Defining HLA-II Ligand Processing and Binding Rules with Mass Spectrometry Enhances Cancer Epitope Prediction.

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.

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target ID in recent decades, the corresponding chemical-based approaches have not changed in over 50 y. Consigned to outdated stoichiometric activation modes, modern target ID campaigns are regularly confounded by poor signal-to-noise resulting from limited receptor occupancy and low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.

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.

Strategies for monitoring cell-cell interactions.

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.

From Basic Biology Discovery to Translational Impact: The Boundless Roles of Chemical Biology.

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.

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.

Design of a Multiuse Photoreactor To Enable Visible-Light Photocatalytic Chemical Transformations and Labeling in Live Cells.

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 in 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.

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system.

Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate.

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.

Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils.

BACKGROUND: Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. OBJECTIVES: To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. METHODS: Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. RESULTS: Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1ß increased the expression of PLA2G10 by eosinophils. CONCLUSIONS: These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2α. The sPLA2-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.

A pan-specific antibody for direct detection of protein histidine phosphorylation.

Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems because of the lack of adequate research tools. We report the development of the first pan-phosphohistidine (pHis) antibody using a stable pHis mimetic as the hapten. This antibody was successfully used in ELISA, western blotting, dot blot assays and immunoprecipitation and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with MS analysis. We also observed that the amount of protein pHis in Escherichia coli lysates depends on carbon source and nitrogen availability in the growth medium. In particular, we found that the amount of pHis on phosphoenolpyruvate synthase (PpsA) is sensitive to nitrogen availability in vivo and that α-ketoglutarate inhibits phosphotransfer from phosphorylated PpsA to pyruvate. We expect this antibody to open opportunities for investigating other pHis proteins and their functions.

A phosphohistidine proteomics strategy based on elucidation of a unique gas-phase phosphopeptide fragmentation mechanism.

Protein histidine phosphorylation is increasingly recognized as a critical posttranslational modification (PTM) in central metabolism and cell signaling. Still, the detection of phosphohistidine (pHis) in the proteome has remained difficult due to the scarcity of tools to enrich and identify this labile PTM. To address this, we report the first global proteomic analysis of pHis proteins, combining selective immunoenrichment of pHis peptides and a bioinformatic strategy based on mechanistic insight into pHis peptide gas-phase fragmentation during LC-MS/MS. We show that collision-induced dissociation (CID) of pHis peptides produces prominent characteristic neutral losses of 98, 80, and 116 Da. Using isotopic labeling studies, we also demonstrate that the 98 Da neutral loss occurs via gas-phase phosphoryl transfer from pHis to the peptide C-terminal α-carboxylate or to Glu/Asp side chain residues if present. To exploit this property, we developed a software tool that screens LC-MS/MS spectra for potential matches to pHis-containing peptides based on their neutral loss pattern. This tool was integrated into a proteomics workflow for the identification of endogenous pHis-containing proteins in cellular lysates. As an illustration of this strategy, we analyzed pHis peptides from glycerol-fed and mannitol-fed Escherichia coli cells. We identified known and a number of previously speculative pHis sites inferred by homology, predominantly in the phosphoenolpyruvate:sugar transferase system (PTS). Furthermore, we identified two new sites of histidine phosphorylation on aldehyde-alcohol dehydrogenase (AdhE) and pyruvate kinase (PykF) enzymes, previously not known to bear this modification. This study lays the groundwork for future pHis proteomics studies in bacteria and other organisms.

Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages.

Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.

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.

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.

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.

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.

Biochemical characterization of selective inhibitors of human group IIA secreted phospholipase A(2) and hyaluronic acid-linked inhibitor conjugates.

We explored the inhibition mode of group IIA secreted phospholipase A(2) (GIIA sPLA(2)) selective inhibitors and tested their ability to inhibit GIIA sPLA(2) activity as chemical conjugates with hyaluronic acid (HA). Analogues of a benzo-fused indole sPLA(2) inhibitor were developed in which the carboxylate group on the inhibitor scaffold, which has been shown to coordinate to a Ca(2+) ligand in the enzyme active site, was replaced with other functionality. Replacing the carboxylate group with amine, amide, or hydroxyl groups had no effect on human GIIA (hGIIA) sPLA(2) inhibition potency but dramatically lowered inhibition potency against hGV and hGX sPLA(2)s. An alkylation protection assay was used to probe active site binding of carboxylate and noncarboxylate inhibitors in the presence and absence of Ca(2+) and/or lipid vesicles. We observed that carboxylate-containing inhibitors bind the hGIIA sPLA(2) active site with low nanomolar affinity, but only when Ca(2+) is present. Noncarboxylate, GIIA sPLA(2) selective inhibitors also bind the hGIIA sPLA(2) active site in the nanomolar range. However, binding for GIIA sPLA(2) selective inhibitors was dependent on the presence of a lipid membrane and not Ca(2+). These results indicate that GIIA sPLA(2) selective inhibitors exert their inhibitory effects by binding to the hGIIA sPLA(2) active site. An HA-linked GIIA inhibitor conjugate was developed using peptide coupling conditions and found to be less potent and selective against hGIIA sPLA(2) than the unconjugated inhibitor. Compounds reported in this study are some of the most potent and selective GIIA sPLA(2) active site binding inhibitors reported to date.