Recently, σ2 receptor ligands have been shown to have promising cytotoxic effects against cancer cells and to modulate the activity of P-glycoprotein (ABCB1) in vitro experiments, but their specific effects and mechanisms remain to be elucidated. We found that A011, a σ2 receptor ligand with the structure of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, showed promising cytotoxicity against breast cancer MCF-7 and adriamycin-resistant MCF-7 (MCF-7/ADR), induced apoptosis, and reversed adriamycin (ADR) and paclitaxel resistance in MCF-7/ADR cells. Furthermore, we demonstrated that A011 increased the accumulation of rhodamine 123 and mitoxantrone in MCF-7/ADR cells. A011 significantly decreased the ATPase activity of the ABCB1 and down-regulated ABCG2 protein expression. In addition, A011, administered alone or in combination with ADR, significantly inhibited tumor growth in the MCF-7/ADR tumor-bearing nude mouse model. A011 may be a potential therapeutic agent for the treatment of tumor resistance.Multidrug resistance (MDR) is thought to be one of the main reasons for the failure of chemotherapy in cancers. ATP-binding cassette subfamily B member 1 (ABCB1) or P-glycoprotein (P-gp) and ATP-binding cassette subfamily G member 2 (ABCG2) play indispensable roles in cancer cell MDR. Sigma-2 (σ Female breast cancer is one of the most common malignancies in the world. Breast cancer surpassed lung cancer to become the most prevalent cancer in 2020, with an estimated 2.3 million new cases . Over thThe ABC transporter superfamily is one of the largest families of transmembrane proteins, consisting of 49 ABC transporter members . Structu2 receptor may be a potential therapeutic target for tumors .The σreported . KO143 w2. MCF-7/ADR cells were maintained using 2 µM ADR. A549/DDP and HepG2/DDP cells were maintained using 2 μg/ml cisplatin. All resistant cells were grown in drug-free medium 2 weeks before experiment.ADR-resistant MCF-7 cells (MCF-7/ADR) and DDP-resistant A549 cells (A549/DDP) were purchased from Shanghai GuYan Biotech Co., Ltd. . DDP-resistant HepG2 cells (HepG2/DDP) were preserved in our laboratory. Their parental cells were originally imported from American Type Culture Collection  and cultured with adriamycin or cisplatin to form MCF-7/ADR cells, A549/DDP and HepG2/DDP. Cells were grown in Minimum Eagle’s medium (for MCF-7/ADR), Dulbecco’s Modified Eagle Medium (for MCF-7) and RPMI 1640 , respectively, containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C, 5% CO3 cells per well were seeded into 96-well plates with 100 μl medium overnight. Subsequently, cells were cultured with different concentrations of A011, adriamycin, cisplatin, paclitaxel and combination, respectively. After 24, 48, and 72 h incubation, 10 μl CCK-8 was added into each well and incubated for 90 min at 37°C. The absorbance was then measured at 450 nm using a microplate reader. IC50 value was calculated as previously described. Survival rate = (ODtreatment—ODblank)/(ODcontrol—ODblank) × 100%, Resistant Fold (RF) = IC50 resistant cells/IC50 parental cells, Reversal Fold (FR) = IC50 Monotherapy/IC50 Combination therapy.The CCK-8 kit was used to determine the cytotoxicity of chemotherapeutic agents in MCF-7/ADR, A549/DDP, HepG2/DDP and their parental cells. In brief, 5 × 103 cells/well) were seeded into 6-well plates overnight. After 24 h incubation, cells were treated with A011  for 24 h and cultured in drug-free medium for 10 days. During this period, cells were washed twice every other day with phosphate buffered saline (PBS) and cultured with fresh medium. Cells were then fixed with 4% paraformaldehyde for 15 min. Crystal violet was used to stain cells for 15 min. The numbers of colonies (cells >50) were counted.MCF-7 and MCF-7/ADR cells  was then added and incubated for 48 h. Subsequently, cells were harvested and washed twice with cold PBS before the addition of Annexin V-FITC and PI for 15 min at room temperature. Flow cytometry was used to detect the apoptotic cells.Annexin V-FITC and propidium iodide (PI) kits were used to determine the apoptotic cells induced by A011 in MCF-7/ADR and parental cells. In short, 2 × 105 cells per well were seeded into 6-well plates. After 24 h incubation, cells were treated with A011  for 48 h. The cells were then washed with PBS and fixed, Hoechst 33,258 was added to stain for 15 min, followed by washing with PBS and photographed under the fluorescence microscope.Hoechst 33,258 fluorescent reagent was used to detect the effect of A011 on apoptosis in MCF-7/ADR and its parental cells. 