Fragments vs LRRK2 with the help of a surrogate and a magic methyl

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in Parkinson’s disease and has thus long been targeted by drug hunters. Dozens of inhibitors have been approved for other kinases, making the protein class appear “easy”, but LRRK2 is particularly challenging. First, it is a large multidomain protein that has resisted crystallography. Second, an inhibitor for a chronic disease such as Parkinson’s will need to be highly selective. Finally, the fact that LRRK2 is in the brain means that inhibitors will need to cross the treacherous blood-brain barrier (BBB), whose function is to exclude anything unusual. A recent J. Med. Chem. paper by Douglas Williamson and collaborators at Vernalis and Lundbeck addresses the first two of these issues.

 

The researchers started by screening 1313 fragments (at 200 µM each) against the disease-relevant G2019S mutant of LRRK2. The screen was run using the DiscoveRx KINOMEscan, which relies on displacement of a kinase from an immobilized ligand. Some 80 hits were then triaged in a kinase activity assay.

 

Rather than banging their heads against the wall that has blocked X-ray structures of LRRK2, the researchers turned to a crystallographic surrogate. The readily crystallizable kinase CHK1 has some similarity to LRRK2, and some inhibitors bind to both kinases. Introducing ten mutations into CHK1 around the ATP-binding site led to a LRRK2 surrogate – an approach we’ve previously mentioned.

 

Among the fragment hits were adenine (compound 7) and two closely related molecules. Crystallization of compound 7 with wild-type CHK1 and the LRRK2 surrogate revealed two different binding modes. Similarity-based searches of in-house and literature compounds led to molecules with nanomolar potency, and subsequent optimization led to compound 17 (all molecules were tested against both wild-type LRRK2 as well as the G2019S and had similar affinities).

 

Interestingly, crystal structures of these molecules revealed them to bind more similarly to the complex of compound 7 bound to wild-type CHK1 rather than the surrogate. Adding a methyl group to compound 17 led to compound 18 with a satisfying 100-fold boost in potency: a true magic methyl, as the other enantiomer had slightly worse affinity than compound 17. Crystallography with the surrogate revealed hydrophobic interactions between the methyl and an alanine side chain. It is worth noting that compound 18 is still fragment-sized and yet has high picomolar affinity for LRRK2.

 

Extensive medicinal chemistry followed, ultimately leading to compound 45. Profiling against 468 kinases in the KINOMEscan assay demonstrated it was quite selective, binding only three other targets with dissociation constants less than 1 µM. The compound was active in cells containing either wild-type or G2019S LRRK2. Unfortunately, while the compound showed good oral bioavailability in dogs, it was not orally bioavailable in rats. Moreover, brain to plasma ratios were low in mice, and the molecule was a substrate for the human protein BCRP, a transporter that pumps small molecules across the BBB.

 

Although these liabilities halted further work on the series, this is nonetheless a nice fragment to lead story that highlights the utility of crystallographic surrogates. But the different binding modes for the initial fragment are a reminder that multiple binding modes are not uncommon, and it is best to employ crystallography not just early but often, when you can.