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Making and Measuring Multivalency

Here’s an unusual paper that’s studying receptor behavior on cell surfaces by use of atomic force microscopy. (Here’s the SI file, which is free to access). The authors took the marketed VEGF inhibitor vandetanib (VD6474) and attached it through linkers to the AFM tip, and then scanned around the surface of live human umbilical vein endothelial cells (HUVECs) sitting on a test surface. Using a tip that was basically saturated with ligand, they were able to detect binding as they got closer to the cell surface, and could map where the effect was strongest by repeatedly pulling the probe back up and repositioning it.

That indicated the strongest binding around the periphery of the cells, which was in line with immunostaining results on the locations of the VEGF receptors. Switching to an AFM tip with much lower compound loading and running a similar set of experiments allowed the team to estimate the force involved in pulling a single vandetanib molecule back out of its VEGF binding pocket (about 45 piconewtons, if you’re wondering). They also estimated that there were about 16 receptors within range of the probe tip, on average (an area of about 250 square nanometers), and the maximum separation between VEGF receptors in that region was about 48 angstroms.

With those figures in mind, the group set about making some multivalent forms of vandetanib, shown at right with the drug structure itself in orange. These had various geometries and spacings between the ligands, and each one was studied in a receptor binding assay. The parent compound has an IC50 of about 47 nM in the assay, for reference, and the bivalent one to its right improves to 1.8 nM. The next one (ZD-3) is identical, at 2.2 nM, and the one at the bottom is only a bit better, at 0.9 nM. These improvements are probably just due to the statistical increase in the likelihood of binding, as expected with a polyvalent ligand, but the ZD-4 structure in the middle seems to hit the spacing just right. That one stands out at 0.025 nM (25 picomolar), a 100-fold improvement over what just multivalency seems to provide and a 2000-fold improvement over the patent drug.

Interestingly, the paper goes on to try that compound out in vivo, comparing radiolabeled versions of the parent compound and the tetravalent form in a U87 xenograft mouse model, dosed i.v. Imaging and histology showed that the tumor uptake of the latter was at least 12-fold higher than the parent drug, and considering the pharmacokinetic challenges of a species like that one, that’s pretty good. There are two comparison PET images in the paper at 24-hour time points, and if I wanted to cavalierly extrapolate from them, I’d say that the parent drug’s radiolabel is showing up more in the urine by then compared to the tetravalent dosing, and the latter seems to be piled up more in the liver (as well as definitely showing higher tumor concentration). But that’s speculation – it would be worthwhile to see a full PK workup on the ZD-4 species itself.

There are a lot of things to be tried out, given these results: how much, for example, does the spacing and density of VEGF receptors vary between cell types and under different conditions? How dynamic is that distribution for even a single cell? What happens if you apply this technique to other cell-surface receptors – how general is it? And there’s surely a lot of variation to be tried on the multivalent chemical structures themselves. One thing that’s clear from the history of such hybrid molecules is that the length and chemical character of the spacing elements is often important, and in ways that are not always easy to rationalize. I’m tempted to say that the team here got very lucky if they did only prepare the four varieties shown in the paper, but there’s nothing wrong with luck, either, is there?

16 comments on “Making and Measuring Multivalency”

  1. Imaging guy says:

    “The ligand-functionalized tip was brought into contact with the surface of the HUVECs where the binding of ZD6474/VEGFR took place. The tip was then withdrawn from the surface, pulling ligand out of the binding pocket.”
    I don’t get it. I thought kinase inhibitors for receptor tyrosine kinases (in this case VEGFR) are supposed to enter the cells and bind to the intracellular domain of receptor tyrosine kinases*. Does this mean that the tip of the AFM penetrates the plasma membrane or what? Another question is whether the drug could still enter the cell if they are made multivalent (× 4).
    * FDA approved anti VEGFR2 antibody (Ramucirumab) binds to the extracellular domain of receptor tyrosine kinase.

