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.

Enthalpy arrays revisited: PDE10A

Two years ago we highlighted enthalpy arrays: very tiny temperature sensors that measure the heat generated during the course of an enzymatic reaction. Molecules that compete with a substrate will alter the kinetics of the reaction and can thus be identified as inhibitors. In the original paper a number of fragment hits were identified against the phosphodiesterase PDE4A, but unfortunately none of these could be structurally characterized. In a new paper in J. Biomol. Screen. Michael Recht and colleagues at Palo Alto Research Center have teamed up with Vicki Nienaber and colleagues at Zenobia to apply enthalpy arrays to the neurological target PDE10A, and this time they were able to obtain numerous crystal structures.

The researchers started by confirming that literature reference compounds behaved as expected. Next, they screened all 16 of the PDE4A hits against PDE10A, including several that were quite weak against PDE4A itself. All of these were active in the enthalpy array assays, with Ki values ranging from 94 to 1400 μM and good ligand efficiencies. In fact, most of the fragments were more potent against PDE10A than the phosphodiesterase against which they were original screened – which perhaps touches on the question of fragment selectivity.

The researchers also screened an additional 85 fragments at a concentration of 2 mM, leading to 8 more hits. All 24 of the hits were then soaked into crystals of PDE10A, yielding 16 crystal structures of bound fragments – a respectable 67% success rate. Interestingly, fragments that produced structures were more potent (average K_I = 590 µM) than those that didn’t (average K_I = 1000 µM), and this difference was statistically significant.

All of the fragments bound at the active site, and fragment growing was used to improve the affinity of two of the fragments. This led to low or sub-micromolar compounds, albeit with a loss in ligand efficiency. These more potent compounds were also selective for PDE10A over PDE4A, though solubility limits precluded testing at very high concentrations.

The paper frankly discusses some of the limits of using enthalpy arrays. For example, since the fragment should be present at a higher concentration than enzyme, very tight binders would require unfeasibly low enzyme concentrations. This limits the practical range of the technique to inhibitors with K_Is ranging from ~500 nM to 2 mM. Also, as Morgen G observed in a comment to the last post, this is more of a biochemical assay (monitoring the heat of an enzymatic reaction) rather than what most people think of when you say the word calorimetry (monitoring the heat of binding, as in the case of isothermal titration calorimetry). Still, enthalpy arrays seem pretty cool; hopefully folks will warm to them.

Fragment Library Vendors (2014 Edition)

We have been updating a lot of lists recently.  One that I think has changed significantly, is the fragment library vendor list, last updated in 2010. As Dan said four years ago, FOB Chris Swain has done a great job of curating who is selling what.  Instead of duplicating efforts, I will just focus on what has changed and making some comments.  I am not going to list companies that have libraries you can access, only those that sell outright their libraries. 

What are the keys for purchasing a good library?  I think minimally, purity and aqueous solubility should be experimentally tested and guaranteed.  I note those vendors who specifically point this out, but one should not assume that those who don't also don't have this data.  Comments can be sent directly to me or made below, and I will update this list.

Some general thoughts: 

• There is no special sauce.  Every library is good and will work for you. It's the choice of screen and how you prosecute it after that makes the difference.
• You don't need no stinkin' IP. 

3DFrag Consortium (New 2014):  I think this ran its course.  While I think by and large it had great ideas I don't think it ever truly answered the question "Do 3D fragments work better (in some areas)?"

Analyticon (New 2014): This is another example of fragments from nature.  The utility of these types of libraries are still up for discussion. 

Asinex: "Inspired by Nature" is its tagline.  However, they do have focused libraries, for such targets as PPIs.  They have 3159 in this library.. 

Chembridge: The collection is now 7000+ compounds (was 5000).  The guarantee greater than 90% purity, but nothing about solubility. 

Chemdiv (New 2014): Their collection is almost 14,000 fragments.

Enamine: More than doubled in size, from 12,000 to more than 28,000. 

Iota: I think they were the first to regularly use nPMI in their compound assesment.  You can only look at their library under CDA. 

Key Organics: They have quite a few specialized fragment libraries: CNS, self-assembly, brominated, fluorinated, and chiral cyclic molecules, in addition to their main libraries.  They guarantee 95% purity, 1mM aqueous solubility, solubility up to 200 mM in DMSO, and with almost no overlap with the Maybridge collections (68 compounds).

Life Chemicals:They are now up to 47,500 fragment molecules (less than 300 MW), of course only 31,000 of these exist, the other 16,000 can be made upon request.  They have 3900 19F fragments.  In terms of those that have experimental solubility, there are 8200.  However, 75% are soluble at 1mM, and 60% at 5mM in PBS.   So, always read the fine print.  They are also the vendor for the Zen-Life library, another library based on nature. 

Maybridge: The grandfather of them all.  30,000 fragments in total.  The 2500 Diversity collection is guranteed soluble at 200 mM in DMSO and 1mM in PBS.  The NMR spectrum is available, but only in organic solvent. It is available in many formats, from powder to DMSO-d6 solution. 

Otava: 8800 fragments in general.  800 19F fragments.  And 575 chelating fragments, if you want a warhead and all the issues they bring with.

Prestwick: 2200 fragments.

