Capillary electrophoresis revisited

Among the various fragment-finding methods, capillary electrophoresis (CE) seems to be among the least-used, at least according to our polls. Indeed, we last wrote about CE in 2012, and since then the company that was popularizing the technique seems to have quietly dropped it from its website. A new paper by Marianne Fillet and collaborators at the University of Liege and the University of Namur in Analytica Chimica Acta presents a how-to guide for CE.

As we previously discussed, the most general CE assay involves filling a capillary with a protein solution as well as a “probe ligand” with affinity for the target protein. Interactions between the probe ligand and the protein will increase the migration time of the probe ligand compared to its progress through a capillary without protein (i.e., the probe ligand will move more slowly through the capillary in the presence of protein).

If a “test ligand” is introduced into the capillary and displaces the probe ligand, the migration time of the probe ligand will again decrease. By changing the concentration of test ligand and measuring the shift in migration time of the probe ligand, the affinity of the test ligand can be determined.

The researchers applied CE to thrombin, a drug target that is often used for validating fragment-finding methods. The low nanomolar inhibitor NAPAP was chosen as the probe ligand due to its strong chromophore (simplifying detection) and positive charge (allowing it to move in the electric field of the capillary). They tested three literature compounds with inhibition constants ranging from high nanomolar to high micromolar and found good agreement with published results.

Next, the researchers applied CE to a small library of fragments, generating several hits. They also describe a method for detecting irreversible binding: this involves screening the protein with an even higher concentration of probe ligand to see whether the test ligand itself can be displaced.

This is a nice study, but it perhaps also illustrates why the technique hasn’t caught on. First and most importantly is throughput; the runs shown are on the order of 14 minutes. Second, it does require a probe ligand. (Screening test ligands directly only works if they are positively charged.) On the other hand, CE can work with native protein, unlike immobilization-based techniques such as SPR and WAC.

Have you tried CE yourself – and if so how did it perform?

WAC vs other methods: all roads lead to good fragments

Among the many ways to find fragments, one of the relatively inexpensive newcomers is weak affinity chromatography, or WAC (see also here). The technique works by immobilizing a target protein onto a column and flowing fragments over it; molecules that bind to the target will elute more slowly than those that don’t. WAC has a number of potential benefits, but as with any technique the question is how well it really works. In a paper published a few months ago in Analytical Chemistry, Sten Ohlson at Linnaeus University and collaborators at Vernalis compared WAC with more established methods.

The protein they chose, HSP90, is sort of the fruitfly of FBLD: just about every technique has been tested on it. It’s also an oncology target with which Vernalis has many years of experience. The researchers chose 111 fragments from the Vernalis library and screened these using WAC. They also screened most of the fragments using surface plasmon resonance (SPR), fluorescence polarization (FP), thermal shift, and NMR (using three techniques: STD, waterLOGSY, and relaxation filtered spectra; only fragments that confirmed in all three NMR assays were considered hits).

The top 27 hits from WAC were also investigated with isothermal titration calorimetry (ITC), and 32 hits were soaked into crystals for X-ray crystallography.

The results were quite encouraging, with good agreement between the different methods:

NMR performed the best, though this could be due in part to the fact that three separate NMR techniques were used. Thermal shift performed the worst, with both false positives as well as false negatives, but even here the agreement was always greater than 50%. It is also important to note that assay conditions varied from technique to technique (for example, the pH ranged from 6.5 to 7.5), which could account for many of the discrepancies.

These results are in sharp contrast to some other comparisons of fragment finding methods (such as here and here), which showed little or no correlation between hits. Why the difference? One possibility is that the folks at Vernalis have worked out all the kinks in their assays and are very adept at separating the true hits from the chaff. Of course, it probably doesn’t hurt that they were working with a well-behaved and extensively characterized target.

The main focus of the paper is WAC, which performed admirably. Compounds could be screened in pools of up to 16 fragments when mass-spectrometry was used as a detection method, and less than 2 milligrams of HSP90 was used to prepare all three of the WAC columns made. One worry with immobilizing your protein is long term stability, but the columns seemed to be stable for at least 6 months through multiple runs.

