Current advances in photocatalytic proximity labeling

Abstract

Understanding the intricate network of biomolecular interactions that govern cellular processes is a fundamental pursuit in biology. Over the past decade, photocatalytic proximity labeling has emerged as one of the most powerful and versatile techniques for studying these interactions as well as uncovering subcellular trafficking patterns, drug mechanisms of action, and basic cellular physiology. In this article, we review the basic principles, methodologies, and applications of photocatalytic proximity labeling as well as examine its modern development into currently available platforms. We also discuss recent key studies that have successfully leveraged these technologies and importantly highlight current challenges faced by the field. Together, this review seeks to underscore the potential of photocatalysis in proximity labeling for enhancing our understanding of cell biology while also providing perspective on technological advances needed for future discovery.

Figure 1.. General principles and activation modes in proximity labeling.

(A) A catalyst (cat) is localized to a biomolecule of interest to generate a reactive intermediate from an inert, pre-reactive species. This reactive probe has a limited range of diffusion such that only proximal biomolecules are covalently tagged for further isolation, identification, and characterization. (B) Chemically triggered proximity labeling, as demonstrated by APEX/HRP peroxidase enzyme catalysts which utilize H₂O₂ to generate phenoxyl radicals from a phenol precursor. (C) Selected examples of photocatalytic proximity labeling manifolds, which are activated by various visible light sources to generate reactive intermediates (radicals, singlet oxygen, carbenes, etc).

Figure 2.. Biological insights generated from cell-surface interactome profiling with photocatalytic proximity labeling.

(A) Cell-surface biomolecules can be probed with a variety of photocatalyst-conjugated targeting agents, including antibodies, recombinant viral proteins, intact pathogens/phages, and small biologics (cytokines, peptides, etc.). Additionally, appropriate catalyst localization can be used to profile synaptic interactions between two cells. (B) Bioorthogonal click chemistry and metabolic incorporation of modified sugars into native glycoproteins enables photoproximity profiling of sialylation-dependent changes in protein microenvironments (GlycoMap).

Figure 3.. Photocatalytic proximity labeling platforms for intracellular interactome profiling.

(A) Two genetically-encodable proximity labeling systems, miniSOG (miniature singlet oxygen generator, PDB 6GPU) and LOV (light oxygen voltage domain, PDB 2Z6C) utilize an engineered flavin-binding protein derived from Arabidopsis thaliana phototropin 2. (B) Flavin cofactors generate singlet oxygen (¹O₂) through blue light excitation and subsequent energy and/or electron transfer to molecular oxygen. (C) Photogenerated reactive oxygen species induce oxidation for labeling nucleic acids and proteins with appropriate capture probes. (D) Photocatalysts can also be chemoenzymatically installed into HaloTag, which self-alkylates molecular payloads containing a chloroalkane linker. (E) Photocatalyst incorporation facilitated through solid phase peptide synthesis of catalyst-containing inteins, which can undergo trans-splicing with a genetically-encodable target protein. In this example, Ir photocatalysts were incorporated into histone tails for subsequent μMap profiling of both wild-type and oncohistone mutations for comparative proteomic profiling.

Figure 4.. Photocatalytic proximity labeling enhances intracellular target identification and binding site mapping of small molecule drugs.

(A) Target and mechanism-of-action identification is key for clinical drug development. (B) Traditional photoaffinity labeling (PAL) probes utilize stoichiometric identification of targets via UV activation of photocrosslinkers, which predominantly undergo unproductive reactivity with water. (C) Appending photocatalysts to small molecules enables multiple labeling events to occur on a target of interest, significantly enhancing signal. (D, E) Quantitative chemoproteomic enrichment of ADORA2A with a diazirine-functionalized (D) or Iridium catalyst-conjugated (E) SCH58261. (F) Photocatalytic labeling and mass spectrometry-based workflow for identifying the protein binding site(s) of small molecules. (G) STAT3 inhibitor MM-206 exhibits an unknown binding mode, which was identified via μMap as an allosteric inhibitor in the coiled-coil domain (CCD) of STAT3 (PDB 6TLC).

Figure 5.. Radius modulation and new controlled activation modes in photocatalytic proximity labeling.

(A) The resolution of different proximity labeling systems can be measured in vitro using protein-coated glass coverslips and super-resolution microscopy visualization of labeled clusters. Both carbene and phenoxyl radical-based labeling systems were compared in microscopy and quantitative chemoproteomics, illustrating the resolution and spatial selectivity differences with both chemistries. (B) The diffusion coefficient of reactive species can be adjusted by increasing the molecular weight of probes, resulting in higher resolution labeling as observed by EGFR labeling on the surface of A549 cells. (C) Photocatalytic proximity labeling can be “activated” through an appropriate quencher coupled with structure-switching DNA aptamers, allowing labeling to occur in user-defined microenvironments.

Figure 6.. Red-shifted photoredox catalysis modes for proximity labeling.

(A) Current activation modes for lightbased proximity labeling are shorter in wavelength and exhibit poor tissue penetration. Longer wavelengths of light can address this need for in vivo proximity labeling applications. (B) μMap-Red photocatalytic activation of aryl azides through single electron transfer (SET) to generate aminyl radicals. (C) Targeted aryl azide activation with Osmium photocatalysts through SET to furnish a triplet nitrene reactive intermediate.