Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1), encoded by the gene INPP5D, is a lipid phosphatase that negatively regulates immune receptor signaling in hematopoietic cells and microglia. Here, we describe a pyridyl-pyrazole-piperidine scaffold and the lead compound 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (32), which demonstrates SHIP1 target engagement, brain exposure, and evidence of a central pharmacodynamic response in vivo. Structure-activity relationship studies, guided by biochemical and cellular assays using multiple human and murine protein constructs and cells, identified SHIP1-active ligands. A thermal shift assay using full-length SHIP1 was used to assess compounds for cellular target engagement, while studies in IL-4 conditioned THP-1 cells was used to demonstrate changes in downstream AKT signaling. Targeted lipidomics revealed changes in the overall phosphoinositide pool consistent with SHIP1 target engagement and reduction of phospho-AKT levels. In a protein-lipid overlay assay, compound 32 induced changes in the relative association of SHIP1 with multiple phosphatidylinositols on a membrane surface. In high-content cellular imaging assays, compound 32 enhanced the uptake of myelin/membrane debris and fibrillar amyloid by primary murine microglia, phenocopying a genetic model with reduced SHIP1 expression. Finally, oral administration of compound 32 resulted in brain exposure sufficient to alter gene expression and reduce IL-1β levels as pharmacodynamic markers of microglial activation and neuroinflammation in an amyloidosis mouse model of Alzheimer's disease. Collectively, these results define a scaffold with SHIP1 target engagement, CNS exposure, and in vivo activity, providing a foundation for the optimization of brain-penetrant SHIP1 ligands suitable for further mechanistic studies and therapeutic development for the treatment of Alzheimer's disease.

Neuroinflammation is increasingly recognized as a central contributor to the pathogenesis and progression of Alzheimer's disease (AD). The apoptosis-associated speck-like protein containing a CARD (ASC), encoded by the PYCARD gene, plays a critical role in the formation of multiple inflammasomes, including NLRP3, a key mediator of inflammation signaling. Beyond its role in inflammasome formation, extracellular ASC specks have been shown to promote amyloid-β aggregation, showing a potential link between inflammation and plaque formation. In this review, we examine the role of ASC in AD pathology and highlight emerging tools to study ASC biology and strategies for ASC targeted drug discovery.

The incidence of Alzheimer's disease (AD) continues to increase, despite decades of effort to develop disease-modifying therapies. In response, the National Institute on Aging (NIA) established the TaRget Enablement to Accelerate Therapy Development for Alzheimer's Disease (TREAT-AD) centers to address the gap between basic research and translational drug discovery. Situated within a robust AD research environment, the Indiana University School of Medicine (IUSM)-Purdue University TREAT-AD Center is one of two National Institutes of Health (NIH)-supported centers funded to accomplish this mission. With a focus on novel biological targets beyond amyloid and tau, our center has assembled the necessary components of a drug discovery engine: project and data management, bioinformatics and computational science, structural biology and biochemistry, assay development and pharmacology, and molecular design and synthesis of small molecules, antibodies, and oligonucleotides. Our objective is to deliver Target Enabling Packages (TEPs) within an open science framework, making data, methods, and research tools broadly accessible through the AD Knowledge Portal. HIGHLIGHTS: The Indiana University School of Medicine (IUSM)-Purdue TREAT-AD Center develops Target Enabling Packages (TEPs) to advance novel targets for the treatment of Alzheimer's disease (AD). The center is overseen by an administrative core and operates through four technical cores - bioinformatics, structural biology, assay development, and medicinal chemistry - within a milestone-driven and open science framework. Multi-omics, systems biology, and machine learning (ML) approaches guide the nomination of high-priority targets beyond amyloid and tau. Cross-core workflows provide structural insights into novel biological targets, validated assays, biomarkers, and molecular probes that enable lead optimization. All data, methods, and tools are openly shared through the AD Knowledge Portal to accelerate global efforts in AD drug discovery.

