In situ identification of cellular drug targets in mammalian tissue

Pang, Zhengyuan; Schafroth, Michael A.; Ogasawara, Daisuke; Wang, Yu; Nudell, Victoria; Lal, Neeraj K.; Yang, Dong Uk; Wang, Kristina; Herbst, Dylan M.; Ha, Jacquelyn; Guijas, Carlos; Blankman, Jacqueline L.; Cravatt, Benjamin F.; Li, Ye

doi:10.1016/j.cell.2022.03.040

Cited by 45 publications

(42 citation statements)

References 56 publications

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“…4a). Importantly, this systematic strategy can be readily adapted to simultaneously profile tropisms of multiple AAV capsid variants or screen various cell-type- specific promoter and enhancer sequences within the same sample by barcoding each variant, enabling cell-type resolved, tissue-level characterization of therapeutics distribution and responses 47 . Such characterization can serve as a screening platform and selection guide for AAV variants to maximize transgene expression in targeted cell types in research and therapeutic applications.…”

Section: Discussionmentioning

confidence: 99%

Spatial Atlas of the Mouse Central Nervous System at Molecular Resolution

Shi

Zhou

et al. 2022

Preprint

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Spatially charting molecular cell types at single-cell resolution across the three-dimensional (3D) volume of the brain is critical for illustrating the molecular basis of the brain anatomy and functions. Single-cell RNA sequencing (scRNA-seq) has profiled molecular cell types in the mouse brain, but cannot capture their spatial organization. Here, we employed an in situ sequencing technique, STARmap PLUS, to map more than one million high-quality cells across the whole adult mouse brain and the spinal cord, profiling 1,022 genes at subcellular resolution with a voxel size of 194 X 194 X 345 nm in 3D. We developed computational pipelines to segment, cluster, and annotate molecularly defined cell types and tissue regions with single-cell resolution. To create a transcriptome-wide spatial atlas, we further integrated the STARmap PLUS measurements with a published scRNA-seq atlas, imputing 11,844 genes at the single-cell level. Finally, we engineered a highly expressed RNA barcoding system to delineate the tropism of a brain-wide transgene delivery tool, AAV-PHP.eB, revealing its single-cell resolved transduction efficiency across the molecular cell types and tissue regions of the whole mouse brain. Together, our datasets and annotations provide a comprehensive, high-resolution single-cell resource that integrates spatial molecular atlas, cell taxonomy, brain anatomy, and genetic manipulation accessibility of the mammalian central nervous system (CNS).

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Section: Discussionmentioning

confidence: 99%

Spatial Atlas of the Mouse Central Nervous System at Molecular Resolution

Shi

Zhou

et al. 2022

Preprint

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“…Recently, Pang et al reported an elegant method to visualize enzyme activity in brain sections using two-step probes. They administered analogues of FAAH inhibitors PF-04457845 and BIA10-2474 equipped with an alkyne to mice, and subsequently coupled the fluorophore ex vivo through CuAAC . They developed a tissue clearing technique termed CATCH that delipidates the brain using sequential hydrogel-based fixation and detergent-based washing to accelerate the CuAAC reaction (Figure A).…”

Section: Visualizing and Controlling Enzyme Activitymentioning

confidence: 99%

“…However, direct visualization of enzyme activity by one-step fluorescent probes avoids the secondary ligation step, simplifies the protocol, and is suitable for live-imaging. Both strategies have been employed to study (sub)cellular localization and distribution of active enzymes with confocal microscopy or flow cytometry. ,− …”

Section: Visualizing and Controlling Enzyme Activitymentioning

confidence: 99%

“…They administered analogues of FAAH inhibitors PF-04457845 and BIA10-2474 equipped with an alkyne to mice, and subsequently coupled the fluorophore ex vivo through CuAAC. 42 They developed a tissue clearing technique termed CATCH that delipidates the brain using sequential hydrogel-based fixation and detergent-based washing to accelerate the CuAAC reaction ( Figure 3 A). They showed that FAAH activity was mainly found in the somas of Ctip2+ excitatory neurons in the somatosensory cortex, but not in astrocytes or inhibitory neurons.…”

Section: Visualizing and Controlling Enzyme Activitymentioning

confidence: 99%

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Chemical Probes to Control and Visualize Lipid Metabolism in the Brain

