Differential scanning fluorimetry (DSF) for covalent ligands

Differential scanning fluorimetry (DSF), also known as a thermal shift assay, is one of the most common techniques for fragment screening according to the most recent methods poll on Practical Fragments. This popularity is in part due to the simplicity of the assay: just mix your protein with a dye such as SYPRO Orange, which binds to the hydrophobic core of unfolded proteins, heat the solution in a PCR thermocycler, and measure the change in fluorescence. Ligands that bind specifically to proteins often stabilize them, increasing the melting temperature. As we noted in 2017, some companies even use DSF to assess the ligandability of new targets.

 

Most of the focus on DSF has involved non-covalent ligands. But the approach also works for covalent ligands, according to a new open-access paper in the British Journal of Pharmacology by Nir London and colleagues at the Weizmann Institute of Science.

 

The researchers acknowledge that they are not the first to explore DSF on covalent ligands; last year we discussed a screen of 47 covalent fragments against 47 kinases, and just last month we highlighted a DSF screen that identified an unexpectedly covalent fragment hit.

 

The new paper starts by performing DSF on five drug targets (BTK, the G12C mutant of KRAS, SARS-CoV-2 MPro, Pin1, and Keap1), each with well-characterized covalent ligands (for example, ibrutinib, acalabrutinib, and evobrutinib for BTK, sotorasib and adagrasib for KRAS^G12C, etc.) For four of the proteins they also tested non-covalent or covalent reversible ligands. Almost all of the ligands increased thermal stability of the bound protein, and covalent ligands tended to have a greater effect.

 

In addition to testing specific ligands, the researchers also performed DSF experiments on the five proteins with three reactive, non-specific covalent ligands: iodoacetamide, ethyl 2-(bromomethyl)acrylate, and a chloroacetamide called RA13. Intact protein mass spectrometry confirmed that these molecules gave essentially complete modification of the five proteins, in some cases at multiple sites.

 

In contrast to the specific ligands, the reactive alkylators tended either to have marginal effects on the melting temperatures of the proteins or to actually destabilize the proteins, sometimes dramatically. That said, there were cases of stabilization. The researchers suggest that “reactive, non-specific, irreversible binders may act as destabilizers, since they form an irreversible covalent bond regardless of molecular recognition, which may result in deformation of the protein if the rest of the binder cannot be accommodated near the modified amino acid.”

 

To assess how well DSF works for covalent fragments more generally, the researchers acquired 2612 acrylamides from Enamine and screened them (each at 8 µM in pools of 5) against Keap1 for 24 hours at 4 ºC. Mass spectrometry showed that just over 100 gave at least 50% modification, which a back of the envelope calculation suggests a covalent efficiency roughly 1 M^-1s^-1. Next, 84 compounds with differing degrees of modification were tested by DSF, and, in contrast to the kinase paper we discussed last year, there was a correlation between extent of modification and either stabilization or destabilization of the protein. But consistent with the specific ligands discussed above, the more reactive fragments (as assessed by glutathione reactivity) were the only ones that caused destabilization.

 

The authors conclude by proposing “DSF as a fast and complementary follow-up approach for electrophilic fragment screening, to prioritize productive and selective covalent binders over promiscuous reactive fragments.” This seems reasonable – what do you think?

Failing honorably and openly on PrP

Many of the posts on Practical Fragments – and indeed much of what appears in the literature – describe successes. This is obvious in the list of fragment-derived clinical compounds, and discoveries of high-affinity tool molecules or even advanceable fragment hits make up a large share of the 750+ posts on this blog. But of course, most of what we do in science fails, and such failures can also be informative. Eric Minikel and 25 collaborators from the Broad Institute and multiple other organizations have just published an illustrative case study on bioRxiv. (Eric also has a detailed and eloquent blog post of his own about the work.)

