Tracking chromatin state changes using nanoscale photo-proximity labelling.

Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects1,2. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health1. However, in the complex environment of the nucleus, it is challenging to determine protein-protein interactions owing to low abundance, transient or multivalent binding and a lack of technologies that are able to interrogate these interactions without disrupting the protein-binding surface under study3. Here, we describe a method for the traceless incorporation of iridium-photosensitizers into the nuclear micro-environment using engineered split inteins. These Ir-catalysts can activate diazirine warheads through Dexter energy transfer to form reactive carbenes within an approximately 10 nm radius, cross-linking with proteins in the immediate micro-environment (a process termed µMap) for analysis using quantitative chemoproteomics4. We show that this nanoscale proximity-labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. µMap improves our fundamental understanding of nuclear protein-protein interactions and, in doing so, is expected to have a significant effect on the field of epigenetic drug discovery in both academia and industry.

Creative approaches using proximity labeling to gain new biological insights.

At its most fundamental level, life is a collection of synchronized cellular processes driven by interactions among biomolecules. Proximity labeling has emerged as a powerful technique to capture these interactions in native settings, revealing previously unexplored elements of biology. This review highlights recent developments in proximity labeling, focusing on methods that push the fundamental technologies beyond the classic bait-prey paradigm, such as RNA-protein interactions, ligand/small-molecule-protein interactions, cell surface protein interactions, and subcellular protein trafficking. The advancement of proximity labeling methods to address different biological problems will accelerate our understanding of the complex biological systems that make up life.

Radius measurement via super-resolution microscopy enables the development of a variable radii proximity labeling platform.

The elucidation of protein interaction networks is critical to understanding fundamental biology as well as developing new therapeutics. Proximity labeling platforms (PLPs) are state-of-the-art technologies that enable the discovery and delineation of biomolecular networks through the identification of protein-protein interactions. These platforms work via catalytic generation of reactive probes at a biological region of interest; these probes then diffuse through solution and covalently "tag" proximal biomolecules. The physical distance that the probes diffuse determines the effective labeling radius of the PLP and is a critical parameter that influences the scale and resolution of interactome mapping. As such, by expanding the degrees of labeling resolution offered by PLPs, it is possible to better capture the various size scales of interactomes. At present, however, there is little quantitative understanding of the labeling radii of different PLPs. Here, we report the development of a superresolution microscopy-based assay for the direct quantification of PLP labeling radii. Using this assay, we provide direct extracellular measurements of the labeling radii of state-of-the-art antibody-targeted PLPs, including the peroxidase-based phenoxy radical platform (269 ± 41 nm) and the high-resolution iridium-catalyzed µMap technology (54 ± 12 nm). Last, we apply these insights to the development of a molecular diffusion-based approach to tuning PLP resolution and introduce a new aryl-azide-based µMap platform with an intermediate labeling radius (80 ± 28 nm).

Small molecule photocatalysis enables drug target identification via energy transfer.

Over half of new therapeutic approaches fail in clinical trials due to a lack of target validation. As such, the development of new methods to improve and accelerate the identification of cellular targets, broadly known as target ID, remains a fundamental goal in drug discovery. While advances in sequencing and mass spectrometry technologies have revolutionized drug target ID in recent decades, the corresponding chemical-based approaches have not changed in over 50 y. Consigned to outdated stoichiometric activation modes, modern target ID campaigns are regularly confounded by poor signal-to-noise resulting from limited receptor occupancy and low crosslinking yields, especially when targeting low abundance membrane proteins or multiple protein target engagement. Here, we describe a broadly general platform for photocatalytic small molecule target ID, which is founded upon the catalytic amplification of target-tag crosslinking through the continuous generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead tag from the small molecule ligand, catalytic signal amplification results in unprecedented levels of target enrichment, enabling the quantitative target and off target ID of several drugs including (+)-JQ1, paclitaxel (Taxol), dasatinib (Sprycel), as well as two G-protein-coupled receptors-ADORA2A and GPR40.

