Therapeutic potential of cis-targeting bispecific antibodies.

The growing clinical success of bispecific antibodies (bsAbs) has led to rapid interest in leveraging dual targeting in order to generate novel modes of therapeutic action beyond mono-targeting approaches. While bsAbs that bind targets on two different cells (trans-targeting) are showing promise in the clinic, the co-targeting of two proteins on the same cell surface through cis-targeting bsAbs (cis-bsAbs) is an emerging strategy to elicit new functionalities. This includes the ability to induce proximity, enhance binding to a target, increase target/cell selectivity, and/or co-modulate function on the cell surface with the goal of altering, reversing, or eradicating abnormal cellular activity that contributes to disease. In this review, we focus on the impact of cis-bsAbs in the clinic, their emerging applications, and untangle the intricacies of improving bsAb discovery and 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.

High-resolution photocatalytic mapping of SARS-CoV-2 spike interactions on the cell surface.

Identifying virus-host interactions on the cell surface can improve our understanding of viral entry and pathogenesis. SARS-CoV-2, the causative agent of the COVID-19 disease, uses ACE2 as a receptor to enter cells. Yet the full repertoire of cell surface proteins that contribute to viral entry is unknown. We developed a photocatalyst-based viral-host protein microenvironment mapping platform (ViraMap) to probe the molecular neighborhood of the SARS-CoV-2 spike protein on the human cell surface. Application of ViraMap to ACE2-expressing cells captured ACE2, the established co-receptor NRP1, and several novel cell surface proteins. We systematically analyzed the relevance of these candidate proteins to SARS-CoV-2 entry by knockdown and overexpression approaches in pseudovirus and authentic infection models and identified PTGFRN and EFNB1 as bona fide viral entry factors. Our results highlight additional host targets that participate in SARS-CoV-2 infection and showcase ViraMap as a powerful platform for defining viral interactions on the cell surface.

Targeted proximity-labelling of protein tyrosines via flavin-dependent photoredox catalysis with mechanistic evidence for a radical-radical recombination pathway.

Flavin-based photocatalysts such as riboflavin tetraacetate (RFT) serve as a robust platform for light-mediated protein labelling via phenoxy radical-mediated tyrosine-biotin phenol coupling on live cells. To gain insight into this coupling reaction, we conducted detailed mechanistic analysis for RFT-photomediated activation of phenols for tyrosine labelling. Contrary to previously proposed mechanisms, we find that the initial covalent binding step between the tag and tyrosine is not radical addition, but rather radical-radical recombination. The proposed mechanism may also explain the mecha-nism of other reported tyrosine-tagging approaches. Competitive kinetics experiments show that phenoxyl radicals are generated with several reactive intermediates in the proposed mechanism-primarily with the excited riboflavin-photocatalyst or singlet oxygen-and these multiple pathways for phenoxyl radical generation from phenols increase the likelihood of radical-radical recombination.

An electroaffinity labelling platform for chemoproteomic-based target identification.

Target identification involves deconvoluting the protein target of a pharmacologically active, small-molecule ligand, a process that is critical for early drug discovery yet technically challenging. Photoaffinity labelling strategies have become the benchmark for small-molecule target deconvolution, but covalent protein capture requires the use of high-energy ultraviolet light, which can complicate downstream target identification. Thus, there is a strong demand for alternative technologies that allow for controlled activation of chemical probes to covalently label their protein target. Here we introduce an electroaffinity labelling platform that leverages the use of a small, redox-active diazetidinone functional group to enable chemoproteomic-based target identification of pharmacophores within live cell environments. The underlying discovery to enable this platform is that the diazetidinone can be electrochemically oxidized to reveal a reactive intermediate useful for covalent modification of proteins. This work demonstrates the electrochemical platform to be a functional tool for drug-target identification.

Targeted activation in localized protein environments via deep red photoredox catalysis.

State-of-the-art photoactivation strategies in chemical biology provide spatiotemporal control and visualization of biological processes. However, using high-energy light (λ < 500 nm) for substrate or photocatalyst sensitization can lead to background activation of photoactive small-molecule probes and reduce its efficacy in complex biological environments. Here we describe the development of targeted aryl azide activation via deep red-light (λ = 660 nm) photoredox catalysis and its use in photocatalysed proximity labelling. We demonstrate that aryl azides are converted to triplet nitrenes via a redox-centric mechanism and show that its spatially localized formation requires both red light and a photocatalyst-targeting modality. This technology was applied in different colon cancer cell systems for targeted protein environment labelling of epithelial cell adhesion molecule (EpCAM). We identified a small subset of proteins with previously known and unknown association to EpCAM, including CDH3, a clinically relevant protein that shares high tumour-selective expression with EpCAM.

Proteomic mapping of intercellular synaptic environments via flavin-dependent photoredox catalysis.

Receptor-ligand interactions play essential signaling roles within intercellular contact regions. This is particularly important within the context of the immune synapse where protein communication at the surface of physically interacting T cells and antigen-presenting cells regulate downstream immune signaling responses. To identify protein microenvironments within immunological synapses, we combined a flavin-dependent photocatalytic labeling strategy with quantitative mass spectrometry-based proteomics. Using α-PD-L1 or α-PD-1 single-domain antibody (VHH)-based photocatalyst targeting modalities, we profiled protein microenvironments within the intercellular region of an immune synapse-forming co-culture system. In addition to enrichment of both PD-L1 and PD-1 with either targeting modality, we also observed enrichment of both known immune synapse residing receptor-ligand pairs and surface proteins, as well as previously unknown synapse residing proteins.

Nanoscale Mapping of EGFR and c-MET Protein Environments on Lung Cancer Cell Surfaces via Therapeutic Antibody Photocatalyst Conjugates.

Receptor tyrosine kinases are involved in essential signaling roles that impact cell growth, differentiation, and proliferation. The overexpression or mutation of these proteins can lead to aberrant signaling that has been directly linked to a number of diseases including cancer cell formation and progression. This has led to intense clinical focus on modulating RTK activity through direct targeting of signaling activity or cell types harboring aberrant RTK behavior. In particular, epidermal growth factor receptor (EGFR) has attracted intense clinical attention due to the impact of inhibiting this RTK on tumor growth. However, mutations incurred through targeting EGFR have led to therapeutic resistance that involves not only direct mutations to the EGFR protein but also the involvement of other RTKs, such as c-MET, that can overcome therapeutic-based EGFR inhibition effects. This has, not surprisingly, led to co-targeting strategies of RTKs such as EGFR and c-MET to overcome resistance mechanisms. While the ability to co-target these proteins has led to success in the clinic, a more comprehensive understanding of their proximal environments, particularly in the context of therapeutic modalities, could further enhance both our understanding of their signaling biology and provide additional avenues for targeting these surface proteins. Thus, to investigate EGFR and c-MET protein microenvironments, we utilized our recently developed iridium photocatalyst-based microenvironment mapping technology to catalog EGFR and c-MET surface environments on non-small cell lung cancer cell lines. Through this approach, we enriched EGFR and c-MET from the cell surface and identified known EGFR and c-MET associators as well as previously unidentified proximal proteins.

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.

Detection of cell-cell interactions via photocatalytic cell tagging.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.