The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility

The assumption that each enzyme expresses a single enzymatic activity in vivo is challenged by the linkage of the neuronal enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) to Parkinson's disease (PD). UCH-L1, especially those variants linked to higher susceptibility to PD, causes the accumulation of alpha-synuclein in cultured cells, an effect that cannot be explained by its recognized hydrolase activity. UCH-L1 is shown here to exhibit a second, dimerization-dependent, ubiquityl ligase activity. A polymorphic variant of UCH-L1 that is associated with decreased PD risk (S18Y) has reduced ligase activity but comparable hydrolase activity as the wild-type enzyme. Thus, the ligase activity as well as the hydrolase activity of UCH-L1 may play a role in proteasomal protein degradation, a critical process for neuronal health

Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line

Neuronal ubiquitin C-terminal hydrolase (UCH-L1) has been linked to Parkinson's disease (PD), the progression of certain nonneuronal tumors, and neuropathic pain. Certain lung tumor-derived cell lines express UCH-L1 but it is not expressed in normal lung tissue, suggesting that this enzyme plays a role in tumor progression, either as a trigger or as a response. Small-molecule inhibitors of UCH-L1 would be helpful in distinguishing between these scenarios. By utilizing high-throughput screening (HTS) to find inhibitors and traditional medicinal chemistry to optimize their affinity and specificity, we have identified a class of isatin O-acyl oximes that selectively inhibit UCH-L1 as compared to its systemic isoform, UCH-L3. Three representatives of this class (30, 50, 51) have IC(50) values of 0.80-0.94 micro M for UCH-L1 and 17-25 micro M for UCH-L3. The K(i) of 30 toward UCH-L1 is 0.40 micro M and inhibition is reversible, competitive, and active site directed. Two isatin oxime inhibitors increased proliferation of the H1299 lung tumor cell line but had no effect on a lung tumor line that does not express UCH-L1. Inhibition of UCH-L1 expression in the H1299 cell line using RNAi had a similar proproliferative effect, suggesting that the UCH-L1 enzymatic activity is antiproliferative and that UCH-L1 expression may be a response to tumor growth. The molecular mechanism of this response remains to be determined

The UCH-L1 Gene Encodes Two Opposing Enzymatic Activities that Affect α-Synuclein Degradation and Parkinson's Disease Susceptibility

AbstractThe assumption that each enzyme expresses a single enzymatic activity in vivo is challenged by the linkage of the neuronal enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) to Parkinson's disease (PD). UCH-L1, especially those variants linked to higher susceptibility to PD, causes the accumulation of α-synuclein in cultured cells, an effect that cannot be explained by its recognized hydrolase activity. UCH-L1 is shown here to exhibit a second, dimerization-dependent, ubiquityl ligase activity. A polymorphic variant of UCH-L1 that is associated with decreased PD risk (S18Y) has reduced ligase activity but comparable hydrolase activity as the wild-type enzyme. Thus, the ligase activity as well as the hydrolase activity of UCH-L1 may play a role in proteasomal protein degradation, a critical process for neuronal health

Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate <i>in Vivo</i> Deamidation of Therapeutic Antibodies

Deamidation of therapeutic antibodies may result in decreased drug activity and undesirable changes in pharmacokinetics and immunogenicity. Therefore, it is necessary to monitor the deamidation levels [during storage] and after <i>in vivo</i> administration. Because of the complexity of <i>in vivo</i> samples, immuno-affinity capture is widely used for specific enrichment of the target antibody prior to LC–MS. However, the conventional use of bead-based methods requires large sample volumes and extensive processing steps. Furthermore, with automation difficulties and extended sample preparation time, bead-based approaches may increase artificial deamidation. To overcome these challenges, we developed an automated platform to perform tip-based affinity capture of antibodies from complex matrixes with rapid digestion and peptide elution into 96-well microtiter plates followed by LC–MS analysis. Detailed analyses showed that the new method presents high repeatability and reproducibility with both intra and inter assay CVs < 8%. Using the automated platform, we successfully quantified the levels of deamidation of a humanized monoclonal antibody in cynomolgus monkeys over a time period of 12 weeks after administration. Moreover, we found that deamidation kinetics between <i>in vivo</i> samples and samples stressed <i>in vitro</i> at neutral pH were consistent, suggesting that the <i>in vitro</i> stress test may be used as a method to predict the liability to deamidation of therapeutic antibodies <i>in vivo</i>

Discovery of Selective LRRK2 Inhibitors Guided by Computational Analysis and Molecular Modeling

Mutations in the genetic sequence of leucine-rich repeat kinase 2 (LRRK2) have been linked to increased LRRK2 activity and risk for the development of Parkinson’s disease (PD). Potent and selective small molecules capable of inhibiting the kinase activity of LRRK2 will be important tools for establishing a link between the kinase activity of LRRK2 and PD. In the absence of LRRK2 kinase domain crystal structures, a LRRK2 homology model was developed that provided robust guidance in the hit-to-lead optimization of small molecule LRRK2 inhibitors. Through a combination of molecular modeling, sequence analysis, and matched molecular pair (MMP) activity cliff analysis, a potent and selective lead inhibitor was discovered. The selectivity of this compound could be understood using the LRRK2 homology model, and application of this learning to a series of 2,4-diaminopyrimidine inhibitors in a scaffold hopping exercise led to the identification of highly potent and selective LRRK2 inhibitors that were also brain penetrable

Selective Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12) with Activity in a Model of Alzheimer’s Disease

Significant data exists to suggest that dual leucine zipper kinase (DLK, MAP3K12) is a conserved regulator of neuronal degeneration following neuronal injury and in chronic neurodegenerative disease. Consequently, there is considerable interest in the identification of DLK inhibitors with a profile compatible with development for these indications. Herein, we use structure-based drug design combined with a focus on CNS drug-like properties to generate compounds with superior kinase selectivity and metabolic stability as compared to previously disclosed DLK inhibitors. These compounds, exemplified by inhibitor <b>14</b>, retain excellent CNS penetration and are well tolerated following multiple days of dosing at concentrations that exceed those required for DLK inhibition in the brain

Scaffold-Hopping and Structure-Based Discovery of Potent, Selective, And Brain Penetrant <i>N</i>‑(1<i>H</i>‑Pyrazol-3-yl)pyridin-2-amine Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12)

Recent data suggest that inhibition of dual leucine zipper kinase (DLK, MAP3K12) has therapeutic potential for treatment of a number of indications ranging from acute neuronal injury to chronic neurodegenerative disease. Thus, high demand exists for selective small molecule DLK inhibitors with favorable drug-like properties and good CNS penetration. Herein we describe a shape-based scaffold hopping approach to convert pyrimidine <b>1</b> to a pyrazole core with improved physicochemical properties. We also present the first crystal structures of DLK. By utilizing a combination of property and structure-based design, we identified inhibitor <b>11</b>, a potent, selective, and brain-penetrant inhibitor of DLK with activity in an in vivo nerve injury model