Elucidating the Cell Surfaceome to Accelerate Cancer Drug Development.

SUMMARY: Cell surface proteins represent ideal therapeutic targets because of their accessibility to antibodies, T cell-directed therapies, and radiotherapies, but there are only 25 therapeutically relevant cell surface targets for which cancer therapies are approved in the United States or European Union. This commentary calls for intensified research into mapping the universe of cell surface proteins - the cell surfaceome - in order to accelerate cancer drug development.

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.

Photochemical Identification of Auxiliary Severe Acute Respiratory Syndrome Coronavirus 2 Host Entry Factors Using µMap.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of the COVID-19 pandemic, remains a global medical problem. Angiotensin-converting enzyme 2 (ACE2) was identified as the primary viral entry receptor, and transmembrane serine protease 2 primes the spike protein for membrane fusion. However, ACE2 expression is generally low and variable across tissues, suggesting that auxiliary receptors facilitate viral entry. Identifying these factors is critical for understanding SARS-Cov-2 pathophysiology and developing new countermeasures. However, profiling host-virus interactomes involves extensive genetic screening or complex computational predictions. Here, we leverage the photocatalytic proximity labeling platform µMap to rapidly profile the spike interactome in human cells and identify eight novel candidate receptors. We systemically validate their functionality in SARS-CoV-2 pseudoviral uptake assays with both Wuhan and Delta spike variants and show that dual expression of ACE2 with either neuropilin-2, ephrin receptor A7, solute carrier family 6 member 15, or myelin and lymphocyte protein 2 significantly enhances viral uptake. Collectively, our data show that SARS-CoV-2 synergistically engages several host factors for cell entry and establishes µMap as a powerful tool for rapidly interrogating host-virus interactomes.

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.

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).

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.

Microenvironment mapping via Dexter energy transfer on immune cells.

Many disease pathologies can be understood through the elucidation of localized biomolecular networks, or microenvironments. To this end, enzymatic proximity labeling platforms are broadly applied for mapping the wider spatial relationships in subcellular architectures. However, technologies that can map microenvironments with higher precision have long been sought. Here, we describe a microenvironment-mapping platform that exploits photocatalytic carbene generation to selectively identify protein-protein interactions on cell membranes, an approach we term MicroMap (µMap). By using a photocatalyst-antibody conjugate to spatially localize carbene generation, we demonstrate selective labeling of antibody binding targets and their microenvironment protein neighbors. This technique identified the constituent proteins of the programmed-death ligand 1 (PD-L1) microenvironment in live lymphocytes and selectively labeled within an immunosynaptic junction.

Difluoromethane as a precursor to difluoromethyl borates.

Difluoromethane (CF2H2) is an ecologically-friendly refrigerant which holds promise as a source of CF2H-. However, the weak acidity (pKa = 35-41) and low stability of the conjugate base have prevented its utilization as a chemical feedstock. In this manuscript, we use a Lewis pair approach to deprotonate CF2H2 and capture CF2H- as R3B-CF2H- adducts. One reagent can be used as a base-free Suzuki reagent in palladium-mediated difluoromethylation, where CF2H- transfer is templated by precoordination to an azaborine derived R3B-CF2H- reagent.

The Difluoromethyl Group as a Masked Nucleophile: A Lewis Acid/Base Approach.

The difluoromethyl group (R-CF2H) imparts desirable pharmacokinetic properties to drug molecules and is commonly targeted as a terminal functional group that is not amenable to further modification. Deprotonation of widely available Ar-CF2H starting materials to expose nucleophilic Ar-CF2- synthons represents an unexplored, yet promising route to construct benzylic Ar-CF2-R linkages. Here we show that the combination of a Brønsted superbase with a weak Lewis acid enables deprotonation of Ar-CF2H groups and capture of reactive Ar-CF2- fragments. This route provides access to isolable and reactive Ar-CF2- synthons that react with a broad array of electrophiles at room temperature. The methodology is highly general in both electrophile and difluoromethyl (hetero)arene and can be applied directly to the synthesis of benzylic difluoromethylene (Ar-CF2-R) linkages, which are useful lipophilic and metabolically resistant replacements for benzylic linkages in medicinal chemistry.

Charge effects regulate reversible CO2 reduction catalysis.

Modular but geometrically constrained ligands were used to investigate the impact of key ligand design parameters (charge and bite angle) on CO2 hydrogenation and formic acid dehydrogenation activity. These studies yielded an optimized catalyst that achieved over 118 000 turnovers in CO2 hydrogenation, 247 000 turnovers in HCO2H dehydrogenation, was applied in a hydrogen storage device used for 6 cycles of hydrogen storage/release without requiring changes in pH or solvent, and generated H2/CO2 gas at a pressure of 190 atm from formic acid.

Borazine-CF3- Adducts for Rapid, Room Temperature, and Broad Scope Trifluoromethylation.

A fluoroform-derived borazine CF3- transfer reagent is used to effect rapid nucleophilic reactions in the absence of additives, within minutes at 25 °C. Inorganic electrophiles spanning seven groups of the periodic table can be trifluoromethylated in high yield, including transition metals used for catalytic trifluoromethylation. Organic electrophiles included (hetero)arenes, enabling C-H and C-X trifluoromethylation reactions. Mechanistic analysis supports a dissociative mechanism for CF3- transfer, and cation modification afforded a reagent with enhanced stability.

Recyclable Trifluoromethylation Reagents from Fluoroform.

We present a strategy to rationally prepare CF3- transfer reagents at ambient temperature from HCF3. We demonstrate that a highly reactive CF3- adduct can be synthesized from alkali metal hydride, HCF3, and borazine Lewis acids in quantitative yield at room temperature. These nucleophilic reagents transfer CF3- to substrates without additional chemical activation, and after CF3 transfer, the free borazine is quantitatively regenerated. These features enable syntheses of popular nucleophilic, radical, and electrophilic trifluoromethylation reagents with complete recycling of the borazine Lewis acid.

Testing the Push-Pull Hypothesis: Lewis Acid Augmented N2 Activation at Iron.

We present a systematic investigation of the structural and electronic changes that occur in an Fe(0)-N2 unit (Fe(depe)2(N2); depe = 1,2-bis(diethylphosphino)ethane) upon the addition of exogenous Lewis acids. Addition of neutral boranes, alkali metal cations, and an Fe2+ complex increases the N-N bond activation (Δ νNN up to 172 cm-1), decreases the Fe(0)-N2 redox potential, polarizes the N-N bond, and enables -N protonation at uncommonly anodic potentials. These effects were rationalized using combined experimental and theoretical studies.

A Proton-Switchable Bifunctional Ruthenium Complex That Catalyzes Nitrile Hydroboration.

A new bifunctional pincer ligand framework bearing pendent proton-responsive hydroxyl groups was prepared and metalated with Ru(II) and subsequently isolated in four discrete protonation states. Stoichiometric reactions with H2 and HBPin showed facile E-H (E = H or BPin) activation across a Ru(II)-O bond, providing access to unusual Ru-H species with strong interactions with neighboring proton and boron atoms. These complexes were found to promote the catalytic hydroboration of ketones and nitriles under mild conditions, and the activity was highly dependent on the ligand's protonation state. Mechanistic experiments revealed a crucial role of the pendent hydroxyl groups for catalytic activity.