Identification and Optimization of PDE10A Inhibitors Using Fragment-Based Screening by Nanocalorimetry and X-ray Crystallography

Abstract

Fragment-based lead discovery (FBLD) is a technique in which, small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/ or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high resolution diffraction with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an x-ray crystallography secondary screen.

Ranking Hits From Saturation Transfer Difference Nuclear Magnetic Resonance–Based Fragment Screening

“…In both cases, crystallographic and thermodynamic data were previously reported. From these reports, kinetic data were calculated assuming diffusion-controlled on-rate kinetics and hence STD amplification factors were determined (Figure 2A; Barelier et al, 2014; Recht et al, 2014). Similar to our experimental screening data, there was no correlation between calculated STD amplification factor and affinity (Pearson's correlation coefficient, p > 0.05).…”

“…(A) STD amplification factors determined with CORCEMA-ST of fragments binding to PDE10A and AmpC. For these fragments, a crystal structure and thermodynamic data were available (Barelier et al, 2014; Recht et al, 2014). (B) Chemical structures of the fragments used to analyze HSP90 (Roughley and Hubbard, 2011) and (C) examples of some of these ligands in complex with protons colored by their normalized STD effect (red being the highest STD effect and blue being the lowest effect).…”

Biophysics in drug discovery: impact, challenges and opportunities

“…Isothermal titration calorimetry. There are only a few examples in the literature using isothermal titration calorimetry (ITC) as a primary assay in fragment-screening campaigns, as this method has a very low throughput and equilibrium dissociation constants of fragment interactions are often too weak to be well determined 22,127,136 .…”

Going Small: Using Biophysical Screening to Implement Fragment Based Drug Discovery

“…Recent research into high-throughput calorimetry offer solutions to researchers wanting to incorporate calorimetry into an earlier stage in their screening cascade. Research into technologies in pursuit of the so-called "lab on a chip" has led to the development of both microfluidic [49] and droplet-based systems [50][51][52]. The droplet-based system has been applied to both binding and kinetics.…”