Dimerization: elegant but not essential

A special case of fragment linking is dimerization, in which two copies of the same fragment bind to adjacent sites in a protein and are subsequently linked together (see for example here, here, and here). A recent example was published in J. Med. Chem. by Bernard Pirotte, Julien Hanson (University of Liège), Lionel Pochet (University of Namur), Jette Kastrup (University of Copenhagen) and their collaborators.

The researchers have for some time been interested in AMPA receptors, critical components in neuronal synaptic transmission. Increasing their activity could be useful for treating diseases such as depression and schizophrenia, but increasing activity indiscriminately is known to be toxic. One approach has been to develop positive allosteric modulators (PAMs), which increase the activity only in the presence of the natural ligand glutamic acid, thus amplifying the normal biological signal.

AMPA receptors themselves are dimers of dimers. Many different PAMs have been reported for AMPA receptors, and some of these are in fact dimeric molecules that span two adjacent binding sites across the dimer interface. A crystal structure of a molecule closely related to compound 35 revealed that each molecule binds to two adjacent protein subunits, so the researchers designed compound 22, which pairs the molecules through a simple ethylene moiety. The strategy paid off with a low nanomolar activator, which crystallography confirmed binds as expected.

Interestingly, conceptually cleaving the bond connecting the two fragments generates a compound (33) which is slightly less active than the initial fragment 35; it is possible the methyl groups are too close to one another when two copies of compound 33 are bound.

As the researchers point out, compound 22 is one of the most potent AMPA receptor PAMs reported. However, it is also quite large, particularly since it needs to cross the blood-brain barrier. No animal data are reported, but a simple metric called the CNS MPO desirability score is reasonably predictive. This score is based on the molecular weight, lipophilicity, total polar surface area, number of hydrogen bond donors, and basicity; higher scores are better. By this measure, compound 22 is predicted not to have high brain penetration, though of course any metric needs to be taken with caution.

However, a separate J. Med. Chem. paper by many of the same researchers revealed that dimerizing the molecules is not essential: simply growing compound 35 could also generate a low nanomolar AMPA receptor PAM (compound 8). Crystallography revealed that the added phenyl group binds where the second molecule of compound 35 would normally bind. Moreover, compound 8 has a higher ligand efficiency as well as a higher CNS MPO desirability score than the dimeric compound 22, suggesting that it is more likely to be able to cross the blood-brain barrier.

In the absence of pharmacological or pharmacokinetic data, if forced to choose I would probably focus on compound 8 rather than compound 22. All of which is to say that although there is a certain elegance to dimerizing molecules, you might be able to replace one of them with a smaller, simpler moiety.

Fragments score a win against WDR5-WIN

Protein-protein interactions (PPIs) can be difficult targets for multiple reasons. First, the contacts often cover large, flattish areas with few “ligandable” pockets. Second, they can involve multiple proteins; imagine trying to disrupt a huge multicomponent machine with a little widget. The protein WDR5 falls into the second category. It serves as a scaffold around which other proteins assemble to regulate epigenetics. One of these proteins, MLL1, is implicated in certain leukemias and binds to WDR5 through the WDR5 INteraction (WIN) motif, making this protein-protein interaction an intriguing anti-cancer target. In a recent paper in J. Med. Chem., Stephen Fesik and colleagues at Vanderbilt University describe their efforts towards this target.

Unlike some PPIs, the WIN motif does contain a nice little pocket which normally recognizes arginine residues. However, since the highly basic guanidine moiety of arginine is undesirable in drugs, the researchers conducted a fragment screen to find new WIN-site binders. A two-dimensional (¹H-¹⁵N HMQC) NMR screen of a large fragment library (>13,800 fragments, more than the majority of respondents in the poll to the right) identified 47 hits that produced similar spectral changes as a peptide that binds in the WIN site. Compound F-1 was the most potent.

A crystal structure of compound F-1 bound to WDR5 revealed that the imidazole moiety binds in the same deep pocket normally occupied by the arginine side chain, with the phenyl ring pointing up out of the pocket. Initial growing off the phenyl ring into nearby hydrophobic pockets produced more potent compounds, but at best these were still micromolar binders. The researchers had more success by targeting a slightly more distant pocket with compounds such as 4a and subsequently compound 4i. A crystal structure of compound 4a bound to WDR5 suggested that the biologically active conformation might not be the lowest energy conformation of the free molecule. Introducing a ring to restrict the conformation led to more potent molecules such as 6e, with sub-nanomolar affinity.

Unfortunately, though potent in biochemical assays, compound 6e and related molecules were about 2800-fold less potent in cell-based assays. The compound is cell permeable and not effluxed, so the disconnect must be due to something else – perhaps the multiple other proteins in the cellular environment. Anyone who has spent much time doing medicinal chemistry will have encountered frustrating situations like this. Perhaps a new chemotype is needed, or perhaps the compounds need to be made even more potent. Indeed, several years ago the Fesik group reported nanomolar binders of MCL-1, but it was not until they improved affinity to picomolar that they saw good cell potency. Stay tuned!

