Fragments vs TLR7/8, starting from HTS

The toll-like receptors TLR7 and TLR8 are closely related proteins that respond to single-stranded RNA, often associated with viral infection, to activate the immune system. While this is useful to ward off disease, when the proteins become overactivated they can lead to autoimmune disorders such as lupus (see here for a recent discussion by Derek Lowe). In a recent ACS Med. Chem. Lett. paper Claudia Betschart and colleagues at Novartis describe advancing a fragment to a potent inhibitor of both proteins.

 

The researchers built a biochemical (specifically, a TR-FRET competition) assay in which they screened 50,000 molecules, each at 20 µM. The campaign yielded some 1500 hits, and this 2020 paper describes the optimization of one of these.

 

The new paper describes the optimization of a completely different molecule, compound 2. This rule-of-three compliant fragment was not only potent in the biochemical assay, it also showed low micromolar cell activity. A crystal structure of the compound bound to TLR8 revealed that it binds at the interface of a homodimer, making hydrogen bonds to both monomers and stabilizing an inactive conformation of the receptor. 

 

A carbon atom in compound 2 was replaced with a nitrogen in compound 3 in the hopes of picking up an additional hydrogen bond, and this led to a ten-fold increase in potency. TLR8 is located in acidic endosomes, and adding a basic piperidine moiety to try to optimize the subcellular localization did in fact improve cellular potency for compound 5. However, basic amines are often associated with hERG binding, which can cause cardiac problems, and this turned out to be the case for this series. This liability was addressed by adding a fluorine to lower the pKa of the amine. Further addition of small moieties to complement the protein led to additional increases in potency, ultimately yielding compound 15.

 

In addition to low nanomolar and even picomolar cellular activity against TLR7 and TLR8, respectively, compound 15 is selective against other TLRs as well as a panel of 100 off-targets. The compound has good DMPK properties in mice and reduced TLR7-dependent interferon-α release in a mouse model.

 

This is a nice medicinal chemistry story focusing on all aspects of optimization, not just potency. Like last month’s Notum and SARM1 posts, it is also another example of a fragment rising to the top of a high-throughput screen. Fragments don't have to be weak.