INTRODUCTION:Microglia are macrophage-like brain resident immune cells known to express numerous Alzheimer's disease risk genes. Here we generated a human induced pluripotent stem cell (iPSC) derived microglia cell culture model for use in neuroimmune modeling and therapeutic testing. METHODS:We generated iPSC lines using episomal reprogramming for subsequent stepwise differentiation of iPSC-derived microglia (iMG) without commercial kits. We characterized the responses of this model to immunogenic stimuli and recombinant TREM2 antibodies. RESULTS:The iMG expressed several key microglia signature genes and are morphologically and transcriptionally dynamic. iMG rapidly phagocytosed myelin debris and strongly changed expression of lipid homeostasis genes. iMG expressed TREM2 and increased TREM2 levels in response to IL-4. Recombinant TREM2 antibody treatment impaired iMG myelin phagocytosis and upregulated chemokines. DISCUSSION:We validated our iMG model system for the evaluation of biological responses of human microglia-like cells to stimuli and pharmacological agents for their transcriptional and functional impacts.

Alzheimer's disease (AD) research has entered a new era where public-private partnerships are reigniting the pursuit of disease-modifying therapies targeting mechanisms beyond amyloid and tau. This perspective outlines how Monument Biosciences was founded to advance novel therapies by translating National Institutes of Health (NIH) -supported discoveries from the TaRget Enablement to Accelerate Therapy Development for AD (TREAT-AD) and the Model Organism Development and Evaluation for Late-onset AD (MODEL-AD) consortia into a venture-backed pipeline focused on neuroinflammation. Monument Bio strategically builds on genetic validation and high-quality, nondilutive-funded research and serves as a case study in how academic research can reduce risk in early-stage biotech development to advance novel therapies. The company exemplifies a novel academic-startup collaboration model, emphasizing robust biomarker strategies to streamline clinical development. Aligned with the framework presented by Richardson et al., in this issue, this approach supports a new generation of neuroscience companies grounded in open science, strong target validation, and disease-relevant endpoints. HIGHLIGHTS: The Alzheimer's field is poised for next-generation therapies beyond amyloid and tau. Monument Bio translates National Institutes of Health (NIH)-supported science into a venture-backed Alzheimer's pipeline. TaRget Enablement to Accelerate Therapy Development for AD (TREAT-AD) and Model Organism Development and Evaluation for Late-onset AD (MODEL-AD) de-risk programs with validated assays, probes, and biomarker tools. Monument Bio shows how TREAT-AD's Target Enabling Packages (TEPs) enable difficult targets. Integrated biomarkers enable predictive translation, faster approval, and investment rationale.

BACKGROUND:The role of microglia in neuroinflammation is widely recognized as a key contributor to the pathogenesis of Alzheimer's disease (AD). Genome-wide association studies have identified PLCγ2 as a key contributor, with specific variants conferring either risk or protection. Notably, the protective PLCγ2•P522R variant is associated with increased mRNA expression, protein levels, and innate activity, whereas the risk variant PLCγ2•M28L shows the opposite trend. Based on these findings, we hypothesize that small molecules capable of enhancing PLCγ2 expression or directly activating the protein could mimic the protective effects of the P522R variant. Such an approach may represent a promising therapeutic strategy to mitigate disease progression and cognitive decline in AD patients. METHOD:We performed high-throughput screening including DNA Encoded Library (DEL) and Affinity Selection Mass Spectrometry (ASMS) using full-length protein to identify novel small molecules which bind to PLCγ2. Target engagement was confirmed using Differential Scanning Fluorimetry (DSF) and Cellular Thermal Shift Assay (CETSA). Structure activity relationship (SAR) studies were performed to synthesize analogs and optimize for binding and cellular pharmacology in IP-One and phagocytosis assays. Top compounds have been studied in vivo to assess pharmacokinetic properties as well as impact on neuroinflammation. RESULT:Novel PLCγ2 activators have been discovered and preliminary optimization has been completed. These compounds have shown positive results for target engagement, biochemical activity, and cellular pharmacology. In silico predictions indicated the molecule structures are suitable CNS drug discovery program starting points. CONCLUSION:Activation of PLCγ2 is a novel therapeutic strategy for treatment of AD. We identified structurally distinct molecular scaffolds capable of enzyme activation and cellular activity. Recommendations for use of probe molecules in target validation studies and the development of lead-like molecules for clinical studies will be made.