Targeting Recycling Endosomes to Potentiate mRNA Lipid Nanoparticles.

mRNA lipid nanoparticles (LNPs) have emerged as powerful modalities for gene therapies to control cancer and infectious and immune diseases. Despite the escalating interest in mRNA-LNPs over the past few decades, endosomal entrapment of delivered mRNAs vastly impedes therapeutic developments. In addition, the molecular mechanism of LNP-mediated mRNA delivery is poorly understood to guide further improvement through rational design. To tackle these challenges, we characterized LNP-mediated mRNA delivery using a library of small molecules targeting endosomal trafficking. We found that the expression of delivered mRNAs is greatly enhanced via inhibition of endocytic recycling in cells and in live mice. One of the most potent small molecules, endosidine 5 (ES5), interferes with recycling endosomes through Annexin A6, thereby promoting the release and expression of mRNA into the cytoplasm. Together, these findings suggest that targeting endosomal trafficking with small molecules is a viable strategy to potentiate the efficacy of mRNA-LNPs.

Metallaphotoredox: The Merger of Photoredox and Transition Metal Catalysis.

The merger of photoredox catalysis with transition metal catalysis, termed metallaphotoredox catalysis, has become a mainstay in synthetic methodology over the past decade. Metallaphotoredox catalysis has combined the unparalleled capacity of transition metal catalysis for bond formation with the broad utility of photoinduced electron- and energy-transfer processes. Photocatalytic substrate activation has allowed the engagement of simple starting materials in metal-mediated bond-forming processes. Moreover, electron or energy transfer directly with key organometallic intermediates has provided novel activation modes entirely complementary to traditional catalytic platforms. This Review details and contextualizes the advancements in molecule construction brought forth by metallaphotocatalysis.

µMap Photoproximity Labeling Enables Small Molecule Binding Site Mapping.

The characterization of ligand binding modes is a crucial step in the drug discovery process and is especially important in campaigns arising from phenotypic screening, where the protein target and binding mode are unknown at the outset. Elucidation of target binding regions is typically achieved by X-ray crystallography or photoaffinity labeling (PAL) approaches; yet, these methods present significant challenges. X-ray crystallography is a mainstay technique that has revolutionized drug discovery, but in many cases structural characterization is challenging or impossible. PAL has also enabled binding site mapping with peptide- and amino-acid-level resolution; however, the stoichiometric activation mode can lead to poor signal and coverage of the resident binding pocket. Additionally, each PAL probe can have its own fragmentation pattern, complicating the analysis by mass spectrometry. Here, we establish a robust and general photocatalytic approach toward the mapping of protein binding sites, which we define as identification of residues proximal to the ligand binding pocket. By utilizing a catalytic mode of activation, we obtain sets of labeled amino acids in the proximity of the target protein binding site. We use this methodology to map, in vitro, the binding sites of six protein targets, including several kinases and molecular glue targets, and furthermore to investigate the binding site of the STAT3 inhibitor MM-206, a ligand with no known crystal structure. Finally, we demonstrate the successful mapping of drug binding sites in live cells. These results establish µMap as a powerful method for the generation of amino-acid- and peptide-level target engagement data.

Photoproximity Labeling of Sialylated Glycoproteins (GlycoMap) Reveals Sialylation-Dependent Regulation of Ion Transport.

Sialylation, the addition of sialic acid to glycans, is a crucial post-translational modification of proteins, contributing to neurodevelopment, oncogenesis, and immune response. In cancer, sialylation is dramatically upregulated. Yet, the functional biochemical consequences of sialylation remain mysterious. Here, we establish a µMap proximity labeling platform that utilizes metabolically inserted azidosialic acid to introduce iridium-based photocatalysts on sialylated cell-surface glycoproteins as a means to profile local microenvironments across the sialylated proteome. In comparative experiments between primary cervical cells and a cancerous cell line (HeLa), we identify key differences in both the global sialome and proximal proteins, including solute carrier proteins that regulate metabolite and ion transport. In particular, we show that cell-surface interactions between receptors trafficking ethanolamine and zinc are sialylation-dependent and impact intracellular metabolite levels. These results establish a µMap method for interrogating proteoglycan function and support a role for sialylated glycoproteins in regulating cell-surface transporters.

