27 May 2014

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.


Kevin Gaffney said...

There seems to be a misunderstanding of the chemical structure of fragment 9. As a sugar, there is the interconversion between cyclic and linear structures. In compound 1, the cyclic form is lock into place through the glycosidic linkage. The removal of this bond allows free interconversion and, as a result, a huge drop in activity. The drawing of fragment 9 as a static single structure is misleading as are conclusions about fragment linking. The use of a carbocylic (THF) analog of structure may provide a more appropriate comparison.

Dan Erlanson said...

Kevin, the structure of Fragment 9 is taken directly from the paper and is, as drawn, a static THF analog and thus unable to equilibrate.

Darren W Begley said...

Unlike synthetic fragments in a biophysical assay, most enzymes tend to be rather selective when it comes to their substrates. Co-evolution in a sea of metabolites has pressured enzymes to be selective; this helps prevent processing the wrong material. At Beryllium (formerly Emerald) we have seen "fragment" selectivity by NMR and crystallography, if the fragment in question is a nucleotide and is (or contained within) the native substrate or not. This mirrors the "small changes in the substrate hav[ing] pronounced effects" by enzyme assay in Sarah's paper. So perhaps the relatively lower selectivity and broader array of protein targets observed for more synthetic aromatic fragments (frequently reported in the literature and in this blog) has to do with such compounds more broadly (not so specifically) mimicking native substrates? Hence the non-fragment, computational approach with all known (exact, specific) metabolites (Suwen Zhao 2013 Nature paper) may have a better chance at divining exact substrates, if the relative docking scores can get there.

Knowing this does not make going about narrowing down the exact function of a protein using fragments any easier, particularly for enzymatic functions as of yet undocumented (think bacterial genomes for organisms which can degrade almost anything). But I think there is still the possibility of a middle ground for fragments to play when the general function is unknown, if the right compounds happen to be in the collection. Maybe not each and every time, but if you're lucky.