TREM2 signaling has been implicated in Alzheimer’s Disease (AD). TREM2 regulates microglial states and functions such as phagocytosis. The most prominent TREM signaling adapter is DAP12, encoded by TYROBP. Understanding functional changes of this complex, and downstream effectors such as SHIP1, PLCG2 and the Scr family kinases Lyn and Hck, is required to evaluate a broad range of therapeutic hypotheses and drug targets for prioritization and enablement. The lack of available, well validated, and openly distributed experimental tools can limit early drug discovery efforts. Therefore, the IUSM Purdue TREAT-AD Center has generated and validated TREM2 activating antibodies to enable the advancement of drug targets in the TREM2/DAP12 signaling pathway. To establish and validate anti-TREM2 agonist antibodies, heavy and light chain variable sequences were identified from multiple publications including patent applications. Antibodies were formatted as either human IgG1, Fc null mutant IgG1 or antibody transport vehicle (ATV) Fc null mutant IgG1. They were expressed in mammalian ExpiCHO cells and tested ex vivo for agonism based on their ability to activate AKT and Syk phosphorylation in THP1 cells and TREM2/DAP12 overexpressing cells respectively. The strongest agonistic candidate was scaled, purified, and further characterized biophysically and functionally. Several agonistic antibodies were identified. AL2p31 antibody showed binding specificity to human versus murine TREM2. Biophysical characterization using biolayer interferometry showed that binding kinetic parameters (KD, Kon, and Koff) were not significantly affected in LALAPG null mutant Fc background. AL2p31 specifically induced Syk phosphorylation in comparison to an isotype control. Analysis of antibodies formatted as bispecific IgG1 targeting both TREM2 and the human transferrin receptor (hTfR), confirmed that RS9-F6 can bind both human and murine TREM2 and revealed the ATV 35-21-16 variant sequence as a binder for the hTfR. The mission of the IUSM Purdue TREAT-AD Center is to enable and advance the next generation of drug targets for the treatment of AD. The validation of anti-TREM2 agonistic antibodies as research tools will enable comprehensive studies of the TREM2/DAP12 signaling and potential drug targets within the pathway including SHIP1, PLCG2 and the Scr family kinase Lyn and Hck.

The goal of the TREAT-AD Center is to enable drug discovery by developing assays and providing tool compounds for novel and emerging targets. The role of microglia in neuroinflammation has been implicated in the pathogenesis of Alzheimer’s disease (AD). Genome-wide association studies, whole genome sequencing, and gene-expression network analyses comparing normal to AD brain have identified risk and protective variants in genes essential to microglial function. among them. The P522R variant of phospholipase C gamma2 (PLCγ2) is associated with reduced risk for AD and has been characterized as a functional hypermorph. Carriers of P522R with mild cognitive impairment exhibited a slower cognitive decline rate. Conversely the M28L variant increases risk. Therefore, activation of the protein PLCγ2 with small molecules has been proposed as a therapeutic strategy to reduce the rate of disease progression and cognitive decline in AD patients. We performed a high-throughput screen using affinity selection mass spectrometry (ASMS) to identify novel small molecules that bind to the full-length protein PLCγ2. A Cellular Thermal Shift Assay (CETSA) was developed to confirm target engagement in cells. A liposomal-based, fluorogenic reporter biochemical assay was implemented to evaluate activity of the enzyme. A high-content imaging assay measuring phagocytosis, cell number, and nuclear intensity was carried out using the BV2 and HMC3 cell lines to characterize cellular pharmacology and cytotoxicity. Structure activity relationship (SAR) studies were performed to synthesize analogs and optimize for binding and cellular pharmacology. Optimized compounds have been studied in vivo to assess pharmacokinetic properties and drug likeness. Novel PLCγ2 activators have been discovered and preliminary optimization has been completed. These compounds have shown positive results for target engagement, biochemical activity, and cellular pharmacology. In silico predictions indicated the molecule structures are suitable CNS drug discovery program starting points. Activation of PLCγ2 is a novel therapeutic strategy for treatment of AD. We identified structurally distinct molecular scaffolds capable of enzyme activation and cellular activity. Recommendations for use of probe molecules in target validation studies and the development of lead-like molecules for clinical studies will be made.

