From substrates to fragments – or not

Recently we highlighted a paper in which enzyme substrates were deconstructed into component fragments and tested against an enzyme with unknown specificity. In a new paper in J. Am. Chem. Soc. a collaboration led by Karen Allen (Boston University), Frank Raushel (Texas A&M), and Brian Shoichet (UCSF) has performed a similar experiment to ask whether fragments could be used to identify substrates.

The researchers chose six enzymes from three different classes and collected various-sized fragments based on known substrates. These were then tested in functional assays to see whether they could be substrates or inhibitors. Stunningly, in most cases the fragments showed no activity against the enzymes; when activity was detectable, it was usually at least 100,000-fold lower than the natural substrate. Even subtle tweaks, such as removing a hydroxyl group, were enough to mess things up, as illustrated for adenosine deaminase (compare compounds 1 and 4). Breaking the substrate in two was sometimes better: compound 8 was turned over slowly by the enzyme, though its complementary fragment 9 had no effect on activity – positive or negative – when added to the assay along with compound 8 or the natural substrate.

Of course, functional assays are less sensitive than biophysical assays, but in the one case where the researchers tried soaking fragments into crystals of the enzyme they found that the fragments bound in a different manner than the substrate – echoing previous work deconstructing synthetic inhibitors of protein-protein interactions.

As the authors note, the remarkably sharp structure-activity-relationships (SAR) observed here could reflect a fact of nature: most enzymes need to be highly selective for their substrates to avoid mucking up cellular metabolism.

Moreover, the notion that two fragments, when properly linked together, can bind more tightly than the sum of their individual binding energies has been a primary motivator behind fragment-based lead discovery for more than 30 years. In a sense, this paper illustrates this principle in reverse. Indeed, it is possible for the energy gained by linking two fragments to exceed the binding energy of an individual fragment.

This is a nice study from which we can draw two lessons, one pessimistic, the other optimistic. On the down side, we are unlikely to be able to use fragments to predict the natural substrates of uncharacterized enzymes, at least on a general basis. As noted previously, this is not surprising: the concept of molecular complexity predicts that fragments should be fairly promiscuous, and we’ve seen time and again that fragment selectivity is not necessarily maintained during optimization.

On the positive side, this study beautifully illustrates that it is possible to achieve massive enhancements in affinity with relatively small changes. Beyond just the magic methyl effect, we’ve got the magic hydroxyl effect, the magic thiophene effect – heck – the magic fragment effect. Of course, these are retrospective analyses, and it’s easier to break things than make them. That said, folks at Astex demonstrated that it is possible to improve the affinity of a millimolar fragment a million-fold by adding just six atoms. Perhaps such opportunities are more general than we have previously dared to dream.