Timtec: No number available, but also can be shipped in DMSO solution. 

Vitas-M: The least helpful website out there.  It's Voldemort Rule compliant and available in multiple formats: mg, mcmol, sets, DMSO solution, dry film.

Zenobia:  Several different collections of very small fragments.

In defense of ligand efficiency – and poll!

Last year we highlighted a provocative article from Michael Shultz in which he took aim at the concept of ligand efficiency (LE). As we noted at the time, he raised some good points, and I am the first to argue that there is value in questioning widespread assumptions.

However, in addition to questioning the utility of LE, Shultz also questioned its mathematical validity. He repeated the attack earlier this year by asserting that ligand efficiency was a “mathematical impossibility.”

This is incorrect.

To set the record straight, Chris Murray (Astex), Andrew Hopkins (University of Dundee), György Keserü (Hungarian Academy of Sciences), Paul Leeson (GlaxoSmithKline), David Rees (Astex), Charles Reynolds (Gfree Bio), Nicola Richmond (GlaxoSmithKline) and I have written a response just published online in ACS Med. Chem. Lett. demonstrating that ligand efficiency is mathematically valid.

One of the criticisms of LE is that it is more sensitive to changes in small molecules (such as fragments) than in larger molecules. However, this is a property of any ratio, and we show that the same behavior applies to more familiar examples such as fuel efficiency: a few blocks of stop-and-go traffic has more of an effect on the overall fuel efficiency of a short trip than a long trip.

Of course, that’s not to say that ligand efficiency and other metrics are perfect or universally applicable; we discuss a number of situations where they may be more or less useful.

In this spirit, Practical Fragments is revisiting a poll from 2011 to see what metrics you use – please vote on the right-hand side of the page, and share your thoughts here. Note that you can vote for multiple metrics, and please check the last box (Polldaddy does not tally individual responses, so this box will track total number of voters to allow us to calculate percentage of respondents who use a given metric).

Keep the comments coming, and check back to see the poll results.

Biofragments: extracting signal from noise, and the limits of three-dimensionality

What does this protein do? Now that any genome can be sequenced, this question gets raised quite often. In many cases it is possible to give a rough answer based on protein sequence: this protein is a serine protease, that one is a protein tyrosine kinase, but figuring out the specific substrates can be more of a challenge. In a recent paper in ChemBioChem, Chris Abell and collaborators at the University of Cambridge and the University of Manchester attempt to answer this question with fragments.

The bacterium Mycobacterium tuberculosis (Mtb), which causes tuberculosis, has 20 cytochrome P450 proteins (CYPs), heme-containing enzymes that usually oxidize small molecules. Although some are essential for the pathogen, it is not clear what many of them do. The researchers used an approach called “biofragments” to try to pin down the substrate of CYP126.

The biofragments approach starts by selecting a collection of fragments based on known substrates. Of course, the specific substrates are not known, so in this case the researchers started with a set of several dozen natural (ie, non-synthetic) substrates of various other CYPs, both bacterial and eukaryotic. They then computationally screened the ZINC database of commercial molecules for fragments most similar to these substrates and purchased 63 of them. Perhaps not surprisingly given their similarity to natural products, these turned out to be more “three-dimensional” than conventional fragment libraries, as assessed both by the fraction of sp³ hybridized carbons and by principal moment-of-inertia.

Next, the researchers screened their fragments against CYP126 using three different NMR techniques (CPMG, STD, and WaterLOGSY). Since they were primarily interested in hits that bind at the active site, they also used a displacement assay in which the synthetic heme-binding drug ketoconazole was competed against fragments. This exercise yielded 9 hits – a relatively high 14% hit rate.

Strikingly, all of the hits are aromatic, and 7 of them could reasonably be described as planar. In other words, even though the biofragment library was relatively 3-dimensional, the confirmed hits were some of the flattest in the library! The researchers interpreted this to mean that “CYP126 might preferentially recognize aromatic moieties within its catalytic site,” but there could be something more general going on – perhaps aromatics are simply less complex, and thus more promiscuous.

Examining the fragment hits more closely, the researchers found that one of them – a dichlorophenol – produced a spectrophotometric shift similar to that produced by substrates when bound to the enzyme. This led them to look for similar structures among proposed Mtb metabolites. Weirdly, pentachlorophenol came up as a possible hit, and a spectrophotometric shift assay reveals that this molecule does have relatively high affinity for CYP126. Whether this is a biologically relevant substrate for the enzyme remains to be seen.

This is an intriguing approach, but I do have reservations. First, in constructing fragment libraries based on natural products, it is essential to avoid anything too “funky”. The Abell lab is one of the top fragment groups out there, well aware of potential artifacts, and has a long history of studying CYPs, but researchers with less experience could easily populate a library with dubious compounds.

More fundamentally though, I wonder about the basic premise of biofragments. The whole point of fragments is that they have low molecular complexity and are thus likely to bind to many targets, so is it realistic to try to extract selectivity data from them? Indeed, as we’ve seen (here and here), fragment selectivity is not necessarily predictive of larger molecules.

That said, the approach is worth trying. Even if it doesn’t ultimately lead to new insights into proteins’ natural substrates, it could lead to new inhibitors.