Rise of the machines for fragment optimization

Our latest poll (please vote on the right-hand side of the page!) is about fragment libraries. Once you have your library, you can screen it using a variety of approaches. But what do you do once you get hits? Computational methods are increasingly being adopted; just this year we’ve discussed two approaches: growing via merging and AutoCouple. A new paper in J. Med. Chem. by Philippe Roche, Xavier Morelli, and collaborators at Aix-Marseille University and several other institutions describes a method that combines virtual screening with automated real-world synthesis in a platform called diversity-oriented target-focused synthesis (DOTS).

The process is best described with an example, and the test case presented is the first bromodomain of BRD4, BRD4(BD1). The researchers, who had previously identified a xanthine-containing series of inhibitors, pared this back to fragment-sized compound F1. Crystallography revealed a nearby pocket, which the researchers attempted to target with DOTS.

The researchers built a virtual library of 576 sulfonamides extending off the para position of the phenyl ring of compound F1. These were then virtually screened against BRD4(BD1) using the S4MPLE molecular modeling tool in which the F1 portion was constrained in the crystallographically observed conformation while the variable bits were allowed to move. The 100 top-scoring molecules were examined more closely, and 17 representatives were chosen to be synthesized on an automated robotic platform. This was actually a fairly modest set, as the Chemspeed system they used can run up to 96 parallel reactions. The crude products were then tested in a fluorescence assay, and all of them showed improved activities compared to the initial fragment. The majority, such as compound 17, showed high nanomolar inhibition.

The 13 submicromolar compounds were then resynthesized, purified, and validated in thermal shift and isothermal titration calorimetry (ITC) assays; these orthogonal methods confirmed their activities. The crystal structure of compound 17 bound to BRD4(BD1) was also solved, and this revealed that – as designed – the initial fragment retained its binding mode while the added portion makes new interactions with the protein.

The fact that 14 of the 17 molecules synthesized were at least an order of magnitude more potent than the initial fragment is satisfying, though it is worth noting that bromodomains are not the most difficult targets. Also, all of the new molecules have lower ligand efficiencies than the initial fragment. Still, advances and combinations of computational and robotic approaches will certainly increase the throughput of synthesis and testing, and I expect to see more of these examples.

Practical Fragments turns ten, and celebrates with a poll on the modern fragment library

Ten years ago today Teddy launched Practical Fragments with a simple question about screening methodologies. More than 660 posts later we've returned to that topic several times, most recently in 2016. But before you can start screening you need a fragment library, which is the subject of our new poll.

Back in 2012 we asked readers the maximum size (in terms of "heavy", or non-hydrogen atoms) they would consider for fragments in their library. The results were mostly consistent with the Rule of 3, so beloved by Teddy that he compared it to a powerful wizard.

There has since been a trend toward smaller fragments, driven in part by empirical findings that smaller fragments have better hit rates, in agreement with molecular complexity theory.

At some point, though, ever smaller fragments will mean lower hit rates: fragments that are too small will bind so weakly they will be difficult to detect. And practical issues arise: organic molecules with just a few non-hydrogen atoms are often volatile.

Therefore, we’re revisiting this question: What is the smallest fragment you would put in your library?

As long as we're on the subject of libraries, how many fragments do you have in your primary screening library, or how many do you screen on a regular basis?

Please vote on the right-hand side of the page. If you have multiple fragment libraries (for example one for crystallographic screening and one for biochemical screening) you can respond for each library; you will need to press "vote" after each answer. Please feel free to leave comments too.

Thanks to all of you for making Practical Fragments a success and for your comments over the years – looking forward to the next decade!

Fragment events in 2018 and 2019

Hard to believe we're already halfway through the year, but there are still some exciting events ahead, and 2019 is already starting to take shape.

2018

August 19-23: The 256th National Meeting of the American Chemical Society, which will be held in Boston, includes a session on "Best practices in fragment-based drug design" on August 20.

September 25-28: CHI's Discovery on Target will also be held in Boston, and there will be lots of presentations of interest to readers of this blog, particularly in the Lead Generation Strategies track. Mary Harner and I will be presenting a FBDD short course over dinner on September 27.

October 7-10: Finally, FBLD 2018 returns to San Diego, where it was born a decade ago. This will mark the seventh in an illustrious series of conferences organized by scientists for scientists. You can read impressions of FBLD 2016, FBLD 2014,  FBLD 2012, FBLD 2010, and FBLD 2009.

2019

March 20-22: Although not exclusively fragment-focused, the Sixth NovAliX Conference on Biophysics in Drug Discovery will have lots of relevant talks, and will be held in lovely Nice. You can read my impressions of the 2018 event here, last year's Strasbourg event here, and Teddy's impressions of the 2013 event here, here, and here.

March 24-26: The Royal Society of Chemistry's Fragments 2019 will be held in the original Cambridge. This is the seventh in an esteemed conference series that alternates years with the FBLD meetings. You can read impressions of Fragments 2013 and Fragments 2009.

April 8-12: CHI’s Fourteenth Annual Fragment-Based Drug Discovery, the longest-running fragment event, will be held in San Diego. You can read impressions of the 2018 meeting here, last year's meeting here, the 2016 meeting here; the 2015 meeting here, here, and here; the 2014 meeting here and here; the 2013 meeting here and here; the 2012 meeting here; the 2011 meeting here; and 2010 here.

November 20-22: If you can't make it to Nice, NovAliX will also be holding a biophysics meeting for the first time in the lovely city of Kyoto.

Know of anything else? Add it to the comments or let us know!