19 June 2017

Fragments vs BRD4, two ways

Bromodomains, epigenetic targets that recognize acetylated lysine residues, have received considerable attention from the fragment community. (I devoted all of last July to the topic, and covered it more recently here.) Of the dozens of bromodomain-containing proteins, the four BET-family members have been highly studied, and the second bromodomain of BRD4 (BRD4-BRDII) in particular has been implicated in cancer and inflammation. In two new papers, researchers from AbbVie describe inhibitors of this target.

In the first Bioorg. Med. Chem. Lett. paper, George Sheppard and colleagues briefly describe a protein-detected (13C-HSQC) NMR screen of BRD4 in which the methyl groups of isoleucine, leucine, valine, and methionine were 13C-labeled. About 18,000 fragments were screened in pools of 30, and hits were then tested individually in NMR and time-resolved fluorescence resonance energy transfer (TR-FRET) assays. Despite extensive work on this target by multiple groups, these screens were able to identify several new fragments, such as the related compounds 1 and 2.


Crystallography of each fragment bound to BRD4 revealed that they bind in the acetyl lysine recognition site and make contacts with the conserved asparagine residue as well as a nearby water molecule. Merging the fragments led to compound 5, with a slight increase in affinity.

Comparison with other BRD4 inhibitors suggested a growth strategy, leading to compound 15, with nanomolar activity in the TR-FRET assay and two cell-based assays. The compound was orally bioavailable but had relatively high clearance, so further medicinal chemistry focused on changing the original core. This ultimately led to compound 38, with improved oral bioavailability, lower clearance, good selectivity against non-BET bromodomains, and activity in a mouse xenograft assay.

The second paper, by Le Wang, John Pratt, and colleagues in J. Med. Chem., starts with a different fragment from the original screen, compound A1. This molecule was even weaker than the fragments described above, but crystallography confirmed that it binds in the same acetyl lysine binding pocket.


Again, comparison with known inhibitors provided ideas for fragment growing, rapidly leading to compound A11. Further medicinal chemistry – which is extensively described in the paper – led to compound A30a, which bears considerable resemblance to the series reported in the previous paper.

Crystal structures of compounds bound to the protein suggested that it might be possible to make a macrocycle, which would in theory increase the affinity by locking the molecule in a low energy conformation. This proved to be synthetically challenging but ultimately worthwhile in the form of compound A74b. (Incidentally, this is the first case I can recall where a fragment led to a macrocycle. It won’t be the last.) Not only was this molecule more potent than the open form, it also showed excellent oral bioavailability and pharmacokinetics, good selectivity against non-BET bromodomains, and even better activity in a mouse xenograft model. 

One lesson from these papers is that fragments can generate new ideas even for heavily pursued targets. A second is that, as we saw in the recent poll, crystallographic information can be critical for advancing fragments to leads. The discovery of new moieties along with clear data on their binding modes can be a powerful combination for creative medicinal chemists.

12 June 2017

Fourth NovAliX Biophysics in Drug Discovery Conference

NovAliX held its fourth Biophysics in Drug Discovery meeting last week in the beautiful city of Strasbourg. This was my first time attending, and although Teddy’s recounting of the first meeting (here, here and here) had given me high expectations they were easily exceeded. With 134 participants from 13 countries, most of whom stayed for the full time, the event felt like a Gordon Research Conference, with lots of lively discussions over excellent food and drink. Rather than trying to summarize all 30+ talks and nearly as many posters, I’ll just provide a few impressions. Conference Chairman Jean-Paul Renaud posted a 2 minute video overview here.

Factors that drive success in FBLD were a theme of Jenny Sandmark (AstraZeneca). She discussed several projects in which fragments were able to generate useful chemical matter, including one (Complement Factor B) where HTS and DNA-encoded libraries both came up short. In the case of neutrophil elastase, SAR on HTS hits was making rapid progress, but chemistry to explore the S1 pocket was difficult. A directed NMR screen of 800 fragments did not yield anything better, but it did save considerable synthetic effort and was thus judged a success.

Tweaking experimental conditions was often essential to get informative results: soaking crystals of neutrophil elastase with fragments dissolved in DMSO produced no hits, while dissolving the fragments in water did. With crystals of Factor XIa, glycerol – not fragments – bound in the active site. Fortunately the researchers recognized this and were able to use a different cryoprotectant.

