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.
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