High-throughput calorimetry for fragment-based lead discovery

Fragment-based lead discovery (FBLD) is an approach in which small, low complexity compounds of 6-15 heavy atoms are screened for binding to, or inhibiting the activity of, a target. The hits from the screen are then elaborated and/or linked to yield early lead compounds. Hits arising from the screen usually bind with low affinity; therefore sensitive detection methods are required. Common approaches to library screening include biophysical methods such as X-ray crystallography, NMR, which require large amounts of material and time. Alterative screening approaches include surface plasmon resonance or biochemical methods using high-concentration HTS assays, but these require target immobilization or labeling. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. The advent of extremely sensitive microcalorimeters have made it increasingly valuable as a tool for hit validation and characterization, but its use in primary screening is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional ITC, particularly for screening libraries of 500-1000 compounds such as those encountered in fragment-based lead discovery. We have developed a calorimetry method using enthalpy arrays, which are arrays of nanocalorimeters, to detect competitive inhibitors of enzymes. We tested the throughput and sensitivity limits of the method by performing fragment screens to identify inhibitors of PDE4A and PDE10A. Several inhibitors with KI<2 mM were identified and moved to X-ray crystallization trials, yielding high-resolution data. The X-ray structural data was supplemented with computational modeling methods to generate lead compounds with sub-micromolar inhibition constants. Out studies show that calorimetry can be an effective prescreening method for FBLD.