A bestiary of fragment hits

What do fragment hits look like, and how do they bind? Fabrizio Giordanetto, David Shaw, and colleagues at D. E. Shaw Research were interested in these questions, and their answers are provided in a recent J. Med. Chem. paper (open access, and also covered well by Derek Lowe).

The researchers started by searching the protein data bank (PDB) for the word “fragment” and selecting higher resolution structures (at least 2.5 Å) with a ligand containing 20 or fewer non-hydrogen atoms. Those of you who have done bibliometric searchers will appreciate that a lot of manual curation is required, and the initial list of 5115 complexes was ultimately winnowed down to 489, with 462 unique fragments and 126 unique proteins, about two-thirds solved to ≤2.0 Å resolution.

In contrast to a previous study, only a minority (18%) of proteins contained more than one binding site, suggesting that secondary (possibly allosteric) sites may be less common than hoped.

As to the fragments themselves, 21 bound in more than one pocket (not necessarily on the same protein), including the universal fragment 4-bromopyrazole. The fragments ranged in size between 6 and 20 non-hydrogen atoms, with 81% having 10 to 16, consistent with our poll last year. Given these sizes, it is perhaps not surprising that the vast majority of fragments conformed to the rule of three.

Roughly two thirds of the fragments were uncharged, while 22% contained a negative formal charge (usually a carboxylic acid) and 11% contained a positive formal charge such as an aliphatic amine. Interestingly, more than 90% of the fragment hits were achiral. Since our 2017 poll found that most fragment libraries contain chiral compounds, these results might suggest lower hit rates for these compounds. Fragment hits also tended to have lower Fsp³ scores than those in a popular commercial library, which is consistent with the observation that “less shapely” fragments give higher hit rates.

Digging more deeply into the chemical structures themselves, nearly a third of the fragments contained a phenyl ring, while 6% contained a pyridine and 5% contained a pyrazole. Thiophenes, indoles, indazoles, piperidines, furans, and pyrrolidines were present in 2-3% of fragment hits. But there was also plenty of diversity: more than half of fragments contained a unique ring system.

So that’s what fragment hits look like. How do they bind? Deeply, for starters: about three quarters of the fragment hits buried more than 80% of their solvent-accessible surface area, and 21 fragments were completely engulfed within a protein.

Not surprisingly, more than 90% of complexes showed at least one polar interaction, such as a hydrogen bond or a coordination bond to a metal ion. Many complexes contained more than one, and one had seven! Interestingly, these polar interactions also tended to be buried. Nitrogen and oxygen atoms from the fragments were equally likely to form hydrogen bonds. Interactions with bound water molecules were considered for high-resolution (≤1.5 Å) structures, and nearly half of these contained a water molecule with at least two hydrogen bonds to the protein and one to a fragment.

Beyond these conventional sorts of polar interactions, there were also less traditional interactions such as arene-mediated contacts, which occurred in nearly half of cases. As we’ve noted, these are often under-appreciated but can be useful for improving affinity. The subject of halogen bonds came up recently, but these turned out to be quite rare, appearing in just 3% of cases. Sulfur-mediated contacts and carbon hydrogen bonds were more common, appearing in 11-12% of complexes, respectively.

All of this has important implications for fragment library design. As the researchers note, this set of 462 fragments could be used as the basis for a library, and laudably all the structures are provided in the supporting information. Generalizing beyond these specific molecules, roughly a quarter of the atoms in the fragment hits are polar (nitrogen or oxygen) and thus more likely to form classic hydrogen bonds. The researchers “strongly suggest” maintaining this ratio in designing new fragments.

The researchers also suggest presenting “a minimum set of individual polar pharmacophoric elements, as opposed to distributing several pharmacophores on a given fragment,” which is essentially the minimal pharmacophore strategy described here.

The one category of data I would have liked to see was affinity. Many binding measurements were probably not reported, and experimental error can be particularly confounding for weaker interactions, but even a subset of the data should allow some conclusions about the strength of various molecular interactions. Hopefully this will be the basis of a follow-up publication.