Last year we discussed the application of dynamic combinatorial chemistry (DCC) to fragment linking. The idea is that a protein will shift the equilibrium of a reversible reaction, selecting the tightest binder. Over the past twenty years practitioners of DCC have generated plenty of papers, some quite nice, but I do not recall seeing examples of the technique generating novel and attractive chemical leads. A new paper in Chem. Eur. J. by Beat Ernst and colleagues at the University of Basel explains why it is so difficult.
The researchers were interested in the bacterial protein FimH, which helps microbes colonize the urinary tract by adhering to human proteins that are decorated with mannose. The chemistry the researchers decided to explore for DCC was the reaction of aldehydes with hydrazides to form acylhydrazones. This reaction is slowly reversible at pH 7, allowing exchange between library members to occur, but it can be essentially frozen by raising the pH.
To try to understand every aspect of their system, the researchers focused on a tiny library. Two aldehydes were chosen, one based on mannose, the other based on glucose. Four (quite similar) commercially available hydrazides were purchased.
The researchers made and tested the affinity of each of the eight possible library members using surface plasmon resonance (SPR). The four acylhydrazones based on mannose had dissociation constants (KD) ranging from 0.33 to 0.76 µM, while the mannose aldehyde came in at 3.2 µM. In contrast, the four acylhydrazones based on glucose had KD values between 152 and 735 µM, comparable to the glucose aldehyde itself (194 µM). Since mannose is the natural ligand for FimH while glucose is not, this was expected.
One challenge of DCC is separating library members from the protein for analysis; releasing bound ligands can be particularly challenging if they bind tightly to the protein. A variety of methods were tested, including microfiltration, but this gave “massive alterations in composition.” Various attempts at protein denaturation and precipitation using organic solvents or heat also failed. The fact that this step was so difficult, even for closely related ligands (the difference between mannose and glucose is the stereochemistry around a single hydroxyl group) underscores the challenge of analyzing DCC mixtures.
The problem was finally solved by using a biotinylated version of FimH which could be captured using commercial streptavidin agarose beads.
The most general approach works as follows.
1. Incubate 100 µM FimH protein with library (with each aldehyde and hydrazide at 50-200 µM) at pH 7 for 3 days in the presence of 10 mM aniline, which catalyzes the acylhydrazone exchange.
2. Raise the pH to 8.5 to stop the reaction, add streptavidin agarose, centrifuge, and discard the supernatant containing the unbound molecules.
3. Resuspend the agarose beads containing the protein, add a competitor to release bound ligands, increase the pH to 12 to ensure release, and analyze the product ratios using HPLC.
Although cumbersome, this protocol does work: mannose-derived compounds were enriched relative to glucose-derived compounds, as expected due to their higher affinities, and the most potent compound was enriched over the less potent ones. That said, the robustness of the results were dependent on the ratios of library components.
So will DCC ever be practical? I’m not so sure. But, as the researchers end hopefully but not hypefully, their work “is a contribution to this challenge.”