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Derek Lowe The 2002 Model

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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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September 12, 2013

Ligands From Nothing

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Posted by Derek

Well, nearly nothing. That's the promise of a technique that's been published by the Ernst lab from the University of Basel. They first wrote about this in 2010, in a paper looking for ligands to the myelin-associated glycoprotein (MAG). That doesn't sound much like a traditional drug target, and so it isn't. It's part of a group of immunoglobulin-like lectins, and they bind things like sialic acids and gangliosides, and they don't seem to bind them very tightly, either.

One of these sialic acids was used as their starting point, even though its affinity is only 137 micromolar. They took this structure and hung a spin label off it, with a short chain spacer. The NMR-savvy among you will already see an application of Wolfgang Jahnke's spin-label screening idea (SLAPSTIC) coming. That's based on the effect of an unpaired electron in NMR spectra - it messes with the relaxation time of protons in the vicinity, and this can be used to determine whatever might be nearby. With the right pulse sequence, you can easily detect any protons on any other molecules or residues out to about 15 or 20 Angstroms from the spin label.

Jahnke's group at Novartis attached spin labels to proteins and used these the find ligands by NMR screening. The NMR field has a traditional bias towards bizarre acronyms, which sometimes calls for ignoring a word or two, so SLAPSTIC stands for "Spin Labels Attached to Protein Side chains as a Tool to identify Interacting Compounds". Ernst's team took their cue from yet another NMR ligand-screening idea, the Abbott "SAR by NMR" scheme. That one burst on the scene in 1996, and caused a lot of stir at the time. The idea was that you could use NMR of labeled proteins, with knowledge of their structure, to find sets of ligands at multiple binding sites, then chemically stitch these together to make a much more potent inhibitor. (This was fragment-based drug discovery before anyone was using that phrase).

The theory behind this idea is perfectly sound. It's the practice that turned out to be the hard part. While fragment linking examples have certainly appeared (including Abbott examples), the straight SAR-by-NMR technique has apparently had a very low success rate, despite (I'm told by veterans of other companies) a good deal of time, money, and effort in the late 1990s. Getting NMR-friendly proteins whose structure was worked out, finding multiple ligands at multiple sites, and (especially) getting these fragments linked together productively has not been easy at all.
But Ernst's group has brought the idea back. They did a second-site NMR screen with a library of fragments and their spin-labeled sialic thingie, and found that 5-nitroindole was bound nearby, with the 3-position pointed towards the label. That's an advantage of this idea - you get spatial and structural information without having to label the protein itself, and without having to know anything about its structure. SPR experiments showed that the nitroindole alone had affinity up in the millimolar range.

They then did something that warmed my heart. They linked the fragments by attaching a range of acetylene and azide-containing chains to the appropriate ends of the two molecules and ran a Sharpless-style in situ click reaction. I've always loved that technique, partly because it's also structure-agnostic. In this case, they did a 3x4 mixture of coupling partners, potentially forming 24 triazoles (syn and anti). After three days of incubation with the protein, a new peak showed up in the LC/MS corresponding to a particular combination. They synthesized both possible candidates, and one of them was 2 micromolar, while the other was 190 nanomolar.
That molecule is shown here - the percentages in the figure are magnetization transfer in STD experiments, with the N-acetyl set to 100% as reference. And that tells you that both ends of the molecule are indeed participating in the binding, as that greatly increased affinity would indicate. (Note that the triazole appears to be getting into the act, too). That affinity is worth thinking about - one part of this molecule was over 100 micromolar, and the other was millimolar, but the combination is 190 nanomolar. That sort of effect is why people keep coming back to fragment linking, even though it's been a brutal thing to get to work.

When I read this paper at the time, I thought that it was very nice, and I filed it in my "Break Glass in Case of Emergency" section for interesting and unusual screening techniques. One thing that worried me, as usual, was whether this was the only system this had ever worked on, or ever would. So I was quite happy to see a new paper from the Ernst group this summer, in which they did it again. This time, they found a ligand for E-selectin, another one of these things that you don't expect to ever find a decent small molecule for.

