Fragments vs ketohexokinase (KHK) deliver a chemical probe

Anyone who has paid any attention to health news will be aware of concerns over high fructose corn syrup. Just a few years ago the stuff was ubiquitous. Today, due to consumer backlash, it is less common, though still widely used as a cheap sweetener in foods and beverages.

In humans fructose metabolism, unlike glucose metabolism, is not regulated by feedback inhibition, so the sugar is metabolized preferentially. Overconsumption of fructose has been correlated with all sorts of metabolic disorders, from insulin resistance to obesity. But even if you avoid consuming any fructose, your body can still convert glucose into fructose.

The rate-determining step in fructose metabolism is the enzyme ketohexokinase (KHK). Mice lacking this enzyme are healthy and resistant to metabolic diseases. Could a pill do the same thing? Although previous KHK inhibitors have been reported – one starting from fragments discussed here – these did not seem suitable for in vivo studies, not least because they are considerably less potent on rat KHK than human KHK. In a recent J. Med. Chem. paper, Kim Huard and her colleagues at Pfizer describe a chemical probe for KHK.

The researchers used STD NMR to screen their 2592-fragment library in pools of 4 or 10 compounds, with each at 240 µM. This resulted in a formidable 451 hits, of which 448 were screened in full dose response curves using SPR. Of these, 179 confirmed, and 114 had affinities better than 100 µM. All of the SPR-validated hits were tested in an enzymatic assay, leading to 23 fragments with IC₅₀ values from 46 to 439 µM. All 23 of these were soaked into crystals of KHK, and all of them yielded structures showing them bound in the ATP-binding pocket. (Incidentally, this is a lovely example of a successful screening cascade using multiple orthogonal methods, though it would be interesting to know what the outcome would have been had the researchers jumped directly to the X-ray screen.)

But what do you do with 23 fragment hits, all with decent ligand efficiencies and experimentally determined binding modes? Rather than focusing on a single fragment, the researchers noticed that many shared common features, for instance a central heterocycle surrounded by various lipophilic substituents, as in compounds 4 and 5. Many, such as compound 4, also contained a nitrile that made a hydrogen bond to a conserved water molecule.

Next, the researchers combed the full Pfizer screening library for compounds that merged common elements of the fragment hits. This led to more potent inhibitors, such as compound 9 (which was present in the library as a racemate – make sure to vote in the poll on the right!). Parallel chemistry around analogs of this and another hit led to compound 12. In contrast to previously reported molecules, this compound is equipotent on rat and human KHK. It also has decent pharmacokinetics, is orally bioavailable, and is quite selective against a broad panel of off-targets. Rat experiments revealed that the compound inhibits fructose metabolism in vivo.

This story is a nice illustration of how lots of different crystal structures can enable fragment merging. There is still some way to go – the potency in particular could be improved. Also, there are actually two human isoforms of KHK, and compound 12 hits both equally – which may or may not be desirable. Nonetheless, this chemical probe should help further elucidate KHK biology, and help to address whether the enzyme is druggable, or merely ligandable.