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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.
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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.
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Péptidos , Proteínas , Ligandos , Proteínas/química , Sitios de Unión , Péptidos/química , Unión ProteicaRESUMEN
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.
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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.
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COVID-19 , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2 , Glicoproteína de la Espiga del Coronavirus , Proteínas Virales/metabolismo , Unión ProteicaRESUMEN
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.
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Descubrimiento de Drogas , Proteínas , Proteínas/metabolismo , Etiquetas de Fotoafinidad/química , Ligandos , FarmacóforoRESUMEN
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.
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Neoplasias del Colon , Luz , Humanos , Molécula de Adhesión Celular Epitelial , CatálisisRESUMEN
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.
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Antígeno B7-H1 , Receptor de Muerte Celular Programada 1 , Ligandos , Proteómica , CatálisisRESUMEN
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.
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Sistemas de Liberación de Medicamentos , Transferencia de Energía , Proteómica , Descubrimiento de Drogas , Espectrometría de MasasRESUMEN
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.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Microambiente TumoralRESUMEN
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.
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Antígeno B7-H1 , Leucocitos Mononucleares , Comunicación Celular , Flavinas , InmunoterapiaRESUMEN
Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.
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COVID-19/prevención & control , Centro Germinal/inmunología , Glicoproteína de la Espiga del Coronavirus/administración & dosificación , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Envejecimiento , Animales , Anticuerpos Antivirales/sangre , Linfocitos B/inmunología , Linfocitos B/metabolismo , COVID-19/virología , Centro Germinal/citología , Centro Germinal/metabolismo , Inmunidad Humoral , Ratones , Ratones Endogámicos C57BL , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Vacunación , Vacunas de Subunidad/inmunologíaRESUMEN
As academia and industry push the boundaries of chemical biology more and more from basic science into impacting human health, we asked experts in the field what uniquely positions chemical biology as a translational science, how and when to maximize its potential, and where the field is headed. We also reflect personally on how chemical biology has impacted our careers in industry and academia.
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Laboratorios , Péptidos/metabolismo , Medicina de Precisión , Humanos , Péptidos/químicaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic that has led to a global economic disruption and collapse. With several ongoing efforts to develop vaccines and treatments for COVID-19, understanding the molecular interaction between the coronavirus, host cells, and the immune system is critical for effective therapeutic interventions. Greater insight into these mechanisms will require the contribution and combination of multiple scientific disciplines including the techniques and strategies that have been successfully deployed by chemical biology to tease apart complex biological pathways. We highlight in this review well-established strategies and methods to study coronavirus-host biophysical interactions and discuss the impact chemical biology will have on understanding these interactions at the molecular level.
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Multicellular organisms depend on physical cell-cell interactions to control physiological processes such as tissue formation, neurotransmission and immune response. These intercellular binding events can be both highly dynamic in their duration and complex in their composition, involving the participation of many different surface and intracellular biomolecules. Untangling the intricacy of these interactions and the signaling pathways they modulate has greatly improved insight into the biological processes that ensue upon cell-cell engagement and has led to the development of protein- and cell-based therapeutics. The importance of monitoring physical cell-cell interactions has inspired the development of several emerging approaches that effectively interrogate cell-cell interfaces with molecular-level detail. Specifically, the merging of chemistry- and biology-based technologies to deconstruct the complexity of cell-cell interactions has provided new avenues for understanding cell-cell interaction biology and opened opportunities for therapeutic development.
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Biología Celular , Comunicación Celular/fisiología , Animales , Comunicación Celular/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Light-powered catalysis has found broad utility as a chemical transformation strategy, with widespread impact on energy, environment, drug discovery and human health. A noteworthy application impacting human health is light-induced sensitization of cofactors for photodynamic therapy in cancer treatment. The clinical adoption of this photosensitization approach has inspired the search for other photochemical methods, such as photoredox catalysis, to influence biological discovery. Over the past decade, light-mediated catalysis has enabled the discovery of valuable synthetic transformations, propelling it to become a highly utilized chemical synthesis strategy. The reaction components required to achieve a photoredox reaction are identical to photosensitization (catalyst, light source and substrate), making it ideally suited for probing biological environments. In this Review, we discuss the therapeutic application of photosensitization and advancements made in developing next-generation catalysts. We then highlight emerging uses of photoredox catalytic methods for protein bioconjugation and probing complex cellular environments in living cells.
