Browsing by Author "Weerawarna, Pathum M."

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    Identification of Chemical Tool Compounds to Investigate the Role of Lyn Kinase in TREM2‐Mediated Microglia Activation and Phagocytosis
    (Wiley, 2025-01-09) Weerawarna, Pathum M.; Robo, Michael T.; Chu, Shaoyou; Mason, Emily R.; Davis, Chris; Angus, Steven P.; Richardson, Timothy I.; Medicine, School of Medicine
    Background: Lyn kinase, a member of the Src family of tyrosine kinases, predominantly phosphorylates ITIM and ITAM motifs linked to immune receptors and adaptor proteins, and is emerging as a target for Alzheimer’s disease (AD). The role of Lyn in TREM2‐mediated microglial activation and phagocytosis, a critical pathway for clearing Aβ plaques, remains unclear and potent, selective, and brain penetrant Lyn inhibitors are unavailable. In this study, we report the characterization of Lyn kinase inhibitors from the literature as well as the establishment of an advanced virtual screening platform at the IUSM‐Purdue‐TREAT‐AD center to identify new type II Lyn inhibitors suitable as molecular probes. Method: We first performed a thorough literature survey and found 14 reported Lyn kinase inhibitors. We then validated their Lyn inhibitor activities and Lyn selectivities using the HotSpot kinase assay. We tested these compounds for microglia activation in a high‐content imaging assay using HMC3 (human) and BV2 (mouse) microglia‐like cell lines. We also performed kinome profiling in these cells to evaluate cellular target engagement and selectivity. Finally, we screened a million‐compounds using a computational pipeline that combined molecular docking, shape‐based screening, and MD simulations to identify novel and potent type II Lyn kinase inhibitors. Result: Our findings revealed that Type I inhibitors, particularly Saracatinib and Bosutinib, potently inhibit Lyn within the picomolar (pM) range. On the other hand, Type II inhibitors, such as Masitinib and Imatinib, displayed pronounced >20‐fold selectivity for Lyn over Hck with low nM Lyn inhibitor activities. Saracatinib and Bosutinib significantly induced phagocytosis in HMC3 cells, whereas Type II inhibitors demonstrated moderate activity in both HMC3 and BV2 cells. Our virtual screening platform identified a new type II Lyn inhibitor with picomolar activity and good Lyn/Hck selectivity. Conclusion: We have successfully evaluated previously reported inhibitors and introduced a novel type II Lyn kinase inhibitor with picomolar (pM) activities suitable for use as chemical probes to investigate the role of Lyn in TREM2‐mediated microglial activation.
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    Inhibition of Lyn kinase: A novel approach to treatment of Alzheimer’s disease
    (Wiley, 2025-01-09) Benitah, Avi L.; Richardson, Timothy I.; Weerawarna, Pathum M.; Robo, Michael T.; Mason, Emily R.; Pharmacology and Toxicology, School of Medicine
    Background: The TREAT‐AD centers aim to improve Alzheimer’s Disease (AD) research by offering free, high‐quality tools and technologies. Lyn is a tyrosine kinase that belongs to the Src family kinases. The expression of Lyn and its activity have been implicated in AD. This class of proteins is involved in TREM2 mediated microglial activation and phagocytosis, a process which is beneficial for clearing neurotoxins such as Aβ oligomers in the brain. Lyn inhibition may activate microglia. Given the relationship between accumulation of Aβ and its exacerbation of neurodegenerative diseases such as AD, selective inhibition of Lyn has been proposed as a novel therapeutic approach to treating early‐onset AD. However, potent, selective, and brain penetrant Lyn inhibitors are unavailable to test this hypothesis. Method: We screened a variety of known kinase inhibitors to determine their activity towards inhibition of Lyn using the biochemical HotSpot kinase assay. With this data in hand, we identified imatinib as a starting point for the design of novel Lyn inhibitors. Structure‐based design and computational docking models were used to propose more active and selective Lyn inhibitors, which were synthesized. The activities were determined, and multiple parameter optimization (MPO) informed iterative Structure Activity Relationship (SAR) studies. The best compounds were evaluated in assays of microglia activation, and their drug metabolism and pharmacokinetic (DMPK) properties were determined. Result: A series of novel type II inhibitors are now available for testing. The results demonstrate a unique tail group provides the novel scaffold with potent activity and selectivity towards inhibition of Lyn, exceeding that of imatinib. Conclusion: Computational models, SAR, and MPO provided potent and selective Lyn inhibitors with good DMPK properties. Further studies are under way to determine the impact of these compounds on TREM2 mediated activation of microglia both in vitro and in vivo.