Last year’s poll revealed that crystallography has become the most popular technique for FBLD. Three recent papers discuss some of the whys and hows.
The first publication, published in Structure by Manfred Weiss and collaborators at Helmholtz-Zentrum Berlin, Philipps-Universität Marburg, Freie Universität Berlin, and Lund University, describes the construction of the F2X-Universal Library for crystallographic screening. Starting from 1.4 million commercially available fragments, the researchers filtered out undesirable molecules such as PAINS and then clustered the remaining compounds by similarity. Among clusters with at least 1000 members they chose one fragment from each. The resulting library, sourced from roughly a dozen vendors, contains 1103 fragments individually dissolved in DMSO at 0.5 M concentration. Outside of the Diamond Light Source, screening 1000 crystals is still a sizable effort, so the researchers have also made a 96-membered subset library called F2X-Entry.
F2X-Entry was screened against two targets, the model protein endothiapepsin (EP) and the spliceosomal protein-protein complex Aar2/RNaseH-like domain of Prp8 (AR). Crystals of each were screened with 100 mM fragments and processed with the assistance of PanDDA (described here).
The results were quite impressive: 29 hits for EP, several of which bound in more than one site, and 20 hits for AR. The overall hit rates of 30% and 21% compare favorably with a previous crystallographic screen against EP that yielded a 20% hit rate. The solvent DMSO sometimes doesn’t play well with crystals, but the researchers were able to obtain 72-75% of the original hits when soaked in the absence of solubilizing DMSO.
One bit of data I would have liked to have seen is how the physicochemical properties of the fragment hits compare to the overall library, similar to what was reported here. Both libraries can be accessed by collaborators at the BESSY II synchrotron, and the smaller library can also be accessed through an MTA, so hopefully as these are screened against more targets this information will be published.
A second paper, in ChemMedChem by Gerhard Klebe and collaborators at Philipps-Universität Marburg and AstraZeneca, also discusses crystallographic screening of a 96-compound library, one sold commercially by Jena Biosciences. The target chosen was tRNA guanine transglycosylase (TGT), an enzyme important for the pathogenicity of the sometimes lethal bacteria Shigella. Soaking each of the compounds at 100 mM yielded 8 structures, 5 of which bound in the active site.
The researchers also screened the library using orthogonal methods, SPR and ligand-detected NMR. As in a previous study from the same group, the results were “puzzling”: none of the hits were detected in all three assays. (The first author, Engi Hassaan, presented some of this work at a CHI meeting last year.) Indeed, only one of the crystallographic hits was found among the 10 hits from SPR, and a different crystallographic hit was found among the 22 NMR hits.
Several plausible reasons for the low overlap are provided, including different buffer compositions and concentrations of fragments. Solubility likely played a role: a dozen fragments could not be screened by NMR at 0.2 mM, and 15 fragments interfered with the SPR assay and thus could not be screened. Indeed, some of the fragments are PAINS, including an eyebrow-raising dinitrocatechol, so the fact that they were not observed by crystallography is perhaps unsurprising.
Finally, in a Biomolecules paper, Gerhard Klebe and collaborators screen the same library against human carbonic anhydrase II (hCAII). This resulted in 9 hits – 8 from the library and 1 from a cryoprotectant used at 2.6 M in some experiments. Among the library hits, four bound at the active site, including a couple hydrazides which make interactions with the catalytic zinc ion. Surprisingly, two fragments bind covalently to the N-terminus of hCAII. One seems to form a formaldehyde-mediated linkage, while the other – an aminomethylheterocycle – has likely oxidized to an aromatic aldehyde that can form an imine linkage.
All of the fragment hits in this case were identified through visual inspection of the electron density maps, and in this system PanDDA was actually not helpful, revealing only three of the fragment hits. The researchers note that they used different soaking times for the different crystals, and suggest that shorter soaking times in particular may not allow time for crystals to equilibrate, as assumed in PanDDA.
So in summary, fragment screening by crystallography is becoming easier and is likely to give high hit rates, particularly when conducted at high concentrations. Also, libraries matter: it is interesting that the F2X-Entry library gave considerably higher hit rates than the second 96-compound library, albeit on different targets. Of course, a fragment hit is only the beginning of a long journey, but at least a structure provides guidance to begin optimization.