Impact of Type II LRRK2 inhibitors on signaling 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 (GZD-824, Rebastinib and Ponatinib) 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. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. Unexpectedly, we 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. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.

A mitochondrial blood-based patient stratification candidate biomarker for Parkinson’s disease

“…The latter has been confirmed in peripheral blood from heterozygous LRRK2 -R1441G mutation carriers with and without PD ( 47 ). Phosphorylation of serine 935 of LRRK2 (pSer935-LRRK2) has historically been an important biomarker site and while it is not a direct autophosphorylation site, does not correlate with LRRK2 kinase activity, it dephosphorylates in response to Type I but not with Type II small molecule LRRK2 inhibitors and likely informs on the confirmation of the LRRK2 kinase domain ( 25, 48 ). We investigated pThr73-Rab10 and pSer935-LRRK2 levels in PBMCs from the MJFF FBN cohort with largely the same subjects that were analyzed for mtDNA damage (Table S6, Figure 5).…”

Impact of 100 LRRK2 variants linked to Parkinson’s Disease on kinase activity and microtubule binding

“…Nine variants [ROC (N1437H, R1441G/C/S/H, A1442P) and COR B (Y1699C, S1761R, L1795F)], increased Ser1292 autophosphorylation 2- to 4-fold (Fig 2A, Fig 3A, D, SFig 1). Previous work revealed that variants that activate LRRK2 kinase activity such as ROC (R1441G/C) and COR B (Y1699C) suppressed LRRK2 biomarker phosphorylation, likely by promoting the closed, active conformation of the LRRK2 kinase domain [25–27]. Consistent with this, 10 activating variants [ROC (N1347H, R1441G/H/S, A1442P, V1447M), COR B (Y1699C, L1795F), kinase (I2020T) and WD40 (G2385R)], displayed >2-fold reduction in phosphorylation of all biomarker sites (Fig 2B, Fig 3A, E, SFig 1).…”

“…This has been proposed to disrupt vesicle trafficking by causing a "roadblock" for microtubule-based motors [6,9]. The closed, active conformation of LRRK2 also leads to the dephosphorylation of a cluster of phosphorylation sites (Ser910, Ser935, Ser955 and Ser973) located between the Hinge helix and LRR domain through an unknown mechanism [25][26][27]. Certain pathogenic mutations such as G2019S (located within the kinase domain) promote autophosphorylation of LRRK2 at Ser1292 [28].…”

LRRK2 mutant knock-in mouse models: therapeutic relevance in Parkinson's disease

“…Type II kinase inhibitors inhibit LRRK2 kinase activity while maintaining an open LRRK2 conformation, therefore complementing the limitation of type I inhibitors. Recently, several type II kinase inhibitors, such as GZD-824 [ 190 ] and Rebastinib [ 191 ], were found to inhibit LRRK2 kinase activity, as they suppressed Rab10 phosphorylation while preventing aberrant LRRK2 binding to microtubule filaments [ 193 ]. However, both GZD-824 and Rebastinib were initially developed for treatments of leukemia [ 194 ], and they show poor selectivity for LRRK2 [ 193 ].…”

“…Recently, several type II kinase inhibitors, such as GZD-824 [ 190 ] and Rebastinib [ 191 ], were found to inhibit LRRK2 kinase activity, as they suppressed Rab10 phosphorylation while preventing aberrant LRRK2 binding to microtubule filaments [ 193 ]. However, both GZD-824 and Rebastinib were initially developed for treatments of leukemia [ 194 ], and they show poor selectivity for LRRK2 [ 193 ]. Therefore, development of novel type II inhibitors with high selectivity towards LRRK2 remains a big challenge for advancement of LRRK2 kinase inhibitors in PD therapeutics.…”