23 August 2009


Dynamic combinatorial chemistry (DCC) has grown alongside of and often intersected with FBDD. In a recent issue of Angewandte Chemie, Jörg Rademann and colleagues at the Leibniz Institute of Molecular Pharmacology describe the latest example.

Put simply, DCC generates new molecules with some desired property by allowing smaller molecules to assemble reversibly under selection pressure. If the selection pressure is binding to a protein target and the molecules undergoing reactions are fragments, DCC can be used for FBDD. As we previously noted, Huc and Lehn published one of the earliest demonstrations of this. DCC has also been used at a few companies, including Astex and Sunesis, and even formed the basis of the (sadly) short-lived Therascope.

Rademann’s approach, "dynamic ligation screening", is based on labeling one fragment with a fluorescent probe and then screening it in a fluorescence polarization assay with other test fragments. If the labeled fragment binds competitively with a test fragment, this implies that the two fragments bind to the same site. However, if the fluorescence polarization signal increases in the presence of the test fragment (indicating increased binding of the labeled fragment), this suggests that the test fragment and the labeled fragment are binding cooperatively.

The researchers applied dynamic ligation screening to the protease caspase-3, a key mediator of apoptosis relevant for many diseases. As their labeled “fragment,” they chose a high-affinity tetrapeptide containing an alpha-ketoaldehyde: the ketone interacts covalently with the catalytic cysteine of the enzyme, while the aldehyde can form imines with amine-containing fragments. Interestingly, this strategy selects for fragments that bind in the S1’ subsite of the enzyme, which has not received as much attention as the tetrapeptide binding sites S1-S4.

A fluorescently labeled version of the tetrapeptide was screened against a library of 7,397 fragments, of which 4,019 contained primary amines. Of these, 78 fragments caused a decrease in the fluorescence polarization signal, suggesting that they compete with the tetrapeptide for binding. These were tested in an enzymatic assay: 21 of them were active at 10 micromolar concentrations, and four had Ki values from 3.1 to 5.5 micromolar; these four molecules have electrophilic carbons, making it likely that they bind to the catalytic cysteine residue.

Of greater interest, 176 fragments were cooperative, increasing the fluorescence polarization (FP) of the labeled tetrapeptide fragment by at least 20%. 50 of these were tested in an enzymatic assay, with the amine shown below emerging as the most potent FP enhancer and a Ki of 120 micromolar alone. A series of experiments guided by mathematical modeling suggested that the protein was templating the formation of an imine bond between the aldehyde of the tetrapeptide and the amine. Moreover, the reduced (amine) version of this conjugate exhibits a very high affinity for caspase-3, with a Ki of 80 picomolar.

Of course, affinity is not everything: with a molecular weight of 767 Da and a clearly peptidic nature, the pharmaceutical properties of this molecule, and even its cell activity, are questionable.

This study is reminiscent of some work we did at Sunesis, using caspase-3 to template the assembly of a non-peptidic inhibitor using Tethering. In that case we built molecules in the S1-S4 pockets, but did not do much work to extend into the S1’ pocket. It would be interesting to see if the fragment Rademann and colleagues discovered also boosts the potency of the molecules we identified.

For dynamic ligation screening to be general it needs to surmount at least two major potential limitations. First, it remains to be seen whether the technique will work with actual fragments, which are likely to have far lower affinities than the 25 nM tetrapeptide used in this study. Second, cooperative binding of the fragments does not translate to synergy in the final molecule: the conjugate has a lower ligand efficiency than either of the fragments, despite the apparent cooperativity of the two fragments binding to the target. This could be because the conjugate contains an amine, whereas the two fragments in solution presumably were linked by an imine; the differences in geometry and chemical nature between these two moieties are profound, and one could imagine that many amine-linked compounds would not be selected as imines, and vice versa.

Still, this is an interesting approach to tackle the long-standing challenge of linking fragments, and it will be fun to watch for new developments.

1 comment:

Lionel said...


I don't think that this example is a real example of an improved fragment linking approach because the aldehyde part is here a peptide and not a fragment. There are two difficulties in the DCC - fragment linking approach :1) find the first fragment that can be modified with an aldehyde group (or an unique amine) and 2) find the second fragment with the DCC technique.

So for me, I think that here it is a VERY GOOD example of how to ADAPT a fluorescence assay to find fragments, but not an example of fragment linking as you say in your conclusion.