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Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: Twitter: Dereklowe

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April 2, 2014

Binding Assays, Inside the Actual Cells

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Posted by Derek

Many readers will be familiar, at least in principle, with the "thermal shift assay". It goes by other names as well, but the principle is the same. The idea is that when a ligand binds to a protein, it stabilizes its structure to some degree. This gets measured by watching its behavior as samples of bound and unbound proteins are heated up, and the most common way to detect those changes in protein structure (and stability) is by using a fluorescent dye. Thus another common name for the assay, DSF, for Differential Scanning Fluorimetry. The dye has a better chance to bind to the newly denatured protein once the heat gets to that point, and that binding even can be detected by increasing fluorescence. The assay is popular, since it doesn't require much in specialized equipment and is pretty straightforward to set up, compared to something like SPR. Here's a nice slide presentation that's up on the web from UC Santa Cruz, and here's one of many articles on using the technique for screening.

I bring this up because of this paper last suumer in Science, detailing what the authors (a mixed team from Sweden and Singapore) called CETSA, the cellular thermal shift assay. They trying to do something that is very worthwhile indeed: measuring ligand binding inside living cells. Someone who's never done drug discovery might imagine that that's the sort of thing that we do all the time, but in reality, it's very tricky. You can measure ligand binding to an isolated protein in vitro any number of ways (although they may or may not give you the same answer!), and you can measure downstream effects that you can be more (or less) confident are the result of your compound binding to a cellular target. But direct binding measurements in a living cell are pretty uncommon.

I wish they weren't. Your protein of interest is going to be a different beast when it's on the job in its native environment, compared to sitting around in a well in some buffer solution. There are other proteins for it to interact with, a whole local environment that we don't know enough to replicate. There are modifications to its structure (phosphorylation and others) that you may or may not be aware of, which can change things around. And all of these have a temporal dimension, changing under different cellular states and stresses in ways that are usually flat-out impossible to replicate ex vivo.

Here's what this new paper proposes:

We have developed a process in which multiple aliquots of cell lysate were heated to different temperatures. After cooling, the samples were centrifuged to separate soluble fractions from precipitated proteins. We then quantified the presence of the target protein in the soluble fraction by Western blotting . . .

Surprisingly, when we evaluated the thermal melt curve of four different clinical drug targets in lysates from cultured mammalian cells, all target proteins showed distinct melting curves. When drugs known to bind to these proteins were added to the cell lysates, obvious shifts in the melting curves were detected. . .

That makes it sound like the experiments were all done after the cells were lysed, which wouldn't be that much of a difference from the existing thermal shift assays. But reading on, they then did this experiment with methotrexate and its enzyme target, dihydrofolate reductase (DHFR), along with ralitrexed and its target, thymidylate synthase:

DHFR and TS were used to determine whether CETSA could be used in intact cells as well as in lysates. Cells were exposed to either methotrexate or raltitrexed, washed, heated to different temperatures, cooled, and lysed. The cell lysates were cleared by centrifugation, and the levels of soluble target protein were measured, revealing large thermal shifts for DHFR and TS in treated cells as compared to controls. . .

So the thermal shift part of the experiment is being done inside the cells themselves, and the readout is the amount of non-denatured protein left after lysis and gel purification. That's ingenious, but it's also the sort of idea that (if it did occur to you) you might dismiss as "probably not going to work" and/or "has surely already been tried and didn't work". It's to this team's credit that they ran with it. This proves once again the soundness of Francis Crick's advice (in his memoir What Mad Pursuitand other places) to not pay too much attention to your own reasoning about how your ideas must be flawed. Run the experiment and see.

A number of interesting controls were run. Cell membranes seem to be intact during the heating process, to take care of one big worry. The effect of ralitrexed added to lysate was much greater than when it was added to intact cells, suggesting transport and cell penetration effects. A time course experiment showed that it took two to three hours to saturate the system with the drug. Running the same experiment on starved cells gave a lower effect, and all of these point towards the technique doing what it's supposed to be doing - measuring the effect of drug action in living cells under real-world conditions.

There's even an extension to whole animals, albeit with a covalent compound, the MetAP2 inhibitor TNP-470. It's a fumagillin derivative, so it's a diepoxide to start off, with an extra chloroacetamide for good measure. (You don't need that last reactive group, by the way, as Zafgen's MetAP2 compound demonstrates). The covalency gives you every chance to see the effect if it's going to be seen. Dosing mice with the compound, followed by organ harvesting, cell lysis, and heating after the lysis step showed that it was indeed detectable by thermal shift after isolation of the enzyme, in a dose-responsive manner, and that there was more of it in the kidneys than the liver.

