Pepstatin-Based Probes for Photoaffinity Labeling of Aspartic Proteases and Application to Target Identification.

Aspartic proteases are a small class of proteases implicated in a wide variety of human diseases. Covalent chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We here report a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin incorporating a minimal diazirine reactive group. The position of this group in the inhibitor determines the labeling efficiency. The most effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Moreover, through chemical proteomics experiments and deep learning algorithms, we identified sequestosome-1, an important player in autophagy, as a direct interaction partner and substrate of cathepsin D.

A photoaffinity labeling strategy identified EF1A1 as a binding protein of cyclic dinucleotide 2'3'-cGAMP.

2'3'-cyclic GMP-AMP (2'3'-cGAMP), generated by cyclic GMP-AMP synthase (cGAS) under activation by cytosolic DNA, has a vital role in innate immune response via its receptor protein stimulator of interferon genes (STING) to fight viral infections and tumors. In order to have a complete understanding of biological functions of 2'3'-cGAMP, it is important to find out whether 2'3'-cGAMP has other unrevealed binding proteins present in mammalian cells and executes unknown functions. Here we report the 2'3'-cGAMP-based photoaffinity probes that capture and isolate 2'3'-cGAMP-binding proteins. These probes enable the identification of some potential 2'3'-cGAMP-binding proteins from HeLa cells. EF1A1, an essential protein regulating protein synthesis, is further validated to associate with 2'3'-cGAMP in vitro and in cells to impede protein synthesis. Thus, our studies provide a powerful approach to enable identification of the 2'3'-cGAMP interactome, discover unknown functions of 2'3'-cGAMP, and understand its physiological/pathological roles in tumor immunity and immune-related diseases.

Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate.

The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.

Adenosine Triggers Larval Settlement and Metamorphosis in the Mussel Mytilopsis sallei through the ADK-AMPK-FoxO Pathway.

Settlement and metamorphosis of planktonic larvae into benthic adults are critical components of a diverse range of marine invertebrate-mediated processes such as the formation of mussel beds and coral reefs, the recruitment of marine shellfisheries, and the initiation of macrobiofouling. Although larval settlement and metamorphosis induced by natural chemical cues is widespread among marine invertebrates, the mechanisms of action remain poorly understood. Here, we identified that the molecular target of adenosine (an inducer of larval settlement and metamorphosis from conspecific adults in the invasive biofouling mussel Mytilopsis sallei) is adenosine kinase (ADK). The results of transcriptomic analyses, pharmacological assays, temporal and spatial gene expression analyses, and siRNA interference, suggest that ATP-dependent phosphorylation of adenosine catalyzed by ADK activates the downstream AMPK-FoxO signaling pathway, inducing larval settlement and metamorphosis in M. sallei. This study not only reveals the role of the ADK-AMPK-FoxO pathway in larval settlement and metamorphosis of marine invertebrates but it also deepens our understanding of the functions and evolution of adenosine signaling, a process that is widespread in biology and important in medicine.

Probes for Photoaffinity Labelling of Kinases.

Protein kinases, one of the largest enzyme superfamilies, regulate many physiological and pathological processes. They are drug targets for multiple human diseases, including various cancer types. Probes for the photoaffinity labelling of kinases are important research tools for the study of members of this enzyme superfamily. In this review, we discuss the design principles of these probes, which are mainly derived from inhibitors targeting the ATP pocket. Overall, insights from crystal structures guide the placement of photoreactive groups and detection tags. This has resulted in a wide variety of probes, of which we provide a comprehensive overview. We also discuss several areas of application of these probes, including the identification of targets and off-targets of kinase inhibitors, mapping of their binding sites, the development of inhibitor screening assays, the imaging of kinases, and identification of protein binding partners.

Synthesis and Characterization of Azido Aryl Analogs of IBNtxA for Radio-Photoaffinity Labeling Opioid Receptors in Cell Lines and in Mouse Brain.

Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice variants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate the analgesic action of novel opioid drugs such as 3'-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that IBNtxA is a potent analgesic effective in a spectrum of pain models but lacks many side-effects associated with traditional opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efficiently radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously, as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specific ligand-protein contacts and its signaling complexes.

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

Coibamide A Targets Sec61 to Prevent Biogenesis of Secretory and Membrane Proteins.

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.

In situ chromatin interactomics using a chemical bait and trap approach.

Elucidating the physiological binding partners of histone post-translational modifications (hPTMs) is key to understanding fundamental epigenetic regulatory pathways. Determining such interactomes will enable the study of how perturbations of these interactions affect disease. Here we use a synthetic biology approach to set a series of hPTM-controlled photo-affinity traps in native chromatin. Using quantitative proteomics, the local interactomes of these chemically customized chromatin landscapes are determined. We show that the approach captures transiently interacting factors such as methyltransferases and demethylases, as well as previously reported and novel hPTM reader proteins. We also apply this in situ proteomics approach to a recently disclosed cancer-associated histone mutation, H3K4M, revealing a number of perturbed interactions with the mutated tail. Collectively our studies demonstrate that modifying and interrogating native chromatin with chemical precision is a powerful tool for exploring epigenetic regulation and dysregulation at the molecular level.

