Fragments vs PDE10A: Astellas’ turn

The 11 members of the phosphodiesterase (PDE) family cleave cyclic nucleotides such as cAMP and cGMP to regulate cell signaling. These enzymes are established drug targets – sildenefil inhibits PDE5, for example. PDE10A inhibitors have been heavily investigated for a variety of neurological disorders, and fragments have played a role in several efforts: we’ve highlighted work from Merck, AstraZeneca, and Zenobia/PARC on this target. A new paper in Chem. Pharm. Bull. by Ayaka Chino and colleagues describes work from Astellas.

A previous HTS screen at the company had led to a series of low nanomolar inhibitors, but these had metabolic liabilities and also inhibited CYP3A4. Thus, the researchers turned to fragments. No details are given as to library size, screening method, or hit rate, though it is worth noting that Astellas has previously reported fragment screening by crystallography. Compound 2 turned out to be a hit, and examination of the crystallographically determined binding mode proved quite useful. (Astute readers will also note the similarity of compound 2 to one of the Merck fragments.)

Because the chlorophenyl moiety was pointing towards solvent, the researchers decided to lop this off  to lower both lipophilicity and molecular weight. Previous publications had also revealed the presence of a “selectivity pocket”, and the researchers therefore grew towards this pocket, yielding molecules such as compound 7. Further tweaking led to compound 13, with low nanomolar potency. In contrast to the HTS-derived lead, this molecule was metabolically stable in vitro and showed negligible inhibition against a panel of 13 CYP enzymes.

This is a nice – albeit brief – example of how fragments can generate new chemical matter even against an extensively explored class of enzymes. Plenty of questions remain around pharmacokinetics, selectivity, and brain penetration, but the paper does end by promising that more will be revealed.

Enthalpy arrays revisited: PDE10A

Two years ago we highlighted enthalpy arrays: very tiny temperature sensors that measure the heat generated during the course of an enzymatic reaction. Molecules that compete with a substrate will alter the kinetics of the reaction and can thus be identified as inhibitors. In the original paper a number of fragment hits were identified against the phosphodiesterase PDE4A, but unfortunately none of these could be structurally characterized. In a new paper in J. Biomol. Screen. Michael Recht and colleagues at Palo Alto Research Center have teamed up with Vicki Nienaber and colleagues at Zenobia to apply enthalpy arrays to the neurological target PDE10A, and this time they were able to obtain numerous crystal structures.

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 K_I = 590 µM) than those that didn’t (average K_I = 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 K_Is 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.

Enthalpy Arrays

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.