Back in the regular assay, they show several examples of this working on other enzymes, but a particularly good one is PARP. Readers may recall the example of iniparib, which was taken into the clinic as a PARP-1 inhibitor, failed miserably, and was later shown not to really be hitting the target at all in actual cells and animals, as opposed to in vitro assays. CETSA experiments on it versus olaparib, which really does work via PARP-1, confirm this dramatically, and suggest that this assay could have told everyone a long time ago that there was something funny about iniparib in cells. (I should note that PARP has also been a testbed for other interesting cell assay techniques).

This leads to a few thoughts on larger questions. Sanofi went ahead with iniparib because it worked in their assays - turns out it just wasn't working through PARP inhibition, but probably by messing around with various cysteines. They were doing a phenotypic program without knowing it. This CETSA technique is, of course, completely target-directed, unless you feel like doing thermal shift measurements on a few hundred (or few thousand) proteins. But that makes me wonder if that's something that could be done. Is there some way to, say, impregnate the gel with the fluorescent shift dye and measure changes band by band? Probably not (the gel would melt, for one thing), but I (or someone) should listen to Francis Crick and try some variation on this.

I do have one worry. In my experience, thermal shift assays have not been all that useful. But I'm probably looking at a sampling bias, because (1) this technique is often used for screening fragments, where the potencies are not very impressive, and (2) it's often broken out to be used on tricky targets that no one can figure out how to assay any other way. Neither of those are conducive to seeing strong effects; if I'd been doing it on CDK4 or something, I might have a better opinion.

With that in mind, though, I find the whole CETSA idea very interesting, and well worth following up on. Time to look for a chance to try it out!

Comments (34) + TrackBacks (0) | Category: Chemical Biology | Drug Assays


1. Anonymous on April 2, 2014 10:10 AM writes...

Why would anyone *want* to measure ligand binding - in a cell, test tube, or otherwise? For intellectual curiosity?

Surely it's better just to screen for drug-related *activity/efficacy* directly, and be done with it. Because that's all that really matters anyway.

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2. John Wayne on April 2, 2014 12:23 PM writes...

@1 It turns out that that is unethical, so we have to look for other surrogate options.

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3. Y.S. on April 2, 2014 12:47 PM writes...

The method works well since PARP-1 is a very abundant protein. I have doubts that the assay window would be usable with most other targets.

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4. SP on April 2, 2014 1:17 PM writes...

"This CETSA technique is, of course, completely target-directed, unless you feel like doing thermal shift measurements on a few hundred (or few thousand) proteins. But that makes me wonder if that's something that could be done. Is there some way to, say, impregnate the gel with the fluorescent shift dye and measure changes band by band?"
Quantitative proteomics, anyone?

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5. P.K. on April 2, 2014 1:27 PM writes...

@1: I guess because "activity/efficacy" doesn't mean "it works the way I want it to". It needn't be just for intellectual curiosity. Maybe you'd want to convince yourself your compound isn’t an artefact, perhaps you want to find a better way to screen for compounds hitting the same mechanism, find a more robust or reproducible assay, or understand the pathway so you can identify other more druggable targets or liabilities. I'm not convinced that CETSA is generic, but surely it isn't controversial to try to understand as much as you can about what you’re doing?

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6. Anonymous on April 2, 2014 2:54 PM writes...

@2: Since when is it unethical to run activity assays in cells, or even isolated tissues?

@5: Sometimes I think we try to understand *too* much, often just leading us down the rabbit hole with more questions than we started with. In the good old days (when we actually used to find new drugs), we'd just stick the stuff in animals to see if it works, rather than try to break it all down to the nth degree of understanding.

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7. annonie too on April 2, 2014 3:23 PM writes...

#1 (6): You need to make more clear and directed comments.

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8. Vaudaux on April 2, 2014 4:17 PM writes...

#2 John Wayne is right. Everything biologists do is a surrogate for efficacy in humans - including animal models, cellular assays, direct measurements of ligand binding, etc.

The trick is keeping in mind exactly what aspect of efficacy your model is a good surrogate for. Then you can try to develop assays that complement each other.

