Development of a highly potent and selective degrader of LRRK2.

The discovery of disease-modifying therapies for Parkinson's Disease (PD) represents a critical need in neurodegenerative medicine. Genetic mutations in leucine-rich repeat kinase 2 (LRRK2) are risk factors for the development of PD, and some of these mutations have been linked to increased LRRK2 kinase activity and neuronal toxicity in cellular and animal models. Furthermore, LRRK2 function as a scaffolding protein in several pathways has been implicated as a plausible mechanism underlying neurodegeneration caused by LRRK2 mutations. Given that both the kinase activity and scaffolding function of LRRK2 have been linked to neurodegeneration, we developed proteolysis-targeting chimeras (PROTACs) targeting LRRK2. The degrader molecule JH-XII-03-02 (6) displayed high potency and remarkable selectivity for LRKK2 when assessed in a of 468 panel kinases and serves the dual purpose of eliminating both the kinase activity as well as the scaffolding function of LRRK2.

A Biochemical Analysis of Patients with COVID-19 Infection.

Several studies have demonstrated that age, comorbidities, and abnormalities in different clinical biomarkers can be important to understand disease severity. Although clinical features of COVID-19 have been widely described, the assessment of alterations of the most common biochemical markers that are reported in patients with COVID-19 still has not been well established. Here, we report clinical and blood biochemical indicators of 100 patients with COVID-19. Throat-swab upper respiratory samples were obtained from patients and real-time PCR was used to confirm SARS-CoV-2 infection. Gender, age, and clinical features such as diabetes mellitus, hypertension, and smoking habits were investigated. Biochemical parameters were categorized and analyzed according to these clinical characteristics. Triglycerides, GPT, and ALP are the biochemical markers that changed the most in the group of hypertension patients. Cholesterol and triglycerides were significantly different (P=0.01; P=0.04, respectively) between diabetic and nondiabetic patients with COVID-19. Potassium levels were significantly different (P=0.03) when comparing smokers with nonsmoker patients. Our results suggest several potential biochemical indexes that changed in patients with COVID-19 and whether certain comorbidity and clinical characteristics influence these markers.

Differential responses to kinase inhibition in FGFR2-addicted triple negative breast cancer cells: a quantitative phosphoproteomics study.

Fibroblast Growth Factor (FGF) dependent signalling is frequently activated in cancer by a variety of different mechanisms. However, the downstream signal transduction pathways involved are poorly characterised. Here a quantitative differential phosphoproteomics approach, SILAC, is applied to identify FGF-regulated phosphorylation events in two triple- negative breast tumour cell lines, MFM223 and SUM52, that exhibit amplified expression of FGF receptor 2 (FGFR2) and are dependent on continued FGFR2 signalling for cell viability. Comparative Gene Ontology proteome analysis revealed that SUM52 cells were enriched in proteins associated with cell metabolism and MFM223 cells enriched in proteins associated with cell adhesion and migration. FGFR2 inhibition by SU5402 impacts a significant fraction of the observed phosphoproteome of these cells. This study expands the known landscape of FGF signalling and identifies many new targets for functional investigation. FGF signalling pathways are found to be flexible in architecture as both shared, and divergent, responses to inhibition of FGFR2 kinase activity in the canonical RAF/MAPK/ERK/RSK and PI3K/AKT/PDK/mTOR/S6K pathways are identified. Inhibition of phosphorylation-dependent negative-feedback pathways is observed, defining mechanisms of intrinsic resistance to FGFR2 inhibition. These findings have implications for the therapeutic application of FGFR inhibitors as they identify both common and divergent responses in cells harbouring the same genetic lesion and pathways of drug resistance.

Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.

There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.

Rab29 activation of the Parkinson's disease-associated LRRK2 kinase.

Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector-binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans-Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29-mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2-mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.

Vomocytosis of live pathogens from macrophages is regulated by the atypical MAP kinase ERK5.

Vomocytosis, or nonlytic extrusion, is a poorly understood process through which macrophages release live pathogens that they have failed to kill back into the extracellular environment. Vomocytosis is conserved across vertebrates and occurs with a diverse range of pathogens, but to date, the host signaling events that underpin expulsion remain entirely unknown. We use a targeted inhibitor screen to identify the MAP kinase ERK5 as a critical suppressor of vomocytosis. Pharmacological inhibition or genetic manipulation of ERK5 activity significantly raises vomocytosis rates in human macrophages, whereas stimulation of the ERK5 signaling pathway inhibits vomocytosis. Lastly, using a zebrafish model of cryptococcal disease, we show that reducing ERK5 activity in vivo stimulates vomocytosis and results in reduced dissemination of infection. ERK5 therefore represents the first host signaling regulator of vomocytosis to be identified and a potential target for the future development of vomocytosis-modulating therapies.

Quantitative Phosphoproteomics Reveals a Role for Collapsin Response Mediator Protein 2 in PDGF-Induced Cell Migration.

The Platelet Derived Growth Factor (PDGF) family of ligands have well established functions in the induction of cell proliferation and migration during development, tissue homeostasis and interactions between tumours and stroma. However, the mechanisms by which these actions are executed are incompletely understood. Here we report a differential phosphoproteomics study, using a SILAC approach, of PDGF-stimulated mouse embryonic fibroblasts (MEFs). 116 phospho-sites were identified as up-regulated and 45 down-regulated in response to PDGF stimulation. These encompass proteins involved in cell adhesion, cytoskeleton regulation and vesicle-mediated transport, significantly expanding the range of proteins implicated in PDGF signalling pathways. Included in the down-regulated class was the microtubule bundling protein Collapsin Response Mediator Protein 2 (CRMP2). In response to stimulation with PDGF, CRMP2 was dephosphorylated on Thr514, an event known to increase CRMP2 activity. This was reversed in the presence of micromolar concentrations of the protein phosphatase inhibitor okadaic acid, implicating PDGF-induced activation of protein phosphatase 1 (PP1) in CRMP2 regulation. Depletion of CRMP2 resulted in impairment of PDGF-mediated cell migration in an in vitro wound healing assay. These results show that CRMP2 is required for PDGF-directed cell migration in vitro.

LAR protein tyrosine phosphatase regulates focal adhesions through CDK1.

Focal adhesions are complex multi-molecular structures that link the actin cytoskeleton to the extracellular matrix through integrin adhesion receptors and play a key role in regulation of many cellular functions. LAR (also known as PTPRF) is a receptor protein tyrosine phosphatase that regulates PDGF signalling and localises to focal adhesions. We have observed that loss of LAR phosphatase activity in mouse embryonic fibroblasts results in reduced numbers of focal adhesions and decreased adhesion to fibronectin. To understand how LAR regulates cell adhesion we used phosphoproteomic data, comparing global phosphorylation events in wild-type and LAR phosphatase-deficient cells, to analyse differential kinase activity. Kinase prediction analysis of LAR-regulated phosphosites identified a node of cytoskeleton- and adhesion-related proteins centred on cyclin-dependent kinase-1 (CDK1). We found that loss of LAR activity resulted in reduced activity of CDK1, and that CDK1 activity was required for LAR-mediated focal adhesion complex formation. We also established that LAR regulates CDK1 activity through c-Abl and Akt family proteins. In summary, we have identified a new role for a receptor protein tyrosine phosphatase in regulating CDK1 activity and hence cell adhesion to the extracellular matrix.

Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study.

Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling.