The Human Ganglioside Interactome in Live Cells Revealed Using Clickable Photoaffinity Ganglioside Probes.

Gangliosides, sialic acid bearing glycosphingolipids, are components of the outer leaflet of plasma membranes of all vertebrate cells. They contribute to cell regulation by interacting with proteins in their own membranes (cis) or their extracellular milieu (trans). As amphipathic membrane constituents, gangliosides present challenges for identifying their ganglioside protein interactome. To meet these challenges, we synthesized bifunctional clickable photoaffinity gangliosides, delivered them to plasma membranes of cultured cells, then captured and identified their interactomes using proteomic mass spectrometry. Installing probes on ganglioside lipid and glycan moieties, we captured cis and trans ganglioside-protein interactions. Ganglioside interactomes varied with the ganglioside structure, cell type, and site of the probe (lipid or glycan). Gene ontology revealed that gangliosides engage with transmembrane transporters and cell adhesion proteins including integrins, cadherins, and laminins. The approach developed is applicable to other gangliosides and cell types, promising to provide insights into molecular and cellular regulation by gangliosides.

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.

Anti-HIV Effects of Baculiferins Are Regulated by the Potential Target Protein DARS.

Baculiferins are a group of marine sponge-derived polycyclic alkaloids with anti-HIV (human immunodeficiency virus) activities. To identify additional baculiferin-based congeners for SAR analysis and to investigate the mode of action, a total of 18 new baculiferin-type derivatives were synthesized. The inhibitory activities of the congeners against the HIV-1 virus were evaluated in vitro, and the relevant SAR was discussed. Compound 18 exerted the most potent activity toward VSV-G-pseudotyped HIV-1 (IC50 of 3.44 µM) and HIV-1 strain SF33 (IC50 of 2.80 µM) in vitro. To identify the cellular targets, three photoaffinity baculiferin probes were simultaneously synthesized. Photoaffinity labeling experiments together with LC-MS/MS data identified aspartate-tRNA ligase (DARS) as a putative target protein of 18. The overexpression and knockdown of DARS in HEK293T cells provided additional data to demonstrate that DARS is a potential target protein in the regulation of HIV virus infection. The modes of antiviral baculiferins 13 and 18 binding to DARS were determined by a molecular docking simulation. Thus, baculiferin 18 is considered a promising lead as a new molecular target for the development of anti-HIV agents.

Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents.

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

Clickable gold-nanoparticles as generic probe precursors for facile photoaffinity labeling application.

Rapid access to appropriately functionalized probes is crucial in chemical labeling approaches to target identification studies. We designed and synthesized clickable gold-nanoparticles as generic probe precursors that enable (1) one-step ligand derivatization by click chemistry, and (2) facile photoaffinity labeling application. Using cholesterol as a model ligand, we successfully demonstrated the utility of the ligand-clicked probe in photoaffinity labeling of endogenously expressed oxysterol-binding protein (OSBP) in cell lysate.

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.

Development of DHQ-based chemical biology probe to profile cellular targets for HBV.

Chronic hepatitis B virus (HBV) infection has been a serious public health burden worldwide. Current anti-HBV therapies could not eliminate HBV ultimately. Considering the characteristics of HBV, it is impossible to be entirely cured based on current therapies. Therefore, it is urgently needed to develop novel therapeutic agents with new mechanism of action. The dihydroquinolizinone (DHQ) derivatives exhibited potent anti-HBV activity by decreasing HBV DNA and HBsAg level in an obscure mechanism of action. In this study, we have optimized the DHQ scaffold, developed the photoaffinity probe, with which to identify potential binding proteins.

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.

Photoaffinity probes for nematode pheromone receptor identification.

Identification of pheromone receptors plays a central role for uncovering signaling pathways that underlie chemical communication in animals. Here, we describe the synthesis and bioactivity of photoaffinity probes for the ascaroside ascr#8, a sex-pheromone of the model nematode, Caenorhabditis elegans. Structure-activity studies guided incorporation of alkyne- and diazirine-moieties and revealed that addition of functionality in the sidechain of ascr#8 was well tolerated, whereas modifications to the ascarylose moiety resulted in loss of biological activity. Our study will guide future probe design and provides a basis for pheromone receptor identification via photoaffinity labeling in C. elegans.

Azi-medetomidine: Synthesis and Characterization of a Novel α2 Adrenergic Photoaffinity Ligand.