For antibiotics, MIC determination by itself (a cell-based assay) is a very poor surrogate for efficacy in an infected patient. The same is true for pharmacokinetics. However, the MIC data together with PK data are extremely predictive of efficacy in animals and often in humans.

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9. once in a blue moon on April 2, 2014 4:19 PM writes...

#6: Even if "sticking things into animals" randomly was now considered ethical and still would be the prime method of choice, 1) it's expensive and 2) there aren't suitable animal models for many human diseases with some of the greatest needs: Dementia, Alzheimer's, cancers, ALS, chronic heart disease, etc. It's no longer simply measuring blood pressure.

Today's researchers have some great technical advantages over previous ones, but also have greater challenges too, not to mention the increased regulatory expectations, and the return-on-investment questions.

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10. gippgig on April 2, 2014 8:04 PM writes...

I don't remember all the details, but DHFR isn't the primary target of methotrexate. One article reported it was AICAR transformylase, but that turned out not to be it either (altho methotrexate does elevate AICAR levels, which might contribute to its anticancer activity). I think it's one of the early enzymes in the purine pathway.

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11. Anonymous on April 2, 2014 8:33 PM writes...

@8: "However, the MIC data together with PK data are extremely predictive of efficacy in animals and often in humans."

Wouldn't efficacy in animals be even more predictive of efficacy in animals?

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12. once in a blue moon on April 2, 2014 9:02 PM writes...

11: Not if there's no good animal model for the disease, and no validated biomarkers either.

How many different ways, different ways do folks have to say it, for you to get it?

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13. Anonymous on April 2, 2014 9:04 PM writes...

@1/6; ahh good old days... of alchemists. They came up with lots of useful things, but I doubt they'd ever be able to make semiconductors. Quantum mechanics would be a rabbit hole for them.

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14. Anonymous on April 2, 2014 10:30 PM writes...

#1 Drug design needs to be done stepwise for the process to be efficient. There are too many variables at play, when you go straight into a cellular assay, that effect efficacy. Because there are so many variables at play, it's difficult to develop trends between structure and activity. Not to say it can't be done. When examining ligand binding, I can make a congeneric series, and more easily (not always) form trends between binding and some physicochemical property that was modified. Once you know what drives binding, you are way ahead of the eightball when you go into a cellular assay.

My 2-cents.

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15. HTSguy on April 2, 2014 11:34 PM writes...

It sounds like an interesting technique. One thing to keep in mind for less potent compounds (e.g. fragment or HTS hits) is that everything sticks to (most) everything else at some concentration. That's one nice thing about radioligand binding assays - you can use a chemically dissimilar ligand as the tracer. Even Biacore and ITC give you some idea of whether the binding is specific by giving you stoichiometry. I wonder if Shoichet-type aggregates provide any protection?

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16. Anonymous on April 3, 2014 2:47 AM writes...

@13: And we'd still be waiting for penicillin with the current approach.

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17. sgcox on April 3, 2014 4:32 AM writes...

This method was used in the recent Nature paper to show crizotinib binding to MTH1 in intact cells:

Looks like it will be popular in future (except with one anonymous person).

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18. Chain & Florey on April 3, 2014 6:43 AM writes...

@16: No we wouldn't.

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19. Anonymous on April 3, 2014 7:42 AM writes...

@18: Well we're still waiting for cures for most diseases based on the current approach, so why do you think we would have discovered penicillin by this approach?

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20. profiler on April 3, 2014 8:57 AM writes...

The potential of this technique is enormous, provided it actually works with more than a few proteins.
Whenever a compound with good target potency and good physchem comes back inactive, in cells, animals, or even clinical trials, someone is bound to ask whether it was actually proven that it did engage the target. Now target engagement is of course dependent on the local concentration of the drug at the site of action (and over time), and its affinity for the endogenous target protein in its cellular context (which may differ from the purified stuff used in the screening).
Both factors are typically not measured because it is hard, often impossible, to do this.
This method offers a way out - if it really works.

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21. Curious Wavefunction on April 3, 2014 11:54 AM writes...

#1: I am always wary of these either/or ("phenotypic screening OR target-based design") debates. Drug discovery is too complex to be left to one kind of approach. Plus the two kinds of approaches address rather different problems. Animal-based screening is a great hit generation and target discovery engine, but it doesn't usually give you drugs, nor does it give you valuable med chem information, nor does it really tell you how to improve PK. Target-based design is what's going to really help in turning a hit into a lead. So both approaches have their place.

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22. Anonymous on April 3, 2014 1:08 PM writes...