2 × 105 cells were seeded into 6-well plates. After 24 h incubation, different concentration of A011 , verapamil (10 μM) or KO143 (10 μM) was separately added to each well and incubated for 2 h at 37°C. Subsequently, each well was washed 3 times with PBS and incubated with Rh123 (5 μg/ml) or mitoxantrone (5 μmol/L) for 2 h at 37°C. Finally, all cells were collected and resuspended with cold PBS. Flow cytometry was applied to measure the fluorescence intensity of intracellular Rh123 and mitoxantrone. Verapamil and KO143 were used as positive agent of ABCB1 and ABCG2, respectively.Flow cytometry was used to detect the intracellular accumulation of Rh123 or mitoxantrone in MCF-7/ADR and its parental cells, with Rh123 and mitoxantrone being the fluorescent substrates for the ABCB1 and ABCG2 transporters, respectively. Briefly, 2 × 105 cells per well) overnight. Cells were then incubated with Rh123 (5 μg/ml) or mitoxantrone (5 μmol/L) for 2 h at 37°C. After 2 h incubation, cells were washed 3 times with PBS and cultured with different concentration of A011 , verapamil (10 μM) or KO143 (10 μM) for 0, 30, 60, 90, 120 min, respectively. Subsequently, cells were collected and washed 3 times with cold PBS. Flow cytometry was used to measure the fluorescence intensity of intracellular Rh123 and mitoxantrone, respectively.Rh123 and mitoxantrone efflux were performed as previously described literature . In shor2+ and ATP reagent was added into each well to trigger the reaction. After 40 min incubation, plates were removed to room temperature and ATP detection reagent was added to initiate luminescence for 20 min at room temperature. Subsequently, luminescence was read on a plate-reading luminometer.The impact of A011 on ABCB1-mediated ATP hydrolysis was measured by the Pgp-Glo™ Assay kits. In brief, ABCB1 membranes were thawed and diluted at 4°C. Membranes were treated with various concentrations of A011 for 5 min at 37°C Mg4 cells per well were seed into 6-well plates overnight and treated with A011 . After 48 h incubation, cells were washed twice with cold PBS and fixed by 4% paraformaldehyde for 15 min. Bovine serum albumin (BSA) (2 mg/ml) was added to block proteins for 1 h, primary antibodies ABCB1 (1:200) or ABCG2 (1:200) were used to incubate proteins for 4 h at 4°C, which were subsequently blocked by second antibodies for 1 h. SlowFadeTM Gold antifade reagent was used to incubate proteins for 5 min. The fluorescence microscope was performed to collect the images.Immunofluorescence assay was performed to detect the influence of A011 on the intracellular localization of ABCB1 and ABCG2 in MCF-7/ADR cells. In brief, 2 × 10Western blot assays were applied to detect the protein expression of ABCB1 and ABCG2 after A011 treatment in MCF-7/ADR cells. Briefly, cells were cultured with or without A011  for 48 h. Radio-immune precipitation assay (RIPA) cell lysis buffer was then used to lysis cells for 30 min on the ice. The total protein concentrations were normalized using the BCA protein assay kit. Subsequently, proteins were load and separated by SDS-PAGE kits, following transferred into polyvinylidene difluoride (PVDF) membrane and blocked by non-fat milk. Primary antibodies  were incubated for overnight at 4°C. Horseradish peroxidase (HRP)-conjugated Mouse or Rabbit antibody was used to co-incubate with PVDF membrane for 1 h at room temperature. The protein bands were visualized by Immobilon Western HRP Substrate Kits and the relative protein expression level was analysis by ImageJ software.in vivo studies. The BALB/c female nude mice  were bought from Guangdong Medical Laboratory Animal Centre . 1 × 107 cells were injected subcutaneously into the right flank of each nude mouse. When the tumor volume reached 100 mm3, nude mice were randomly divided into five groups (6 mice in each group):(1) Control , 2) A011 (1 mg/kg), 3) ADR (5 mg/kg), 4) A011 (5 mg/kg), 5) A011 (1 mg/kg) plus ADR (5 mg/kg) (A011 + ADR). The drugs were administered intraperitoneally every 3 days for 21 days and the body weight and tumor volume (V) of nude mice were measured according to the following formula: V = 0.5a × b2 [“a” is the length (mm) and “b” is the width (mm)]. The nude mice were killed by spinal dislocation and the tumor tissues were dissected and weighed. The liver, kidney and tumor samples from nude mice were fixed and preserved. All animal experiments complied with the China Animal Welfare Guide. The protocol was reviewed and approved by the Experimental Animal Research Committee of Guangdong Medical University.The MCF-7/ADR cells inoculated nude mice xenograft model was used for The above tissue samples were dehydrated in ethanol, embedded in paraffin and sectioned with a microtome (4 μm). Sections were stained with hematoxylin and eosin for hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) staining, respectively. The immunohistochemistry (IHC) staining was performed with anti-ABCG2 or anti-ABCB1 antibody. The treated samples were observed under a microscope and photographed and the images were analyzed.p value was less than 0.05.All experiments were conducted in at least three independent experiments. All data were analyzed using GraphPad Prism’s one-way ANOVA and shown as mean ± SD. Differences were considered statistically significant when the 50 values of A011 were 3.79 ± 0.13 μM, 5.35 ± 0.42 μM, RF value 1.41, 4.59 ± 0.35 μM, 7.74 ± 0.32 μM, RF value 1.69, 4.07 ± 0.25 μM, 8.29 ± 0.04 μM, RF value 2.04 for MCF-7, MCF-7/ADR, A549, A549/DDP, HepG2 and HepG2/DDP cells, respectively   . In addi < 0.05) . These rHoechst 33,258 staining showed that MCF-7/ADR and MCF-7 cells exhibited increased cytoplasmic density, nuclear consolidation, nuclear membrane nucleolus fragmentation and increased apoptotic vesicles in a dose-dependent manner after A011 treatment compared to cells from control group with intact cell structure . FurtherTo investigate the mechanism by which A011 reversed drug resistance in tumor cells, we determined the effect of A011 on the function of ABCB1 and ABCG2 transporters. Verapamil and KO143 were inhibitors of the ABCB1 and ABCG2 transporters respectively, and were able to inhibit the transport of substrates outside the cell membrane by the ABCB1 and ABCG2 transporters, so they were used as positive control. In addition, Rh123 and mitoxantrone were fluorescent substrates for the ABCB1 and ABCG2 transporters respectively, and were able to be quantified by flow cytometry. The results showed that the accumulation of Rh123 or mitoxantrone in MCF-7/ADR cells was significantly lower than their accumulation in parental cells. A011 significantly increased the accumulation of Rh123 and mitoxantrone in MCF-7/ADR cells. Moreover, the intracellular accumulation of Rh123 was higher in the A011 group compared to the verapamil group . In addiTo further investigate the role of A011 on the function of the ABCB1 transporter, we measured the effect of A011 on the ATPase activity of the ABCB1 transporter. The results showed that verapamil increased the activity of ABCB1 transporter ATPase, which was in agreement with the description of previous studies . In contGiven reducing the expression of transporter proteins and altering their localization in cells is one of the mechanisms to overcome MDR, we further investigated the effect of A011 on ABCB1 and ABCG2 proteins in MCF-7/ADR cells by immunofluorescence and western blot. Immunofluorescence assay showed that A011 did not alter the localization of ABCB1 and ABCG2 proteins in MCF-7/ADR cells . And wesin vivo, we established a xenograft model with MCF-7/ADR cells in nude mice. A011 (1 mg/kg) or ADR (5 mg/kg) alone showed low growth inhibitory activity against MCF-7/ADR tumors, with growth inhibition rates of 6.94% and 22.58%, respectively. However, when ADR (5 mg/kg) was co-administered with A011 (1 mg/kg), the anti-tumor activity of ADR was significantly enhanced with the growth inhibition rate of 58.43%. A011 (5 mg/kg) alone showed good anti-tumor activity against MCF-7/ADR tumors with the growth inhibition rate of 43.13%  and A011 combined with ADR groups. In HE staining of tumor tissue, approximately 60% and 70% of tumor cells were necrotic in A011 (5 mg/kg) and A011 combined with ADR respectively, compared to 40% in the control group . TUNEL sIntrinsic MDR and acquired MDR are two of the major barriers to tumor treatment, seriously threatening the survival and affecting the lives of cancer patients . Due to 2 receptor has been found to be highly expressed in rapidly proliferating tumors such as breast cancer and is considered to be one of the potential targets for the treatment of tumors  alone or A011 (1 mg/kg) co-administered with ADR showed promising anti-tumor activity, significantly inhibiting the growth of MCF-7/ADR tumors without significant toxicity, suggesting that A011 has the potential to overcome MDR.The σf tumors . Most ofactivity , and thoactivity . Moreovet tumors . In this. Dysregulation of apoptosis is one of the hallmarks of cancer and is associated not only with tumorigenesis and progression, but also with tumor resistance to chemotherapeutic agents  and are expected to be used for in vivo measurement of tumor proliferation status (2 receptor ligands displayed cytotoxic effects on a variety of human cancer cells, and inhibited tumor growth (2+ levels in MCF-7 cells and induced endoplasmic reticulum stress and autophagy (2 receptor and A011 may be a candidate for cancer treatment either alone or in combination with other anticancer agents.A variety of σn status . Studiesr growth . We prevutophagy . In addi2 ligand A011, containing a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline structure, exhibited excellent anti-breast cancer MDR activity both in vivo and in vitro, either alone or in combination with ADR. A011 demonstrated anti-MDR activity by inhibiting the transporting function of ABCB1 and ABCG2 transporters and thus was a potential therapeutic agent for the treatment of tumor resistance.In this study, we elucidated that the σ