The role of microglia in neuroinflammation is widely recognized as a key contributor to the pathogenesis of Alzheimer’s disease (AD). Genome-wide association studies have identified PLCγ2 as a key contributor, with specific variants conferring either risk or protection. Notably, the protective PLCγ2•P522R variant is associated with increased mRNA expression, protein levels, and innate activity, whereas the risk variant PLCγ2•M28L shows the opposite trend. Based on these findings, we hypothesize that small molecules capable of enhancing PLCγ2 expression or directly activating the protein could mimic the protective effects of the P522R variant. Such an approach may represent a promising therapeutic strategy to mitigate disease progression and cognitive decline in AD patients. We performed high-throughput screening including DNA Encoded Library (DEL) and Affinity Selection Mass Spectrometry (ASMS) using full-length protein to identify novel small molecules which bind to PLCγ2. Target engagement was confirmed using Differential Scanning Fluorimetry (DSF) and Cellular Thermal Shift Assay (CETSA). Structure activity relationship (SAR) studies were performed to synthesize analogs and optimize for binding and cellular pharmacology in IP-One and phagocytosis assays. Top compounds have been studied in vivo to assess pharmacokinetic properties as well as impact on neuroinflammation. Novel PLCγ2 activators have been discovered and preliminary optimization has been completed. These compounds have shown positive results for target engagement, biochemical activity, and cellular pharmacology. In silico predictions indicated the molecule structures are suitable CNS drug discovery program starting points. Activation of PLCγ2 is a novel therapeutic strategy for treatment of AD. We identified structurally distinct molecular scaffolds capable of enzyme activation and cellular activity. Recommendations for use of probe molecules in target validation studies and the development of lead-like molecules for clinical studies will be made.

The TaRget Enablement to Accelerate Therapy Development of Alzheimer’s Disease (TREAT-AD) Centers are dedicated to identifying and validating targets from the NIH Accelerating Medicines Partnership for Alzheimer’s Disease (AMP-AD). The centers develop Target Enabling Packages (TEPs) to explore new therapeutic target hypotheses, moving beyond the traditional focus on amyloid or tau pathologies. In accordance with open science principles, data, methods, and tools are freely shared with the research community via an open-access platform, the AD Knowledge Portal. The Indiana University School of Medicine and Purdue University TREAT-AD (IUSM Purdue TREAT-AD) Center comprises four technical cores: Bioinformatics and Computational Biology (BCB), Structural Biology and Biophysics Core (SBB), Assay Development and High Throughput Screening (ADHTS), and Medicinal Chemistry and Chemical Biology (MCCB). These cores collaborate to develop research tools that are used to validate biological targets and assess their druggability with an initial focus on understanding the role of neuroinflammation in AD. The BCB Core supports target selection and validation with data and analysis. The SBB Core provides proteins for assay development, biophysical assays, and structural studies to aid in mode of action and Structure Activity Relationship (SAR) studies. The ADHTS Core develops in vitro and in vivo assays for SAR studies and translational PD biomarker strategies to assist in determining early phase clinical dosing regimens. The MCCB Core selects therapeutic modalities (small molecules, antibodies, siRNA) and discovers pharmacological tools, employing strategies for SAR studies to balance pharmacological and drug-like properties. Target Enabling Packages (TEPs) are now available via the AD Knowledge Portal for microglia targets that were prioritized for early drug discovery studies. TEPs include bioinformatics analysis, biological reagents and protocols, protein production methods, and recommended chemical probes with detailed information (Figure 1). Novel small molecule hits and leads were identified for SHIP1, PLCG2, SHP1 and LYN/HCK. A pipeline of prioritized microglia targets were selected and enabled for early drug discovery. The IUSM Purdue TREAT-AD Center is now working with AMP-AD researchers to explore biological hypothesis in addition to the role of neuroinflammation in AD.