@21: What percentage of false positives from the first screen do you rule out by running the second? Is it truly worth the time and investment, or just a case of doing every experiment possible, because you can?

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23. sgcox on April 3, 2014 1:53 PM writes...

#22 Sometimes 100%. YMMV

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24. Anonymous on April 3, 2014 2:13 PM writes...

@24: Well then just do the second screen, not the first.

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25. sgcox on April 3, 2014 2:20 PM writes...

# Or third, or forth. Even better! So lets start with Phase III on some random chemical. Drug discovery is easy after all.

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26. Anonymous on April 3, 2014 3:49 PM writes...

Well now you're just being silly.

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27. Anonymous on April 3, 2014 3:58 PM writes...

@23: That would imply your second screen is better than a Phase 3 trial, so clearly you just made that up. Game Set & Match! :-)

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28. sgcox on April 3, 2014 4:57 PM writes...

OK, your trolling win. Whatever, bye.

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29. Anonymous on April 4, 2014 2:03 AM writes...

I'm testing it in cellular lysates, and it works pretty well. Very usefull with proteins that are difficult to express and purify as recombinant (high MW/membrane proteins). Of course you need quite potent compounds (IC50 at least 100nM).
Next step will be to try the in-cell method. Finger crossed!

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30. UCSD on April 4, 2014 8:15 AM writes...

@29: Im also running the method, it works great as you said. I haven't experienced that I need particularly potent compounds to get shifts in Tagg. I have screened libraries of compounds and seen stabilization effects with high micromolar binders. I tried out a natural compound and did the ITDRF experiments that the inventors present and I saw stabilisation at low millimolar concentrations of the described target protein (about a dozen other reference proteins were left unstabilized in my controls)
As someone else said previously here, eventually certain compounds will/could stick unspecifically also to your target of interest. Im aware of this but it didn't seem to be a problem in my study.
If your compound is cell-permeable I can't see why it shouldn't work for you 29. Unless the protein is behaving really different or interacting with other proteins in a cellular environment, but I guess that is a result and information too...

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31. Anonymous on April 4, 2014 11:12 PM writes...

@4 and 29

This technique is very similar to FSEC and FSEC-TS from the Gouaux group. The only difference seems to be that this was done by western and FSEC is done by SEC with a fluorescent probe. Westerns are more high throughput compatible though... Why doesn't anyone make a 96-well FSEC instrument yet?

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32. Anonymous on April 5, 2014 8:17 AM writes...

I don't see more similarities between these methods than that the sample is heated (and that you do in pretty much all TSAs). Is FSEC working in complex mixtures (lysates etc)? A quite important difference is that fsec needs over expressed and/or genetically modified target protein, addition of GFP is not a small perturbation to a system and I would be careful to call that biologically relevant. However, it could come in handy if you already have a GFP reporter expression system and work with large amounts of protein and study buffer effects and detergents for membrane proteins. Also, I think Waldo and some others showed that fluorescent probes like GFP that Gouaux uses, could very well affect the stability of the target protein and keep it in solution longer, this would for sure mask any stabilisation that a small molecule would infer. Adding a smaller tag (for ex a His-tag) to your protein construct would enable you to simply add a anti-His fluorescent probe, Im suspecting, though, that you might need to purify your protein anyway since an anti-his tag sticks to many other proteins with exposed histidines. Furthermore you would need to wash of unbound probe. All these manoeuvres would for sure give you less information about the binding of a compound in the cellular environment. Couldn't you use the AKTA micro system with a fluoreader for your applications? That one takes 96 well plates and runs it on for ex 3 ml GF columns, it is really quick and sensitive!

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33. Anonymous on April 5, 2014 11:16 PM writes...

Yes, FSEC is used in complex mixtures. FSEC also only requires a fluorescent signal, which can come from a number of places, not just GFP. The Lumio tag is a simple example. As you hypothesize, there are also fluorescent small molecules/peptides that bind histidine tags quite specifically. Since they are all running through SEC, you don't need to remove unbound probe, it simply elutes much later due to its size.

I think you're right that the micro would work for this! Thanks for bringing it to my attention!

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34. Morten G on April 12, 2014 5:19 PM writes...

This sounds really interesting! I haven't read the paper yet but it seems like a simplification of Fast Parallel Protealysis where thermolysin is mixed with cell lysate and heated. Western blots show you at what temperature the protein of interest unfolds so the protease can digest it.

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