27 July 2015

Fragments vs DDR1/2: a chemical probe

Our last post was about the utility of chemical probes: small molecules with sufficient potency and selectivity to be able to address specific biological questions. A recent paper in ACS Med. Chem. Lett. by Chris Murray and colleagues at Astex describes an excellent example of finding a new probe for the discoidin domain receptors (DDR1 and DDR2). Previous publications had suggested a role for these receptor tyrosine kinases in certain types of lung cancer, but some of this work had relied on non-selective inhibitors.

The researchers started with a thermal shift assay of their 1500 fragment library against DDR1, followed by crystallographic screening, resulting in around 50 fragment-protein complexes. Not surprisingly most of the fragments bound in the hinge-binding region of the kinase, but around 10 bound in the so-called “back pocket”, with the protein in the inactive DFG-out conformation. As the researchers point out, it is rare to see fragments binding here.

Compound 1 was of particular interest. At first glance, it's not impressive: with 18 heavy atoms and MW > 250 Da, it is on the large end for an Astex fragment, and it had low activity in a biochemical assay. However, its binding mode revealed potential areas for improvements, and the methylene was an unusual feature in a back-pocket binder.

The first step in improving affinity was to add a hinge binder. This was done with the aid of an in-house program called AstexMerge, based on the program BREED, which superimposes a set of ligands. The user chooses a starting molecule, and the program tries to merge that with other molecules while taking account of bond angles and distances. This process led to the design of compound 2, and a few tweaks quickly led to compound 4.

Although compound 4 was potent against DDR1 and 2 and showed good cell-based activity in a phosphorylation assay, it did potently inhibit some other kinases too, most notably c-kit. That problem was fixed with further medicinal chemistry, notably addition of a methyl group to the previously mentioned methylene and replacement of the urea, leading to compound 9, which was potent, selective, and showed good pharmacokinetics in mice.

Although compound 4 was not completely selective, it was more so than some of the previously described molecules, so the researchers tested it in lung cancer cells and found that, despite the fact that it inhibited DDR2 phosphorylation, it showed no effect on cell proliferation. Thus, “the project was halted in favor of more attractive targets.”

Clearly the researchers didn't start out trying to disprove the role of DDR1/2 in squamous cell lung cancer, but their efforts will save others from pursuing the same course. The publication introduces some attractive chemical probes for interrogating the biology of these receptors; hopefully one of these molecules will be added to the Chemical Probes Portal as an alternative to the less-selective probes that have been used in previous studies. Who knows, perhaps someone will find another indication for which DDR1/2 inhibitors are just the ticket.

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