Genome-wide association studies, whole genome sequencing, and gene-expression network analyses comparing normal to Alzheimer’s disease (AD) brain tissue have identified risk and protective variants in genes essential to microglial function. Among these genes is phospholipase C gamma2 (PLCγ2). The P522R variant of PLCγ2 is associated with reduced risk for AD and has been characterized as a functional hypermorph. Carriers of P522R with mild cognitive impairment exhibited a slower cognitive decline rate. Therefore, activation of the protein PLCγ2 with small molecules has been proposed as a therapeutic strategy to reduce the rate of disease progression and cognitive decline in AD patients. We evaluated analogs of a reported phospholipase activator and performed a high-throughput screen using full-length PLCγ2 and affinity selection mass spectrometry (ASMS). Soluble substrate and liposomal-based biochemical assays were developed to evaluate activation and inhibition of the enzyme. A Cellular Thermal Shift Assay (CETSA) was developed to confirm target engagement in cells. A high-content imaging assay measuring phagocytosis, cell number, and nuclear intensity was implemented using the BV2 and HMC3 cell lines to characterize cellular pharmacology and cytotoxicity. Results were recapitulated in primary murine microglia. Active compounds predicted to have drug-like properties were subjected to assays measuring solubility, cellular permeability, and mouse microsomal stability. Structurally distinct, novel PLCγ2 activators have been discovered. In preparation for in vivo studies in mice, in vitro absorption, distribution, metabolism, and excretion (ADME) assays have been carried out for activators with sufficient cellular potency and drug-like properties. Activation of PLCγ2 is a novel therapeutic strategy for treatment of AD. We identified structurally distinct molecular scaffolds with varying degrees of enzyme activation and cellular activity. Recommendations for use of probe molecules in target validation studies and the development of lead-like molecules for clinical studies will be made.

INTRODUCTION:The risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target. METHODS:We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies. RESULTS:SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration. DISCUSSION:3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies. Highlights:Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain-contaning inositol phosphatase 1 (SHIP1) target engagement and on-target activity in cellular assays.A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health.SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic.The chemical probe 3-((2,4-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine is recommended to explore SHIP1 pharmacology.

Recent evidence including GWAS and differential gene expression comparing normal to affected Alzheimer’s brain tissue have identified risk and protective variants in genes such as TREM2, PLCG2 and INPP5D that are essential to microglia function. INPP5D encodes SHIP1, a multi-domain protein with a phosphatase that converts PI(3,4,5)P 3 to PI(3,4)P 2 , a SH2 domain that interacts with receptor ITAMs and competes with SYK, and a proline rich region that binds many other proteins. SHIP1 therefore limits microglia activation in multiple ways. Inhibition of SHIP1 early in disease would increase microglial protective functions and reduce the rate of disease progression and cognitive decline in Alzheimer’s patients. We performed a screen of 50K compounds at the SHIP1 phosphatase, analyzed a publicly available fragment-based screen, and evaluated inhibitors reported in the literature. We utilized the malachite green assay with PtdIns(3,4,5)P 3 -diC8 and SHIP1 Ptase-C2 to measure inhibitory potency. A Cellular Thermal Shift Assay was used to confirm target engagement in cells. A high-content imaging assay measuring phagocytosis, cell number, and nuclear intensity was implemented using the BV2 and HMC3 cell lines to characterize cellular pharmacology and cytotoxicity. Mouse microglia were assayed to demonstrate similar activity in primary cells. Inhibitors predicted to have drug-like properties were subjected to assays measuring solubility, cellular permeability, and mouse microsomal stability. A physiological based pharmacokinetic model was compared to measured exposure in vivo for select compounds upon oral administration in mice. SHIP1 inhibitors have been compared head-to-head in a set of assays relevant to both enzyme inhibition and microglia activation. Structurally distinct, novel, and selective SHIP1 inhibitors have been discovered. The enzyme mode of action, cellular activity and drug-like properties were determined. Pharmacokinetic profiles were determined for inhibitors with sufficient cellular potency and drug-like properties for in vivo studies in mice. Inhibition of SHIP1 is a novel therapeutic strategy for treatment of Alzheimer’s. We identified structurally distinct molecular scaffolds with varying degrees of enzyme inhibition, cellular activity, and exposure in mice. Recommendations for use of probe molecules in target validation studies and the development of lead-like molecules for clinical studies will be made.

Animal model systems play a fundamental role in the development and evaluation of novel treatments for Alzheimer’s disease (AD). The examination of safety and tolerability in animal models is a necessary first step prior to any human clinical trials. Equally important, preclinical testing of novel therapeutics in disease relevant models is required for the determination if a potential therapeutic should advance. There are a number of important considerations in the preclinical workflow that range from selection of the most appropriate animal model related to drug mechanism of action, as well as what AD-relevant measures are to be evaluated to determine if a candidate therapy should advance. In this chapter we highlight the process of preclinical animal model testing for novel therapeutics in AD, as well as detail several of the models utilized and the measures relevant to AD. We also include the emerging approaches to provide better AD animal models (MODEL-AD) as well as emerging approaches to refine the process of identifying new treatments (TREAT-AD).