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Start Small, Start Right

There’s an excellent paper in the most recent issue of Chemistry and Biology that illustrates some of what fragment-based drug discovery is all about. The authors (the van Aalten group at Dundee) are looking at a known inhibitor of the enzyme chitinase, a natural product called argifin. It’s an odd-looking thing – five amino acids bonded together into a ring, with one of them (an arginine) further functionalized with a urea into a sort of side-chain tail. It’s about a 27 nM inhibitor of the enzyme.
(For the non-chemists, that number is a binding affinity, a measure of what concentration of the compound is needed to shut down the enzyme. The lower, the better, other things being equal. Most drugs are down in the nanomolar range – below that are the ulta-potent picomolar and femtomolar ranges, where few compounds venture. And above that, once you get up to 1000 nanomolar, is micromolar, and then 1000 micromolar is one millimolar. By traditional med-chem standards, single-digit nanomolar = good, double-digit nanomolar = not bad, triple-digit nanomolar or low micromolar = starting point to make something better, high micromolar = ignore, and millimolar = can do better with stuff off the bottom of your shoe.
What the authors did was break this argifin beast up, piece by piece, measuring what that did to the chitinase affinity. And each time they were able to get an X-ray structure of the truncated versions, which turned out to be a key part of the story. Taking one amino acid out of the ring (and thus breaking it open) lowered the binding by about 200-fold – but you wouldn’t have guessed that from the X-ray structure. It looks to be fitting into the enzyme in almost exactly the same way as the parent.
And that brings up a good point about X-ray crystal structures. You can’t really tell how well something binds by looking at one. For one thing, it can be hard to see how favorable the various visible interactions might actually be. And for another, you don’t get any information at all about what the compound had to pay, energetically, to get there.
In the broken argifin case, a lot of the affinity loss can probably be put down to entropy: the molecule now has a lot more freedom of movement, which has to be overcome in order to bind in the right spot. The cyclic natural product, on the other hand, was already pretty much there. This fits in with the classic med-chem trick of tying back side chains and cyclizing structures. Often you’ll kill activity completely by doing that (because you narrowed down on the wrong shape for the final molecule), but when you hit, you hit big.
The structure was chopped down further. Losing another amino acid only hurt the activity a bit more, and losing still another one gave a dipeptide that was still only about three times less potent than the first cut-down compound. Slicing that down to a monopeptide, basically just a well-decorated arginine, sent the activity down another sixfold or so – but by now we’re up to about 80 micromolar, which most medicinal chemists would regard as the amount of activity you could get by testing the lint in your pocket.
But they went further, making just the little dimethylguanylurea that’s hanging off the far end. That thing is around 500 micromolar, a level of potency that would normally get you laughed at. But wait. . .they have the X-ray structures all along the way, and what becomes clear is that this guanylurea piece is binding to the same site on the protein, in the same manner, all the way down. So if you’re wondering if you can get an X-ray structure of some 500 micromolar dust bunny, the answer is that you sure can, if it has a defined binding site.
And the value of these various derivatives almost completely inverts if you look at them from a binding efficiency standpoint. (One common way to measure that is to take the minus log of the binding constant and divide by the molecular weight in kilodaltons). That’s a “bang for the buck” index, a test of how much affinity you’re getting for the weight of your molecule. As it turns out, argifin – 27 nanomolar though it be – isn’t that efficient a binder, because it weighs a hefty 676. The binding efficiency index comes out to just under 12, which is nothing to get revved up about. The truncated analogs, for the most part, aren’t much better, ranging from 9 to 15.
But that guanylurea piece is another story. It doesn’t bind very tightly, but it bats way above its scrawny size, with a BEI of nearly 28. That’s much more impressive. If the whole argifin molecule bound that efficiently, it would be down in the ten-to-the-minus nineteenth range, and I don’t even know the name of that order of magnitude. If you wanted to make a more reasonably sized molecule, and you should, a compound of MW 400 would be about ten femtomolar with a binding efficiency like that. There’s plenty of room to do better than argifin.
So the thing to do, clearly, is to start from the guanylurea and build out, checking the binding efficiency along the way to make sure that you’re getting the most out of your additions. And that is exactly the point of fragment-based drug discovery. You can do it this way, cutting down a larger molecule to find what parts of it are worth the most, or you can screen to find small fragments which, though not very potent in the absolute sense, bind very efficiently. Either way, you take that small, efficient piece as your anchor and work from there. And either way, some sort of structural read on your compounds (X-ray or NMR) is very useful. That’ll give you confidence that your important binding piece really is acting the same way as you go forward, and give you some clues about where to build out in the next round of analogs.
This particular story may be about as good an illustration as one could possibly find – here’s hoping that there are more that can work out this way. Congratulations to van Aalten and his co-workers at Dundee and Bath for one of the best papers I’ve read in quite a while.