The researchers describe a five-year effort to find small-molecule binders of the prion protein, PrP, which misfolds and forms aggregates that lead to neurodegeneration. The hope was that binders could be turned into PrP stabilizers or perhaps even degraders. PrP has been studied for decades and there are plenty of literature reports of small molecules that seem to interact with the target, but none of these have been convincingly validated. Moreover, the crystallographic structure of the protein does not reveal attractive binding pockets.

Fragment-based methods have succeeded for other difficult targets, so the researchers performed STD and ¹⁹F NMR screens. Of 6630 pooled fragments, 238 initial hits were retested by STD NMR, leading to 80 hits that were then assessed by two-dimensional (TROSY) NMR. This led to a single hit, a substituted benzimidazole. Unfortunately the binding site could not be determined: chemical shift perturbations were spread across the protein. Differential scanning fluorimetry (DSF) showed the molecule caused a slight decrease in thermal stability. Both of these results suggest some sort of pathological mechanism, but the researchers did multiple experiments to rule out aggregation. A dose-response suggested a dissociation constant well above 1 mM, and none of 54 analogs tested proved any better. Soaking crystals of PrP with 20 of these was also unsuccessful.

Next, the researchers performed a thermal shift assay of just over 30,000 compounds (not necessarily fragments), which yielded both stabilizers and destabilizers. Unfortunately, none of 93 tested by two-dimensional NMR (HSQC) revealed any sign of binding. A DNA-encoded library (DEL) screen of 256,000 macrocycles also didn’t yield any confirmed hits (though more on that below), nor did a computational screen of just under 7 million molecules.

Given this experience, the researchers cautiously conclude that their “results may hint toward relative rarity of PrP binders in chemical space.” They do suggest several alternative approaches, such as screening the more biologically-relevant membrane-bound form of the protein. It may also be worth doing a high-concentration crystallographic screen. Finally, the researchers note that one of the DEL scaffolds “was judged to be a likely covalent binder and was not pursued further.” This decision may be worth revisiting. Indeed, covalent approaches have led to clinical compounds against another formerly undruggable target, KRAS.

According to the blog post, the researchers have pivoted to oligonucleotide therapeutics, where they seem to be making good progress. This makes sense, and I wish them luck. But I hope someone returns to PrP itself with new tools. If they do, the assays established and described here will prove invaluable.

Assessing ligandability by thermal scanning

Ligandability refers to the ability to find small-molecule leads against a target. A protein might be ligandable but not druggable if, for example, potent inhibitors of the target do not affect a disease state. But knowing in advance whether a target is ligandable can be useful, both to decide whether to embark on a campaign and to plan the resources it will likely require. Fragment screens by NMR have been shown to be good predictors of ligandability, but not everyone has access to this technology. Computational methods (such as FTMap) are also useful, but require a structure of the target. In a recent paper in J. Med. Chem., Stefan Geschwindner and colleagues at AstraZeneca describe high-throughput thermal scanning (HTTS) for assessing ligandability.

Thermal scanning (alternately called, as the researchers note, thermal shift, differential scanning fluorimetry (DSF), or thermofluor) relies on the preferential binding of a fluorescent dye to protein that is heat-denatured. Since ligands generally stabilize a protein against denaturation, an increase in melting temperature (Tm) is taken as an indication of binding. The assays can be plate-based and thus very fast.

The researchers chose 16 diverse targets (mostly enzymes) and screened their 763-ligandability fragment set (described here) at 1 mM by HTTS. Hits were defined as compounds that increased  thermal stability at least 3-fold above the standard deviation of controls. Targets were then categorized as follows:

Low ligandability: hit rate < 1.5%

Medium ligandability: hit rate between 1.5 and 4.5%

High ligandability: hit rate > 4.5%

Nine targets ranked low, and all of these failed high throughput screening (HTS), while 5 out of the 7 targets ranked medium or high by HTTS yielded useful HTS hits. Of course, failure in an HTS does not preclude target advancement by other means – including FBLD. Ultimately all but three targets (including all of those ranked medium or high and 6 of 9 ranked low) went on to enter hit-to-lead optimization programs.

