Fragments vs wild-type GPCRs by SPR

Membrane proteins such as G-protein coupled receptors (GPCRs) represent a large fraction of drug targets. These are mostly overlooked by the fragment community, for two reasons. First, assays for low affinity binders are difficult to develop. Second, the proteins usually lack structural information useful for advancing fragment hits. Earlier this year Heptares provided a lovely solution to both problems by generating stabilized mutant GPCRs, which could be screened using surface plasmon resonance (SPR) and characterized crystallographically. In a new paper in ACS Med. Chem. Lett., Iva Navratilova, Andrew Hopkins, Robert Lefkowitz, and a multinational team at the University of Dundee, Duke University, and the University of North Carolina Chapel Hill report using SPR to screen fragments against a wild-type GPCR.

The researchers chose the human β2 adrenergic receptor, which has served as a model GPCR for a variety of biological and biophysical studies. They expressed this with a His₁₀ tag on the C-terminus and used a conventional nickel chip to immobilize the protein in the presence of detergent. The immobilized protein was able to bind to a known agonist and antagonist with dissociation constants similar to those reported in the literature, suggesting that it was folded correctly.

Next, 656 fragments were screened against the protein at 50 micromolar each. Using a surface containing β2 adrenergic receptor blocked with a known high-affinity, slowly dissociating agonist as a reference, the researchers looked for fragments that bound selectively to the surface containing the unblocked protein. A total of 81 fragments were then examined more closely in dose-response curves, yielding five confirmed hits, with dissociation constants ranging from 17 nM to 22 micromolar.

All five of these hits were tested in a conventional radioligand competition assay, confirming their binding. Interestingly, four of the five ligands were N-arylpiperazines, a class of molecules that the Heptares team also found as ligands for the β1 adrenergic receptor. When tested against this GPCR most were not selective, but one did show some selectivity for β2 adrenergic receptor against a panel of 27 GPCRs.

The fragment hits were then tested for activity in a cell-based assay, and all of them inhibited signaling. This illustrates a general complication with binding (versus functional) assays: with simple enzymes, once you’ve found a binder, it is probably either an inhibitor or has no effect. With GPCRs, a binder could be an agonist, an antagonist, a partial agonist, an inverse agonist, a neutral antagonist, or something else entirely; you need to go into cells quickly to figure out what you’ve got.

I do wonder whether it would be possible to screen at higher concentrations to look for weaker ligands, particularly for more challenging GPCRs for which no small molecule ligands are known. Still, the fact that SPR works as well as it does for a native GPCR is quite impressive. I suspect that we’ll see more and more fragment screening by SPR on membrane proteins. Whether folks will be comfortable optimizing fragment hits in the absence of high-resolution structures, though, remains to be seen.

FBDD Down Under

Fragment-Based Drug Design Down Under was held at Monash University in Melbourne, Australia earlier this month. The first dedicated FBDD conference in this country was full of enthusiasm: I had the impression many of the 100 or so participants, most of them Australian, were happily surprised to meet so many other fragment aficionados. With 18 oral presentations, nearly as many posters, and a lively panel discussion I can only touch on some of the broader themes here, so please weigh in with your own observations.

Fragment library design received considerable attention, which was nice as this is an area that is all too often ignored in conferences. Pete Kenny's name came up a couple times in helping to put together the CSIRO fragment library. Craig Morton gave an excellent overview of the SVIMR fragment library and some of the challenges constructing it: of roughly 1600 fragments purchased, 450 were either not sufficiently soluble in water or DMSO or not sufficiently pure to be included. David Chalmers of MIPS presented an analysis of the physicochemical properties of approved drugs, noting that roughly three quarters are ionizable, with potential implications for library design.

