Impact of Type II LRRK2 inhibitors on signalling and mitophagy

Abstract

Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinson's disease. In this study we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (Ponatinib and GZD-824) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Unexpectedly, we strikingly find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open-closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.

Discovery of XL01126: A Potent, Fast, Cooperative, Selective, Orally Bioavailable, and Blood–Brain Barrier Penetrant PROTAC Degrader of Leucine-Rich Repeat Kinase 2

“…Rab10 5,49 is one of the bona fide substrates of LRRK2, whose phosphorylation status is directly affected by LRRK2 kinase activity and protein level. Phosphorylation of LRRK2 at Ser935 is a well-studied biomarker site used to assess the efficacy of type 1 LRRK2 inhibitors 30 . HG-10-102-01 based PROTACs can potentially dephosphorylate LRRK2 at Ser935 through both LRRK2 degradation and inhibition.…”

“…These unintended effects may interfere with vesicle trafficking and could underlie undesirable on-target side-effects observed on lungs and kidneys 24,25 . Alternative LRRK2 targeting strategies, such as G2019S LRRK2 selective inhibitors 26,27 , LRRK2 Type II inhibitors, LRRK2 dimerization inhibitors 28 , GTPase inhibitors, antisense oligonucleotide 29 , type 2 LRRK2 kinase inhibitors 30 , and LRRK2 proteolysis targeting chimeras (PROTACs) [31][32][33][34] , have therefore been proposed and are under active exploration.…”

Discovery of XL01126: A Potent, Fast, Cooperative, Selective, Oral bioavailable and Blood Brain Barrier Penetrant PROTAC Degrader of Leucine Rich Repeat Kinase 2 (LRRK2)

“…Rab10 5,48 is one of the bona fide substrates of LRRK2, whose phosphorylation status is directly affected by LRRK2 kinase activity and protein level. Phosphorylation of LRRK2 at Ser935 is a well-studied biomarker site used to assess the efficacy of type 1 LRRK2 inhibitors 29 . HG-10-102-01 based PROTACs can potentially dephosphorylate LRRK2 at Ser935 through both LRRK2 degradation and inhibition.…”

“…These unintended effects may interfere with vesicle trafficking and could underlie undesirable on-target side-effects observed on lungs and kidneys 23,24 . Alternative LRRK2 targeting strategies, such as G2019S LRRK2 selective inhibitors 25,26 , LRRK2 dimerization inhibitors 27 , GTPase inhibitors, antisense oligonucleotide 28 , type 2 LRRK2 kinase inhibitors 29 , and LRRK2 proteolysis targeting chimeras (PROTACs) [30][31][32][33] , have therefore been proposed and are under active exploration. As one of the most promising disease-modifying targets, LRRK2 lies at the nexus of an emerging signaling network of high relevance for understanding and developing treatments for PD 34 .…”

Molecular mechanisms defining penetrance of LRRK2-associated Parkinson’s disease

“…These residues have received a lot of attention as potential biomarker sites as they are dephosphorylated in response to LRRK2 inhibition [53], which has been widely used as in vivo pharmacokinetic markers, especially Ser935, for small molecule LRRK2 kinase inhibitor compounds [54][55][56]. It is also clear that phosphorylation of these residues does not correlate with intrinsic LRRK2 kinase activity [29], and emerging structural [57] and LRRK2 kinase inhibitor profiling [58] studies suggest that these biomarker sites report on LRRK2 conformation in an either inactive 'open' or active 'closed' conformation. Furthermore, various pathogenic variants such as p.R1441G, p.Y1699C and p.I2020T suppress the phosphorylation of Ser910 and Ser935 [57,59] likely via mutation-induced conformational changes of LRRK2.…”