Encouragingly, HTTS and NMR agreed perfectly for low and high ligandability targets, but NMR assigned three targets as medium where HTTS assigned them as low. The researchers thus set out to increase the sensitivity of HTTS.

It turns out that entropically-driven binders tend to cause greater thermal shifts than enthalpically driven binders. The observation that most fragments bind largely enthalpically, and with low affinity too, makes them particularly challenging to detect. To try to shift the balance, the researchers repeated the HTTS assay for three of the low-scoring targets in D₂O instead of H₂O, which enhances entropic interactions at the expense of enthalpic interactions. Indeed, all three targets showed enhanced hit rates, and two moved from low to medium ligandability.

Another way to improve sensitivity of a thermal shift assay is to add urea, which destabilizes proteins by lowering the unfolding enthalpy. Adding non-denaturing amounts of urea (0.8 to 2.4 M concentration) to the three low-scoring targets above did indeed increase the hit rate for two of them.

One interesting tidbit is the observation that particularly stable targets, with unfolding temperatures >70 °C, tend to produce lower hit rates in HTTS than less stable targets. This could account for the very different experiences people have had with the technique.

This is a nice paper, and the approach may be worth implementing, as the researchers note has already happened at AstraZeneca. Although HTTS is unlikely to ever be as robust as SPR, NMR, or crystallography, it is hard to beat the low cost and high speed.

Disrupting constitutive protein-protein interfaces

Protein-protein disruptions are notoriously difficult because the interfaces between proteins tend to be large and flat, with few of the deep pockets where small molecules prefer to bind. That's not to say they're impossible: the second approved fragment-derived drug targets a protein-protein interaction. This interaction, as with most others studied (see here, here, and here, for example), is transient: two proteins come together to transmit a biological signal, then dissociate. But many proteins form constitutive dimers or oligomers, and these tend to be even more challenging to disrupt. This is the class of targets discussed in a paper just published in J. Am. Chem. Soc.

Wei-Guang Seetoh and Chris Abell (University of Cambridge) were interested in the protein kinase CK2, a potential anti-cancer target. The enzyme is a tetramer containing two identical catalytic subunits (CK2α) and two identical regulatory units (CK2β). Previous experiments had shown that introducing mutations into CK2β that disrupted dimer formation decreased enzymatic activity and increased protein degradation. Would it be possible to find small molecules that did this?

Chris Abell is a major proponent of the thermal shift assay, in which a protein is heated in the presence of a dye whose fluorescence changes when it binds to denatured protein. The way this assay is normally conducted, small molecules are added, and if they bind to the protein they stabilize it, thus increasing the melting temperature (see here for an interesting counterexample).For oligomeric proteins, one might expect that anything that disrupts the oligomers would destabilize the proteins, thus lowering the thermal stability, and indeed this turned out to be the case in a couple model systems. Thus, the researchers screened dimeric CK2β against 800 fragments, each at the (very high) concentration of 5 mM. No fragments significantly increased the melting temperature, but 60 decreased the stability by at least 1.5 °C.

Best practice for finding fragments includes using multiple orthogonal methods, so all 60 hits were tested (at 2 mM each) in three different ligand-detected NMR assays: STD, waterLOGSY, and CPMG. Impressively, 40 of these showed binding in all three assays. There was no correlation between the binding affinity and the magnitude of thermal denaturation, which is not surprising because the thermal shift incorporates not just the enthalpy change of ligand binding but also the enthalpy change of protein unfolding. Thus, as the researchers note, “the extent of thermal destabilization cannot be used as a measure of its binding affinity.”

Next, all 40 confirmed fragments were tested at 2 mM to see whether they caused CK2β dimer dissociation, as assessed by native state electrospray ionization mass spectrometry (ESI-MS). 18 fragments shifted the equilibrium to monomeric protein, though interestingly no protein-fragment complexes could be observed. These 18 fragments also decreased dimerization in an isothermal titration calorimetry (ITC) assay.

