Browsing by Author "Robo, Michael T."

Now showing 1 - 4 of 4
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    Exploring Class‐II PI3K Inhibition for the treatment of Alzheimer’s Disease: Virtual Screening for PI3KC2A Inhibitors
    (Wiley, 2025-01-09) Jadala, Chetna; Robo, Michael T.; Richardson, Timothy I.; Pharmacology and Toxicology, School of Medicine
    Background: Focusing on novel AD treatments, the TREAT‐AD centers offer an array of free research tools, shared via the AD Knowledge Portal in a Target Enablement Package (TEP). This abstract showcases the research conducted by the IUSM‐Purdue TREAT‐AD Center, specifically focusing on Targeting class‐II PI3K’s as a potential breakthrough in AD therapy. Endocytosis within the brain encompasses diverse pathways for internalizing extracellular cargoes and receptors into cells. The prominent routes include clathrin‐mediated endocytosis and phagocytosis. Endocytosis plays a crucial role in processing amyloid precursor protein (APP) leading to abnormal production of Aβ peptides. Recycling endosomes are vital for delivering and eventually releasing Aβ into the brain. Recent research emphasizes the pivotal role of PI3K‐C2α, a class II PI3K member, in regulated endocytosis through its clathrin‐binding domain. Its localization spans clathrin‐coated pits, endocytic vesicles, early endosomes, and the trans‐Golgi network, generating phosphatidylinositol 3‐phosphate (PtdIns(3)P) and/or phosphatidylinositol 3,4‐bisphosphates (PtdIns(3,4)P2) in vivo. Targeting clathrin‐mediated endocytosis by inhibiting PI3K‐C2α, a key regulator in clathrin coated vesicle formation, could be a potential therapeutic strategy against Alzheimer’s disease. Method: We conducted extensive virtual screenings of vast compound libraries to determine potent small molecules inhibiting PI3K‐C2α. Employing shape‐based screening, and clustering techniques, we identified leading compounds for subsequent in vitro kinase assays. Compounds exhibiting nanomolar activity were selected for further investigation. Leveraging these findings, we conducted Structure‐Activity Relationship (SAR) studies, optimizing analogs to enhance binding affinity and cellular pharmacology. Result: We have identified novel PI3K‐C2α inhibitors and are in the initial stages of optimization. These compounds exhibit promising target engagement, pending further assessment for biochemical activity and cellular pharmacology. In silico assessments suggest their structures are ideal for CNS drug discovery plans. Conclusion: Inhibiting PI3K‐C2α stands as a promising therapeutic approach for Alzheimer’s disease. We've discovered unique molecular structures that inhibit the enzyme. Our findings suggest potential probe molecules for validating the target and developing lead compounds for clinical investigations.
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    Fast free energy estimates from λ-dynamics with bias-updated Gibbs sampling
    (Springer Nature, 2023-12-21) Robo, Michael T.; Hayes, Ryan L.; Ding, Xinqiang; Pulawski, Brian; Vilseck, Jonah Z.; Biochemistry and Molecular Biology, School of Medicine
    Relative binding free energy calculations have become an integral computational tool for lead optimization in structure-based drug design. Classical alchemical methods, including free energy perturbation or thermodynamic integration, compute relative free energy differences by transforming one molecule into another. However, these methods have high operational costs due to the need to perform many pairwise perturbations independently. To reduce costs and accelerate molecular design workflows, we present a method called λ-dynamics with bias-updated Gibbs sampling. This method uses dynamic biases to continuously sample between multiple ligand analogues collectively within a single simulation. We show that many relative binding free energies can be determined quickly with this approach without compromising accuracy. For five benchmark systems, agreement to experiment is high, with root mean square errors near or below 1.0 kcal mol-1. Free energy results are consistent with other computational approaches and within statistical noise of both methods (0.4 kcal mol-1 or less). Notably, large efficiency gains over thermodynamic integration of 18-66-fold for small perturbations and 100-200-fold for whole aromatic ring substitutions are observed. The rapid determination of relative binding free energies will enable larger chemical spaces to be more readily explored and structure-based drug design to be accelerated.
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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.