Glyn Williams (Astex) has been doing FBLD since the earliest days, and discussed some of the lessons learned. Although the current Astex fragment library consists of about 1800 compounds, some 8000 fragments have been evaluated over the years. Small fragments in particular can be quite volatile, so Astex stores its library at -80 °C under nitrogen. Researchers also do rigorous quality control (QC) and maintain “fragment CVs”, which summarize analytical and screening data for each molecule. Astex is heavily invested in synthesizing novel fragments to explore specific regions of chemical space, and this means being constantly on guard for risks such as redox cyclers.

Understanding the enemy is always useful, so Martin Redhead (UCB) has constructed a library of bad actors – including PAINS, chelators, and metals – to stress-test his assays. But even robust assays show plenty of false positives. A screen of 20,000 molecules against a protein-protein interaction revealed 105 stabilizers, only 3 of which turned out to be real, but four times as many inhibitors, none of which were legitimate. Some of the stabilizers could subsequently be modified to inhibitors, and Martin suggested that screening for stabilizers initially could be a general way of improving signal to noise.

NMR is uniquely capable of providing extensive QC information about both proteins and ligands, and Alvar Gossert (ETH) discussed how a “validation cross” confirming both integrity and binding can improve confidence. Alvar also discussed using dynamic nuclear polarization to reduce the number of spectra required to assign a protein from five collected over two weeks to one obtained in under three days. This requires a specialized setup involving a second magnet, but it does appear powerful. Another unusual NMR configuration was described by Ad Bax (NIH), who is studying protein folding by rapidly (< 1 msec) increasing the pressure of a sample to 2500 bar – the equivalent energy of “discharging a small handgun into your NMR.”

Plenty of more accessible technologies were also described, including some interesting new commercial offerings. Matyas Vegh (Creoptix) discussed using waveguide interferometry to study protein-ligand interactions. This is similar to surface plasmon resonance (SPR) but with higher sensitivity and lower bulk effect. Their new four-channel instrument can be temperature controlled from 4-45 °C and is capable of accurately measuring kinetics even for weak binders, such as the 2.79 s-1 off-rate for the binding of methylsulfonamide (MW = 95 Da, Kd = 419 µM) to carbonic anhydrase II. And Sven Malik (Sierra Sensors) described a sensitive new SPR instrument with 8 channels, each with 4 spots, capable of running 384 samples in under 3 hours.

Several talks or posters highlighted an instrument from Biodesy which is capable of studying sub-Ångström conformational changes in dye-labeled, surface-immobilized proteins using second harmonic generation. Their plate-based instrument can measure 20,000 samples per week. Elizabeth Vo (UCSF) is using this to study the protein K-Ras, and has identified a number of active fragments which are being further characterized using orthogonal methods. It will be fun to see how these compare with previously reported K-Ras binders.

Finally, the keynote lecture was delivered by Jean-Pierre Changeux (Collège de France and Institut Pasteur), who described some of the highlights – many of them quite recent – from a career that spans nearly six decades. Jean-Pierre literally invented the model for allostery at a time when the three dimensional structures of only two proteins were known; today the Protein Data Bank contains more than 130,000 structures, and at least 90 marketed drugs are known to work through allosteric mechanisms. It is humbling to be reminded of how far we've come, yet how little we still know.

Strasbourg is a wonderful city, but with today’s travel budgets it can be difficult to access for some researchers, so next year the conference will head to Boston (June 12-15), returning to Strasbourg in 2019 before moving on to Kyoto in 2020. Hope to see you there!

05 June 2017

Poll results: what structural information is needed to optimize fragments

Our latest poll asked “how much structural information do you need to begin optimizing a fragment?” Over the past few weeks we received 143 responses, and the results are shown here.
Just over a third of you said that you wouldn’t work on a project without a crystal structure, while nearly a quarter said you’d settle for an NMR-based model. In other words, more than half of you demanded fairly detailed structural data to embark on a fragment optimization campaign.

But consistent with continuing improvements in modeling, almost a fifth said that a computational model would be just fine.

And perhaps most surprisingly, fully one quarter of you said that SAR alone would be sufficient to begin optimizing a fragment. Presumably this is driven at least somewhat by internal successes, and I look forward to seeing these disclosed in meetings and publications.

All of these approaches are rapidly developing, and it will be fun to revisit this poll in a few years to see whether crystallography maintains its lead, or whether lower resolution methods gain dominance.

In the meantime, are there other topics you’d like to see polled?