The researchers started by confirming that literature reference compounds behaved as expected. Next, they screened all 16 of the PDE4A hits against PDE10A, including several that were quite weak against PDE4A itself. All of these were active in the enthalpy array assays, with Ki values ranging from 94 to 1400 μM and good ligand efficiencies. In fact, most of the fragments were more potent against PDE10A than the phosphodiesterase against which they were original screened – which perhaps touches on the question of fragment selectivity.
The researchers also screened an additional 85 fragments at a concentration of 2 mM, leading to 8 more hits. All 24 of the hits were then soaked into crystals of PDE10A, yielding 16 crystal structures of bound fragments – a respectable 67% success rate. Interestingly, fragments that produced structures were more potent (average KI = 590 µM) than those that didn’t (average KI = 1000 µM), and this difference was statistically significant.
All of the fragments bound at the active site, and fragment growing was used to improve the affinity of two of the fragments. This led to low or sub-micromolar compounds, albeit with a loss in ligand efficiency. These more potent compounds were also selective for PDE10A over PDE4A, though solubility limits precluded testing at very high concentrations.
The paper frankly discusses some of the limits of using enthalpy arrays. For example, since the fragment should be present at a higher concentration than enzyme, very tight binders would require unfeasibly low enzyme concentrations. This limits the practical range of the technique to inhibitors with KIs ranging from ~500 nM to 2 mM. Also, as Morgen G observed in a comment to the last post, this is more of a biochemical assay (monitoring the heat of an enzymatic reaction) rather than what most people think of when you say the word calorimetry (monitoring the heat of binding, as in the case of isothermal titration calorimetry). Still, enthalpy arrays seem pretty cool; hopefully folks will warm to them.