Of course, no technique is perfect, and one area where WAC gets whacked is in determining dissociation constants. The correlation between K_D values measured by SPR and ITC was excellent (R² = 0.91) but much worse for WAC versus ITC (R² = 0.38) and nonexistent for WAC versus SPR (R² = 0.016), though some of this could possibly be explained by differences in buffer conditions.

Overall it looks like WAC is a great way to find fragments, though you may want to use other methods to actually quantify binding. This paper provides a detailed guide for using WAC, as well as good descriptions of other fragment-finding methods.

Purifying hydrophilic fragments

Lipophilicity is a topic that comes up periodically. Lipophilic molecules are increasingly viewed as problematic from a drug development standpoint. Even if the correlation studies indicting lipophilicity are not as strong as they appear, at the end of the day we would prefer most of our drugs to be nicely water soluble.

That said, many of the molecules we make are on the greasy side. GDB-17, Jean-Louis Reymond’s recent computational enumeration of small molecules with 17 or fewer heavy atoms, reveals that most potential molecules tend to be much more polar than similarly sized compounds that have actually been made. One likely reason for this is that purifying highly water-soluble molecules is difficult; it’s hard to wash away inorganic reagents, and they often stick to the normal silica gel that chemists use to purify conventional molecules. Reverse-phase HPLC is useful, but can be tedious and low throughput.

In a recent issue of Drug Discovery Today, Andrew Hobbs and Robert Young of GlaxoSmithKline provide practical tips on using reverse-phase flash chromatography as an alternative to HPLC. They report working at scales from milligrams to tens of grams and are able to separate some very polar molecules. There’s a lot of good stuff in this paper on choosing columns, solvents, and loading techniques. A lot of these details get pretty nuanced, so it’s nice to have them in one place. If you’re trying to isolate hydrophilic molecules, definitely check it out.

WACing GAK and thrombin (faster)

Last year we highlighted a fragment-finding technique called weak affinity chromatography, or WAC. Those of you who were at FBLD 2012 saw some nice updates on the approach, but if you missed the meeting you can read two new papers. In the first of these (in Anal. Bioanal. Chem.), Elinor Meiby and Sten Ohlson at Linnaeus University, in collaboration with researchers at Oxford University, describe the use of WAC against a kinase.

Recall that WAC works by immobilizing a protein onto an HPLC column and then flowing ligands through the column; ligands that bind to the protein will have longer retention times compared to their retention times in a column without bound protein. Small amounts of fragments can be injected (2 picomoles in this case), and the fragments are at low concentration, minimizing potential artifacts.

Here the researchers were interested in cyclin G-associated kinase (GAK), a potential target for Parkinson’s disease. They used less than 1 mg of protein to prepare their column and used adenosine as a positive control to show that at least 22% of the protein was still active. They then performed a virtual screen of 3200 fragments (from TimTec), looking for molecules that would bind to the ATP site of five different proteins; the 170 highest-scoring fragments were then tested by WAC in mixtures of 13 compounds. Despite the use of mixtures, run times were long (140 minutes per injection), so this was not a high-throughput approach, although more compounds could potentially be injected simultaneously (see below).

Mass-spectrometry was used as a detection method to identify fragments. The change in retention time between the protein column and reference column is related to the dissociation constant, and the researchers found that 78 of the fragments had an estimated Kd of better than 0.2 mM, a fairly high hit rate. (30 of the 170 molecules could not be detected on both the GAK and reference column, so over half of the assessed molecules tested positive.) However, it is possible that some of the fragments bind to but don’t inhibit the protein. In fact, 23 fragments eluted more slowly from the reference column than from the protein-containing column, illustrating that non-specific effects are certainly possible (see also below). That said, many of the best fragments are structurally similar to known kinase inhibitors.

One of the coolest features of WAC is that a couple of the fragments were racemic, and these gave double peaks on the GAK column but only single peaks on the reference column, suggesting that one of the two enantiomers binds more tightly than the other.

