16 September 2019

Fragments find flexibility in fascin 1

Protein flexibility can be both an opportunity and a barrier – quite literally, when a solid wall of protein seems to block opportunities for fragment growing. But like secret doorways, protein domains can yawn open to expose tunnels and cavities. An example of this was published earlier this year in Bioorg. Med. Chem. Lett. by Stuart Francis and collaborators at the CRUK Beatson Institute.

The researchers were interested in fascin 1, which increases the invasiveness of multiple cancers by helping pack filamentous actin into bundles important for cell migration. The team began their search for an inhibitor by performing a surface plasmon resonance (SPR) screen of 1050 fragments, generating an impressive 53 hits. Although a number of these were reported to bind to multiple sites on the protein, only one is discussed.

Compound 1 binds between two domains of the protein in a pocket that does not exist in unbound fascin. However, the fact that the pocket completely envelopes the fragment “hampered attempts to develop the series.” Fortunately, the researchers were following the patent literature, and when they characterized compound 2 (not a fragment, and reported by a different group), they discovered that while it binds in the same pocket as compound 1, additional conformational changes occur to accommodate the larger molecule.


Next, the researchers looked for analogs of compound 2 and also performed a virtual screen against the enlarged pocket. Of 110 commercial compounds tested, three gave dissociation constants better than 100 µM, including compound 3. The researchers recognized that compound 3 lacks the halogens found on both the original fragment and compound 2, and by adding these they were able to improve the affinity more than ten-fold. Further optimization ultimately led to BDP-13176, with mid-nanomolar affinity by SPR and ITC as well as activity in a functional assay. Although the molecule has reasonable solubility and stability against liver microsomes, it has low permeability and high efflux.

This is a nice structure-based design story, and while the fragment did provide some information about the binding site, one could argue that the real breakthrough came with determining the binding mode of compound 2. Indeed, without this information, it would have been all too easy to assume that the pocket was not ligandable. This is an important reminder that crystal structures usually only reveal one form of a protein. The system is also a good test case for modelers who want to see how their algorithms perform against a dynamic protein. Breakthroughs are often unexpected, and it is always worth making a few compounds that don’t look like they’ll fit.

09 September 2019

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!

02 September 2019

Fragment vs hematopoietic prostaglandin D2 synthase: a chemical probe

Six years ago we highlighted work out of GlaxoSmithKline and Astex describing some of their efforts to find inhibitors of hematopoietic prostaglandin D2 synthase (H-PGDS), an enzyme implicated in asthma, lupus, and multiple other inflammatory diseases. A recent paper in Bioorg. Med. Chem. by David Deaton and collaborators describes another chemical series from that program.

As noted in the earlier publication, the researchers were graced with 76 crystallographic fragment hits, of which compound 1a was a weak but ligand-efficient member. Several other fragments that bound in the same region contained a methoxy group, and a quick survey of commercially available analogs led to compound 1b, with a nice bump in potency. Replacing the nitrile with an amide (compound 1d) improved activity further.


What do you do when you’ve got an amide? Make lots of them! This was effective, and the researchers show more than 60 analogs leading to low nanomolar inhibitors such as compound 1bg. One problem with amides is that they can be enzymatically cleaved in vivo, but this challenge was surmounted by tweaking the substituents.

The researchers also noted that the compounds are quite electron-rich, potentially leading to phototoxicity, and in fact some of the molecules degraded upon exposure to UV light. Also, methoxy substituents are prone to dealkylation in vivo. The researchers solved both problems by replacing the methyl with a difluoromethyl group, leading to GSK2894631A.

This molecule was put through a battery of tests and found to be orally bioavailable (at least in mice) with good pharmacokinetics. It is selective against related enzymes as well as a larger panel of receptors and transporters. Encouragingly, the compound showed potent activity in a mouse model of acute inflammation. In other words, this looks to be a useful chemical probe to explore the biology of prostaglandin signaling.

This is a nice story on several levels, and it also illustrates an important point that younger researchers and folks in academia sometimes overlook: it can take ages before work done in industry sees the light of day. Indeed, one of the authors on the paper left Astex more than five years ago, so the work described is likely several years older than that. Still, better late than never. Good science is always worth publishing, even if – like another paper we recently highlighted – it happened some time ago.