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.
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