    1. Derek Lowe says:

      Now that’s a good point. The kinase domain is indeed intracellular. The compound is attached to the AFM tip by a tether, although it’s a bit hard to picture that being long enough to allow it to dip through the membrane and interact with the ATP site. The schematic illustrations in the paper are all cartoons of extracellular binding, and the strings “intra” or “kinase” do not appear anywhere. Worth a query to the author, which I’ll send today. . .

      1. GB says:

        Any reply or further information on this Derek?

  2. Barry says:

    reaching down through the phospholipid bilayer to access the ATP site gives a whole new meaning to “fishing expedition”

  3. Anonymous says:

    Whitesides studied tether length on vancomycin multimers in the 90’s-00s. Tether length was definitely a correlated factor. (No lit access to look up some papers I seem to recall. Maybe later.) There are other examples of tethered multimers, as well, but there is a tendency to remember the more famous names; The Matthew Effect.

    1. Some idiot says:

      Vancomycin is an excellent case in point. The group of the late DH Williams at Cambridge did a lot of work on that group of antibiotics. The short version is that they all dimerise at the surface of the bacteria, and that the dimerisation is critical for activity. The short version is that you lose serious orders of magnitude of activity (through lost cooperativity) when there is no dimerisation. Sort of nature’s take on tethering. Nice work in the paper, though!

    2. Barry says:

      but vancomycin’s target is extracellular; it’s the construction of the wall itself. You’re not asking the umbilicus to span the phospholipid bilayer to access a cytosolic active-site

      1. Some idiot says:

        Oh absolutely, but my point was not intra/extra, but just tethering… Vancomycin is a sort of self-tethered compound (non-covalent) which gains its activity through cooperative interactions.

  4. tlp says:

    >> What happens if you apply this technique to other cell-surface receptors – how general is it?
    Making homo- and heterodimeric GPCR ligands used to be quite popular (see e.g. 6-years old review https://pubs.acs.org/doi/10.1021/jm4004335) especially because their dimerization seems to have functional significance – from subtype selectivity to agonism/antagonism (other cool example from Gmeiner lab: https://www.nature.com/articles/srep33233)
    The tricky part here is to figure out what effects are due to stronger binding and which are due to altering receptors’ oligomerization state.

    1. Barry says:

      at least for many growth factor receptors, altering the receptors’ oligomerization state (well, causing dimerization) is necessary for signal transduction. I.e. this would not be artifactual

      https://www.ncbi.nlm.nih.gov/books/NBK22539/

  5. Inoutinbetween says:

    Cool paper in principle, multi-valent compound data aside, but the intra/extra-cellular issue seems to be a fatal flaw. If they are not penetrating the membrane how can they be measuring binding? If they are penetrating the membrane, what are they in fact measuring…? Who reviewed this?

  6. eub says:

    “allowed the team to estimate the force involved in pulling a single vandetanib molecule back out of its VEGF binding pocket”

    mind = blown

    Fascinating question what they’re doing about the cell membrane though.

  7. Nick K says:

    I hope the authors can resolve the question of intra- vs intercellular interaction. If so, we will have a fascinating bridge between the molecular and macroscopic worlds.

  8. Andy II says:

    Multi-valency vs. affinity story remind me of the important work by Dr. YC Lee at Hopkins in ’80s besides the Whitesides work. He studied carbohydrate (N-linked oligos) and lectins (carbohydrate binding proteins). N-linked oligos are typically multivalent and the distance among the terminal sugars are geometrically optimized to fit their specific receptors. A good summary is in FASEB J. 1992 Oct;6(13):3193-200. He also created artificial multivalent glyco-derivatives and proved that multivalency tremendously increase the binding.

  9. nonspecific says:

    A control with the hinge binding motif blocked couldn’t have hurt

    1. ScientistSailor says:

      Actually, it might have hurt their ability to publish…

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