In this case, it's still not what an organic chemist would be likely to call a "decent small molecule", because they started with something akin to sialyl Lewis, which is already a funky tetrasaccharide. Their trisaccharide derivative had roughly 1 micromolar affinity, with the spin label attached. A fragment screen against E-selectrin had already identified several candidates that seemed to bind to the protein, and the best guess what that they probably wouldn't be binding in the carbohydrate recognition region. Doing the second-site screen as before gave them, as fate would have it, 5-nitroindole as the best candidate. (Now my worry is that this technique only works when you run it with 5-nitroindole. . .)

They worked out the relative geometry of binding from the NMR experiments, and set about synthesizing various azide/acetylene combinations. In this case, the in situ Sharpless-style click reactions did not give any measurable products, perhaps because the wide, flat binding site wasn't able to act as much of a catalyst to bring the two compounds together. Making a library of triazoles via the copper-catalyzed route and testing those, though, gave several compounds with affinities between 20x and 50x greater than the starting structure, and with dramatically slower off-rates.

They did try to get rid of the nitro group, recognizing that it's only an invitation to trouble. But the few modifications they tried really lowered the affinity, which tells you that the nitro itself was probably an important component of the second-site binding. That, to me, is argument enough to consider not having those things in your screening collection to start with. It all depends on what you're hoping for - if you just want a ligand to use as a biophysical tool compound, then nitro on, if you so desire. But it's hard to stop there. If it's a good hit, people will want to put it into cells, into animals, into who knows what, and then the heartache will start. If you're thinking about these kinds of assays, you might well be better off not knowing about some functionality that has a very high chance of wasting your time later on. (More on this issue here, here, here, and here). Update: here's more on trying to get rid of nitro groups).

This work, though, is the sort of thing I could read about all day. I'm very interested in ways to produce potent compounds from weak binders, ways to attack difficult low-hit-rate targets, in situ compound formation, and fragment-based methods, so these papers push several of my buttons simultaneously. And who knows, maybe I'll have a chance to do something like this all day at some point. It looks like work well worth taking seriously.

Comments (20) + TrackBacks (0) | Category: Analytical Chemistry | Chemical News | Drug Assays


1. milkshaken on September 12, 2013 10:58 AM writes...

replacing nitroaryl: the replacement bit needs to be electron deficient aryl, probably with H-acceptor group. Cyanopyridine, pyridazine and benzfurazane would be the first to try.

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2. pgwu on September 12, 2013 11:11 AM writes...

To some degree they look like the binding of bi- or tri-antennary glycopeptides, with more flexible bonds.

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3. The Aqueous Layer on September 12, 2013 1:03 PM writes...

...and your chicks for free.

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4. barry on September 12, 2013 1:27 PM writes...

but a med. chemist looks at the result and says unhesitatingly that it came from fragment-based-design. I.e. it's the sort of ligand from which no one knows how to get a drug.
190nM is an achievement to be proud of in a very tough game. And as long as your targets are serum-exposed, a compound that will never get out of the serum compartment (Vss=150mL/Kg) may even be desireable. But what's the market for a "drug" that has to be injected and that flushes out of the body as fast as the kidneys can filter?

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5. rhodium on September 12, 2013 1:42 PM writes...

Whenever you post one of your "Things I won't work with" essays people always seem to want more. I would be happy with more of these.

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6. Basho on September 12, 2013 1:50 PM writes...

@1. milkshaken

Embrace a nitro
Shining as bond acceptor -
Isostere pitfall?

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7. Teddy Z on September 12, 2013 1:53 PM writes...

For me, this is another example of "And then what?"
What academics don't realize is that all of the assumptions in these methods just make them impractical: known ligand with modest affinity is just the first one that you can add a big spin label to.
I can go on and on, but the more fun part is NMR acronyms. A brief recollection of NMR acronyms is in my Link.

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8. milkshaken on September 12, 2013 2:17 PM writes...

@6: its not just H-bond acceptor (because simple amid in its place usually performs poorly): el deficient nitroaromatics are excellent for pi-stacking with tyrosines; charge transfer is favorable if one partner is very el rich and the other el deficient

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9. barry on September 12, 2013 2:28 PM writes...

I've also seen pi-stacks of nitroarenes with electron-rich side chains (Tyr/Trp) in x-ray structures when I can get 'em . I wonder if there's a signature of charge transfer in the spectroscopy for this pi-stack visible even when I can't get the co-crystal?
Like iodo-arenes, nitro-arenes can be fiendishly hard to replace without losing a lot of affinity.