µMap-Red: Proximity Labeling by Red Light Photocatalysis.

Modern proximity labeling techniques have enabled significant advances in understanding biomolecular interactions. However, current tools primarily utilize activation modes that are incompatible with complex biological environments, limiting our ability to interrogate cell- and tissue-level microenvironments in animal models. Here, we report µMap-Red, a proximity labeling platform that uses a red-light-excited SnIV chlorin e6 catalyst to activate a phenyl azide biotin probe. We validate µMap-Red by demonstrating photonically controlled protein labeling in vitro through several layers of tissue, and we then apply our platform in cellulo to label EGFR microenvironments and validate performance with STED microscopy and quantitative proteomics. Finally, to demonstrate labeling in a complex biological sample, we deploy µMap-Red in whole mouse blood to profile erythrocyte cell-surface proteins. This work represents a significant methodological advance toward light-based proximity labeling in complex tissue environments and animal models.

Reactive intermediates for interactome mapping.

The interactions of biomolecules underpin all cellular processes, and the understanding of their dynamic interplay can lead to significant advances in the treatment of disease through the identification of novel therapeutic strategies. Protein-protein interactions (PPIs) in particular play a vital role within this arena, providing the basis for the majority of cellular signalling pathways. Despite their great importance, the elucidation of weak or transient PPIs that cannot be identified by immunoprecipitation remains a significant challenge, particularly in a disease relevant cellular environment. Recent approaches towards this goal have utilized the in situ generation of high energy intermediates that cross-link with neighboring proteins, providing a snapshot of the biomolecular makeup of the local area or microenvironment, termed the interactome. In this tutorial review, we discuss these reactive intermediates, how they are generated, and the impact they have had on the discovery of new biology. Broadly, we believe this strategy has the potential to significantly accelerate our understanding of PPIs and how they affect cellular physiology.

Selective C-F Functionalization of Unactivated Trifluoromethylarenes.

Fluorinated organic molecules are pervasive within the pharmaceutical and agrochemical industries due to the range of structural and physicochemical properties that fluorine imparts. Currently, the most abundant methods for the synthesis of the aryl-CF2 functionality have relied on the deoxyfluorination of ketones and aldehydes using expensive and poorly atom economical reagents. Here, we report a general method for the synthesis of aryl-CF2R and aryl-CF2H compounds through activation of the corresponding trifluoromethyl arene precursors. This strategy is enabled by an endergonic electron transfer event that provides access to arene radical anions that lie outside of the catalyst reduction potential. Fragmentation of these reactive intermediates delivers difluorobenzylic radicals that can be intercepted by abundant alkene feedstocks or a hydrogen atom to provide a diverse array of difluoalkylaromatics.

Catalytic Strategy for Regioselective Arylethylamine Synthesis.

A mild, modular, and practical catalytic system for the synthesis of the highly privileged phenethylamine pharmacophore is reported. Using a unique combination of organic catalysts to promote the transfer of electrons and hydrogen atoms, this system performs direct hydroarylation of vinyl amine derivatives with a wide range of aryl halides (including aryl chlorides). This general and highly chemoselective protocol delivers a broad range of arylethylamine products with complete regiocontrol. The utility of this process is highlighted by its scalability and the modular synthesis of an array of bioactive small molecules.

Radical Hydroarylation of Functionalized Olefins and Mechanistic Investigation of Photocatalytic Pyridyl Radical Reactions.

We report the photoredox alkylation of halopyridines using functionalized alkene and alkyne building blocks. Selective single-electron reduction of the halogenated pyridines provides the corresponding heteroaryl radicals, which undergo anti-Markovnikov addition to the alkene substrates. The system is shown to be mild and tolerant of a variety of alkene and alkyne subtypes. A combination of computational and experimental studies support a mechanism involving proton-coupled electron transfer followed by medium-dependent alkene addition and rapid hydrogen atom transfer mediated by a polarity-reversal catalyst.

