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Protein-Protein Compounds – The Flip Side

The topic of protein-protein inhibitor compounds has come up around here several times. It’s the classic “undruggable” target, although that adjective isn’t quite accurate. Let’s leave it at “definitely harder than the usual stuff”; no one could argue with that.
But there’s a flip side to this area that people don’t think about so much. What about a compound that would make two proteins interact more tightly? A conversation with a reader of the site got me to thinking about this, and it turns out that there’s a good review of the concept here, from 2012. The compounds that are known to really do this sort of thing all seem to be natural products, which I don’t suppose should come as a surprise. The most well-worked-out of the group is (as some readers will have guessed) FK506 (tacrolimus). Very few drug research organizations have been brave enough to tackle a mechanism like this, so you’re not going to see many examples of synthetic compounds. How small (and drug-like) a compound can be and still work through a mechanism like this is an open question.
In principle, it shouldn’t be that hard a screen to run – you could imagine an assay where you watch a FRET signal hang around instead of disappearing (once you’re sure that hanging all the FRET thingies off the protein partners didn’t mess with the binding event, of course). You’d probably be able to see this effect by biophysical techniques as well – NMR, SPR (if you could recapitulate the protein-protein interaction with an immobilized partner on a chip), etc. You’d want a lot of structural information – seeing some sort of plausible binding surface that spans the two proteins would help to settle the nerves a bit.
You’d also want some targets, but there are probably more of them than we’re used to thinking about. That’s because we’re don’t tend to think about this mode of action at all, and if you’re not keeping it in mind, you won’t spot opportunities for it. The whole gain-of-function side of the business is hard to work in, for good reasons. I’m not aware of endogenous small molecules that work this way, so it’s not like there are a lot of highly evolved binding pockets waiting for us to fill them. Come to think of it, I’m not aware of endogenous small molecules that work as protein-protein inhibitors, either – those processes seem to get regulated by modifications on the proteins themselves, by local concentration, or by intervention of still other proteins to rearrange binding surfaces. The scarce evolutionary record of this sort of thing might be an accident, or it might be telling us (believably) that this isn’t an easy thing to do.
So I would not necessarily pin all my hopes for next year’s new targets portfolio on one of these, but it would be interesting to screen and see what might turn up. Who wants to be first?
Update: here’s an example from the recent literature for you!

27 comments on “Protein-Protein Compounds – The Flip Side”

  1. ex GSK says:

    Difficult for a small molecule could be easier with a bispecific antibody
    http://www.nature.com/nm/journal/v18/n10/full/nm.2942.html

  2. lcollia says:

    This subject is a very interesting one.
    When we want to do this kind of work, I think it is important to differentiate 2 possibilities:
    1- the compound will be in between the 2 proteins parterns. In this case I can cite cyclosporin A that first bind cyclophilin and then through cyclosporin, the complex will bind calcineurin.
    This type of molecule is difficult to make.
    2- the compound will bind one protein in an alosteric site and it will modify the geometry of the protein that will be able to bind a second protein partner (but not through the small molecule). This is the same way as for an allosteric binder that can block or enhance an activity.
    Lionel

  3. Orthogon says:

    Jeffery Kelly has done something similar by stabilizing tetramers of transthyretin. The drug is approved in Europe and is called tafamidis.

  4. chucky says:

    I would differentiate the topic in a similar but distinct fashion as @1:
    1-The compound induces a PPI that would not otherwise occur. This I believe is reputedly the case for cyclosporin and FK506. Although I have to admit I’m been skeptical of that whole story for awhile now. Are we so sure that tacrolimus doesn’t function simply as a prolyl isomerase inhibitor?
    2-The compound stabilizes an existing PPI, either allosterically or by binding at the interface. I’m not aware of any examples but it appears quite plausible.

  5. IC50 says:

    “In principle, it shouldn’t be that hard a screen to run – you could imagine an assay where you watch a FRET signal hang around instead of disappearing (once you’re sure that hanging all the FRET thingies off the protein partners didn’t mess with the binding event, of course). You’d probably be able to see this effect by biophysical techniques as well – NMR, SPR (if you could recapitulate the protein-protein interaction with an immobilized partner on a chip), etc. You’d want a lot of structural information – seeing some sort of plausible binding surface that spans the two proteins would help to settle the nerves a bit.”
    Only the first technique really has the throughput to do anything other than a focussed library and I definitely wouldn’t want to be the person trying to develop that SPR assay! Although I have no idea how you show that the “FRET thingies” aren’t affecting your assay without a whole battery of tool cpds at your disposal – apart from running your screen and then putting in the effort to deconvolute.
    Would be really interesting to see if it works though.