Olaparib-Based Photoaffinity Probes for PARP-1 Detection in Living Cells.

The poly-ADP-ribose polymerase (PARP) is a protein from the family of ADP-ribosyltransferases that catalyzes polyadenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair machinery to sites of DNA damage. The inhibition of PARP activity by olaparib can cause cell death, which is of clinical relevance in some tumor types. This demonstrates that quantification of PARP activity in the context of living cells is of great importance. In this work, we present the design, synthesis and biological evaluation of photo-activatable affinity probes inspired by the olaparib molecule that are equipped with a diazirine for covalent attachment upon activation by UV light and a ligation handle for the addition of a reporter group of choice. SDS-PAGE, western blotting and label-free LC-MS/MS quantification analysis show that the probes target the PARP-1 protein and are selectively outcompeted by olaparib; this suggests that they bind in the same enzymatic pocket. Proteomics data are available via ProteomeXchange with identifier PXD018661.

Photo-affinity pulling down of low-affinity binding proteins mediated by post-translational modifications.

Weak and transient protein-protein interactions (PPIs) mediated by the post-translational modifications (PTMs) play key roles in biological systems. However, technical challenges to investigate the PTM-mediated PPIs have impeded many research advances. In this work, we develop a photo-affinity pull-down assay method to pull-down low-affinity binding proteins, thus for the screen of PTM-mediated PPIs. In this method, the PTM-mediated non-covalent interactions can be converted to the covalent interactions by the photo-activated linkage, so as to freeze frame the low-affinity binding interactions. The fabricated photo-affinity magnetic beads (PAMBs) ensure high specificity and resolution to capture the interacted proteins. Besides, the introduction of PEG passivation layer on PAMB has significantly reduced the non-specific interaction as compared to the traditional pull-down assay. For proof-of-concept, by using this newly developed assay method, we have identified a set of proteins that can interact with a specific methylation site on Flap Endonuclease 1 (FEN1) protein. Less interfering proteins (decreased over 80%) and more proteins sub-classes are profiled as compared to the traditional biotin-avidin pull-down system. Therefore, this new pull-down method may provide a useful tool for the study of low-affinity PPIs, and contribute to the discovery of potential targets for renewed PTM-mediated interactions that is fundamentally needed in biomedical research.

Structure-Guided Design and In-Cell Target Profiling of a Cell-Active Target Engagement Probe for PARP Inhibitors.

Inhibition of the poly(ADP-ribose) polymerase (PARP) family of enzymes has become an attractive therapeutic strategy in oncology and beyond; however, chemical tools to profile PARP engagement in live cells are lacking. Herein, we report the design and application of PARPYnD, the first photoaffinity probe (AfBP) for PARP enzymes based on triple PARP1/2/6 inhibitor AZ9482, which induces multipolar spindle (MPS) formation in breast cancer cells. PARPYnD is a robust tool for profiling PARP1/2 and is used to profile clinical PARP inhibitor olaparib, identifying several novel off-target proteins. Surprisingly, while PARPYnD can enrich recombinant PARP6 spiked into cellular lysates and inhibits PARP6 in cell-free assays, it does not label PARP6 in intact cells. These data highlight an intriguing biomolecular disparity between recombinant and endogenous PARP6. PARPYnD provides a new approach to expand our knowledge of the targets of this class of compounds and the mechanisms of action of PARP inhibitors in cancer.

Affinity-Based Protein Profiling Reveals Cellular Targets of Photoreactive Anticancer Inhibitors.

Affinity-based protein profiling has proven to be a powerful method in target identification of bioactive molecules. Here, this technology was applied in two photoreactive anticancer inhibitors, arenobufagin and HM30181. Using UV irradiation, these photoreactive reagents can covalently cross-link to target proteins, leading to a covalent binding with target proteins. Moreover, the cellular on/off targets of these two molecules, including ATP1A1, MDR1, PARP1, DDX5, NOP2, RAB6A, and ERGIC1 were first identified by affinity-based protein profiling and bioimaging approaches. The protein hit, PARP1, was further validated to be involved in the function of the anticancer effects.

Fast and Efficient Fc-Specific Photoaffinity Labeling To Produce Antibody-DNA Conjugates.

Antibody-DNA conjugates are powerful tools for DNA-assisted protein analysis. Growing usage of these methods demands efficient production of high-quality conjugates. We developed an easy and fast synthesis route yielding covalent antibody-DNA conjugates with a defined conjugation site and low batch-to-batch variability. We utilize the Z domain from protein A, containing the unnatural amino acid 4-benzoylphenylalanine (BPA) for photoaffinity labeling of the antibodies' Fc region. Z(xBPA) domains are C-terminally modified with triple-glycine (G3)-modified DNA-oligonucleotides via enzymatic Sortase A coupling. We show reliable modification of the most commonly used IgG's. To prove our conjugates' functionality, we detected antibody-antigen binding events in an assay called Droplet Barcode Sequencing for Protein analysis (DBS-Pro). It confirms not only retained functionality for both conjugate parts but also the potential of using DBS-Pro for quantifying protein abundances. As intermediates are easily storable and our approach is modular, it offers a convenient strategy for screening various antibody-DNA conjugates using the same starting material.