As noted earlier, this campaign was not high-throughput, and in the second paper (in J. Biomol. Screen) Minh-Dao Duong-Thi, Sten Ohlson, and collaborators at Linnaeus and AstraZeneca sought to speed things up. They took 590 fragments from their larger collection and pooled them into 11 groups of 35-65 members in DMSO, with final concentrations of each fragment as low as 0.023 mM. Fragments likely to be positively charged were pooled together, and negatively charged fragments were pooled separately to facilitate mass spectrometry analysis. Also, fragments in each pool were chosen to have unique molecular weights to facilitate unambiguous identification.

These researchers looked for hits against thrombin, a protein they had previously used in developing WAC. The mixtures were screened in 20-minute runs, so all 590 fragments were screened in under 4 hours. 60 fragments were not observed in the mixtures, but 36 of these could be detected when injected singly under more optimized conditions.

The 30 best hits were then confirmed by injecting them individually. To assess whether they bind to the active site of thrombin, the enzyme was inactivated with an irreversible inhibitor, and the fragments retested. Remarkably, only a single fragment showed a significant reduction in binding to the inactivated protein, suggesting that the other fragments were either promiscuous or bound to regions outside the active site. The selective fragment contains an amidine moiety, a known thrombin-binding motif.

This paper demonstrates that WAC can be done in a fairly high-throughput manner. Although the number of non-selective binders does raise concerns, WAC could still be a valuable primary screening method, particularly given that it can be done using standard laboratory equipment.

Microscale Thermophoresis (MST)

Practical Fragments has a soft spot for new biophysical methods to identify fragments, many of which are given unfortunately non-descriptive initialisms. To a list that includes SPR, ITC, STD, MS, TINS, CEfrag, and WAC, we can now add Microscale Thermophoresis (MST), described in a new paper in Angew. Chem. Int. Ed. by Philippe Baaske and colleagues at NanoTemper Technologies as well as academic collaborators.

Thermophoresis, also referred to as the Soret effect, occurs when particles move in response to a temperature gradient. In this case, the “particles” are proteins, whose movements depend on size, charge, conformation, and solvation, and can be altered by factors such as ligand-binding.

In MST, a fixed concentration of protein is incubated with varying concentrations of ligand in small capillaries. An infrared laser rapidly heats a spot on the capillary, and an ultraviolet light source excites aromatic residues within the protein. The fluorescence in the heated spot changes as the protein moves along the temperature gradient. This movement is affected by ligand binding, and so measurements at different ligand concentrations can be used to construct a binding curve.

The researchers used MST to study the binding of ligands to several proteins, including ionotropic glutamate receptors (iGluRs), p38-alpha MAP kinase, thrombin, and even the calcium sensor Syt1. The dissociation values determined by MST were mostly comparable to literature values, and the researchers could also perform competition studies in which adding an excess of one ligand blocked a different ligand for the same site.

A nice feature of the technology is that, since it uses native protein, one doesn’t need to worry about the effects of immobilization or conjugation, factors that researchers using SPR, TINS, and WAC must consider. On the other hand, the fluorescence signal relies on native amino acid residues (tryptophan in the examples here), which can be obscured by many compounds. Also, in its current incarnation MST doesn’t appear particularly high-throughput, though it also doesn’t use much protein

Still, this seems like a pretty cool approach. I’ve started seeing NanoTemper at more conferences (such as FBLD 2012), so hopefully you will have a chance to check them out and let us know what you think.

Capillary Electrophoresis

One of the fun aspects of fragment-based lead discovery is the number of ingenious biophysical methods for finding low-affinity fragments. In a recent issue of J. Biomol. Screen., Carol Austin and colleagues at Selcia describe their approach, capillary electrophoresis, which they term CEfrag.

Capillary electrophoresis itself has been around for quite a while. It involves applying a high voltage across a thin capillary filled with liquid; a solution to be analyzed is injected, and the voltage causes migration of analytes (for example, proteins or small molecules). Analyte movement through the capillary depends on charge and “hydrodynamic radius,” which is a function of molecular size and shape. In the case of CEfrag, the idea is to start with a reporter molecule that can be readily detected, for example via UV absorbance. Under a standard set of conditions, this “probe ligand” will have a characteristic mobility. If an excess of protein that binds to the probe ligand is present in the running buffer, the migration time will shift. If an inhibitor is also present in the running buffer, this will prevent the probe ligand from binding to the protein, also causing the migration time to change. By running different concentrations of inhibitor and measuring the changes in mobility, the inhibition constant can be determined.