Three dimensional fragments have been much discussed lately, and Martin Drysdale of the Beatson Institute described a UK consortium, 3Dfrag.org, to put together a library of 3-dimensional fragments, as defined by having a principal moment of inertia closer to a sphere (think adamantane) as opposed to a plane (benzene) or a rod (2-butyne). The project is still in its early stages, with about 200 fragments acquired thus far. Martin also described an interesting collaboration with the Broad Institute to use existing DOS-derived fragment-sized molecules for screening.

There were several talks on fragment screening methods, especially NMR and SPR. In an intriguing comparison, Jerome Wielens of SVIMR described parallel efforts on HIV integrase, both using essentially the same (Maybridge) library. STD NMR screening produced more than 50 hits, ultimately yielding 15 co-crystal structures, while SPR screening (also discussed later by Tom Peat of CSIRO) produced 16 hits and ultimately 6 crystal structures, yet few of the hits were in common. There were differences in the protein constructs and pH, and some of the NMR hits may have been artifactual, while the use of a reference protein in SPR may have weeded out some true positives. All of which underlines the fact that using multiple biophysical methods is ideal.

STD NMR came under scrutiny from others as well: San Lim of MIPS described compounds that showed a signal when screened in mixtures but not when tested individually, and Martin Drysdale discussed one target that gave a 36% hit rate using the technique, leading him to pick SPR as a primary screening method. Still, there are some interesting possibilities: Thomas Haselhorst of Griffith University discussed using STD NMR not just for screening membrane proteins but for screening viruses, cells, and even fungal spores!

Markku Hämäläinen of GE Healthcare discussed the use of both SPR (specifically Biacore) and ITC. In the case of SPR, he termed one class of problematic compounds “selective promiscuous binders”: for example, a positively charged protein may cause negatively charged fragments to aggregate around it, giving anomalously high signals. Using a positive control and setting a maximum Rmax in fitting the data can help weed these out and provide more accurate dissociation constants. In a collaboration with Merck Serono on a kinase target, 105 hits from a 1920-fragment library gave an 80% confirmation rate when tested in ITC, and 41 of 48 produced co-crystal structures.

But as we are increasingly seeing, Biacore is no longer the only name in the SPR game: Olan Dolezal of CSIRO described Bio-Rad’s ProteOn instrument and found that, while it was less sensitive than Biacore, its higher throughput made it an attractive primary screening instrument.

There were also a couple interesting talks on in-situ methods for fragment assembly, including MS-based methods described by Sally-Ann Poulsen of Griffith and click-based methods discussed by William Tieu at the University of Adelaide. One of the problems with assembling a high-affinity molecule in situ is product release: a molecule made in situ might bind so tightly it never leaves the protein, which essentially stops production once a stoichiometric amount of the inhibitor is made. In Tieu’s case, the problem was cleverly overcome by introducing a mutation to lower the affinity.

Finally, Jonathan Baell of MIPS gave an excellent (though disturbing) talk on PAINS – a topic which is still unfortunately not sufficiently appreciated. In one illuminating example, he found that an in-house screen of a histone acetyltransferase produced only a single legitimate hit, along with a plethora of PAINS.

One common theme both in the presentations and offline discussions was the relative lack of chemistry support; definitely a pity, since there are certainly plenty of chemists looking for new opportunities. Of course, funding chemistry is a problem not unique to the Southern Hemisphere.

Australia is clearly a new world for fragments, and it will be fun to see how the field develops there. And on a personal note, I found Aussies to be some of the warmest, most genuine people I have met in any country. I definitely look forward to finding an excuse to return.

Fragments vs membrane proteins with SPR

Membrane proteins such as GPCRs account for something like half of all drug targets, but they present a serious challenge for fragment-based approaches. This is partly because the biophysical methods usually used for fragment screening often don’t work for membrane proteins, and partly because, in the absence of structural information, it’s hard to know what to do with fragment hits. But there is progress. We’ve highlighted a couple papers that use functional screens or TINS to find fragments against membrane proteins, and in a recent issue of Biochem. Pharmacol. U. Helena Danielson and colleagues at Beactica and two academic institutes show how surface-plasmon resonance (SPR) can also be applied.

