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Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.

Genetic mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to autosomal dominant Parkinson's disease. The most prevalent mutation, G2019S, results in enhanced LRRK2 kinase activity that potentially contributes to the etiology of Parkinson's disease. Consequently, disease progression is potentially mediated by poorly characterized phosphorylation-dependent LRRK2 substrate pathways. To address this gap in knowledge, we transduced SH-SY5Y neuroblastoma cells with LRRK2 G2019S via adenovirus, then determined quantitative changes in the phosphoproteome upon LRRK2 kinase inhibition (LRRK2-IN-1 treatment) using stable isotope labeling of amino acids in culture combined with phosphopeptide enrichment and LC-MS/MS analysis. We identified 776 phosphorylation sites that were increased or decreased at least 50% in response to LRRK2-IN-1 treatment, including sites on proteins previously known to associate with LRRK2. Bioinformatic analysis of those phosphoproteins suggested a potential role for LRRK2 kinase activity in regulating pro-inflammatory responses and neurite morphology, among other pathways. In follow-up experiments, LRRK2-IN-1 inhibited lipopolysaccharide-induced tumor necrosis factor alpha (TNFα) and C-X-C motif chemokine 10 (CXCL10) levels in astrocytes and also enhanced multiple neurite characteristics in primary neuronal cultures. However, LRRK2-IN-1 had almost identical effects in primary glial and neuronal cultures from LRRK2 knockout mice. These data suggest LRRK2-IN-1 may inhibit pathways of perceived LRRK2 pathophysiological function independently of LRRK2 highlighting the need to use multiple pharmacological tools and genetic approaches in studies determining LRRK2 function.

In vivo gene therapy shows promise as a treatment for both genetic and acquired disorders. The hepatic asialoglycoprotein receptor (ASGPr) binds asialoorosomucoid-polylysine-DNA (ASOR-PL-DNA) complexes and allows targeted delivery to hepatocytes. The tris(N-acetylgalactosamine aminohexyl glycoside) amide of tyrosyl(glutamyl) glutamate [YEE(GalNAcAH)3] has been previously reported to have subnanomolar affinity for the ASGPr. We have used an iodinated derivative of YEE(GalNAcAH)3 linked to polylysine and complexed to the luciferase gene (pCMV-Luc) in receptor-binding experiments to establish the feasibility of substituting ASOR with the synthetic glycopeptide for gene therapy. Scatchard analyses revealed similar Kd values for both ASOR and the glycopeptide. Binding and internalization of 125I-Suc-YEE(GalNAcAH)3 were competitively inhibited with either unlabeled ASOR or glycopeptide. The reverse was also true; 125I-ASOR binding was competed with unlabeled YEE(GalNAcAH)3 suggesting specific binding to the ASGPr by both compounds. Examination of in vivo delivery revealed that the 125I-labeled glycopeptide complex mimicked previous results observed with 125I-ASOR-PL-DNA. CPM in the liver accounted for 96% of the radioactivity recovered from the five major organs (liver, spleen, kidney, heart, and lungs). Cryoautoradiography displayed iodinated glycopeptide complex bound preferentially to hepatocytes rather than nonparenchymal cells. In vitro, as well as in vivo, transfections using the glycopeptide-polylysine-pCMV-luciferase gene complex (YG3-PL-Luc) resulted in expression of the gene product. These data demonstrate that the YEE(GalNAcAH)3 synthetic glycopeptide can be used as a ligand in targeted delivery of DNA to the liver-specific ASGPr.

Anti-topoisomerase I autoantibodies (anti-topo I) are associated with proximal scleroderma and are of prognostic significance in patients with Raynaud's phenomenon. Polyclonal anti-idiotypic sera were raised against affinity-purified anti-topo I from 2 patients with scleroderma (EM, SG) and 1 healthy individual (NM). All 3 anti-topo I preparations expressed immunodominant private Ids in or near the antigen binding site of the autoantibody. Further analysis of Id-EM showed isotypic restriction to IgG and a stable Id-expression over the course of 9 years. Id-SG and Id-NM were expressed on IgG and on IgA. The idiotypic character of anti-topo I closely resembles that of anti-centromere autoantibodies which are associated with the CREST syndrome of scleroderma. The data suggest an antigen-driven process in the origin of autoantibodies in scleroderma.

Specific DNA delivery has been achieved via interactions between an asialoorosomucoid-polylysine conjugate and the asialoglycoprotein receptor. We have now extended this technology to another cell type. In order to achieve DNA delivery uniquely to T cells, we have employed an antibody-polylysine conjugate which binds and is internalized via CD5. Binding analyses of the T101 monoclonal antibody to Jurkat cells and freshly isolated human peripheral T lymphocytes were performed and Scatchard plots revealed Kd values of 1.4 and 1.2 pM, respectively. To introduce DNA into the T cell, a complex of T101-polylysine and the luciferase plasmid was formed (T101-PL-DNA). 125I-labeled antibody alone or T101-PL-DNA complexes were both shown to internalize. Subcellular fractionation indicated that the complex remained in the endosomal compartment of the cell for up to 90 min. However, with the addition of adenovirus particles, there was a decrease of labeled complex in the endosomal fraction over time suggesting it was no longer ‘tethered’ to the endosome vesicle. In vitro transfections confirmed this result showing the addition of adenovirus particles during incubation resulted in increased expression of the luciferase protein. Without adenovirus, there was limited expression of the transduced gene. These data revealed that T101 can deliver DNA via an antibody-PL conjugate. The addition of adenovirus allowed the DNA to escape the endosome enabling expression of the reporter gene.