Here we report biophysical and biochemical studies on JMJD7 from Drosophila melanogaster (dmJMJD7). Notably, crystallographic analyses reveal that the unusual dimerization mode of JMJD7, which involves interactions between both the N- and C-terminal regions of both dmJMJD7 monomers and disulfide formation, is conserved in human JMJD7 (hsJMJD7). The results further support the assignment of JMJD7 as a lysyl hydroxylase and will help enable the development of selective inhibitors for it and other JmjC oxygenases.The JmjC family of 2-oxoglutarate dependent oxygenases catalyse a range of hydroxylation and demethylation reactions in humans and other animals. Jumonji domain-containing 7 (JMJD7) is a JmjC ( In total, there are 60–70 human 2OG oxygenases, some of which have been developed as drug targets. Notably, hypoxia inducible factor (HIF) prolyl hydroxylase (PHD1-3) inhibitors have been approved for the treatment of anaemia in chronic kidney diseases, because transcription of the erythropoietin gene is upregulated by HIF5. Catalysis by the PHDs signals for proteasome regulated degradation of HIF-α isoforms5. The PHDs belong to a structural subfamily of prolyl hydroxylases which extends from humans to some bacteria5. A second type of human 2OG oxygenase, Factor Inhibiting HIF (FIH), also negatively regulates HIF activity by reducing its affinity for transcription promoting histone acetyl transferases6. FIH also plays a role in the context-dependent regulation of the sets of HIF target genes that are upregulated in response to hypoxia6. Crystallographic analyses have revealed that FIH belongs to the JmjC subfamily of 2OG oxygenases3. Subsequently, the largest identified family of histone Nε-methyl lysine demethylases (KDMs) was also shown to have a JmjC type fold (JmjC KDMs)7.2-Oxoglutarate (2OG) and Fe(II) dependent oxygenases catalyse hydroxylations, demethylations and other two-electron oxidations on a range of substrates, including proteins, nucleic acids, small molecules, and lipids in humans and other animals3. Different subfamilies of 2OG oxygenases are characterised by modifications and additions to this core DSBH fold. Within the set of JmjC 2OG oxygenases, two broad subfamilies have been identified based on structural similarities and the types of reactions catalysed3. One of these is the JmjC KDMs, some of which also have N-methyl arginine demethylase (RDM) activity8, which typically  are monomeric and which normally have multiple other domains, at least some of which are often involved in chromatin binding3. By contrast, the JmjC hydroxylases usually do not have multiple domains and, at least most of them do not have KDM or RDM activity7. As exemplified by FIH, the JmjC hydroxylases typically catalyse hydroxylation of methylene groups on protein residue side chains to give stable alcohol products, though at least one ‘JmjC hydroxylase’ modifies RNA3.Like all other identified 2OG oxygenases, both the PHDs and the JmjC KDMs have a core distorted double-stranded β-helix (or cupin) fold10. DRG1 and 2 contain a guanine nucleotide-binding (G) domain, which is flanked by HTH (helix-turn-helix) and TGS  domains that can recruit auxiliary protein(s) for efficient GTP hydrolysis12  class GTPases that are present in archaea, eukaryotes and plants and have roles in protein synthesis, signal transduction, cell motility, and transport13. The yeast orthologue of DRG1 (Rbg1) is reported to directly interact with the 80S ribosome , to ‘switch on’ their regulatory functions. Indeed, phosphorylation of a threonine (T100) located on the GTP binding domain switch I loop in DRG1 substantially promotes DRG1 catalysed GTP hydrolysis19.DRG1 was first identified in the developing murine brain and frog embryos and was shown to play a role in animal developmentS)-lysyl hydroxylation of both human DRG1 (at K22) and DRG2 (at K21) is catalysed by the 2OG oxygenase JmjC (Jumonji-C) domain-containing 7 (JMJD7)  catalyses lysyl hydroxylation of DRG1 and 2 expands the interface between oxygen and the regulation of protein functionMus musculus (90% sequence identity), Danio rerio (66%), Drosophila melanogaster (45%) and Glycine max (40%)  that