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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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September 8, 2009

Right Where You Want Them

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Posted by Derek

Imagine a drug molecule, and imagine it's a really good one. That is, it's made it out of the gut just fine, out into the bloodstream, and it's even slipped in through the membrane of the targeted cells. Now what?

Well, "cells are gels", as Arthur Kornberg used to say, and he was right. There's not a lot of bulk water sloshing around in there. It's all stuck to and sliding around with enzymes, structural proteins, carbohydrates, and the like, and that's what any drug molecule has to be able to do as well. And there's no particular reason for most of them to go anywhere particular inside the cell, once they're inside. They just diffuse around until they hit their targets, to which they stick (which is something they'd better do).

What if things didn't work this way? What if you could micro-inject your drug right into a particular cell compartment, or have it target a particular cell structure, instead of having to mist it all over the place? We now have a good answer to that question, but how much good it's going to do us drug discoverers is another thing entirely.

I'm referring to this paper from JACS, from a group at the University of Tokyo. They're targeting the important signaling enzyme PI3K. That's downstream of a lot of things, and in this case they used the PDGFR receptor in the cells, and a phosphorylated peptide that's a known ligand. To make the peptide go where they wanted, though, they further engineered both the ligand and the cells. The cells got modified by expression of dihydrofolate reductase (DHFR) in their plasma membranes, and the peptide ligand was conjugated to trimethoprim (TMP). TMP has a very strong association with DHFR, so this system was being used as an artificial targeting method. (It's as if the cell had been built up with hook-bearing Velcro on the inside of its plasma membrane, and the PI3K ligand was attached to a strip of the fuzzy side). Then to see what was going on, they also attached a fluorescent ligand to the peptide ligand as well.

Of course, this ligand-TMP-fluorescent fusion beast wasn't the best candidate for getting into a cell on its own, so the team microinjected it. And the results were dramatic. Normally, stimulating the PDGFR receptor in these cells led to downstream signaling in less than one minute. In cells that didn't have the DHFR engineered into their membranes, the fluorescent ligand could be seen diffusing through the whole cytosol, and giving a very weak PDGFR response. But in the cells with the targeting system built in, the ligand immediately seemed to stick to the inside of the plasma membrane, as planned, and a very robust, quick response was seen.

The paper details a number of control experiments that I'm not going into here, and I invite the curious to read the whole thing. I'm convinced, though, that the authors are seeing what they hoped to see. In other words, ligands which aren't worth much when they have to diffuse around on their own can be real tigers when they're dragged directly to their site of action. It makes sense that this would be true, but it's nice to see it demonstrated for real. I'll quote the last paragraph of the paper, though, because that's where I have some misgivings:

In summary, we have demonstrated that it is feasible to rapidly and efficiently activate an endogenous signaling pathway by placing a synthetic ligand at a specific location within a cell. The strategy should be applicable to other endogenous proteins and pathways through the choice of appropriate ligand molecules. More significantly, this proof-of-principle study highlights the importance of controlling the subcellular locales of molecules in the design of new synthetic modulators of intracellular biological events. There might be a number of compounds (not only activators but also inhibitors) that have been dismissed but may acquire potent biological activities when they are endowed with subcellular-targeting functions. Our next challenge is to develop cell-permeable carriers capable of delivering cargo ligands to specifically defined regions or organelles inside cells.

Where they lost me was in pointing out how important this is in designing new compounds. The problem is, these are very artificial, highly engineered cells. Everything's been set up to make them do just what you want them to do. If you don't cause them to express boatloads of DHFR in their membrane, nothing works. So what lessons does this have for a drug discovery guy like me? I'm not targeting cells that have been striped with convenient Velco patches.

And even if I find something endogenous that I can use, I can't make molecules that have to be delivered through the cell membrane by microinjection. You can see from the last sentence, though, that the authors realize that part as well. But that "next challenge" they speak of is more than enough to keep them occupied for the rest of their working lives. These kinds of experiments are important - they teach us a lot about cell biology, and there's sure a lot more of that to be learned. But the cells won't give up their secrets without a fight.

Comments (9) + TrackBacks (0) | Category: Biological News


1. Jose on September 8, 2009 8:50 AM writes...

I can only hope the authors wrote that last paragraph as a matter of course, of record, of necessity, and not out of any real scientific hope in the next century or two..... or maybe they just spit-shining it for a new round of grant applications?

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2. Ty on September 8, 2009 8:54 AM writes...

If the beast was microinjected to the cytosol, how was the targeting system superior to the ligand by itself? It must diffuse to the target, too, anyway. Peptides diffuse better in the cell? [not likely] TMP-DHFR affinity is much higher than the ligand-PI3K binding? [not sufficient] Maybe it's simply because there are so many DHFR molecules on the target "organnelle". If that's the case, there's no way this will work in the real system. Unless the carrier-target machinery utilizes a distinct mechanism other than plain diffusion-based affinity, whatever can make it more efficient than ligand-target binding?

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3. qetzal on September 8, 2009 10:13 AM writes...

I don't have access to the full text. Is this anything more than increasing the local concentration of the ligand near the receptor? Can you get the same effect in cells without DHFR by simply using a higher concentration of the ligand?

Also, did the authors try microinjecting the unmodified ligand? I imagine attaching TMP and a fluorophore could affect the ligand's binding. The fact that they can increase the modified ligand's activity using DHFR is pretty interesting, but is the end result actually better than microinjecting the unmodified ligand?

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4. barry on September 8, 2009 10:15 AM writes...

The result should surprise no one. Raf is effectively inactive as a kinase as long as it's floating in the cytosol, and is activated by recruitment to the plasma membrane by association with Ras. That activation can be mimicked by attaching a prenyl anchor to Ras.

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5. Knut Jacksworthy on September 8, 2009 10:25 AM writes...

There is a huge graveyard of chemical methods out there that have tombstones reading "Worked in principle"

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6. Flash on September 8, 2009 10:36 AM writes...

Peter Wipf and collaborators have reported the mitochondrial targeting of selective electron scavengers and inhibitors of nitric oxide synthase to the mitochondria using truncated fragments of the cell penetrating peptide gramicidin S.

J. Am. Chem. Soc. 2005, 127, 12460-12461

Org. Biomol. Chem. 2007, 5, 307-309

No microinjection, no engineered cells....

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7. Morten G on September 8, 2009 2:01 PM writes...

What if you were targeting two enzymes in the same metabolic pathway? Two proteins in the same metabolic pathway are usually located close to each other. Two nanomolar inhibitors linked together with something like a PEG?
Linking up two bits targeting the same protein has been a resounding failure as far as I know but I haven't really heard of anyone doing it for different proteins. Except for the article Derek wrote about of course and they weren't trying to inhibit DHFR. Of course a linked thingie is probably never getting from the gut to the blood or from the blood into the cell but it would probably be easier to get through the FDA than two compounds in one pill.

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8. weirdo on September 8, 2009 4:34 PM writes...

What? What? "Linked thingies will never work"? Fah! You've clearly never heard of "Advanced Medicine"! Or, whatever they're called now. Theravance, maybe? ("to more accurately reflect our Mission Statement").

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9. emjeff on September 9, 2009 9:01 AM writes...

Why is any of this surprising? You rig a cell up with the genetic machinery to respond to your molecule, then you introduce it into the cell in a highly artificial way , and shockingly, you get a response. In other news, the sun is rising tomorrow...

I'm with Derek on this one, this does nothing for those of us in the real world, trying to get molecules to go where we want them to go.

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