Fragments in China

The 2017 International Symposium on Fragment Based Lead Discovery (pdf here) was held in Shanghai, China last week. I was fortunate to be able to attend what I believe was the first significant FBLD meeting in Asia. Antimicrobials were a major theme, particularly against drug-resistant pathogens. The two days were filled with nearly 20 talks, so I’ll just try to capture a few impressions.

Ian Gilbert discussed the fragment-based efforts underway at the University of Dundee, focusing especially on library design. Among initially purchased commercial compounds, only 56% passed quality control, with 26% insufficiently soluble (at least 2 mM in water) and most of the rest either unstable or impure, similar to what has been seen by others. Ian has also enlisted undergraduate students to make “capped” fragments ready for optimization, as well as novel heterocycles.

Biophysics was a major theme of the conference, and Ian made a strong case for biolayer interferometry (BLI), one of the lesser-used fragment finding techniques. A screen can be completed in just a few days with less than a milligram of protein. In particular, BLI may be useful for assessing ligandability: Ian tested 31 targets, 13 known to be ligandable and 5 known to be not ligandable, and found good agreement with previous research. Ligandable targets generally gave primary hit rates >4.5%.

Ismail Moarefi (Crelux, now part of WuXi AppTec) highlighted microscale thermophoresis (MST) and differential scanning fluorimetry (DSF). NMR had identified ten hits against Pim1, but only six had yielded crystal structures, despite considerable effort. Of the four that didn’t, three had no activity by MST, while the fourth was very weak. Ismail also discussed the Prometheus nanoDSF instrument, which is sufficiently sensitive that it can resolve two-stage melting curves for a two-domain protein.

Another lesser used fragment-finding technique, affinity mass spectrometry, was described by Wenqing Shui (ShanghaiTech University). This uses ultrafiltration to separate protein-bound ligands from unbound molecules and mass spectrometry to identify hits; up to 1000 molecules can be screened in a single assay! Wenqing provided several success stories, including fragment hits with very weak (millimolar) affinity. She also demonstrated that the technique works against a membrane preparation of a GPCR.

Among more common biophysical methods, NMR was represented by Ke Ruan (University of Science and Technology of China). The challenge was characterizing a low-solubility ligand which caused extensive line-broadening of the protein due to intermediate exchange rates. This was solved by examining the distance between a fluorinated ligand and a paramagnetic label on the protein and using this to model the binding mode.

But by far the star of the show was crystallography. We’ve previously mentioned the high-throughput capabilities developed at the Diamond Light Source, and part of the impetus for this conference was to bring these technologies to China. Frank von Delft (Diamond and University of Oxford) noted that since the XChem platform launched in late 2015 more than 50,000 crystals have been screened against more than 40 targets, resulting in more than 1000 fragment structures. The group is committed to removing barriers and bottlenecks and today can process 1000 crystals per week through compound soaking, harvesting, data collection, and processing (using specially developed programs such as PanDDA). More than 30 external groups have used the facility, and every target has yielded at least one hit.

Of course, to collect data on 1000 crystals requires you to reproducibly grow lots of well-diffracting crystals that can handle the rigors of soaking, and Diamond has released a handy list of tips and tricks. Getting the right crystals was also the theme of two talks, one by Sheng Ye (Chinese Academy of Sciences) and the other by Carien Dekker (Novartis). Sheng emphasized the importance of optimizing the protein construct, which could include trimming flexible termini or disordered loops, mutating flexible surface residues, or considering different species. He also noted that adding heavy metal ions can actually improve the quality of the crystals as well as making the structures easier to solve. Carien also emphasized the importance of getting the construct right and discussed how seeding (crushing a hard-won crystal and using this to seed new drops) can be very useful. As we’ve noted, screening fragments at extremely high concentrations seems to be the current state of the art, with Novartis moving to 50 mM in the final soak and Diamond going beyond 200 mM! (In contrast to other types of screens at high concentrations, crystallography should not yield false positives, though hits might bind so weakly as to be undetectable by any other method.)

Such a wealth of structures can be daunting, and Anthony Bradley (Diamond) described the construction and use of a “poised library” for follow-up studies. The 768 fragments are (mostly) soluble to 500 mM in DMSO and are designed such that simple chemistry could generate 1.4 million analogs based on reagents currently in stock at Enamine. Potential analogs can be searched using the Fragment Network approach described here, and I was happy to see that Diamond has released their own open-source version (updated link as of 3 Jan 2018).

Jianhua He (Chinese Academy of Sciences) described the facilities at the Shanghai Synchrotron Radiation Facility (SSRF). This is the first third-generation synchrotron in China and has hosted more than 200 research groups since it opened in 2009. Feng Ye, who works at SSRF, gave a talk (in Mandarin) about screening a bacterial protein at XChem; the movies showing liquid handling and robotics would be impressive in any language. Renjie Zhang (Diamond), who also spoke in Mandarin, gave a talk describing (I’m told) not just XChem but how outside users can apply for access. Although there is currently a long waiting list, this should be addressed within the next year or so when SSRF gains Diamond status.

At the 2015 Pacifichem meeting there were only a few speakers from China. Given the level of interest and expertise I saw last week, I predict that the 2020 meeting will see many more.