has > 80% sequence identity to human DRG1/2  Fig. a. D. mel1/2 Fig. b.Escherichia coli and tested the purified enzyme for its ability to hydroxylate Drosophila GBP as well as human DRG1 and 2 peptide substrate fragments that are efficiently hydroxylated by hsJMJD720. The results of mass spectrometric (MS) assays reveal that dmJMJD7 efficiently hydroxylates D. melanogaster GBP and both human DRG1 and DRG2 peptide fragments in the presence of Fe(II), 2OG and ascorbate, though the latter is not essential for JMJD7 catalysis. The MS fragmentation analyses of hydroxylated GBP and DRG1/2 show that dmJMJD7 catalysed hydroxylation occurs at the same DRG1 and DRG 2 lysine-residues (K22 for DRG1 and K21 for DRG2) as the hsJMJD7 catalysed reactions and occurs on the analogous lysine residue in the Drosophila GBP peptide (K22)  Figs. , S3. Giv2) Figs. c, implyi20. We therefore carried out crystallographic analyses on dmJMJD7. We obtained dmJMJD7 crystals of His6-dmJMJD7.Mn complexed with its co-substrate 2OG or co-product succinate; catalytically inert Mn(II) was used instead of the cofactor Fe(II) to block catalysis under aerobic conditions, as was done for hsJMJD720. The His6-dmJMJD7.Mn(II) 2OG complex structure was solved by molecular replacement using PHASER26 using a hsJMJD7 crystal structure (PDB: 5NFO) with two dmJMJD7 molecules per asymmetric unit in the P21 space group. All other dmJMJD7.ligand structures were subsequently solved using the His6-dmJMJD7.Mn.2OG structure (PDB: 7YXG) as the initial search model.The observation that both hsJMJD7 and dmJMJD7 catalyse DRG/GBP hydroxylation raises the question as to their evolutionary relationship from a structural perspective, given that they share only ~ 45% sequence identity Fig. a20. We t6-dmJMJD7.Mn structures reveal that the overall fold of dmJMJD7 is very similar to that of hsJMJD7  revealed an unprecedented dimerization mode, with interactions between monomers involving both the N- and C-terminal regions; the hsJMJD7 dimer is further stabilised by a disulphide link between two Cys47 residues (located on α2) from each monomer  and N–N′ (α2–α2′) dimerization regions has a combined buried surface area of ~ 1756 Å2, compared to ~ 1700 Å2 for hsJMJD7  dimerization interface involves hydrophobic (Leu34–Leu34′ on α2) and electrostatic (Tyr41-Lys43′ on α2) interactions between the monomers. Notably, the two monomers are covalently linked by a disulfide bond (Cys37–Cys37′ on α2), as observed in crystallographic analysis of hsJMJD720. The buried surface area and complementarity of residues at the dimer interface is significant enough for JMJD7 dimerization as observed in solution20.Crystallographic analysis of the Hismer Fig. b20. As wner Fig. a. The dmJD7 Fig. a. Two me27) conserved metal-binding triad of residues  , Tyr123 (non-DSBH aa), Thr172 (βII), and Tyr183 (βIII). The conservation of 2OG C5 carboxylate binding mode of hsJMJD7 in dmJMD7 is particularly notable since this feature is uniquely characteristic of JMJD7 amongst the structurally characterised 2OG oxygenases  than elsewhere Fig. a, both hses Fig. .5 and JmjC demethylases being pursued for the treatment of cancer28. Most reported 2OG oxygenase inhibitors bind to the active site Fe(II) and compete with 2OG. Comprehensive knowledge of how such 2OG mimetics bind to different human 2OG oxygenases is of interest both with respect to optimising potency for medicinal targets and small-molecule probes of oxygenase function as well as minimising off-target inhibition. We thus attempted to obtain structures of hsJMJD7 and dmJMJD7 with 2OG analogues and found that dmJMJD7 was particularly amenable to such studies. We obtained His6-dmJMJD7.Mn crystal structures in complex with the near isosteric 2OG analogue N-oxalylglycine  peptide substrate), N-oxalyl-D-alanine (IC50 4.14 μM), N-oxalyl-D-phenylalanine, and pyridine-2,4-dicarboxylate   prolyl hydroxylases (PHD1-3) inhibitors being approved for the treatment of anaemiaG1 aa 16–0 peptidetrans to H175 and D177 and by interacting with the 2OG C5 carboxylate binding residues in a closely analogous bidentate manner to that observed for 2OG itself  or a polyethylene glycol fragment  located within 4.0 Å of the 2OG C4 methylene group . Notably, in the 2OG/succinate/N-oxalylglycine complexes, Trp164 is positioned to form a stacking interaction with the methylene groups of 2OG/succinate/N-oxalylglycine; the C4-methylene substitution in N-oxalyl-D-alanine or N-oxalyl-D-phenylalanine compared to N-oxalylglycine prevents this interaction. Tyr123 is one of four dmJMJD7 residues positioned to form H-bonds with the 2OG/succinate/N-oxalylglycine C5-carboxylate  located N-terminal to the DSBH β-strands, which normally forms a loop around the dmJMJD7 active site and is likely involved in induced-fit during substrate binding. Compared to that observed in the dmJMJD7.Mn.2OG/succinate/N-oxalylglycine/2,4-PDCA structures, this loop adopts a different conformation and forms a non-DSBH β-strand extending the major DSBH β-sheet in the N-oxalyl-te Figs. . The int7. 