The protein p97 is important in regulating protein homeostasis, and thus a potential anti-cancer target. But this is no low-hanging fruit: the protein has three domains and assembles into a hexamer. Two domains, D1 and D2, are ATPases. The third (N) domain binds to other proteins in the cell. All the domains are dynamic and interdependent. Oh, and crystallography is tough. Previous efforts have identified inhibitors of the D2 domain, but not the others. Not to be put off by difficult challenges, a group of researchers at the University of California San Francisco (UCSF) led by Michelle Arkin and Mark Kelly have performed fragment screening against the D1 and N domains, and report their adventures in J. Biomol. Screen.
Within UCSF, the Small Molecule Discovery Center (SMDC) has assembled a fragment library of 2485 commercial compounds from Life, Maybridge, and Asinex. These have an average molecular weight of 207 Da and 15 heavy atoms, with ClogP ~1.5. The researchers used both biophysical and virtual screening.
For the physical screening, the researchers started with surface plasmon resonance (SPR), with each fragment at 0.25 mM. This resulted in 228 primary hits – a fairly high hit rate. Full dose response studies revealed that 160 of theses fragments showed pathological behavior such as concentration-dependent aggregation or superstoichiometric binding. A further 30 showed weak or no binding, 13 were irreversible, and 5 bound nonspecifically to the reference surface, leaving only 20 validated hits which were then repurchased.
The 228 primary hits were also assessed by STD NMR, each at 0.5 mM when possible (some fragments were not sufficiently soluble). Of these, 84 gave a strong STD signal, and 14 of these were also among the 20 SPR-validated hits.
The 20 repurchased fragments were further tested by both SPR and STD NMR, and 13 of them reconfirmed by both methods. The paper includes a table listing all 20 compounds, and one observation that struck me was the fact that all but one of the hits – which had dissociation constants ranging from 0.14 to 1.7 mM – are larger than the library average. Such results could argue for including larger fragments in libraries, though this goes against both molecular complexity theory as well as extensive experience at groups such as Astex.
Next, the researchers sought to discover information on the binding sites. Three fragments could be competed by ADP, suggesting that they bind in the nucleotide-binding site of D1. To narrow things down further, the researchers turned to 13C-1H-methyl-TROSY NMR, in which specific side chain methyl groups of Ile, Leu, Met, Val, and Ala were labeled, and chemical shifts were examined in the presence and absence of fragments. Two of the proposed nucleotide-binding site fragments showed similar shifts as AMP or ADP, further supporting a common binding mode (the third was too weak to test). This was not an easy experiment: the hexamer has a mass of 324 kDa, well above where most people do protein-detected NMR.
Independent of all the biophysical screens, virtual screens were conducted using Glide XP, which suggested that the nucleotide binding site would be the hottest hot spot. Happily, all three fragments that appear to bind to this site scored highly in the in silico work, with two of these within the top 100 fragments. However, the binding sites for the other ten confirmed fragments remain obscure.
This paper serves as a useful guide for how fragment screening is performed on a tough target in a top-tier research group. Although difficult, it is not impossible to advance fragments in the absence of structure. While it remains to be seen whether that will be the case for any of these fragments, the researchers have provided a wealth of data for those who wish to try.