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Chemical Biology

Controlling Proteins, One by One

Here’s what looks like a very useful method for turning protein function on and off, reported in a new paper in Nature Chemistry. (This PDF link may work for you). The authors, from the MRC Molecular Biology labs at Cambridge (right there on Francis Crick Avenue), have a neat system for optical switching.

Here we introduce genetically directed bioorthogonal ligand tethering (BOLT) and demonstrate selective inhibition (iBOLT) of protein function. In iBOLT, inhibitor–conjugate/target protein pairs are created where the target protein contains a genetically encoded unnatural amino acid with bioorthogonal reactivity and the inhibitor conjugate contains a complementary bioorthogonal group. iBOLT enables the first rapid and specific inhibition of MEK isozymes, and introducing photoisomerizable linkers in the inhibitor conjugate enables reversible, optical regulation of protein activity (photo-BOLT) in live mammalian cells.

This (to my eyes) is an offshoot of the “tethering” approach that Sunesis used to do. (Dan Erlanson, ex-Sunesis, blogs over at Practical Fragments, and may well have some comment on this). What the Cambridge group has done is to engineer a protein (MEK, in their first example) to incorporate a strained cyclooctyne-containing amino acid (as introduced by the Bertozzi lab), to have a “spring-loaded” partner for cycloaddition. After finding a working MEK variant with this in place, they then took a known MEK inhibitor and hung a spacer group off of it, with a tetrazine on the end (the cycloaddition partner). That’s the inverse-electron-demand Diels-Alder bioconjugation system introduced by the Fox lab at Delaware.
So far, this is nicely done (but by now relatively well-worked-out) chemical biology. The authors show that their tagged inhibitor does not work well against native MEK, but that it shows rapid and potent inhibition against the engineered protein. They also make sure that that protein really is covalently labeled, as predicted, and that the tetrazine-containing compound doesn’t wander around doing other things to the proteome (it looks clean). They also tried some interesting variations, changing the core inhibitor structure and also changing the linker length. The tethered SAR followed the known inhibitor SAR pretty well, which makes sense. Switching the inhibitor structure to sunitinib (with an added tetrazine) gave a compound that wasn’t much of an inhibitor of wild-type MEK, but did indeed go after several of the engineered ones. (Interestingly, the profile of it and the other tetrazine-conjugated inhibitor species were different across several of the enzyme variants, for reasons that are not yet clear). And the linker length didn’t affect things much, except the longer linkers were worse (entropic penalty?) I should note that this is in some contrast to what George Whitesides and his group found in a model system a few years ago – they didn’t observe much of a dropoff with longer linking groups.
Now comes the funky part. The group put in a linker with an azobenzene group, because azobenzenes are well known to undergo cis/trans isomerization photochemically. And that double bond switch really changes the position of the group out on the end, as you can easily picture. (Here’s that same trick being worked on some receptor ligands). Hitting it with 360nm light flips it from the usual trans to the cis – you can flip it back with 440nm light, or wait three or four hours for it to naturally isomerize back on its own.
Six of the group’s MEK variants were inhibited by the trans-azobenzene linked compound. One of those also turned out to be switchable via the different wavelengths described above. In live cells, MEK activity could be turned on and off by irradiation, or the normal activity could just be allowed to recover by itself.
The authors then took their sunitinib species and tried it out on LCK. Now sunitinib itself hits a lot of kinase, but LCK isn’t one of its better ones (this paper has it as about 1.2 micromolar, compared to single-digit nanomolar on several others). Picking a similar site to put in the cyclooctyne amino acid in LCK as was used for MEK did indeed allow many of these mutants to be inhibited by that species, while that tetrazine compound did not have any activity on wild-type LCK.
I would like to see a profile of the sunitinib/tetrazine compound against a wide kinase panel – if it really is dead, this points a possible way to affect a variety of proteins that don’t necessarily have good small-molecule ligands for them. Building in the tethering, and that extra kick of affinity that it gives, might point the way to that. The optical switching is another area with a lot of promise, too. Spatiotemporal control of protein function is the only way we’re going to unravel a lot of these networks, and we need all the help we can get to do that. These techniques are not easy – when you read the paper, you see that if you had just given it one reasonable at every step, you probably wouldn’t have gotten anywhere. There are a lot of constructs, a lot of mutants, and a lot of shots on goal. But it’s a very worthy goal, for sure.

5 comments on “Controlling Proteins, One by One”

  1. Anon says:

    Title here seems like a bit of hype. Still need a selective inhibitor to tether, which is the real problem for most of these targets…

  2. Witold says:

    Interestingly, I did something similar in grad school with Caspases. Came up with a redox-activatable Caspase that was tuned to be inactive in serum pH but active in cytosol.

  3. Derek Lowe says:

    #1 – that’s the thing. Sunitinib isn’t a selective inhibitor, but its conjugate with the tetrazine seems to be a selective inhibitor for the mutant LCKs, if I’m reading this correctly.

  4. kinaguy says:

    Maybe I am missing the data that shows sunitinib-tetrazine is selective? They show selectivity for the LCK-mutant over wild-type LCK, but we know sunitinib is a bad inhibitor of LCK.
    The dimethyl amine of sunitinib is solvent-exposed (both in LCK and sunitinib’s actual targets) and sticks out of the kinase. Many other TKIs have been modified in that area (e.g., with BODIPY) and do not lose binding to their target kinase.
    I would bet a lot of money sunitinib-tetrazine hits the same targets sunitinib does.
    The method is neat, but I agree with #1 that a selective inhibitor is needed. Unless you don’t care about all the off-targets, of which sunitinib has many.

  5. milkshake says:

    1) Sutent hits about one third of all known human kinases at or below 100 nanomolar IC50. 2) Substituting a non-basic sidechain inplace of diethylamine will somewhat hurt the potency but not more than one order of magnitude – especially if there is H-bond acceptor (such as in this case). The main role of amine substitution in Sutent is making it soluble (the scaffold is a typical kinase inhibitor brick) and increasing the volume of distribution.

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