2OG oxygenase catalysed hydroxylation to give stable alcohol products has been identified on the side chains of multiple protein residues, including prolyl, asparaginyl, aspartyl, lysyl, arginyl, and histidinyl residues in eukaryotic proteins. The enzymes catalysing such reactions appear to operate with varying degrees of selectivity, at least in cellular and in vitro studies, with FIH, AspH, and Jumonji domain-containing 6 (JMJD6) being rather promiscuous. By contrast, other protein hydroxylases appear to be much more selective, e.g. the HIF-α hydroxylating PHDs34. Lysine-residues are particularly notable for their propensity to undergo 2OG oxygenase catalysed modifications. In addition to Nε-methyl group demethylation, the methylene groups of lysyl side chains can be modified by hydroxylation at the C5, C4, and, as catalysed by JMJD7, C3 positions, giving rise to independent PTMs3. Lysyl-residue hydroxylation can occur to give (5R)-hydroxylated products as catalysed by the procollagen lysyl hydroxylases (PLODs)35, (5S)-hydroxylated products as catalysed by JMJD637, and a C4 hydroxylated product as catalysed by Jumonji domain-containing 4 (JMJD4)38.Protein hydroxylation and N-methyl demethylation via hydroxylation, as catalysed by 2OG oxygenases, have emerged as post-translational modifications of fundamental importance in eukaryotic biologyε-methyl lysine demethylation, and the TET enzymes catalyse 5-methylcytosine modifications39. The activity of the hypoxia-inducible transcription factor is tightly controlled by prolyl- and asparaginyl-hydroxylases34. 2OG oxygenases are also involved in the regulation of RNA; thus, JMJD6 is proposed to be involved in the regulation of mRNA splicing by catalysing hydroxylation of splicing factors40, and in animals, the fat mass and obesity protein (FTO) and AlkB homologue 5 (ALKBH5) are N6-methyladenine hydroxylases/demethylases, and TWY5 and ALKBH8 hydroxylate the anticodon loops of some tRNAs39. Recent work suggests that the translational machinery is also a major target of 2OG oxygenases. MYC-induced nuclear antigen (MINA53/ RIOX2) and nucleolar protein 66 (NO66/ RIOX1) are closely related JmjC oxygenases that catalyse histidinyl hydroxylation of ribosomal proteins Rpl27a and Rpl8, respectively42. OGFOD1 is a prolyl hydroxylase of Rps23 that controls decoding during translation43. JMJD4 regulates translational termination via lysyl hydroxylation of the termination translation factor, eukaryotic release factor 1 (eRF1)38.2OG oxygenases have established roles in regulating chromatin function/ transcription, e.g. the JmjC KDMs catalyse NS)-lysyl hydroxylation of both human DRG1 and 2, plays essential roles in cellular processes, including protein synthesis (amongst others)20. The unique (to date) ability of JMJD7 to catalyse lysyl C3 hydroxylation coupled with its role in regulating protein synthesis means JMJD7 is a JmjC enzyme of particular interest. The JMJD7 gene, located on human chromosome 15, can form a naturally occurring read-through transcript with the neighbouring PLA2G4B gene to encode a JMJD7-PLA2G4B fusion protein overexpressed in cancers, including breast, prostate, and thyroid cancer24. JMJD7 variants have been linked to patients with severe intellectual disabilities (M160V)23 and autism (R260C)22. These links to cancer and autism make JMJD7 a compelling enzyme to study from the perspective of linking oxygenase activity to physiology and disease, including by using appropriate models to study JMJD7 function at an organismal level.It has been reported that hsJMJD7, a JmjC 2OG-oxygenase catalysing  of sequence similarity, our substrate selectivity, structural and inhibition results collectively imply remarkable conservation between hsJMJD7 and dmJMJD7 in terms of their catalysis and biochemistry. Thus, Drosophila appears to be a suitable model organism for further ‘on-target’ pharmacodynamic (PD) studies for probing JMJD7 function. Indeed, Drosophila has been used as a model to study cancer and autism45, both of which have been linked with JMJD724.Our results provide direct evidence that dmJMJD7 catalyses oxygen-dependent hydroxylation of human DRG (1 and 2) and their counterpart in 20 reveal the unusual dimerization mode of JMJD7, which involves interactions between the N- and C-terminal regions of both dmJMJD7 monomers and is