  6. barry says:

    no a priori reason to stop at protein-protein interactions. There’s a lot of evidence implicating transcription factors in human disease. So we want to modulate a protein-DNA interaction. But Tularik spent a small fortune screening (Yeast two-hybrid?) for small molecules that did this and found no leads in 2million cmpds.
    Maybe not undruggable, but dauntingly close.

  7. JAB says:

    The dominant class of natural products that inhibit protein-protein interactions would be tannins, which are quite non-specific protein binders and non-druggable, though folks have tried (Crofelmer was approved by FDA recently). The FK506 type of things are likely to be rare indeed.

  8. David Borhani says:

    It is a very nice review.
    I’ve called the concept (in presentations I have developed beginning in late 2005) “Block or Lock”. The link is to my 2011 revision, which details several examples of Blocking or Locking compounds, including actual drugs), that act directly (at the interface; my “Type 1”) or allosterically (“Type 2”), in the pharmacopeia. Interestingly, natural products that impact protein-protein interactions tend to favor Lockers, whereas most of us in Pharma have (unwisely?) pursued Blockers.
    Until very recently, the literature largely discussed modulation of protein-protein interactions almost exclusively in terms of Blocking (which is why I initially asked the question, in 2005, how do the PPI modulators we already know about actually work?).

  9. weirdo says:

    “I’m not aware of endogenous small molecules that work this way, so it’s not like there are a lot of highly evolved binding pockets waiting for us to fill them”
    I’ll take nuclear receptors for 500, please, Alec.

  10. Chrispy says:

    The fundamental problem with finding these kinds of compounds is the near-sighted, target-based approach we have to running screens and finding leads. Derek, you allude to it when you mention a FRET-based assay. It seems that we always want to reduce biology to a single, simple event for a screen. Far better would be a phenotypic screen, where any number of protein-protein interactions could affect your output. (Recall that cyclosporine was discovered in a phenotypic screen — putting fungal extracts into animals, of all things.) A combination of Lipinski jingoism and target focus has left this industry where it is now. No wonder biologics will overtake small molecules over the next few years — in biologics it makes sense to be target-based, and inhibiting protein-protein interactions (antibodies) or inducing new protein-protein interactions (BITEs) is easy.

  11. Anon plant guy says:

    If you’re looking for a place to probe protein-protein interactions with small molecules, humans are the wrong model organism. Plants use an endocrine system at least as complex as yours, but instead of protein hormones, they’re very much biased towards small molecules filling the same roles. Medicinal plants are particularly known for replacing signaling pathways served by proteins with ones mediated by secondary metabolites, which makes sense.
    The old school method for exploring this in vivo is feeding radiolabelled compound, then measuring radioactivity and protein composition of coimmunoprecipitation pellets.
    I don’t think you’ll find many protein-protein interactions you can recapitulate in vitro. Chemists imagine protein A holding hands with protein B, and a binding spot between them where their drug goes. That’s not really how proteins interact. Especially in signalling, it looks more like a cluster of a dozen structural, regulatory, and kinase proteins all tangled up together, exchanging partners and making transient low energy interactions.

  12. patentgeek says:

    @3: “Are we so sure that tacrolimus doesn’t function simply as a prolyl isomerase inhibitor?”
    FK506 analogs which potently inhibit FKBP PPIase activity but are truncated in the “effector domain” and do not interact with calcineurin are inactive as immunosuppressants (JACS 1991, 113, 8045). Ditto for rapamycin analogues that are potent PPIase inhibitors but do not interact with TOR. (Biochem. Biophys. Res. Commun. 1993, 192, 1340).

  13. D says:

    This type of mindset is something that our lab heavily focuses on. We have found many small molecules that stabilize proteins and oligomeric interactions of proteins. One method of determining this effect is by using Thermofluor and DSC to measure free energy changes and deltaTm.
    Likewise, we are working on finding peptides that bind to target proteins with a higher affinity then their natively unfolded biological counterpart. In this way, we can make mutants with a higher affinity for their target and look for phenotypic effects.
    I think this concept will become more popular in the near future.

  14. Sideline Chemist says:

    Another avenue in this area is small molecules that help to stabilize protein conformation. CFTR in cystic fibrosis immediately pops to mind. I wish Derek could comment on Vertex’s work in that area, but understand the legalities. When Vertex is able to tell the story of how the current clinical candidates were found, it will be illuminating.

