Fragments vs β-glucocerebrosidase

The protein β-glucocerebrosidase, also called GCase and GBA, is a lysosomal enzyme that cleaves glucosylceramide. People with inactivating mutations in both copies of GCase develop Gaucher’s Disease, which can be treated with a recombinant form of GCase. Heterozygous mutations increase risk for Parkinson’s Disease and for dementia with Lewy bodies, and though the mechanism is unclear, stabilizing the enzyme and/or boosting activity of residual GCase might help. This approach is described in a recent J. Med. Chem. paper by Nick Palmer and colleagues at Astex Pharmaceuticals.

 

The researchers started with a crystallographic screen of 440 fragments, resulting in a whopping 91 hits. In parallel, 1800 fragments (including the aforementioned 440) were screened using ligand-observed NMR, SPR, and thermal shift assays, and hits were confirmed crystallographically to yield another 15 structures. Astex has previously reported that multiple ligand binding sites are common in proteins, and GCase is no exception, with the 106 ligands binding to 13 distinct sites.

 

With this embarrassment of riches, prioritization became critical. Sites formed by crystal packing and shallow solvent-exposed sites were deprioritized, along with those near the active site, since ligands binding there might inhibit the enzyme. SPR was not well-suited to measuring ligand affinities due to non-specific binding, and ligand-observed NMR was similarly complicated due to multiple binding sites. However, isothermal titration calorimetry (ITC) proved to be effective, and this technique was used to narrow in on two binding sites.

 

Site A was particularly attractive: it had 31 fragment hits, one of which has a respectable dissociation constant of 12 µM. Screening of analogs did not lead to anything better, but merging this fragment with another Site A fragment led to compound 15. Interestingly, crystallography revealed that this molecule binds not at Site A but at Site B. Although the affinity is low, the ligand efficiency is respectable. The fragment also makes several polar interactions and has multiple vectors for growing the molecule.

 

 

Testing analogs of compound 15 led to compound 16, and growing led to compound 17, with low micromolar affinity. Further structure-based design ultimately led to compound 22, with low nanomolar affinity. The molecule increased GCase activity in a cellular assay, albeit at a fairly high (mid-micromolar) concentration. The molecule was found to be cell permeable with no efflux, so the source of the disconnect between affinity and cell activity is unclear.

 

This lovely example of structure-guided fragment-based ligand design holds several lessons. First, as noted above, finding fragments is often the easy part; selecting among them and figuring out what to do next can be challenging. Second, especially at the earliest stages of optimization, fragments can change not just their binding mode but their binding site entirely.

 

Finally, figuring out which sites will be best for high-affinity allosteric ligands isn’t necessarily straightforward. Of the 105 fragment hits at 13 sites, only four bound in Site B, yet this site turned out to be more fruitful than Site A, which had many more bound fragments. The researchers note that Site B had previously been identified as ligandable by FTMap, supporting the utility of computational approaches.

 

The researchers conclude, “we hope that our findings will be of use to the wider community.” Certainly from a best practices perspective the paper succeeds. And although the most advanced molecules described do not meet all the criteria for robust chemical probes, and it is unclear whether they will work with mutant proteins, they could still be useful to better understand the complicated biology of GCase.