Correlative light-electron microscopy

Stages

1. 1.
   Enzyme-focused probe and inhibitor discovery(broad_screen)

   This stage identifies selective chemical matter against key endocannabinoid enzymes and establishes perturbation tools for downstream biology.

   Selection: Use activity-based probes to guide discovery of highly selective and in vivo active inhibitors of biosynthetic and metabolic endocannabinoid enzymes.

2. 2.
   Chemical proteomic guidance during drug discovery and development(functional_characterization)

   This stage refines and supports translational inhibitor development after initial probe-enabled discovery.

   Selection: Apply ABPP and chemical proteomics to guide development of MAGL- and FAAH-targeting compounds such as ABX-1431 and PF-04457845.

3. 3.
   Spatial visualization of enzyme activity in brain slices(secondary_characterization)

   This stage adds spatial and cell type-specific context to enzyme activity beyond inhibitor discovery alone.

   Selection: Use activity-based probes to visualize lipid-metabolizing enzyme activity with high spatial resolution in brain slices.

4. 4.
   Photoresponsive lipid probing of transport, release, and interaction partners(functional_characterization)

   The review explicitly states this stage is needed because activity-based probes cannot capture transport, release, and uptake of signaling lipids in a spatiotemporally controlled manner.

   Selection: Deploy bio-orthogonal lipids with photoreactive, photoswitchable, or photocaged groups to study transport, release, uptake, interaction partners, and rapid functional responses.

5. 5.
   Real-time endocannabinoid sensing across preparations(confirmatory_validation)

   This stage provides direct dynamic readout of endocannabinoid release across increasingly physiological systems.

   Selection: Use genetically encoded sensors to monitor real-time endocannabinoid release with high spatiotemporal resolution in cultured neurons, acute brain slices, and in vivo mouse models.

Steps

1. 1.
   Use activity-based probes to discover selective and in vivo active enzyme inhibitors

   Generate selective perturbation tools against biosynthetic and metabolic endocannabinoid enzymes.

   The review presents enzyme inhibition as an initial route to study endocannabinoid signaling and as the basis for later disease-model and translational work.

2. 2.
   Apply ABPP and chemical proteomics to guide development of translational compounds

   Support drug discovery and development decisions for MAGL- and FAAH-targeting compounds.

   After initial inhibitor discovery, the review places chemical proteomic guidance as essential for advancing compounds such as ABX-1431 and PF-04457845.

3. 3.
   Switch to photoresponsive bio-orthogonal lipids when transport and release questions cannot be answered by activity-based probes

   Address transport, release, uptake, and interaction-partner questions with spatiotemporal control.

   The abstract explicitly states that activity-based probes cannot capture these lipid processes in a spatiotemporally controlled manner, motivating a change in tool class.

4. 4.
   Use genetically encoded sensors for real-time monitoring across cultured neurons, brain slices, and in vivo mouse models

   Directly monitor endocannabinoid release dynamics with high spatiotemporal resolution.

   The review presents sensors as the tool class that enables real-time monitoring after earlier perturbation and visualization approaches establish enzyme and lipid-control context.