There is still a long way to go: all the fragments are very weak, and preliminary SAR studies were unable to find analogs with significantly improved activity. Indeed, it is unclear where the fragments bind, or whether the binding site(s) are even ligandable. Still, the combined use of biophysical techniques on a particularly gnarly target make this an interesting study on the frontiers of molecular recognition.

Review of 2013 reviews

The year is coming to an end, and as we did last year, Practical Fragments is looking back at notable events as well as reviews that we haven’t previously highlighted.

The fragment calendar started in March in Oxfordshire, at the RSC Fragments 2013 conference, closely followed in April by CHI’s FBDD meeting in San Diego (here and here). Closing out the year for conferences that Teddy or I attended was the Novalix conference on Biophysics in Drug Discovery in Strasbourg (here, here, and here).

There weren’t any new books published (though the special issue of Aus. J. Chem. practically counts as one), but there were several notable reviews.

Stephen Fesik and colleagues at Vanderbilt University published “Fragment-based drug discovery using NMR spectroscopy” in J. Biomol. NMR. This is an excellent overview that covers library design, NMR screening methodologies, and compound optimization. The researchers make an interesting case for including multiple similar compounds and allowing for larger, more lipophilic fragments, while always being careful to avoid “bad actors”. They also do a good job of summarizing the various NMR techniques, including their strengths and limitations, in language accessible to a non-spectroscopist. Finally, the section on fragment linking discusses the theoretical gains in affinity, the practical challenges to achieving these, and strategies to overcome them.

Turning to the other high-resolution structural technique, Rocco Caliandro and colleagues at the CNR-Istituto di Cristallografia in Italy published “Protein crystallography and fragment-based drug design” in Future Med. Chem. This provides a fairly technical description of X-ray crystallography and its role in FBDD, along with a table summarizing around 30 examples, five of which are discussed in some detail.

Of course, it’s always best to use multiple techniques for finding fragments, so it’s well worth perusing “A three-stage biophysical screening cascade for fragment-based drug discovery,” published in Nature Protocols by Chris Abell and colleagues at the University of Cambridge. This expands on a gauntlet of biophysical assays (involving differential scanning fluorimetry (DSF), NMR, crystallography, and isothermal titration calorimetry (ITC)) that we discussed earlier this year. Nature Protocols are highly detailed, with lots of troubleshooting tips, so this is a great resource if you’re exploring any of these techniques.

Finally, Christopher Wilson and Michelle Arkin at the University of California San Francisco published “Probing structural adaptivity at PPI interfaces with small molecules” in Drug Discovery Today: Technologies. Protein-protein interactions are frequent targets for FBLD: see for example here, here, here, here, and here – and that’s just for 2013! The current review gives a nice overview of the technology called Tethering, focusing on the cytokine IL2 and an allosteric site on the kinase PDK1.

And with that, Practical Fragments thanks you for reading and says goodbye to 2013. May your 2014 be happy and fulfilling!

Fragments vs Tankyrases: DSF shines again

The two human tankyrase isoforms, TNKS1 and TNKS2, are members of the PARP family of proteins, which has received considerable attention as a pool of potential anticancer targets. In a recent paper in J. Med. Chem., a team of researchers from Sweden and Singapore use fragment-based methods to discover potent, selective inhibitors of the tankyrases. This is a nice example of FBLD from academia.

The researchers used differential scanning fluorimetry (DSF) to screen 500 fragments at 1 mM concentration each against TNKS2. In this assay, the protein is mixed with a fragment and a fluorescent dye that binds to the denatured form of the protein. When the mixture is heated, fragments that bind the protein should stabilize it against thermal denaturation. Thus, fragment binding can be detected by an increase in melting temperature (which is itself inferred by an increase in fluorescence). As noted previously, people have very different opinions of DSF; some folks swear by it, while others find that it produces too many false positives and negatives.

