Zachary Kevin Sweeney

Ser1292 Autophosphorylation Is an Indicator of LRRK2 Kinase Activity and Contributes to the Cellular Effects of PD Mutations

LRRK2 autophosphorylation on Ser1292 may be a useful indicator of kinase activity, providing a readout for screening candidate LRRK2 inhibitors. LRRK2 Inhibitor Heralds a Happier Song Genetic polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) are the most common causes of familial Parkinson’s disease (PD) and are also linked to idiopathic PD. The most prevalent LRRK2 PD mutation G2019S imbues the kinase with a gain of function, suggesting that blocking LRRK2 activity may be a therapeutic strategy for reversing the pathogenic effects of LRRK2 mutations in PD. However, the mechanistic link between LRRK2 kinase activity and the cellular effects of PD mutations remains elusive, and there has been no reliable way to monitor LRRK2 kinase activity in vivo. Using quantitative mass spectrometry and subsequent phospho-specific antibody approaches, Sheng et al. now report that LRRK2 phosphorylates itself on Ser1292 in vitro and in vivo (Ser1292 autophosphorylation). Five of the six confirmed familial LRRK2 PD mutations increased Ser1292 autophosphorylation when transiently expressed in heterologous cells, suggesting increased Ser1292 autophosphorylation as a common feature of LRRK2 PD mutations. Elimination of the Ser1292 autophosphorylation site abrogated the defects on neurite outgrowth caused by LRRK2 PD mutations in cultured rat embryonic neurons. Using Ser1292 autophosphorylation as the readout of kinase activity, Sheng et al. developed assays to monitor LRRK2 kinase activity in cultured cells and rodents. These assays were used to profile the potencies of hundreds of LRRK2 kinase inhibitors derived from high-throughput compound screening. A potent and selective compound that effectively inhibited LRRK2 kinase activity in mouse brains and reversed cellular effects of LRRK2 PD mutations in cultured primary neurons was identified. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson’s disease (PD). Although biochemical studies have shown that certain PD mutations confer elevated kinase activity in vitro on LRRK2, there are no methods available to directly monitor LRRK2 kinase activity in vivo. We demonstrate that LRRK2 autophosphorylation on Ser1292 occurs in vivo and is enhanced by several familial PD mutations including N1437H, R1441G/C, G2019S, and I2020T. Combining two PD mutations together further increases Ser1292 autophosphorylation. Mutation of Ser1292 to alanine (S1292A) ameliorates the effects of LRRK2 PD mutations on neurite outgrowth in cultured rat embryonic primary neurons. Using cell-based and pharmacodynamic assays with phosphorylated Ser1292 as the readout, we developed a brain-penetrating LRRK2 kinase inhibitor that blocks Ser1292 autophosphorylation in vivo and attenuates the cellular consequences of LRRK2 PD mutations in vitro. These data suggest that Ser1292 autophosphorylation may be a useful indicator of LRRK2 kinase activity in vivo and may contribute to the cellular effects of certain PD mutations.

Discovery of highly potent, selective, and brain-penetrable leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors.

There is a high demand for potent, selective, and brain-penetrant small molecule inhibitors of leucine-rich repeat kinase 2 (LRRK2) to test whether inhibition of LRRK2 kinase activity is a potentially viable treatment option for Parkinsons disease patients. Herein we disclose the use of property and structure-based drug design for the optimization of highly ligand efficient aminopyrimidine lead compounds. High throughput in vivo rodent cassette pharmacokinetic studies enabled rapid validation of in vitro-in vivo correlations. Guided by this data, optimal design parameters were established. Effective incorporation of these guidelines into our molecular design process resulted in the discovery of small molecule inhibitors such as GNE-7915 (18) and 19, which possess an ideal balance of LRRK2 cellular potency, broad kinase selectivity, metabolic stability, and brain penetration across multiple species. Advancement of GNE-7915 into rodent and higher species toxicity studies enabled risk assessment for early development.

Effect of selective LRRK2 kinase inhibition on nonhuman primate lung

LRRK2 kinase inhibitors, under development for Parkinson’s disease, have an effect on type II pneumocytes in nonhuman primate lung, suggesting that pulmonary toxicity may be a critical safety liability. A lung phenotype for LRRK2 inhibitors Human genetic evidence implicates leucine-rich repeat kinase 2 (LRRK2) as a high-priority drug target for Parkinson’s disease. However, the benefit and risk of inhibiting the kinase activity of LRRK2 is unknown and is currently untested in humans. Using two selective LRRK2 kinase inhibitors, Fuji et al. report a safety liability in nonhuman primates characterized by morphological changes in lung. These results are consistent with observations in mice lacking LRRK2. These safety observations offer a cautionary note for pharmacological modulation of LRRK2 in humans. Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson’s disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.

