1 mM) that is difficult to measure with many established techniques, yet it is beneficial to identify the fragments with a high ligand efficiency (-ΔG/number of heavy atoms) to take through lead optimization. In principle, isothermal titration calorimetry (ITC) can be used to characterize the thermodynamics of fragment binding to targets, but its use in FBS is severely hampered by the need for large samples (≈0.2-1.5 mL), long measurement times, and high fragment solubility in the injectant. Our enthalpy array technology enables measurements with 250 nL drops that only take a few minutes. The technology enables the possibility to determine accurate inhibitor constants for competitive inhibitors from single measurements with a label-free readout. In this talk we will discuss the application of enthalpy arrays to confirm and rank hits identified in a fragment-based screen against two related proteases and describe both the potential and the limitations of the technology. Future applications for enthalpy array technology in drug discovery include secondary assays and thermodynamic characterization of leads. The presentation will cover: Capabilities of this label-free technology Enzyme assays for protease and kinase inhibitors Enthalpy array application in fragment-based drug discovery.