Fragments on Glass

I’m always a fan of new fragment technologies, and Hioryuki Osada and colleagues at RIKEN have just published a very intriguing one which they call a “fragment combination array,” or FCA.

The approach involves immobilizing fragments onto a specially prepared glass slide using a photogenerated carbene reaction; this can be done as an array of microscopic spots. Next, the glass slide is treated with a fluorescently labeled protein and washed. If the protein sticks to the small molecule, it will show up as a fluorescent spot. Appealingly, if the protein of interest is genetically fused to a fluorescent protein, crude cell lysates can be used, simplifying the assay. The researchers previously demonstrated that natural products and drugs could successfully be immobilized to a treated glass chip using this method, and that the molecules retained their ability to bind to protein targets, despite the covalent linkage to the chip.

Of course, binding interactions between drugs and their targets are generally much stronger than between fragments and their targets. Also, because fragments are so small, there is a higher probability that the part of the fragment used to attach to the glass will be critical to but inaccessible for binding. Fortunately, the carbene chemistry is fairly non-selective, inserting into C-H and O-H bonds at random; thus, the likelihood is that at least some of the fragments will bind in a productive fashion. The technique is conceptually similar to the SPR-based methodology used by Graffinity, though to my knowledge Graffinity screens individual fragments as opposed to binary pairs.

But does it actually work? An initial proof-of-concept used FKBP12 ligands, the same ones previously described in the first famous “SAR by NMR” paper. In that example, a low micromolar pipecolinic acid derivative was linked to a high micromolar benzanilide derivative to generate a nanomolar binder to FKBP12. In the current case, the immobilized pipecolinic acid derivative was able to capture fluorescently-labeled FKBP12, while the benzanilide was not. However, co-spotting the two fragments led to a much stronger signal (more fluorescence) than the pipecolinic acid spot alone, suggesting synergy between the two immobilized fragments.

Having shown this, the researchers next turned to the protein carbonic anhydrase II (CAII), which has a predilection for sulfonamides. They created an array from four aromatic sulfonamide-containing fragments (and one negative control) and ten diverse non-sulfonamide-containing fragments. A screen of these 50 different mixtures against fluorescently-labeled CAII revealed a number of hits, and by merging elements of one of the diverse compounds onto a sulfonamide “anchor” fragment, the researchers were able to improve the potency of the sulfonamide from 435 nM to 29 nM.

Of course, there are limits: clearly the technique is not as sensitive as many other fragment-detection methods, as illustrated by the inability to detect benzanilide binding to FKBP12, an interaction with a Kd in the mid to high micromolar range. In fact, both test cases involve protein targets that have been shown to be highly amenable to fragment-based methods, and both start with known fragments with relatively high affinities. Moreover, the covalent immobilization methodology won’t work for all fragments; acetazolamide, a fragment-like high-affinity binder of CAII, didn’t work in this assay, likely due to poor geometry or sterics of the immobilized fragment.

Still, FCA is a neat and potentially very rapid method for finding a second fragment once a first has been identified. It will be fun to watch how the technique evolves.