The present paper is an excellent resource for those wanting to try DSF for themselves; it provides clear experimental details and discussion of some of the things that can go wrong. One recommendation is to validate hits from the initial single-point assay by running dose-response curves over a wide concentration, such as 5-4000 micromolar. Another interesting tip is to add the protease chymotrypsin to the mix; doing so gave cleaner data, presumably by chewing up mis-folded contaminants.

The 500-fragment DSF screen identified two fragments against TNKS1, both of which were characterized crystallographically. The researchers chose to pursue fragment 2 since the structure suggested that this had good vectors for growing. Interestingly, removing the methyl group caused a complete loss in activity – another example of the power of methyl groups, and a sobering reminder of how subtle changes could make the difference between finding a fragment or not.

Fragment 2 was already quite potent, and adding an aryl substituent as in compound 11 further increased activity. Replacing the fluorine with a chlorine was even better, but at the cost of solubility, so the researchers added solubilizing groups and obtained potent, soluble molecules such as compound 17. Compounds 11 and 17 were also soaked into crystals of TNSK2, and the resulting structures overlay nicely with each-other as well as with the structure of the initial fragment.

Dissociation constants and kinetic parameters of the more potent molecules were determined by SPR, and although in general improvements in affinity were driven by decreases in k_off rates, k_on rates started to play a role with the more potent compounds. In another nice vindication for DSF, the thermal shift correlated nicely with both the IC₅₀ and K_d values.

Compounds 11 and 17 bind differently than other PARP inhibitors, so the researchers tested compound 11 against six other PARPs and found it to be quite selective. In fact, it is even 16-fold selective for TNSK2 over TNSK1. While that property may ultimately not be desirable in a therapeutic, it should be useful for exploring the biology.

Overall this is a lovely piece of work, and it does make a good case for the utility of DSF. The fragments identified are quite potent; perhaps the technique really shines at finding these exceptional fragments.

Multiple methods find fragments on MEK1, but fluorimetry shines

The tendency of fragments found in one assay to reproduce – or not – in another assay is a frequent topic at Practical Fragments. In a recent paper in Bioorg. Med. Chem. Lett., researchers at Sanofi describe their experience screening the oncology-associated kinase MEK1.

The researchers were interested in the ATP-binding site of MEK1, and they started with a virtual screen (using Glide-SP) of a 10,000 compound library. The top 196 hits were then tested experimentally by differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), leading to 30 and 44 hits, respectively, with 12 in common. A subsequent biochemical assay of the same 10,000 compound library yielded 106 hits, only 13 of which were in common with the virtual screen. 158 different fragments were identified by one or more of the three experimental methods.

Of 13 hits selected for follow-up experiments, crystallography ultimately yielded structures for 7 of them, of which 5 had been identified in the virtual screen. Interestingly, SPR had only confirmed 2 of these molecules, while DSF had confirmed all of them. Thus, in contrast to some reports, the Sanofi folks are quite sanguine about DSF and advocate using it widely and early in a project (as indeed many people do seem to be doing). The technique is fast and easy, and in this case the researchers were able to run the DSF screen before they had finished developing their biochemical assay.

The paper includes detailed comparisons between virtual screening, DSF, SPR, biochemical, and X-ray approaches, and is well worth examining if you are putting together a screening cascade.

The researchers conclude:

There is no gold-standard method for screening fragments. The general approach is to conduct a primary screen and then follow this up with at least another method to confirm hits, which are subsequently prioritised for structure determination. Different groups adopt different methods based on availability of materials, in-house expertise and prior experiences screening fragments.

In other words, multiple methods can find fragments. Ultimately, you’ll probably find real hits whatever methods you use, as long as you’re careful.