The researchers demonstrated their approach using that old work-horse of FBLD, the cancer target Hsp90. The known Hsp90 inhibitor radicicol was used as the probe ligand. A total of 609 fragments were screened individually at an initial concentration of 0.5 mM, yielding 42 fragments that reproducibly inhibited radicicol mobility by 20% or more. This ~7% hit rate is similar to that found by others for this target.

Only 12 of the 42 hits identified by CEfrag were also detected in a confirmatory fluorescence polarization (FP) assay, of which only 5 gave measurable IC₅₀ values. However, FP is not ideal for evaluating fragments. In fact, one of the CE hits that didn’t reproduce by FP was ethamivan, the starting fragment for the program that ultimately led to Astex’s AT13387, now in a phase 2 clinical trial for GIST.

To get a better sense of the quality of the CE hits, the researchers put 6 fragments into crystallography trials: 3 hits from both CE and FP, two from CE alone, and one that hit neither. The negative control didn’t produce a structure, whereas two of the FP-confirmed hits produced co-crystal structures (the one that did not had solubility issues). One of the two CE-only hits (ethamivan) also did.

With a throughput of 100 compounds per day per instrument, this is not a high-throughput method, but it is comparable to many other biophysical approaches. Also, the low protein consumption and ability to use unmodified protein are selling points. Have you tried CEfrag? If so, what do you think?

Weak affinity chromatography (WAC)

There are many ways to find fragments: NMR and X-ray crystallography are old favorites, but SPR is quickly catching on. There are also more specialized approaches, such as ITC, MS, TINS, and biochemical screening. Now another 3-letter abbreviation has joined the list: a paper published online by Sten Ohlson and colleagues at Linnaeus University in Sweden in Analytical Biochemistry describes weak affinity chromatography, or WAC.

The principle is remarkably simple. First, a protein of interest is covalently immobilized onto a chromatography column packed with modified silica gel. This can be done on a standard high-performance liquid chromatograph (HPLC). Then each fragment to be tested is injected in buffer; those that have affinity for the immobilized protein will stay on the column longer than they would if they lacked affinity. The fragments can be detected with either UV spectrometry or mass spectrometry.

To demonstrate the technique, the researchers used two model enzymes, thrombin and trypsin, and a couple dozen fragments ranging in mass from 93 to 307 Da. Most of these fragments contained an amidine, a moiety known to bind to both proteins. Columns without any immobilized protein served as controls. Of course, fragments may associate non-specifically with proteins, so the researchers also treated protein-containing columns with irreversible or potent reversible inhibitors; a fragment that comes out later from a column containing an active protein than from a column containing an inactive protein is presumably binding specifically to the active site.

Remarkably, the technique appears to work: most of the amidine-containing fragments were retarded in columns containing active protein compared to columns containing inhibited protein. Moreover, the relative affinity ranking correlated with the inhibitory activity of fragments in enzymatic assays. Some of the fragments were quite weak, with calculated dissociation constants around 1 mM.

The researchers also demonstrated that they could screen a mixture of 11 fragments, using mass-spectrometry to follow each fragment, and that the change in retention time was comparable to that observed when running each fragment individually. In this case it was important to use low fragment concentrations so as to avoid saturating the protein active sites.

As with any technique, there are bound to be limitations. The immobilized protein needs to be stable for an extended time; in the current case there was some degradation in performance, albeit over the course of months and more than 200 injections. A more serious constraint is the need for a proper reference. Inactivating an enzyme provides an ideal solution, but one that won’t be so easily generalized to all targets.

In some ways WAC could be seen as a low-price cousin of TINS: both methods rely on an immobilized protein, but while TINS uses a custom modified NMR spectrometer, WAC can get by with a much less pricey HPLC (though a mass-spectrometer seems nearly indispensible). It will be fun to see how WAC develops, and in particular whether it can be used to discover novel fragments against more challenging targets.