  15. RKN says:

    My work involves identifying candidate disease markers, small (usually n

  16. Boghog says:

    Another advantage of stabilizing protein-protein interactions is that you are much more likely to find high affinity concave binding pockets near these protein-protein interfaces compared to monomeric protein surfaces which tend to be convex. See for example:
    PMID: 22355140
    “The distribution of ligand-binding pockets around protein-protein interfaces” (see especially Figure 1)

  17. Broadie says:

    I saw a couple cool examples of this at the recent High Throughput Chemistry & Chemical Biology GRC (highly recommended conference, by the way). Sam Gerritz from BMS presented this work on Influenza (http://www.pnas.org/content/108/37/15366.full) and MG Finn from Georgia Tech showed a similar story on HCV capsid formation (http://jvi.asm.org/content/80/22/11055.full).

  18. daen says:

    Slightly off topic, but Derek’s mention of FK506 immediately reminded me of Barry Werth’s excellent book, “The Billion Dollar Molecule”. I was handed a copy on day one of working for a small Danish biotech, and from then on, I was hooked.

  19. diverdude says:

    I mentioned this on another thread but…
    I worked on a compound with this general type of mechanism of action in the early nineties and what we found when we got into patients was that what we mostly generated was a hapten.
    Raising antibodies to endogenous proteins is not the wisest thing you can do.

  20. RKN says:

    My work involves identifying candidate disease markers, small (usually n .le. 10) PPI sub-networks dys-regulated between test and control. It occurs to me that locking or blocking specific interactions in these sub-networks, in a relevant cell or animal model, might be a dandy proteomic validation. Thanks for the link to the review.

  21. Ex Intercalator says:

    Camptothecin stabilises the complex of topoisomerase I and DNA, it was discovered through screening of natural products for cytotoxicity. I’m sure there are more examples, there are certainly plenty of natural and unnatural ligands that disrupt protein DNA interactions. Have we not found small molecules that do similar things with protein protein interactions because we haven’t looked or not known how to?

  22. Christophe Verlinde says:

    Here’s some work along these lines. On top of that we made use of the concept of multivalency.
    J Am Chem Soc. 2005 Feb 23;127(7):2044-5.
    Protein heterodimerization through ligand-bridged multivalent pre-organization: enhancing ligand binding toward both protein targets.
    Liu J, Zhang Z, Tan X, Hol WG, Verlinde CL, Fan E.
    Source
    Biomolecular Structure Center, Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
    Abstract
    Structure-based design of a bifunctional ligand for two protein pentamers, cholera toxin B pentamer (CTB) and human serum amyloid P component (SAP), leads to multivalent dimerization of CTB and SAP in solution. This multivalent heterodimerization of proteins significantly enhances the affinity of the bifunctional ligand toward both target proteins.
    PMID: 15713072 [PubMed – indexed for MEDLINE]

  23. jbosch says:

    we are in the process of publishing such an example, thanks for the review link.

  24. TPO receptor agonist says:

    TPOr agonist (and marketed drug) Promacta is another example of a PPI agonist. It was a screening hit (that many companies found) and is thought to stabilize/bias an “on” conformation of the receptor dimer.
    Published here:
    Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation. Matthews EE, Thévenin D, Rogers JM, Gotow L, Lira PD, Reiter LA, Brissette WH, Engelman DM. FASEB J. 2011 Jul;25(7):2234-44. doi: 10.1096/fj.10-178673. Epub 2011 Mar 14.
    Modelling atypical small-molecule mimics of an important stem cell cytokine, thrombopoietin.
    Tarasova A, Winkler DA. ChemMedChem. 2009 Dec;4(12):2002-11. doi: 10.1002/cmdc.200900340.
    Single Pass TM receptor dimers are a great place to be looking for small molecule PPI agonists:
    Orientation-specific signalling by thrombopoietin receptor dimers.
    Staerk J, Defour JP, Pecquet C, Leroy E, Antoine-Poirel H, Brett I, Itaya M, Smith SO, Vainchenker W, Constantinescu SN. EMBO J. 2011 Sep 2;30(21):4398-413. doi: 10.1038/emboj.2011.315.

  25. Michael McG says:

    Don’t the taxanes and vinca alkaloids act by interfering with microtubule (dis)assembly through binding at the NTP site and not being hydrolyzable?

  26. David Borhani says:

    @25 – They bind at a different site. See my pdf (link in update #8)

  27. John says:

    You forgot another, better, more recent example:
    http://www.ncbi.nlm.nih.gov/pubmed/23698361

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