Fragment screening by photo-CIDNP

Last year we highlighted a talk by Félix Torres in which he described photochemically induced dynamic nuclear polarization (photo-CIDNP) as a rapid, sensitive method for fragment screening. He, Roland Riek, and collaborators at the Swiss Federal Institute of Technology and the Latvian Institute of Organic Synthesis have just published details (open access) in J. Am. Chem. Soc.

 

The discovery of photo-CIDNP dates back to 1967, which is a useful reminder that technology advancement does not necessarily happen rapidly. The physics and mathematics are a bit complicated, but in essence the process requires a photosensitizer molecule that is excited by light and can also form a radical pair with a given ligand molecule. This “hyperpolarized” ligand is easily detectable by NMR. If the ligand is bound to a protein, the ligand is less able to be hyperpolarized, and thus conducting experiments in the presence and absence of protein reveals whether a small molecule binds to a protein of interest.

 

In practice, the researchers used fluorescein as the photosensitizer. To prevent quenching of the excited state by dissolved oxygen, the samples also included glucose (at 2.5 mM) and the enzymes glucose oxidase and catalase. Samples were illuminated with a 450 nm laser whose light was fed into a 600 MHz NMR instrument via an optical fiber.

 

So, what do you get for all this elaborate setup? Speed and sensitivity. The hyperpolarization allows ligands to be detected with a single scan taking just 2 seconds, as opposed to a typical STD NMR experiment which can take tens of minutes. Moreover, compound and protein concentration can be reduced, which both saves on precious materials and reduces the risk of aggregation.

 

But to realize these benefits, the researchers needed to construct a fragment library suitable for photo-CIDNP. Only about 30 molecules had been reported to be suitable for photo-CIDNP, but these included aromatic moieties frequently found in drugs such as indole, phenol, and imidazole rings. The researchers tested over 1300 fragments and selected a set of 212 that were rule-of-three compliant and showed at least five-fold signal-to-noise enhancement in photo-CIDNP.

 

This “NMhare” library was screened against the enzyme PIN1, which has been implicated in cancer and other diseases. Each fragment was screened individually at 50 µM with or without 25 µM PIN1. Although each experiment took only 2 seconds, changing the samples took longer, and the entire set of 424 experiments took 11 hours. The researchers described a flow-based system that could potentially screen 5000 compounds per day.

 

After visual inspection and quality control, twenty hits were identified. Remarkably, all twenty of these confirmed as binders using protein-detected (¹⁵N,¹H-HSQC) NMR, with fragments at 200 µM and isotopically labeled protein at 50 µM. Two of the fragments had been previously reported as binders, and the researchers were able to determine dissociation constants for these in the low millimolar range. Moreover, they were able to demonstrate that photo-CIDNP could detect one of these fragments at just 5 µM in the presence of 2 µM PIN1.

 

Overall this is neat technology, though as it requires some engineering I’m not sure where it falls under the “practical” descriptor of this blog. That said, if it proves sufficiently useful I’m sure vendors will supply off-the-shelf solutions. I look forward to hearing what NMR aficionados have to say.