likely conserved in all animals making JMJD7. The JMJD7 dimerization mode contrasts with those observed for other JmjC dimers. FIH and TYW5 dimerise via bundles of α-helices located at their C-termini, while MINA53 and NO66 form dimers via interactions between α-helices located between their JmjC and winged-helix domains41. In the case of JMJD7 dimerization, the presence of a conserved disulfide which appears to help link the two monomers, is an unusual feature in 2OG oxygenase oligomers. Given that protein disulfide isomerase is the β-component of the α2β2 collagen prolyl hydroxylase46, it is possible that the JMJD7 disulfide is part of a similar mechanism for regulating its activity.Notably, the crystallographic analyses on dmJMJD7 combined with previously reported crystal structures for hsJMJD747. To enable studies on the roles of hsJMJD7 and dmJMJD7, we are presently undertaking structure–activity relationship studies employing 2OG mimetics. The combined observations further validate the general utility of 2OG mimetics for use in JMJD7 inhibition and suggest that inhibitor derivatives substituted at the 2OG C4 or analogous position may be of particular utility with JMJD7, as has been the case for the JmjC hydroxylase FIH29 and JmjC KDMs48. However, the different conformations for the sidechains of key JMJD7 active site residues observed for closely related inhibitors  suggest that care should be taken in assuming conserved binding modes even within the same series, as has also been found to be the case for inhibition of the PHDs where induced conformational changes can manifest on inhibitor (and substrate) binding32. Thus, it would seem to be important to combine structure/ computationally guided and empirical approaches to achieve potent and selective inhibitors of specific human 2OG oxygenases, such as JMJD7.The structural results presented here on JMJD7 complexes will enable research on the development of selective JMJD7 inhibitors, including via computationally guided approachesD. melanogaster JMJD7 gene  was cloned into the pET28a vector (Novagen) by GenScript and used for the production of N-terminally His6-tagged dmJMJD7. The required construct was verified by DNA sequencing. Recombinant dmJMJD7 protein was produced in E. coli Rosetta™ (DE3) pLysS cells (Novagen). Cells were grown in 2TY media at 37 °C to an OD600 of 0.6–0.8. Protein production was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (ITPG); cells were grown overnight at 18 °C. Protein was purified by nickel affinity and size exclusion chromatography in buffer containing 50 mM HEPES pH 7.5, 200 mM NaCl and 5% glycerol. Protein purity was assessed by SDS-PAGE analysis. Highly purified fractions  were then pooled and concentrated (25 mg/mL).The 6-dmJMJD7 were incubated for 5–60 min at 37 °C with 200 µM Fe(II), 300 µM 2OG  and 4 mM ascorbate20. Reactions were quenched by adding 1% (v/v) formic acid; 1 µL of assay sample was then mixed with 1 µL of α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution  before being spotted onto a Waters 96-spot MALDI plate. The samples were analysed using MALDI-MS on a Waters Micromass™ MALDI micro MX™ instrument in the positive ion reflectron mode.Substrate assay mixtures  containing 50 µM substrate (as defined above) peptide and 10 µM His6-dmJMJD7dm activity was assessed by SPE-MS using the DRG1 peptide substrate: ARTQKNKATAHHLGLLKARLAKLRR . Enzyme and substrate solutions were prepared in the assay buffer  phosphine (TCEP); reagents were dispensed using a Thermo Multidrop combi dispenser (Thermo Scientific) equipped with a small volume dispensing cassette. All compounds were prepared as a 20 mM stock solution in DMSO. For IC50 determinations, an 11-point and threefold serial dilution was prepared in DMSO using an ECHO550 acoustic dispenser (Labcyte); compound serial dilutions were dry dispensed (0.25 µl per well) into a 384-well polypropylene assay microplate (Greiner Bio-One). His6-dmJMJD7dm (0.1 µM) was prepared in the assay buffer and 25.0 µl transferred into each well. JMJD7 was pre-incubated with the potential inhibitor for 15 min at room temperature. 