LRRK2 Kinase activity: Tergetting the kinase

The unambiguous identification of a protein included in the so-called druggable genome clearly linked with PD susceptibility provides the opportunity to exploit existing technology to move beneficial molecules expediently to the clinic.
For example, large panels of active recombinant protein kinases are now commercially available to help define the specificity of kinase inhibitors early in the development process. Protein kinase inhibitors have proven efficacy in the
treatment of human disease since the successful application of the first approved kinase inhibitor trastuzumab in cancer therapies. After the approval and successful implementation of the small-molecule imatinib kinase inhibitor spurred the
formation of many kinase inhibitor discovery programs that are now a ubiquitous part of the modern pharmaceutical industry. However, protein kinases are far from ideal targets because problems with specificity plague the safety record of
inhibitory compounds. Additional problems such as loss of sensitivity of the drug due to acquired mutations in cancer cell targets would presumably not present an issue for the treatment of PD. However, an LRRK2 inhibitor may need
to be administered for the remainder of the patient’s life, requiring
a difficult-to-achieve level of safety from a potential inhibitory compound in the more vulnerable group of older individuals with PD. In addition, compounds would have to cross the blood–brain barrier freely to target the cells of interest.

The unique biology of the LRRK2 protein presents both an opportunity for specificity and unique challenges in identifying possible LRRK2 kinase inhibitors. Classic
protein kinase inhibitors might be grouped together as ATP-competitive inhibitors and irreversible inhibitors. The ATP-binding pocket within a protein kinase is an ideal target for compounds that possess drug-like properties, although ATP-binding pockets tend to encode some of the highest sequence homology found between different protein kinases. Residues critical to the formation of the ATP-binding pocket are conserved between kinases but not necessarily near in amino acid sequence. The majority of kinases, including LRRK2, have not been described on a structural level.
Description of the structure of the LRRK2 ATP-binding pocket and comparison with known ATP-binding pockets of other protein kinases should help shed light on whether ATP competitive compounds will be feasible for an LRRK2-based therapy. The activation loop of protein kinases, almost always defined as the sequence lying between the DFG…APE canonic sequence motif, have been used as targets for small
molecules because the activation loop is critical to kinase activity. The p38 protein kinase inhibitor BIRB796 causes a switch from a ‘‘DFG-in’’ conformation to a ‘‘DFG-out’’ conformation, leading to a steric clash with the phosphate groups
of ATP. LRRK2 possess a unique activation-loop sequence ‘‘DYG,’’ distinct from the nearly ubiquitous ‘‘DFG’’ found in protein kinase activation loops. The PD-causing
G2019S mutation further disrupts this motif to ‘‘DYS,’’ and proves the importance of these particular residues for LRRK2 kinase activity. Theoretically, a small molecule might exist that possesses activity similar to that of BIRB796 by taking
advantage of the unique structure of the LRRK2 activation loop in blocking kinase activation in a highly specific manner. Likewise, a small molecule could preferentially interact with the DYS motif in patients that carry the G2019S mutation for customized therapy, in case inhibition of LRRK2 as a whole causes intolerable side effects in humans. LRRK2 protein resides as membrane-associated and
freely soluble protein in the cytosol so that LRRK2 ATP pocket binding compounds must compete with intracellular ATP concentrations to 10mM to achieve inhibition. In addition to ATP-competitive inhibitors, irreversible inhibitors also represent a viable option for a LRRK2 kinase inhibitor, but the usual concerns of specificity and safety with irreversible inhibitors limit desirability. The safety of inhibiting
LRRK2 kinase activity and potentially LRRK1 kinase activity in humans can obviously not be fully described without highly selective and potent inhibitory molecules. In limiting undesirable effects due to the loss of LRRK activity, the available target-validation data and human genetic discoveries suggest that the LRRK2 kinase may not have to be fully inactivated to provide neuroprotection in PD, because the most common pathogenic mutations induce a relatively mild upregulation (around twofold in vitro) of kinase activity. Relevant model systems will prove invaluable in
this regard.
If the relatively unique structure of the LRRK2 kinase domain suggests that specific small-molecule inhibitors might exist, the numerous potential mechanisms for disruption of LRRK2 kinase activity through nonclassic inhibition offer another level of opportunity. In vitro data suggest that disruption of GTP-binding or nucleotide exchange within the LRRK2 GTPase domain would necessarily disrupt kinase
activity. Likewise, allosteric modulators that block a necessary conformational state, such as protein dimerization, could potentially inhibit LRRK2 kinase activity in a highly specific way (78). Substrate-competitive inhibitors would rely on the discovery of robust LRRK2 kinase targets in cells. As yet, even the sites for LRRK2 autophosphorylation have not been mapped, because of the notoriously low activity
of LRRK2 in cells and in vitro. However, the numerous technical challenges that will certainly be resolved with time are dwarfed by the potential of LRRK2 as the most exciting and viable target yet identified in PD.