Interrogating Pd(II) Anion Metathesis Using a Bifunctional Chemical Probe: A Transmetalation Switch.

Ligand metathesis of Pd(II) complexes is mechanistically essential for cross-coupling. We present a study of halide→OH anion metathesis of (Ar)PdII complexes using vinylBPin as a bifunctional chemical probe with Pd(II)-dependent cross-coupling pathways. We identify the variables that profoundly impact this event and allow control to be leveraged. This then allows control of cross-coupling pathways via promotion or inhibition of organoboron transmetalation, leading to either Suzuki-Miyaura or Mizoroki-Heck products. We show how this transmetalation switch can be used to synthetic gain in a cascade cross-coupling/Diels-Alder reaction, delivering borylated or non-borylated carbocycles, including steroid-like scaffolds.

Strategy for Conditional Orthogonal Sequential CuAAC Reactions Using a Protected Aromatic Ynamine.

A method for conditional control of orthogonal sequential Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reactions is reported. The inherent reactivity of an aromatic ynamine is controlled by a silyl protecting group that allows the selective CuAAC reaction of less reactive alkynes. Alternatively, the same protected ynamine undergoes selective CuAAC reaction via silyl deprotection in situ to give the ynamine click products. This allows complete orthogonal control of dialkyne systems and provides a unifying strategy for chemoselective CuAAC ligations in multialkyne/azide systems.

Determining the Origin of Rate-Independent Chemoselectivity in CuAAC Reactions: An Alkyne-Specific Shift in Rate-Determining Step.

We report a kinetic and spectroscopic analysis of alkyne-dependent chemoselectivity in the copper-catalyzed azide-alkyne click (CuAAC) reaction. Studies of six alkyne subtypes reveal that the rate-determining step (RDS) of an aromatic ynamine class is shifted from acetylide formation to the azide ligation/migratory insertion event allowing chemoselectivity independent of overall rate.

Speciation control during Suzuki-Miyaura cross-coupling of haloaryl and haloalkenyl MIDA boronic esters.

Boronic acid solution speciation can be controlled during the Suzuki-Miyaura cross-coupling of haloaryl N-methyliminodiacetic acid (MIDA) boronic esters to enable the formal homologation of boronic acid derivatives. The reaction is contingent upon control of the basic biphase and is thermodynamically driven: temperature control provides highly chemoselective access to either BMIDA adducts at room temperature or boronic acid pinacol ester (BPin) products at elevated temperature. Control experiments and solubility analyses have provided some insight into the mechanistic operation of the formal homologation process.

A modular synthesis of functionalised phenols enabled by controlled boron speciation.

A modular synthesis of functionalised biaryl phenols from two boronic acid derivatives has been developed via one-pot Suzuki-Miyaura cross-coupling, chemoselective control of boron solution speciation to generate a reactive boronic ester in situ, and oxidation. The utility of this method has been further demonstrated by application in the synthesis of drug molecules and components of organic electronics, as well as within iterative cross-coupling.

Tandem Chemoselective Suzuki-Miyaura Cross-Coupling Enabled by Nucleophile Speciation Control.

Control of boronic acid speciation is presented as a strategy to achieve nucleophile chemoselectivity in the Suzuki-Miyaura reaction. Combined with simultaneous control of oxidative addition and transmetalation, this enables chemoselective formation of two C-C bonds in a single operation, providing a method for the rapid preparation of highly functionalized carbogenic frameworks.

Chemoselective boronic ester synthesis by controlled speciation.

Control of boronic acid solution speciation is presented as a new strategy for the chemoselective synthesis of boronic esters. Manipulation of the solution equilibria within a cross-coupling milieu enables the formal homologation of aryl and alkenyl boronic acid pinacol esters. The generation of a new, reactive boronic ester in the presence of an active palladium catalyst also facilitates streamlined iterative catalytic C-C bond formation and provides a method for the controlled oligomerization of sp(2) -hybridized boronic esters.