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 Parkinsons 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.

Discovery of Highly Potent, Selective, and Brain-Penetrant Aminopyrazole Leucine-Rich Repeat Kinase 2 (LRRK2) Small Molecule Inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has drawn significant interest in the neuroscience research community because it is one of the most compelling targets for a potential disease-modifying Parkinsons disease therapy. Herein, we disclose structurally diverse small molecule inhibitors suitable for assessing the implications of sustained in vivo LRRK2 inhibition. Using previously reported aminopyrazole 2 as a lead molecule, we were able to engineer structural modifications in the solvent-exposed region of the ATP-binding site that significantly improve human hepatocyte stability, rat free brain exposure, and CYP inhibition and induction liabilities. Disciplined application of established optimal CNS design parameters culminated in the rapid identification of GNE-0877 (11) and GNE-9605 (20) as highly potent and selective LRRK2 inhibitors. The demonstrated metabolic stability, brain penetration across multiple species, and selectivity of these inhibitors support their use in preclinical efficacy and safety studies.

Small-Molecule Inhibitors of Store-Operated Calcium Entry

Molecules that inhibit store‐operated calcium entry (SOCE) are potentially useful immunomodulating agents. The identification of proteins involved in this pathway may further enable the identification of selective inhibitors. Herein we document some examples of the small‐molecule inhibitors of SOCE that have been reported to date. We also describe methods that were used to characterize the mechanism of action of these inhibitors.

Discovery of Novel PI3-Kinase δ Specific Inhibitors for the Treatment of Rheumatoid Arthritis: Taming CYP3A4 Time-Dependent Inhibition

PI3Kδ is a lipid kinase and a member of a larger family of enzymes, PI3K class IA(α, β, δ) and IB (γ), which catalyze the phosphorylation of PIP2 to PIP3. PI3Kδ is mainly expressed in leukocytes, where it plays a critical, nonredundant role in B cell receptor mediated signaling and provides an attractive opportunity to treat diseases where B cell activity is essential, e.g., rheumatoid arthritis. We report the discovery of novel, potent, and selective PI3Kδ inhibitors and describe a structural hypothesis for isoform (α, β, γ) selectivity gained from interactions in the affinity pocket. The critical component of our initial pharmacophore for isoform selectivity was strongly associated with CYP3A4 time-dependent inhibition (TDI). We describe a variety of strategies and methods for monitoring and attenuating TDI. Ultimately, a structure-based design approach was employed to identify a suitable structural replacement for further optimization.

Structure-Based Drug Design of RN486, a Potent and Selective Bruton's Tyrosine Kinase (BTK) Inhibitor, for the Treatment of Rheumatoid Arthritis.

Structure-based drug design was used to guide the optimization of a series of selective BTK inhibitors as potential treatments for Rheumatoid arthritis. Highlights include the introduction of a benzyl alcohol group and a fluorine substitution, each of which resulted in over 10-fold increase in activity. Concurrent optimization of drug-like properties led to compound 1 (RN486) ( J. Pharmacol. Exp. Ther. 2012 , 341 , 90 ), which was selected for advanced preclinical characterization based on its favorable properties.

Potent and highly selective benzimidazole inhibitors of PI3-kinase delta.

Inhibition of PI3Kδ is considered to be an attractive mechanism for the treatment of inflammatory diseases and leukocyte malignancies. Using a structure-based design approach, we have identified a series of potent and selective benzimidazole-based inhibitors of PI3Kδ. These inhibitors do not occupy the selectivity pocket between Trp760 and Met752 that is induced by other families of PI3Kδ inhibitors. Instead, the selectivity of the compounds for inhibition of PI3Kδ relative to other PI3K isoforms appears to be due primarily to the strong interactions these inhibitors are able to make with Trp760 in the PI3Kδ binding pocket. The pharmacokinetic properties and the ability of compound 5 to inhibit the function of B-cells in vivo are described.

Potent and selective inhibitors of PI3Kδ: obtaining isoform selectivity from the affinity pocket and tryptophan shelf.

A potent inhibitor of PI3Kδ that is ≥ 200 fold selective for the remaining three Class I PI3K isoforms and additional kinases is described. The hypothesis for selectivity is illustrated through structure activity relationships and crystal structures of compounds bound to a K802T mutant of PI3Kγ. Pharmacokinetic data in rats and mice support the use of 3 as a useful tool compound to use for in vivo studies.