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Despite the growing use of visible-light photochemistry in both chemistry and biology, no general low-heat photoreactor for use across these different disciplines exists. Herein, we describe the design and use of a standardized photoreactor for visible-light-driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we performed photoredox reactions across multiple visible light wavelengths, a high-throughput photocatalytic cross-coupling reaction, and inâ vitro labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings.
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Biotina/análisis , Luz , Fotobiorreactores , Tiramina/química , Catálisis , Línea Celular Tumoral , Diseño de Equipo , Humanos , Estructura Molecular , Procesos Fotoquímicos , Tiramina/análogos & derivados , Tiramina/síntesis químicaRESUMEN
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.
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Antígeno B7-H1/metabolismo , Membrana Celular/metabolismo , Microambiente Celular , Linfocitos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Catálisis , Membrana Celular/efectos de la radiación , Transferencia de Energía , Humanos , Células Jurkat , Linfocitos/efectos de la radiación , Metano/análogos & derivados , Metano/química , Metano/efectos de la radiación , Procesos Fotoquímicos , Rayos UltravioletaRESUMEN
Increasing evidence indicates CD4+ T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that will be presented by human leukocyte antigen class II molecules (HLA-II), hindering efforts to optimally target them therapeutically. Obstacles include inaccurate peptide-binding prediction and unsolved complexities of the HLA-II pathway. To address these challenges, we developed an improved technology for discovering HLA-II binding motifs and conducted a comprehensive analysis of tumor ligandomes to learn processing rules relevant in the tumor microenvironment. We profiled >40 HLA-II alleles and showed that binding motifs were highly sensitive to HLA-DM, a peptide-loading chaperone. We also revealed that intratumoral HLA-II presentation was dominated by professional antigen-presenting cells (APCs) rather than cancer cells. Integrating these observations, we developed algorithms that accurately predicted APC ligandomes, including peptides from phagocytosed cancer cells. These tools and biological insights will enable improved HLA-II-directed cancer therapies.
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Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Cáncer/inmunología , Mapeo Epitopo/métodos , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Inmunoterapia/métodos , Espectrometría de Masas/métodos , Neoplasias/terapia , Algoritmos , Alelos , Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Conjuntos de Datos como Asunto , Antígenos HLA/genética , Antígenos HLA-D/metabolismo , Humanos , Neoplasias/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Programas InformáticosRESUMEN
Lower glycolysis involves a series of reversible reactions, which interconvert intermediates that also feed anabolic pathways. 3-phosphoglycerate (3-PG) is an abundant lower glycolytic intermediate that feeds serine biosynthesis via the enzyme phosphoglycerate dehydrogenase, which is genomically amplified in several cancers. Phosphoglycerate mutase 1 (PGAM1) catalyzes the isomerization of 3-PG into the downstream glycolytic intermediate 2-phosphoglycerate (2-PG). PGAM1 needs to be histidine phosphorylated to become catalytically active. We show that the primary PGAM1 histidine phosphate donor is 2,3-bisphosphoglycerate (2,3-BPG), which is made from the glycolytic intermediate 1,3-bisphosphoglycerate (1,3-BPG) by bisphosphoglycerate mutase (BPGM). When BPGM is knocked out, 1,3-BPG can directly phosphorylate PGAM1. In this case, PGAM1 phosphorylation and activity are decreased, but nevertheless sufficient to maintain normal glycolytic flux and cellular growth rate. 3-PG, however, accumulates, leading to increased serine synthesis. Thus, one biological function of BPGM is controlling glycolytic intermediate levels and thereby serine biosynthetic flux.