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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: derekb.lowe@gmail.com Twitter: Dereklowe

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October 5, 2010

Chemical Biology: Plastic Antibodies?

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Posted by Derek

Here's an interesting example of a way that synthetic chemistry is creeping into the provinces of molecular biology. There have been a lot of interesting ideas over the years around the idea of polymers made to recognize other molecules. These appear in the literature as "molecularly imprinted polymers", among other names, and have found some uses, although it's still something of a black art. A group at Cal-Irvine has produced something that might move the field forward significantly, though.

In 2008, they reported that they'd made polymer particles that recognized the bee-sting protein melittin. Several combinations of monomers were looked at, and the best seemed to be a crosslinked copolymer with both acrylic acid and an N-alkylacrylamide (giving you both polar and hydrophobic possibilities). But despite some good binding behavior, there are limits to what these polymers can do. They seem to be selective for melittin, but they can't pull it out of straight water, which is a pretty stringent test. (If you can compete with the hydrogen-bonding network of bulk water that's holding the hydrophilic parts of your target, as opposed to relying on just the hydrophobic interactions with the other parts, you've got something impressive).

Another problem, which is shared by all polymer-recognition ideas, is that the materials you produce aren't very well defined. You're polymerizing a load of monomers in the presence of your target molecule, and they can (and will) link up in all sorts of ways. So there are plenty of different binding sites on the particles that get produced, with all sorts of affinities. How do you sort things out?

Now the Irvine group has extended their idea, and found some clever ways around these problems. The first is to use good old affinity chromatography to clean up the mixed pile of polymer nanoparticles that you get at first. Immobilizing melittin onto agarose beads and running the nanoparticles over them washes out the ones with lousy affinity - they don't hold up on the column. (Still, they had to do this under fairly high-salt conditions, since trying this in plain water didn't allow much of anything to stick at all). Washing the column at this point with plain water releases a load of particles that do a noticeably better job of recognizing melittin in buffer solutions.

The key part is coming up, though. The polymer particles they've made show a temperature-dependent change in structure. At RT, they're collapsed polymer bundles, but in the cold, they tend to open up and swell with solvent. As it happens, that process makes them lose their melittin-recognizing abilities. Incubating the bound nanoparticles in ice-cold water seems to only release the ones that were using their specific melittin-binding sites (as opposed to more nonspecific interactions with the agarose and the like). The particles eluted in the cold turned out to be the best of all: they show single-digit nanomolar affinity even in water! They're only a few per cent of the total, but they're the elite.

Now several questions arise: how general is this technique? That is, is melittin an outlier as a peptide, with structural features that make it easy to recognize? If it's general, then how small can a recognition target be? After all, enzymes and receptors can do well with ridiculously small molecules: can we approach that? It could be that it can't be done with such a simple polymer system - but if more complex ones can also be run through such temperature-transition purification cycles, then all sorts of things might be realized. More questions: What if you do the initial polymerization in weird solvents or mixtures? Can you make receptor-blocking "caps" out of these things if you use overexpressed membranes as the templates? If you can get the particles to the right size, what would happen to them in vivo? There are a lot of possibilities. . .

Comments (15) + TrackBacks (0) | Category: Analytical Chemistry | Chemical Biology | Chemical News | Drug Assays


COMMENTS

1. retread on October 5, 2010 9:43 AM writes...

"At RT, they're collapsed polymer bundles, but in the cold, they tend to open up and swell with solvent." I don't understand why this should happen. Do any of the cognescenti reading this blog? One of the pleasures of being retired, is the ability to admit that you don't understand something.

Permalink to Comment

2. CurryWorks on October 5, 2010 9:52 AM writes...

They take advantage of lower critical solution temperature (LCST) behavior of the polymer matrix.

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3. Wavefunction on October 5, 2010 9:54 AM writes...

@retread: A wild guess, but something akin to cold denaturation of proteins? There is still debate about the exact mechanism but it's believed to involve favorable hydration of non-polar groups inside the protein which would usually be unfavorable at RT. Interestingly, some of the conclusions about cold denaturation have been drawn from studies with N-alkyl amides, probably similar to the polymers in this study.

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4. Hap on October 5, 2010 10:15 AM writes...

At lower T, maybe the loss of entropy of solvent molecules bound to polymer is less (because it's at least linearly dependent on temperature) so that the enthalpy of solvation can more easily compensate for it, leading to greater solvation of the polymer. I don't actually know, though.

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5. Anonymous on October 5, 2010 12:09 PM writes...

This technology appears superfluous now that we have photonic imprints.

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6. lt on October 5, 2010 1:56 PM writes...

Then there's affinity vs selectivity... What else could those purified particles bind?

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7. metalate on October 5, 2010 3:52 PM writes...

Can I make a petty complaint? Figure 2 of this paper (the only important data presented) is entirely illegible, especially the inset graphs, without blowing the thing up to 400%. I pity anyone who tries to read this on paper. Can't JACS enforce some minimum font size standards?

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8. PlatoMolloy on October 5, 2010 5:51 PM writes...

@It,
Selectivity in MIPs is usually an order of magnitude better for a target compound than for any close analogues. Proteins are a bit trickier. Usually only a characteristic sequence is imprinted. This often allows for increased selectivity. Affinity is usually the problem in these cases.

Permalink to Comment

9. metalate on October 5, 2010 6:19 PM writes...

As I re-read the paper, it seems that these are NOT MIPs. i.e., no attempt was made to imprint the polymer NPs with mellitin. They just have a random mixture of NPs, some of which happen to bind mellitin. The yield of high affinity (nM) NPs is said to be "several percent" (rather unscientific sounding). So apparently a random synthesis of polymer NPs gives them several percent of nanomolar binders. Far better than a small-molecule library screen, I would think. Unless I'm missing something...

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10. Donna on October 5, 2010 8:54 PM writes...

A previous study using a MIP against melittin in in mice

http://pubs.acs.org/cen/news/88/i19/8819news4.html

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11. gippgig on October 5, 2010 9:21 PM writes...

Melittin is amphiphilic, meaning that one side of the molecule is hydrophobic while one side is hydrophilic (3D structure JBC 257 6016). This is precisely the kind of physical property that makes it easy for a "clumsy" system to recognize a molecule.

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12. cliffintokyo on October 6, 2010 12:00 AM writes...

Derek: Excellent description of how these polymer particles are working, and the neat technologies used to sift out those with highest affinity.
Might inspire some readers to come up ideas for making other *designer receptors* for biological ligands.
Selectivity also needs to be addressed, as already pointed out (#6).

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13. European Chemist on October 6, 2010 5:50 AM writes...

Care to comment on the Nobel Prize awardees? ;-)

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14. bad wolf on October 6, 2010 7:01 AM writes...

"The Giants win the pennant! The Giants win the pennant!"

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15. jonathan on October 6, 2010 8:04 AM writes...

Therefore the rate of dissolution in these cases becomes slower than if it was short branching, where the interaction between chains is practically Polymer solubility this will also useful to all

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