The researchers were interested in GABA_A receptors, a class of ion channels involved in multiple physiological functions. The receptors normally form hetero-pentamers, but for simplicity the researchers used a homo-oligomeric receptor, consisting of five β3 subunits. Each β3 subunit carried a tag containing eight histidine residues that could be recognized by antibodies immobilized to the surface of the SPR chip. The GABA_A receptors were detergent-solubilized; control channels contained antibodies and detergent with no receptors. The resulting GABA_A-modified chips were quite stable; the researchers report being able to run roughly 200 samples over the course of 20 hours with a single chip. (The specific detergents and conditions are critical, so if you’re interested in pursuing this yourself the experimental section is invaluable.)

A set of 51 histaminergic and 15 GABAergic ligands were tested for binding, resulting in nearly two dozen hits with dissociation constants (K_Ds) between 13 and 300 micromolar. Some of these are exceptionally small: for example, histamine, with a molecular weight of 111 g/mol and just 8 heavy atoms showed a K_D of 98 micromolar, which is consistent with published results using different methods. A number of other ligands were also identified, some for the first time, though other previously reported ligands did not repeat in this system.

It will be fun to see the screening results of a larger, unbiased library. Of course, finding fragment hits against a membrane protein is only the first step to developing drugs – one still needs to figure out how to improve potency, most likely in the absence of structure. But, to paraphrase Churchill, at least this paper and related research represent the end of the beginning.

Surface Plasmon Resonance (SPR)

Fragment-based drug discovery took off with NMR in the 1990s and went mainstream with X-ray crystallography in the 2000s. Now surface plasmon resonance (SPR) is becoming increasingly popular as a primary means of identifying hits. The technique has been mentioned more than a dozen times on Practical Fragments, but we’ve never devoted an entire post to it until now.

This post follows up on two recent publications. The first is an excellent summary of SPR by our friends at FBDD-Lit. Peter Kenny gives an overview of the technique and reports on a workshop given by SPR mavens Dave Myszka and Rebecca Rich. He also covers some of the seminal papers in the field.

The second report is in the brand new journal ACS Medicinal Chemistry Letters. In it, Iva Navratilova and Andrew Hopkins of the University of Dundee provide practical advice on using SPR for fragment-screening.

The authors describe their work on using SPR to identify fragments that bind to carbonic anhydrase II, a popular target for proof-of-concept studies. They screened a library of 656 fragments with molecular weights between 94 to 341 Da, with an average of 187 Da, or about 13 non-hydrogen atoms. The entire screen, which was done at three concentrations (16.6, 50, and 150 micromolar) took 4 weeks from assay development to hit confirmation on a Biacore T100, and consumed a total of 27 micrograms of protein.

Importantly, Navratilova and Hopkins were keenly aware of the potential for false positives or nonspecific binders (of which there were 230 at the highest concentration!) One way they controlled for such artifacts was to include an unrelated reference protein; data could be corrected by subtracting the response to the reference protein from the response to the target protein. Another analytical method to reduce the number of false positives was to only consider compounds that exceeded a minimum threshold for ligand efficiency (a metric invented by Hopkins and co-workers), a decision justified here given the often high affinities observed for carbonic anhydrase inhibitors. After these filters, an examination of the stoichiometry of binding revealed a dozen specific binders and four non-specific binders, a hit rate of 1.8%.

My one reservation with this paper is that carbonic anhydrase is a particularly easy test case, unlikely to fairly represent many of targets that people screen. Indeed, the confirmed hits (all of which contain sulfonamides), have affinities from 0.13 to 14 micromolar – far better than a typical fragment screen, and comparable to many HTS screens. Still, the tools and analyses described should apply to more challenging targets.

Finally, it is worth noting that if you want access to SPR technology but don’t have the resources or expertise to do it yourself, at least a couple companies (Beactica and Graffinity) specialize in applying SPR to FBDD.