Eleventh Annual Fragment-based Drug Discovery Meeting

The first major fragment event of 2016, CHI’s Drug Discovery Chemistry, was held last week in San Diego. FBDD was the main focus of one track, and fragments played starring roles in several of the others as well, including inflammation, protein-protein interactions, and epigenetics. Also, for the first time this year the event included a one-day symposium on biophysical approaches, which also included plenty of fragments.

In agreement with our polls, surface plasmon resonance (SPR) received at least a mention in most of the talks. John Quinn (Genentech) gave an excellent overview of the technique, packed with lots of practical advice. At Genentech fragments are screened at 0.5 mM in 1% DMSO at 10°C using gradient injection, which permits calculation of affinities and ligand efficiencies directly from the primary screen. Confirmation of SPR hits in NMR is an impressive 80%. A key source of potential error in calculating affinities is rebinding, in which a fragment dissociates from one receptor and rebinds to another. That problem can be reduced by increasing the flow rate and minimizing the amount of protein immobilized to the surface. Doing so also lowers the signal and necessitates greater sensitivity, but happily the baseline noise has decreased by 10-fold in the past decade.

Some talks focused on using SPR for less conventional applications. Paul Belcher (GE) described using the Biacore S200 to measure fragments binding to wild-type GPCRs. In some cases this provided different hits than those detected against thermally stabilized GPCRs. And Phillip Schwartz (Takeda) described using SPR to characterize extremely potent covalent inhibitors for which standard enzymatic assays can produce misleading results. These screens require exotic conditions to regenerate the chip, so it helps that the SensiQ instrument has particularly durable plumbing.

In theory, SPR can be used to measure the thermodynamics of binding by running samples at different temperatures, but John Quinn pointed out that enthalpic interactions dominate for most fragments, so the extra effort may not be worthwhile. Several years ago many researchers felt that enthalpically driven binders might be more selective or generally superior. Today more people are realizing that thermodynamics is not quite so simple, and Ben Davis (Vernalis) may have put the nail in the coffin by showing that, for a set of 22 compounds, enthalpy and entropy of binding could vary wildly simply by changing the buffer from HEPES to PBS! (Free energy of binding remained the same with either buffer.)

Thermal shift assays (TSA or DSF) continued to be controversial, with Ben finding lack of agreement between the magnitude of the shift and affinity, though there was a correlation with success in crystal trials. In contrast, Mary Harner (BMS) reported good agreement between thermal shift and affinity. She also found that it seemed to work better when the fragments bound in deep pockets than when they bound closer to the surface. However, Rumin Zhang (Merck), who has tested more than 200 proteins using TSA, mentioned that some HCV protease inhibitors could be detected despite the shallow active site. Rumin also pointed out that a low response could indicate poor quality protein – if most of the protein is unfolded it might be fine for biochemical assays but not for TSA. Negative thermal shifts are common and, according to Rumin, sometimes lead to structures, though others found this to be the case less often.

What to do when assays don’t agree was the subject of lively discussion. Mary Harner noted that out of 19 targets screened in the past two years at BMS using NMR, SPR, and TSA, 45% of the BMS library hit in at least 1 assay. However, 68% of hits showed up in only a single assay. Retesting these did lead to more agreement, but even many of the hits that didn’t confirm in other assays ultimately led to leads. All techniques are subject to false negatives and false positives, so lack of agreement shouldn’t necessarily be cause for alarm. Indeed, Ben noted that multiple different soaking conditions often need to be attempted to obtain crystal structures of bound fragments.

Crystallography in general is benefiting from dramatic advances in automation. Jose Marquez described the fully automated system at the EMBL Grenoble Outstation, which is open to academic collaborators. And Radek Nowak (Structural Genomics Consortium, Oxford) discussed the automated crystal harvesting at the Diamond Light Source, which is capable of handling 200 crystals per hour. He also revealed a program called PANDAA (to be released soon) that speeds up the analysis of crystallographic data.

Crystallography was used as a primary screen against KEAP1, as discussed by Tom Davies (Astex). A subset of 330 of the most soluble fragments was tested in pools of four, which revealed several hot spots on the protein. Interestingly, an in-house computational screen had not identified all of these hot spots, though Adrian Whitty (Boston University) noted that they could be detected with FTMap. The fragments themselves bound exceptionally weakly, but intensive optimization led to a low nanomolar inhibitor.

Another case in which extremely weak fragments turned out to be useful was described by Matthias Frech (EMD Serono). A full HTS failed to find any confirmed hits against cyclophilin D, but screening by SPR produced 168 fragments, of which six were characterized crystallographically. Although these were all mM, with unimpressive ligand efficiencies, they could be linked or merged with known ligands to produce multiple leads – a process which took roughly one year from the beginning of the screen. Matthias noted that sometimes fragment efforts are started too late to make a difference, and that it is essential to not be dogmatic.

Huifen Chen discussed Genentech's MAP4K4 program. Of 2361 fragments screened by SPR, 225 had affinities better than 2 mM. Crystallography was tough, so docking was used instead, with 17 fragments pursued intensively for six months, ultimately leading to two lead series (see here and here), though one required bold changes to the core. This program is a nice reminder of why having multiple fragment hits can be useful, as the other 15 fragments didn’t pan out.

Finally, George Doherty (AbbVie) gave a good overview of the program behind recently approved venetoclax, which involved hundreds of scientists over two decades. He also described intensive medicinal chemistry which led to a second generation compound, ABT-731, with improved solubility and oral bioavailability.

We missed Teddy at this meeting, and there is plenty more to discuss, so please add your comments. If you did not attend, several excellent events are still coming up this year. And mark your calendar for 2017, when CHI returns to San Diego April 24-26.