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DBL%20Hendrix%20small.png College chemistry, 1983

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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March 24, 2009

Grabbing Onto A Protein's Surface

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Posted by Derek

I’ve written here before about the "click" triazole chemistry that Barry Sharpless’s group has pioneered out at Scripps. This reaction has been finding a lot of uses over the last few years (try this category for a few, and look for the word "click"). One of the facets I find most interesting is the way that they’ve been able to use this Huisgen acetylene/azide cycloaddition reaction to form inhibitors of several enzymes in situ, just by combining suitable coupling partners in the presence of the protein. Normally you have to heat that reaction up quite a bit to get it to go, but when the two reactants are forced into proximity inside the protein, the rate speeds up enough to detect a product.

Note that I said “inside the protein”. My mental picture of these things has involved binding-site cavities where the compounds are pretty well tied down. But a new paper from Jim Heath’s group at Cal Tech, collaborating with Sharpless and his team, demonstrates something new. They’re now getting this reaction to work out on protein surfaces, and in the process making what are basically artificial antibody-type binding agents.

To start with, they prepared a large library of hexapeptides out of the unnatural D-amino acids, in a one-bead-one-compound format. (Heath’s group has been working in this area for a while, and has experience dealing with these - see this PDF presentation for an overview of their research). Each peptide had an acetylene-containing amino acid at one end, for later use. They exposed these to a protein target: carbonic anhydrase II, the friend of every chemist who’s trying to make proteins do unusual things. The oligopeptide that showed the best binding to the protein’s surface was then incubated with the target CA II protein and another library of diverse hexapeptides. These had azide-containing amino acids at both ends, and the hope was that some of these would come close enough, in the presence of the protein, to react with the anchor acetylene peptide.

Startlingly, this actually worked. A few of the azide oligopeptides did do the click triazole-forming reaction. And the ones that worked all had related sequences, strongly suggesting that this was no fluke. What impresses me here is that (1) these things were lying on top of the protein, picking up what interactions they could, not buried inside a more restrictive binding site, and (2) the click reaction worked even though the binding constants of the two partners must not have been all the impressive. The original acetylene hexapeptide, in fact, bound at only 500 micromolar, and the other azide-containing hexapeptides that reacted with them were surely in the same ballpark.

The combined beast, though, (hexapeptide-triazole-hexapeptide) was a 3 micromolar compound. And then they took the thing through another round of the same process, decorating the end with a reactive acetylene and exposing it to the same azide oligopeptide library in the presence of the carbonic anhydrase target. The process worked again, generating a new three-oligopeptide structure which now showed 50 nanomolar binding. This increase in affinity over the whole process is impressive, but it’s just what you’d expect as you start combining pieces that have some affinity on their own. Importantly, when they made a library on beads by coupling the whole list of azide-containing hexapeptides with the biligand (through the now-standard copper-catalyzed reaction), the target CA II protein picked out the same sequences that were generated by the in situ experiment.

So what you have, in the end, is a short protein-like thing (actually three small peptides held together by triazole linkers) that has been specifically raised to bind a protein target – thus the comparison to antibodies above. What we don't know yet, of course, is just how this beast is binding to the carbonic anhydrase protein. It would appear to be stretched across some non-functional surface, though, because the triligand didn't seem to interfere with the enzyme's activity once it was bound. I'd be very interested in seeing if an X-ray structure could be generated for the triligand complex or any of the others. Heath's group is now apparently trying to generate such agents for other proteins and to develop assays based on them. I look forward to seeing how general the technique is.

This result makes a person wonder if the whole in situ triazole reaction could be used to generate inhibitors of protein-protein interactions. Doing that with small molecules is quite a bit different than doing it with hexapeptide chains, of course, but there may well be some hope. And there's another paper I need to talk about that bears on the topic; I'll bring that one up shortly. . .

Comments (7) + TrackBacks (0) | Category: Biological News | Chemical News


1. lazybratsche on March 24, 2009 9:17 AM writes...

When can I order a custom "synthetic antibody" for my westerns? I bet someday, this could be a lot faster and cheaper than bleeding some poor rabbit that's been injected with your protein of choice. Here, binding to some relatively unimportant bit of the protein surface could even be a feature, instead of a bug.

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2. cientifico on March 24, 2009 9:47 AM writes...

A target-guided synthesis approach (the umbrella term covering the triazole reaction and others) has been tested with protein-protein interactions, but using N-acylsulfonamide formation from thio acids and sulfonyl azides rather than triazole formation. Manetsch used this to generate a compound with a nanomolar IC50 for Bcl-xL (DOI: 10.1021/ja802683u), but it's an old Abbott lead compound.

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3. Derek Lowe on March 24, 2009 10:32 AM writes...

Bingo! That Manetsch paper is the one that I'm referring to in the last paragraph - a post on it is coming up shortly.

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4. Ben on March 24, 2009 10:56 AM writes...

Cool find -- it reminds me a lot of Sunesis's disulfide tethering system.

Derek, do you have a sense of how "drug-like" the resultant compounds are? It seems like these compounds clearly fail Lipinski's rules (although what drug today doesn't? :-D), but throwing massive unoptimized peptide-like fragments and expecting the new dissociation constants to somehow be a combination of the old ones doesn't strike me as a very promising drug discovery route...

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5. Practical Fragments on March 24, 2009 11:12 AM writes...

The Manetsch paper was also a nice illustration of fragment-based ligand discovery. Practical Fragments did a blog posting on it (linked) a few months back.

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6. Harry on March 25, 2009 6:43 AM writes...

Got to hear Sharpless talk about this click chemistry at Informex a few years back. Very interesting talk, although he tended to go so quickly that I was a few slides back trying to keep up. Probably just me.

Seems to me that this has potential anyhow. Hopefully it won't get oversold like combinatorial chemistry, genomics, etc., etc.

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7. Anonymous on March 29, 2009 8:03 AM writes...

Great post and good link.

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