Agonists at the α2 adrenergic receptor produce sedation, increase focus, provide analgesia, and induce centrally mediated hypotension and bradycardia, yet neither their dynamic interactions with adrenergic receptors nor their modulation of neuronal circuit activity is completely understood. Photoaffinity ligands of α2 adrenergic agonists have the potential both to capture discrete moments of ligand-receptor interactions and to prolong naturalistic drug effects in discrete regions of tissue in vivo. We present here the synthesis and characterization of a novel α2 adrenergic agonist photolabel based on the imidazole medetomidine called azi-medetomidine. Azi-medetomidine shares protein association characteristics with its parent compound in experimental model systems and by molecular dynamics simulation of interactions with the α2A adrenergic receptor. Azi-medetomidine acts as an agonist at α2A adrenergic receptors, and produces hypnosis in Xenopus laevis tadpoles. Azi-medetomidine competes with the α2 agonist clonidine at α2A adrenergic receptors, which is potentiated by photolabeling, and azi-medetomidine labels moieties on the α2A adrenergic receptor as determined by mass spectrometry in a manner consistent with a simulated model. This novel α2 adrenergic agonist photolabel can serve as a powerful tool for in vitro and in vivo investigations of adrenergic signaling.

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.

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.

Structurally Diverse Histone Deacetylase Photoreactive Probes: Design, Synthesis, and Photolabeling Studies in Live Cells and Tissue.

Histone deacetylase (HDAC) activity is modulated in vivo by post-translational modifications and formation of multiprotein complexes. Novel chemical tools to study how these factors affect engagement of HDAC isoforms by HDAC inhibitors (HDACi) in cells and tissues are needed. In this study, a synthetic strategy to access chemically diverse photoreactive probes (PRPs) was developed and used to prepare seven novel HDAC PRPs 9-15. The class I HDAC isoform engagement by PRPs was determined in biochemical assays and photolabeling experiments in live SET-2, HepG2, HuH7, and HEK293T cell lines and in mouse liver tissue. Unlike the HDAC protein abundance and biochemical activity against recombinant HDACs, the chemotype of the PRPs and the type of cells were key in defining the engagement of HDAC isoforms in live cells. Our findings suggest that engagement of HDAC isoforms by HDACi in vivo may be substantially modulated in a cell- and tissue-type-dependent manner.

Clickable photoaffinity ligands for the human serotonin transporter based on the selective serotonin reuptake inhibitor (S)-citalopram.

To date, the development of photoaffinity ligands targeting the human serotonin transporter (hSERT), a key protein involved in disease states such as depression and anxiety, have been radioisotope-based (i.e., 3H or 125I). This letter instead highlights three derivatives of the selective serotonin reuptake inhibitor (SSRI) (S)-citalopram that were rationally designed and synthesized to contain a photoreactive benzophenone or an aryl azide for protein target capture via photoaffinity labeling and a terminal alkyne or an aliphatic azide for click chemistry-based proteomics. Specifically, clickable benzophenone-based (S)-citalopram photoprobe 6 (hSERT Ki = 0.16 nM) displayed 11-fold higher binding affinity at hSERT when compared to (S)-citalopram (hSERT Ki = 1.77 nM), and was subsequently shown to successfully undergo tandem photoaffinity labeling-biorthogonal conjugation using purified hSERT. Given clickable photoprobes can be used for various applications depending on which reporter is attached by click chemistry subsequent to photoaffinity labeling, photoprobe 6 is expected to find value in structure-function studies and other research applications involving hSERT (e.g., imaging).

Diazirine-containing tag-free RNA probes for efficient RISC-loading and photoaffinity labeling of microRNA targets.

We designed and synthesized a photo-reactive and tag-free RNA probe for the identification of microRNA (miRNA) targets. To synthesize the RNA probe, we designed a novel nucleoside analog 1-O-[3-ethynyl-5-(3-trifluoromethyl-3H-diazirine-3-yl)]benzyl-ß-d-ribofuranose containing aryl trifluoromethyl diazirine and ethynyl moieties. The RNA probe containing this analog was observed to form crosslinks with complementary RNA by UV irradiation and was rapidly tagged by Cu-catalyzed azide alkyne cycloaddition (CuAAC). In addition, the tag-free and photo-reactive miRNA-145 probe showed comparable gene silencing activity to that of unmodified miRNA-145. Therefore, miRNA probes containing the nucleoside analog are promising candidates for the identification of target mRNAs of miRNAs.

Fishing for Drug Targets: A Focus on Diazirine Photoaffinity Probe Synthesis.

Target identification is a high-priority, albeit challenging, aspect of drug discovery. Diazirine-based photoaffinity probes (PAPs) can facilitate the process by covalently capturing transient molecular interactions. This can help identify target proteins and map the ligand's interactome. Diazirine probes have even been incorporated by cellular machinery into proteins. Embarking on the synthesis of customized PAPs, containing either an aliphatic or trifluoromethyl phenyl diazirine, can be a considerable endeavor, particularly for medicinal chemists and chemical biologists new to the field. This review takes a synthetic focus, aiming to summarize available routes, propose new avenues, and illuminate recent advances in diazirine synthesis. Select examples of diazirine photoaffinity labeling applications have been included throughout to provide instructive definition of the advantages and limitations of the technology while simultaneously highlighting how these reagents can be applied in a practical sense.

Selective Photoaffinity Probe That Enables Assessment of Cannabinoid CB2 Receptor Expression and Ligand Engagement in Human Cells.

Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB2R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB2R-selective bifunctional probe that covalently captures CB2R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB2R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.