Allosteric Inhibition of Parkinson’s-Linked LRRK2 by Constrained Peptides

Abstract

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multidomain protein with dual kinase and GTPase function that is commonly mutated in both familial and idiopathic Parkinson’s Disease (PD). While dimerization of LRRK2 is commonly detected in PD models, it remains unclear whether inhibition of dimerization can regulate catalytic activity and pathogenesis. Here, we show constrained peptides that are cell-penetrant, bind LRRK2, and inhibit LRRK2 activation by downregulating dimerization. We further show that inhibited dimerization decreases kinase activity and inhibits ROS production and PD-linked apoptosis in primary cortical neurons. While many ATP-competitive LRRK2 inhibitors induce toxicity and mislocalization of the protein in cells, these constrained peptides were found to not affect LRRK2 localization. The ability of these peptides to inhibit pathogenic LRRK2 kinase activity suggests that disruption of dimerization may serve as a new allosteric strategy to downregulate PD-related signaling pathways.

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

“…Over the past years, several LRRK2 kinase inhibitors have been developed, including LRRK2-IN-1, HG-10-102-01, MLi-2, PF-06447475, and DNL201 and DNL151, which are the first two LRRK2 kinase inhibitors in clinical trials. , However, all of these inhibitors are ATP-competitive type 1 kinase inhibitors, which preferably bind to the closed active conformation of LRRK2, leading to dephosphorylation of Ser935 and other biomarker sites, LRRK2 aggregation, and mislocalization to microtubules. , These unintended effects may interfere with vesicle trafficking and could underlie undesirable on-target side effects observed on lungs and kidneys. , Alternative LRRK2-targeting strategies, such as G2019S LRRK2 selective inhibitors, , LRRK2 dimerization inhibitors, GTPase inhibitors, antisense oligonucleotide, type 2 LRRK2 kinase inhibitors, and LRRK2 proteolysis targeting chimeras (PROTACs), − have therefore been proposed and are under active exploration.…”

“…22,23 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 dimerization inhibitors, 28 GTPase inhibitors, antisense oligonucleotide, 29 type 2 LRRK2 kinase inhibitors, 30 and LRRK2 proteolysis targeting chimeras (PROTACs), 31−34 have therefore been proposed and are under active exploration.…”

Structure-Guided Discovery of Aminoquinazolines as Brain-Penetrant and Selective LRRK2 Inhibitors

“…The LRRK2 gene encodes a large (286 kDa) multifunctional protein comprised of GTPase and kinase domains flanked by several protein–protein interacting regions. A major step forward in our understanding of LRRK2 took place over the past year with the publication of three papers on the structures of its soluble, microtubule-associated, and full-length forms. − As with many protein kinases, LRRK2 self-interacts through mechanisms that may regulate enzymatic activity, and evidence would suggest that kinase activity resides with the dimeric state. − Missense mutations within the activation loop such as G2019S (glycine to serine substitution at amino acid 2019) have been found to increase kinase activity (gain of function) and represent the most prevalent genetic risk factor for Parkinson’s disease (PD), − with mutation-driven forms of the disease presenting as clinically indistinguishable from sporadic PD. , Therefore, moderation of LRRK2 kinase activity via design of small-molecule inhibitors, − among other approaches, − has been a central focus in the pursuit of PD-modifying therapies. This endeavor confronts several fundamental challenges, two of which include (1) the exquisite safety profiles required of small-molecule kinase inhibitors for the treatment of non-oncologic diseases , and (2) the well-documented fact that design strategies aimed at optimizing brain permeability align in diametric opposition to the physicochemical properties which typically characterize ATP-competitive kinase inhibitors. , …”

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

“…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 .…”