X-ray crystallography is the most popular fragment-finding method according to our latest poll. This is in part due to the previously unmatched level of detail a crystal structure can provide, and in part due to the increasing speed and automation for data collection and processing. But many proteins can never be coaxed into crystals, and while it is possible to advance fragments in the absence of structure, it is rarely easy.
In the past few years, cryogenic electron microscopy (cryo-EM) has come to rival crystallography in terms of resolution (see here for details). The first mention of the technique on Practical Fragments was in 2013, when Teddy wrote that he “could not figure out how you would use [it] in screening/FBHG. However, the point of emerging technology is to emerge…” And emerge it has. At FBLD 2018 researchers from Astex presented the first cryo-EM structure of a bound fragment, and in a new paper in Drug Disc. Today Harren Jhoti and collaborators at Astex and Isohelio provide details for two proteins.
The first target, β-galactosidase (Bgal), is a model protein that has previously been characterized by cryo-EM. The researchers solved the bound structures of three small molecules, two of them fragment-sized, to resolutions of 2.2-2.3 Å. The quality of the maps is such that they could easily be mistaken for crystallographic data: the density is clear and includes ordered water molecules. Induced conformational changes are evident, stereochemistry is unambiguous for all the ligands, and one piperidine ring even shows a hole in the middle. Even more impressive, the structures were solved using automated software.
But three structures do not a screen make. For this, the researchers chose the oncology target pyruvate kinase 2 (PKM2). They designed a small library of 68 highly soluble fragments and screened these at 5 mM. The structures of two complexes are shown, and while the resolution is lower than for Bgal, clear contacts with the protein are evident. Next, the researchers screened cocktails of four fragments, each at 25 mM. Fragments in mixtures were chosen to have diverse shapes, and two structures are shown demonstrating that the technique can distinguish the binders.
Throughput is still an issue: each round of data collection and processing – whether individual compound or cocktail – took about a day. However, the researchers expect that improvements in software and hardware should enable 400 fragments to be screened in less than a month. Indeed, given that much of the focus of cryo-EM has been on generating novel structures, one can imagine various shortcuts for screening, such as collecting smaller numbers of images to look for any evidence of bound ligands. I wonder too if something like PanDDA could be developed for cryo-EM.
Both proteins described have been previously characterized by crystallography, but these are still early days, so it is only a matter of time before we see fragment structures in proteins that haven’t been crystallized. That will open thousands of proteins heretofore inaccessible for structure-based design. The researchers conclude by predicting that “cryo-EM will have a transformative impact on the pharmaceutical industry in the near future.” I would not bet against this.