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.

Click here to link

Fragment linking is a topic we’ve discussed several times. One of the more interesting approaches is template-directed synthesis, in which a protein causes two fragments in close proximity to react with one another (see here for example). In a recent issue of Angew. Chem. Int. Ed., Beat Ernst and colleagues at the University of Basel provide a new variant of this theme, without requiring structural information about the protein.

The researchers were interested in a protein called myelin-associated glycoprotein, or MAG, which blocks axonal regrowth. They started with an NMR screen to determine which members of a fragment library bind to MAG as assessed by a phenomenon known as transverse magnetization decay; essentially, small molecules that bind to a protein behave like large molecules in showing a rapid decay in magnetization, so an increased magnetization decay of fragments in the presence of protein suggests binding. A number of fragments were identified as binders, but the site of binding was not determined.

To find molecules that could be linked, the researchers took a known ligand, the sialic acid derivative 1 (see figure – albeit larger than a fragment), and modified this to contain a spin-label. Spin-labels are small moieties that contain an unpaired electron and, just like large molecules, cause an increase in magnetization decay, but only to molecules within close proximity. The two effects are additive, and thus the researchers could determine which fragments bind to the protein in close proximity to the spin-label-containing derivative of compound 1. In fact, the distance dependence is so pronounced that different protons on the fragment can show different effects, thus indicating which portion of the fragment is close to the spin label (see here for a similar approach using ILOE). In this case, the researchers found that a nitroindole fragment (see figure) had its 5-membered ring positioned closer to the spin label than its 6-membered ring.



Knowing the relative positions of the two ligands, the researchers modified them so they could be linked together. They added functional groups with different linker lengths to create several analogs, replacing the spin label with an alkyne and adding an azide to the nitroindole fragment. They then incubated all the analogs together in the presence of the protein. Analysis of the reaction by HPLC-MS after three days at 37 degrees revealed one prominent product, with a mass consistent with compound 7. Two isomers of this product can be formed, with syn and anti configurations around the triazole, and the researchers synthesized both of them. Interestingly, the anti isomer (shown) had a Kd for MAG of 190 nM, while the syn isomer bound roughly 10-fold more weakly.

Although the ligand efficiency of the final compound is low, sugar-based molecules typically have low ligand efficiencies, and maintaining the same efficiency as starting compound 1 is impressive. However, the ligand efficiency of the final molecule is probably lower than the second-site ligand: the researchers don’t report its affinity, but since it would likely need to be 10 mM or better to be detected its ligand efficiency is probably at least 0.23 kcal/mol/atom.

Still, the final product is sufficiently potent that it could make a useful biological probe. Moreover, the approach is notable in not requiring structure of the protein – a rare and attractive feature for fragment linking.