Click first, ask questions later

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.