Fragments vs sepiapterin reductase, via 19F NMR

It has been two years since we’ve had a post devoted to fluorine NMR. Though I don't share Teddy’s “fetish” for ¹⁹F-based screening, I do think the technique can be quite powerful, as demonstrated in a recent J. Med. Chem. paper by Jo Alen, Markus Schade, and their colleagues at Grünenthal GmbH.

The researchers were interested in sepiapterin reductase, which is abbreviated as SPR but which I’ll spell out to avoid confusion with surface plasmon resonance. This enzyme performs the last step in the production of tetrahydrobiopterin, an essential cofactor for multiple enzymes, including some that synthesize neurotransmitters and produce nitric oxide. Sepiapterin reductase has been proposed as a target for non-opioid-based pain medications.

The primary assay involved displacement of a fluorine-containing inhibitor that binds in the substrate site of the enzyme; thus, the researchers could use ¹⁹F NMR without requiring fluorinated fragments. A total of 4750 fragments were screened at 250 µM, initially in pools of 12. The 26 hits were then tested in an enzymatic assay, and 21 showed activity better than 75 µM. The best, compound 3, was sub-micromolar.

Crystal structures were obtained for six compounds, including compound 3, and all bound in the substrate pocket as predicted from the original displacement assay. The phenolate of compound 3 makes hydrogen bonds to two critical catalytic residues. Not surprisingly, capping this moiety with a methyl group led to an inactive compound. The researchers made dozens of variants, but aside from compound 26, most of these were disappointingly less active. Compound 26 does show good solubility and permeability, though no cell data are provided, and the phenol will likely be glucuronidated in vivo.

This is a nice story that illustrates a not-infrequent frustration: after identifying the initial nanomolar hit from a small library, the researchers likely thought improving potency still further would be easy. Instead, it took more than 60 analogs just to gain another order of magnitude. That said, 57 nM is nothing to sneeze at. And this situation is certainly preferable to the more common alternative of starting with a weak fragment that remains weak no matter what you do to it!