12 comments on “Start Small, Start Right”

  1. MTK says:

    10 to the minus 19th would be in the 100’s of zeptomoles.
    femto = minus 15
    atto = minus 18
    zepto = minus 21
    yocto = minus 24
    I don’t know if there are prefixes below that.

  2. SP says:

    You also missed picomolar- below nanomolar is picomolar then femtomolar then attomolar. I don’t think things go below yocto because you’re talking about less than one discrete molecule.

  3. Sili says:

    Stuff like this makes me regret – once again – that I never learnt anything about practical protein crystallography.
    Doesn’t make it any better that I never even got my degree in small molecule crystallography – not many of those jobs around.

  4. Derek Lowe says:

    Fixed the picomolar/femtomolar thing – that’s what I get for writing this stuff on the train when I’m not completely awake!

  5. Lowly_Modeler says:

    I am wondering, how expensive is it to do a study like this?

  6. David Young says:

    In some ways, it stands to reason that a more potent drug, one where the dose in nanomolar concentration is sufficient to inhibit an enzyme, or kill a cancer cell is better than one in the millimolar range. After all, a drug that fits the groove in the enzyme so effectivelty that it takes only low nanomolar concentrations to work would seem to be less likely to inhibit some other vital process whether that be a cardiac pump, renal tubule, nerve axon or liver cell.
    But for some drug classes, it is the therapeutic ratio that is most important. I have wondered if medical science has occasionally missed the best drug, if, perhaps the best drug in a class is one that requires a slightly higher dose to effect that anticancer action but at the same time has less side effects. Hard to know. One can’t exactly test all 350 analogues in human studies. We make our best estimate as to the best drug and go with it.
    David

  7. weirdo says:

    Um, it took a study like this to demonstrate the guanylurea was the key binding anchor?

  8. streptorsay says:

    Interesting post, as usual, but I think that some “argifin” became “arginin”.

  9. DLIB says:

    That Med-Chem trick has consequences down the line.
    Much easier to develop resistance to pre-rigidified compounds ( that minimize entropy loss on binding ). If only you guys actually used thermodynamic data when you optimize — you’d know why things happen.

  10. Morten says:

    @ #9 DLIB
    So there are no pharmas that use ITC at all? Wow.

  11. DLIB says:

    @ Morten
    Oh, they do…just too late and too little ( mostly due to instrument issues — material quantity ). I just gave a talk at one on this very issue. I think this type of data needs to be utilized before it even gets into the hands of med chem. There are some technical issues ( that IMHO 😉 I’ve a good idea how to surrmount. One of the problem I think is one of education. These systems are rarely introduced in undergrad O-chem / Biochem labs. And rarely in the labs of O-chem grad students too. Mass Spec/NMR/IR/HPLC/GC…all very familiar techniques and bread and butter instruments. People tend to gravitate to techniques they know and are comfortable with. If they knew what they were missing, I think there’d be more pressure to try to get systems in that’d do the trick ( using much less material ) . Just an opinion.

  12. Shane says:

    Perhaps the small guanylurea has a high binding efficiency because it is at the bottom of a deep cleft with interactions all around it? Very low affinity ligands tend to look like this (like biotin in avidin). So each enzyme architecture would have a limit to how low affinity can be as the ligand is built up with increasing molecular mass. As the ligand gets bigger the deep clefts run out and you are left with less sticky flat surfaces.

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