2022

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Conspectus Signaling lipids, such as the endocannabinoids, play an important role in the brain. They regulate synaptic transmission and control various neurophysiological processes, including pain sensation, appetite, memory formation, stress, and anxiety. Unlike classical neurotransmitters, lipid messengers are produced on demand and degraded by metabolic enzymes to control their lifespan and signaling actions. Chemical biology approaches have become one of the main driving forces to study and unravel the physiological role of lipid messengers in the brain. Here, we review how the development and use of chemical probes has allowed one to study endocannabinoid signaling by (i) inhibiting the biosynthetic and metabolic enzymes; (ii) visualizing the activity of these enzymes; and (iii) controlling the release and transport of the endocannabinoids. Activity-based probes were instrumental to guide the discovery of highly selective and in vivo active inhibitors of the biosynthetic (DAGL, NAPE-PLD) and metabolic (MAGL, FAAH) enzymes of endocannabinoids. These inhibitors allowed one to study the role of these enzymes in animal models of disease. For instance, the DAGL–MAGL axis was shown to control neuroinflammation and the NAPE-PLD–FAAH axis to regulate emotional behavior. Activity-based protein profiling and chemical proteomics were essential to guide the drug discovery and development of compounds targeting MAGL and FAAH, such as ABX-1431 (Lu AG06466) and PF-04457845, respectively. These experimental drugs are now in clinical trials for multiple indications, including multiple sclerosis and post-traumatic stress disorders. Activity-based probes have also been used to visualize the activity of these lipid metabolizing enzymes with high spatial resolution in brain slices, thereby showing the cell type-specific activity of these lipid metabolizing enzymes. The transport, release, and uptake of signaling lipids themselves cannot, however, be captured by activity-based probes in a spatiotemporal controlled manner. Therefore, bio-orthogonal lipids equipped with photoreactive, photoswitchable groups or photocages have been developed. These chemical probes were employed to investigate the protein interaction partners of the endocannabinoids, such as putative membrane transporters, as well as to study the functional cellular responses within milliseconds upon irradiation. Finally, genetically encoded sensors have recently been developed to monitor the real-time release of endocannabinoids with high spatiotemporal resolution in cultured neurons, acute brain slices, and in vivo mouse models. It is anticipated that the combination of chemical probes, highly selective inhibitors, and sensors with advanced (super resolution) imaging modalities, such as PharmacoSTORM and correlative light-electron microscopy, will uncover the fundamental basis of lipid signaling at nanoscale resolution in the brain. Furthermore, chemical biology approaches enable the translation of these fundamental discoveries into clinical solutions for brain dis...

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“…In the recent report by Pang et al., 5 we developed Clearing Assisted Tissue click CHemistry (CATCH) by combining tissue clearing with in situ click chemistry. This allowed us to visualize covalent drug binding at subcellular resolution directly.…”

mentioning

confidence: 99%

Illuminating spatial pharmacology with in situ drug imaging

Pang

2022

Clinical & Translational Med

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In pharmacology, absorption, distribution, metabolism, excretion (ADME) describes in vivo drug actions. Typically, distribution is measured as organ-specific drug concentrations, with the notion that drugs could have various tendencies to enter and retain in different organs. Such variability can stem from on-and off-target engagement, chemical property of the drug (size, charge, polarity, etc.), or the property of the organ (lipid content, vasculature, blood-brain barrier, etc.). 1 While the inter-organ difference in drug distribution is well-recognized, cellular diversity of drug engagement within an organ is not easily accessible with conventional pharmacokinetics (PK) and pharmacodynamics (PD) studies. This knowledge gap is becoming increasingly important as we begin to understand how single-cell level heterogeneity could significantly affect organ physiology and pathology. 2 A barrier to tracking drug engagement in vivo across tissue compartments and cell types is the lack of tools to visualize drug molecules in situ. Conventional bulk tissue analysis loses spatial and cellular information. Although it retains spatial information, positron emission tomography generally lacks sufficient resolution to resolve cell type identities. 3 Fluorescence-based imaging has been widely used with antibodies or messenger ribonucleic acid (mRNA) probes to visualize endogenous biomolecules, but such tags are too bulky to fit small molecule drugs. To circumvent these barriers, we turned to copper(I)-catalyzedThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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In situ identification of cellular drug targets in mammalian tissue

Cited by 45 publications

References 56 publications

Spatial Atlas of the Mouse Central Nervous System at Molecular Resolution

Spatial Atlas of the Mouse Central Nervous System at Molecular Resolution

Chemical Probes to Control and Visualize Lipid Metabolism in the Brain

Illuminating spatial pharmacology with in situ drug imaging

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