As regular readers know, there are lots of ways to find fragments, each with its own strengths and weaknesses. Isothermal titration calorimetry (ITC) is useful for being able to extract thermodynamic values from an experiment, but it tends to be low-throughput and is thus used more as a secondary rather than a primary assay. To make calorimetry more convenient, Michael Recht and colleagues at Palo Alto Research Center have constructed nanocalorimeters. They describe using these “enthalpy arrays” for fragment screening in a paper just published online in the Journal of Biomolecular Screening.
In a typical ITC experiment, a protein is mixed with a ligand and the tiny temperature change that occurs upon binding is detected. In the case of nanocalorimeters, up to 96 detectors are arranged on a plate, and sample volumes are typically a few hundred nanoliters. At this scale, the enthalpy of binding becomes very challenging to measure, but it is possible to measure the heat generated during the course of a reaction as an enzyme processes its substrate. This allows one to follow the reaction in real time without any dyes, labels, or artificial substrates. Also, since an inhibitor will change the reaction profile in a predictable manner, one can determine its mechanism of action.
Each detector in the enthalpy array is set up such that droplets are rapidly mixed together in sets of two. In the experimental set, one droplet contains the protein of interest while the other contains a substrate and a fragment. (Each detector also incorporates a control pair – one droplet containing the substrate/fragment mixture and another containing bovine serum albumin.) In the current paper, the researchers were looking for competitive inhibitors of PDE4A10, a phosphodiesterase implicated in inflammatory disorders. The protein was present at a final concentration of 5 micromolar, substrate was at 2 mM, and each fragment was present at up to 2 mM. 160 very small fragments (average molecular weight only 154 Da) were screened individually, resulting in 11 competitive hits with Ki < 2 mM; 2 other hits displayed more complex kinetics.
The 11 competitive hits were characterized in more detail; the most potent had a Ki of 0.32 mM and the most ligand efficient had LE = 0.43 kcal/mol-atom. In collaboration with Vicki Nienaber and colleagues at Zenobia, all 11 of these were taken into crystallography experiments. This proved challenging: unliganded PDE4A10 crystals suitable for fragment soaking could not be grown, necessitating more labor-intensive co-crystallography. Unfortunately, although some crystals were obtained, they did not diffract at high enough resolution to unambiguously fit the electron density of the fragments, though there was evidence for binding in the active site. The researchers were able to crystallize PDE4A10 with pentoxifylline, a known phosphodiesterase inhibitor. Since many of the fragments have structural features reminiscent of other phosphodiesterase inhibitors, this suggests starting points for modeling.
As described in this paper, enthalpy arrays could be used as a primary screen for fragment hits with defined modes of action before follow-up by slower methods. Although in this particular case crystallography was not successful determining co-crystal structures of the novel fragments, in a recent talk Michael described a related system which did yield good crystal structures. I look forward to seeing additional applications of this approach.