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10. Anonymous on September 12, 2013 2:37 PM writes...

@8. milkshaken

Who said hydrogen?
Pi stack binding through the fog
Attractive forces

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11. milkshaken on September 12, 2013 2:41 PM writes...

there should be shift to higher wavelength in the UV-vis spectra of the nitro compound (many charge-transfer complexes of nitroaromatics are deeply colored) but I do not know how to quantify it.

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12. cynical1 on September 12, 2013 2:50 PM writes...

I think your link to the "new paper" is not to the new Earnst paper which is JACS (2013), 135(26), 9820-9828.

With regard to their earlier work on MAG, did they ever actually show that the new compound with enhanced binding affinity actually led to more potent concomitant reduction of the inhibition of MAG towards axonal regeneration versus the monovalent glycoside alone? They didn't in that paper. That's really the important question isn't it? Otherwise, they just made something that sticks to MAG better but doesn't exert any pharmacological effect that the low potency binders did show. How do you know that having two binding sites didn't perturb the activity of only binding at one site?

Same thing with the new e-selectin paper. It's all binding affinity. So it sticks to e-selectin better. Got it. Show me an experiment that proves that these compounds exert an increased pharmacological effect other than just enhanced binding. It doesn't have to be an in vivo experiment but I'd like to see a pharmcodynamic readout in a cell line. Otherwise, I think I'm going to stay on the fence for now.

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13. Practical Fragments on September 12, 2013 6:10 PM writes...

Practical Fragments also highlighted these papers (click the name for the post). It's nice work, but one thing that struck me was the failure of in situ click chemistry. It was good of the authors to mention this; I wonder how many other false negatives go unreported?

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14. Nick K on September 12, 2013 11:51 PM writes...

Nifedipine contains a nitrophenyl group. Why are nitroaryls always considered to be unacceptable as pharmacophores?

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15. DrSnowboard on September 13, 2013 1:42 AM writes...

Nick K

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16. Nick K on September 13, 2013 4:32 AM writes...

DrSnowboard: Any evidence for the reduction of the nitro group in nifedipine to an aniline? From what I've seen, nifedipine is metabolized almost exclusively oxidatively by CYP.

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17. DrSnowboard on September 13, 2013 6:25 AM writes...

Nick K: No offence, but I don't care about nifedipine. You asked why nitroaryls will get you a wince in a drug discovery setting. LMGTFY....

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18. Nick K on September 13, 2013 6:37 AM writes...

Dr Snowboard: But you do accept that a nitroaryls CAN make valuable drugs? Nifedipine (Adalat) is still widely prescribed in cardiology more than thirty years after its discovery.

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19. Sulphonamide on September 13, 2013 7:46 AM writes...

I'm not quite sure with which side of the argument I agree - but might have a better idea if I have to spend a second batch of 5 years trying to replace an aliphatic nitro in a hit compound. It is by far and away the most potent compound against the target in vitro, works beautifully in vivo in all sorts of indications with unmet medical needs...but, when even academics choose not to patent something that apparently does everything we could possibly ask, it is pretty telling that we know there is a problem. It mightn't be so bad if the chemistry weren't so difficult that each bioisostere is many months work (apart from the carboxylic acid, nothing else took less than a year). Still, if it were easy chemistry, or we knew exactly what to do about a nitro, then I guess there wouldn't be the opportunity or the satisfaction of a worthy challenge.

Alternatively, in 5 years I might well agree that it would indeed have been better for me (not the biologists who have no reason to care if it isn't a drug as the grants keep rolling in) if it had never been in the screening collection.

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20. DrSnowboard on September 14, 2013 6:58 AM writes...

Nick K: Of course there are valuable drugs with nitro groups. I am the last person to say never include x in your molecule because such molecules are ALL useless. However, good luck persuading your development organisation that you have thoroughly eliminated the possibility of liver injury or bladder cancer rearing its ugly head in P3/4. Good luck persuading the VC's expert that that nitro group is essential and non-value crippling in your programme. Pointing to a drug that's been on the market for 30years and saying 'see, it can work' , not very persuasive.

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