The synthesis and characterization of a clickable-photoactive NAADP analog active in human cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing second messenger which triggers Ca2+ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca2+ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This 'all-in-one-clickable' NAADP (AIOC-NAADP) elicited Ca2+ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca2+ release. In mammalian cell homogenates, incubation with low concentrations of [32P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [32P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [32P]5-N3-NAADP.

Design, Synthesis, and Biological Application of Novel Photoaffinity Probes of Dihydropyridine Derivatives, BAY R3401.

To explore the molecular mechanisms of BAY R3401, four types of novel photoaffinity probes bearing different secondary tags were synthesized. Their potency for glycogenolysis was evaluated in primary human liver HL-7702 cells and HepG2 cells. Probe 2d showed the best activity in primary human liver HL-7702 cells and HepG2 cells, with IC50 values of 4.45 µM and 28.49 µM, respectively. Likewise, probe 5d showed IC50 values of 6.46 µM in primary human liver HL-7702 cells and 15.29 µM in HepG2 cells, respectively. Photoaffinity labeling experiments were also performed and protein bands larger than 170 kDa were specifically tagged by probe 2d. The results suggest that the synthesized probe 2d might be a very promising tool for the isolation of the target proteins of BAY R3401.

Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells.

Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.

Photoaffinity palladium reagents for capture of protein-protein interactions.

Protein-protein interactions (PPIs) are indispensable in almost all cellular processes. Probing of complex PPIs provides new insights into the biological system of interest and paves the way for the development of therapeutics. Herein, we report a strategy for the capture of protein-protein interactions using photoaffinity palladium reagents. First, the palladium-mediated reagent site specifically transferred a photoaffinity modified aryl group to the designated cysteine residue. Next, the photoaffinity group was activated by UV radiation to trap the proximal protein residue for the formation of a crosslink. This strategy was used to capture the PYL-ABA-PP2C interaction, which is at the core of the abscisic acid (ABA) signalling pathway. Our results indicated that this palladium-mediated strategy can serve as an alternative for incorporating an increasing number of diverse substrates for protein crosslinking through cysteine modifications and can be explored for use in mapping protein-peptide or protein-protein interaction surfaces and in trapping potential interacting partners.

Crenolanib-Derived Probes Suitable for Cell- and Tissue-Based Protein Profiling and Single-Cell Imaging.

Crenolanib (CP-868,596), a potent inhibitor of FLT3 and PDGFRα/ß, is currently under phase III clinical investigation for the treatment of acute myeloid leukemia. However, the protein targets of Crenolanib in cancer cells remain obscure, which results in difficulties in understanding the mechanism of actions and side effects. To alleviate this issue, in this study, a photoaffinity probe and two fluorescent probes were created based on Crenolanib, followed by competitive protein profiling and bioimaging studies, with the aim of characterizing the cellular targets. A series of unknown protein hits, such as MAPK1, SHMT2, SLC25A11, and HIGD1A, were successfully identified by means of pull-down/LC-MS/MS; these might provide valuable clues for understanding drug action and potential toxicities. Moreover, the fluorescent probes are suitable for imaging drug distribution at the single-cell level.

Antibiotic Discovery with Synthetic Fermentation: Library Assembly, Phenotypic Screening, and Mechanism of Action of ß-Peptides Targeting Penicillin-Binding Proteins.

In analogy to biosynthetic pathways leading to bioactive natural products, synthetic fermentation generates mixtures of molecules from simple building blocks under aqueous, biocompatible conditions, allowing the resulting cultures to be directly screened for biological activity. In this work, a novel ß-peptide antibiotic was successfully identified using the synthetic fermentation platform. Phenotypic screening was carried out in an initially random fashion, allowing simple identification of active cultures. Subsequent deconvolution, focused screening, and structure-activity relationship studies led to the identification of a potent antimicrobial peptide, showing strong selectivity for our model system Bacillus subtilis over human HEK293 cells. To determine the antibacterial mechanism of action, a peptide probe bearing a photoaffinity tag was readily synthesized through the use of appropriate synthetic fermentation building blocks and utilized for target identification using a quantitative mass spectrometry-based strategy. The chemoproteomic approach led to the identification of a number of bacterial membrane proteins as prospective targets. These findings were validated through binding affinity studies with penicillin-binding protein 4 using microscale thermophoresis, with the bioactive peptide showing a dissociation constant ( Kd) in the nanomolar range. Through these efforts, we provide a proof of concept for the synthetic fermentation approach presented here as a new strategy for the phenotypic discovery of novel bioactive compounds.