Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson's disease

Abstract

The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinson’s disease patients, but little is known about its regulation and function. A common mutation changing Gly2019 to serine enhances catalytic activity, suggesting that small-molecule inhibitors might have utility in treating Parkinson’s disease. We employed various approaches to explore the substrate-specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower Km and nearly 2-fold higher Vmax than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating threonine over serine. We also observed that several ROCK (Rho kinase) inhibitors such as Y-27632 and H-1152, suppressed LRRK2 with similar potency to which they inhibited ROCK2. In contrast, GSK429286A, a selective ROCK inhibitor, did not significantly inhibit LRRK2. We also identified a mutant LRRK2[A2016T] that was normally active, but resistant to H-1152 and Y-27632, as well as sunitinib, a structurally unrelated multikinase inhibitor that, in contrast with other compounds, suppresses LRRK2, but not ROCK. We have also developed the first sensitive antibody that enables measurement of endogenous LRRK2 protein levels and kinase activity as well as shRNA (short hairpin RNA) methods to reduce LRRK2 expression. Finally, we describe a pharmacological approach to validate whether substrates are phosphorylated by LRRK2 and use this to provide evidence that LRRK2 may not be rate-limiting for the phosphorylation of the proposed substrate moesin. The findings of the present study will aid with the investigation of LRRK2.

Roco kinase structures give insights into the mechanism of Parkinson disease-related leucine-rich-repeat kinase 2 mutations

“…It is speculated that the ATP-binding site is the direct target for many of the inhibitors, but the exact binding mechanism is unknown. We were able to cocrystallize the Roco4 kinase domain with H1152, which originally was identified as a rho-associated protein kinase (ROCK) inhibitor but recently was reported to have nearly the same inhibitory effect on LRRK2 (35). H1152 also was found to inhibit Roco4 kinase activity, and binding is ATP competitive (Fig.…”

“…To date, several relatively nonspecific kinase inhibitors, such as H1152, staurosporine, sunitinib, and GW5074, and more specific LRRK2 inhibitors, such as LRRK2-IN-1, have been identified (34,35). It is speculated that the ATP-binding site is the direct target for many of the inhibitors, but the exact binding mechanism is unknown.…”

Biochemical and molecular features of LRRK2 and its pathophysiological roles in Parkinson's disease

“…An analysis of autophosphorylated LRRK2 by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometery revealed that Ser1403, Thr1404, Thr1410, Thr1491 in the ROC domain, as well as Thr1967 and Thr1969 in the kinase domain, can all be autophosphorylated (60). Kinase assays of LRRK2 using various synthetic peptides showed that the enzyme prefers to phosphorylate Thr over Ser residues, and one study developed a synthetic peptide, Nictide, as a LRRK2 substrate for in vitro kinase assays (61).…”

“…Because LRRK2 mutations are inherited in an dominant manner and the most prevalent mutation, G2019S, increases kinase activity, it will be easier to develop a LRRK2 kinase inhibitor instead of developing a drug to enhance function of the FPD recessive genes. Two recent papers tested LRRK2 kinase activity with several known kinase inhibitors (61,103). Nichols et al tested the effectiveness of three Rho kinase (ROCK) inhibitors and showed that Y-27632 and H-1152, but not GSK429286A, suppressed LRRK2 with similar potency as their activity against ROCK (61).…”

Assaying the Kinase Activity of LRRK2 <em>in vitro</em>

“…Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations 7,8 . Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool [9][10][11] . In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.…”