Three years ago Beat Ernst and his colleagues at the University of Basel described using NMR to identify
two molecules that bind next to one another on the protein MAG. They then used
in situ “click chemistry” to link these together to obtain a more potent
binder. In a recent issue of J. Am. Chem.
Soc. they have taken a similar approach to the protein E-selectin, but without the in situ part.
The selectins are cell adhesion proteins involved in a
variety of biological processes, notably inflammation and tumor metastases.
They bind to carbohydrates on the surface of leukocytes, but the affinities of
any one selectin for a given carbohydrate tends to be low – often only
millimolar. In the current paper, the researchers started with a reasonably
potent modified carbohydrate, compound 3.
The researchers performed an NMR screen of 80 fragments in
which they looked for increased relaxation of protons in the fragments upon
binding to protein. This led to five hits. To determine whether these bound
near compound 3, the researchers modified compound 3 with a “spin-label,” a
moiety that would increase the rate of relaxation of nearby molecules and so
make them detectable (see the previous post for more details). Two of the
fragments appeared to bind near compound 3, and the researchers chose to pursue
compound 4 – the very same fragment they had pursued previously for MAG.
At this point the researchers replaced the spin label with an alkyne (attached via spacers of various lengths) and added azide groups (again, with various spacers) to compound 4 and attempted to perform in situ click chemistry in the presence of the protein, as they had done previously. Nothing happened. Having come this far, they used more conventional conditions (ie, without the protein present) to make a small library of 20 triazoles and tested these for binding, leading to 5 hits with nanomolar activity, such as compound 43.
Given the high affinity of compound 43 for E-selectin, why
didn’t it form in situ? The researchers suggest that:
Given its flat binding
site, E-selectin does not act as an effective supramolecular catalyst for the
alkyne-azide cycloaddition, because even upon simultaneous binding of first-
and second-site ligands their azide- and acetylene-substituted linkers are not
sufficiently preorganized to accelerate the cycloaddition reaction.
This is an example of a false negative from in situ fragment
assembly. We previously wrote about another case in which in situ click
chemistry yielded the less potent of two regioisomers due to trace amounts of
contaminating copper. There is something conceptually beautiful about having a
protein template the formation of its own inhibitor, but how often does it
really work?
Ending on a positive note, the NMR approach described here
is an example of linking without the need for protein structure. You just might
want to click before you assay.
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