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.

Poll results: fragment libraries in 2023

Our latest poll on fragment libraries suggests the field is settling into some standard practices. The poll ran from April 9 through May 26. Of the 59 participants, all but one answered all the questions (there was one skip for the last question). This is slightly down from previous years; perhaps people are sick of internet polls? We also don’t know how many organizations the respondents represent; it is possible several people voted from one company or university, which might skew the results. Nonetheless, we think this survey gives a reasonable snapshot of how people construct and maintain fragment libraries.

 

Our first question asked about library size, and the results are similar to when we last asked this question in 2018, with the average library having between 1001 and 2000 fragments.

 

 

Next, we asked about the size of fragments themselves, specifically the minimum and maximum number of non-hydrogen atoms allowed in a fragment. The minimum hasn’t really changed from 2018, averaging 7-8 heavy atoms. However, the fraction of respondents who include the tiniest fragments has doubled (albeit from a low number), perhaps due to increasing interest in MiniFrags and MicroFrags.

 

Unlike in 2018, the maximum size of fragments seems to be bimodal, with some folks drawing the line at 15-16 heavy atoms (consistent with this analysis from Astex) while others allow larger fragments. It will be interesting to see whether this bifurcation represents a true shift, though even fragments with 22 heavy atoms are likely to be under 300 Da, consistent with the rule of three, which is twenty years old this year.

 

 

We then asked about the presence of chiral molecules. There was little change from 2017, with most respondents stating that they have racemic compounds in their library, though there was a slight increase in the number of respondents excluding chiral fragments.

 

 

A new question for this poll asked whether synthetic tractability was considered at the outset of library design. This was a consideration for 85% of people who took the poll; more than a third said they considered progressability for every fragment in the library.

 

 

 

 

 

 

 

 

 

Finally, we asked about library storage conditions. As was the case when we asked this question nine years ago, more than two-fifths of respondents said they store their library at -20 ˚C. However, the fraction of respondents who store their library at room temperature dropped, while those who store their libraries at -80 ˚C increased.

 

 

 

 

 

Although some changes are noticeable over the years, it seems that best practices have been established and widely adopted in fragment library design. What do you think – does anything surprise you?