Halogenated fragments stabilize mutant p53

Practical Fragments recently discussed using fluorinated fragments for ¹⁹F NMR, but there are other halogens out there – are these useful for constructing fragment libraries? SGX Pharmaceuticals had a collection of fragments enriched with bromine atoms, the thought being that this atom would facilitate crystallography. Halogens can also make productive interactions with proteins, including so-called “halogen bonds” to backbone carbonyl atoms or pi-systems. With this in mind, Andreas Joerger at Cambridge University and Frank Boeckler at Eberhard-Karls University and their colleagues have assembled and screened a “halogen-enriched fragment library.” Their results are reported in a recent issue of J. Am. Chem. Soc.

The library consists of 79 non-reactive, soluble aromatic compounds containing bromine or iodine. Because these elements are so large, the researchers used a modified rule of 3 – instead of a molecular weight limit of 300, they limited the fragments to no more than 22 heavy atoms (see also our recent post on this topic here). They then screened this library against the Y220C mutant form of p53, which contains a surface crevice that destabilizes the protein and contributes to cancer cell survival. Thermal shift assays were used as the primary screen, with hits being confirmed by 2D NMR and ITC. This resulted in the discovery of compound 3, which crystallography confirmed was making a halogen bond to a backbone carbonyl.

Modifying the amine substituent improved potency modestly, and building off the phenyl ring towards a nearby pocket improved the potency further, albeit at a cost in ligand efficiency. Still, this compound (PhiKan5196) does represent the most potent Y220C binder reported, and represents an order of magnitude improvement over previous work. Moreover, the molecule induces apoptosis in p53 Y220C containing human cancer cell lines but not in matched wild-type p53 cell lines. (Unfortunately the compound also appears to be generally cytotoxic.)

This library is an interesting approach in part because it is somewhat heretical: for various reasons most library designers exclude molecules containing bromine or, especially, iodine. That said, the thyroid hormones do contain iodine aplenty, and MEK kinase seems to have a predilection for bromine or iodine as well. What do you think? Are halogenated fragments a useful tool for certain targets, or an unproductive diversion?

Stabilizing p53 with a little help from fragments

The protein p53 is inactivated in a large fraction of cancer cells and has long been of interest for oncology. Mutations of the gene frequently lead to a destabilized form of the protein. For example, substitution of cysteine for tyrosine at position 220 causes the mutant protein to rapidly denature at body temperature and also opens a reasonably large and hydrophobic crevice on the surface of the protein at lower temperatures. If molecules could be identified that bind in this crevice, the protein might be stabilized, restoring its function. In a recent paper* in Chemistry and Biology, Alan Fersht and colleagues at Cambridge University have targeted this crevice using fragment screening.

The researchers assembled a fragment library of 1895 molecules from three commercial vendors (ChemBridge, Life Chemicals, and Maybridge). They then used two orthogonal screening methods, NMR (WaterLOGSY) and thermal denaturation scanning fluorimetry, to identify fragment hits. These were then confirmed using two-dimensional HSQC NMR. WaterLOGSY identified 70 confirmed hits, while thermal screening identified only 17; oddly, only three of these were in common. The authors suggest that fluorescence quenching may lead to a higher false negative rate for the thermal denaturation method, but it is also possible that the NMR method is identifying fragments that bind so weakly as to show no effect on protein stability.

Of the 84 hits, three fragments could subsequently be characterized bound to p53 crystallographically. They all fit in the Y220C crevice, though each sits in a somewhat different location.

There is still a long way to go for these molecules: the most potent fragment has a Kd of 105 micromolar. Still, with a ligand efficiency of 0.33 kcal/mol per atom, this compares favorably to the best molecule the authors had previously identified from an in silico screen of 2.7 million molecules (Kd roughly 150 micromolar, ligand efficiency 0.29 kcal/mol per atom).

Although it is still not clear that stabilizing mutant p53 will be a viable approach for treating cancer, the identification of a number of diverse fragments suggests that the Y220C site may be druggable. Moreover, the fragments themselves are potential starting points for developing more potent molecules.

*Thanks to Mauro Angiolini for bringing this publication to our attention on LinkedIn.