25 µl of the assay mixture, consisting of Fe(II) (20 µM), L-ascorbic acid (200 µM), 2OG (10 µM) and DRG1peptide (10 µM), was prepared in the assay buffer. Reactions were initiated by dispensing the DRG1 substrate (25.0 µl) into each well. After 15 min at room temperature, reactions were stopped by adding 5 µl of 10% (v/v) aqueous formic acid. Assay plates were transferred to a RapidFire RF360 robot coupled to an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer operated in the positive ion mode .Small molecule inhibition of HisSamples were aspirated under vacuum for 400 ms, loaded onto a C4 SPE cartridge, and non-volatile buffer salts were removed by washing the cartridge with 0.1% (v/v) formic acid in LCMS grade water at a flow rate of 1.5 ml/min for 5.0 s. The DRG1 peptide was then eluted into the mass spectrometer with 85% (v/v) acetonitrile, 15% (v/v) LCMS grade water, 0.1% (v/v) formic acid at a flow rate of 1.25 ml/min for 5.0 s. The cartridge was re-equilibrated with water for 500 ms. The mass spectrometer was operated with the following parameters: drying gas temperature (350 °C), gas flow rate (12 L/minute), capillary voltage (3500 V), fragmentor voltage (150 V). Ion chromatogram data were extracted for the + 7-charge state for the substrate peptide, and the hydroxylated peptide product and peak area data for extracted ion chromatograms were integrated using RapidFire Integrator software . % conversion of the non-hydroxylated peptide to hydroxylated peptide product was calculated using the equation:50 data were determined from nonlinear regression curve fit using GraphPad Prism 5.IC6-dmJMJD7.Mn(II).ligand complexes  were obtained in sitting drops at 25 °C in buffer conditions containing 0.1 M Bis–Tris pH 5.4, 0.3 M magnesium chloride, 18–23% polyethylene glycol 3350, 0.1 M ammonium acetate  and 0.002 M manganese chloride. Crystals were cryo-protected with 20% ethylene glycol before flash freezing in liquid nitrogen. Data were collected using single crystals at 100 K at the Diamond Light Source (DLS) beamlines and processed using HKL200049. The dmJMJD7.2OG structure was solved by molecular replacement using PHASER26 with 2 molecules in the asymmetric unit using a reported hsJMJD7 crystal structure (PDB: 5NFO)20. All other dmJMJD7.ligand structures were subsequently solved using the dmJMJD7.2OG structure (PDB: 7YXG) as the initial model. The structures were refined by CNS50/ PHENIX51 until Rfactor/Rfree converged with iterative cycles model building using COOT52. MOLPROBITY53 was used to monitor the geometric quality of the models between refinement cycles. Water molecules were added to peaks (> 1.5σ 2FoFc) in electron density maps that were within H-bonding distance to protein atoms with a reasonable geometry to form H-bonds.Crystals of HisSupplementary Information.


Here, we describe that CGP37157, aninhibitor of a mitochondrial sodium (Na+)/Ca2+ exchanger, induces paraptosis in Jurkat cells via intracellularpathways similar to those induced by 4. The findingsallow us to suggest that the induction of paraptosis by 4 and CGP37157 is associated with membrane fusion between mitochondriaand the ER, subsequent Ca2+ influx from the ER to mitochondria,and a decrease in the mitochondrial membrane potential (ΔΨm). On the contrary, celastrol, a naturally occurringtriterpenoid that had been reported as a paraptosis inducer in cancercells, negligibly induces mitochondria-ER membrane fusion. Consequently,we conclude that the paraptosis induced by 4 and CGP37157(termed paraptosis II herein) proceeds via a signaling pathway differentfrom that of the previously known paraptosis induced by celastrol,a process that negligibly involves membrane fusion between mitochondriaand the ER (termed paraptosis I herein).We previously reported that a cyclometalatediridium (Ir) complex-peptidehybrid (IPH)  Amongthem, paraptosis is a relatively new type of non-apoptotic PCD, inwhich cytoplasm and intracellular organelles undergo vacuolizationby the dilation of mitochondria and/or the endoplasmic reticulum (ER),12 possibly due to the transfer of calcium (Ca2+) from the ER to mitochondria.13 Although various inducers of paraptosis, including viruses,14 natural products,23 organic molecules,25 and metal complexes,34 have been reported, the mechanisms responsible for this processare complicated and remain unclear. It is assumed that there are somedifferent intracellular pathways in paraptosis, which are stimulatedby these different types of paraptosis inducers, and that paraptosiscould be classified into some different types.Programmedcell death (PCD)is an essential mechanism for the control of intracellular homeostasisfor cell survival and proliferation and is also recognized as a cellularsuicide, which is one of the strategies for anticancer therapeutics.fac-Ir(tpy)31a [tpy = 2-(4′-tolyl)pyridine]and fac-Ir(ppy)31b (ppy= 2-phenylpyridine) have high photophysical properties that includelong Stokes shifts, high quantum yields, and long emission lifetimes.37 Therefore, Ir complexes have been proposed as attractive candidatesas phosphorescent materials such as in organic light-emitting diodes(OLEDs),39 photoredox catalysts,41 bioimaging probes,47 anticancer agents,49 and related tools. We previouslyreported Ir complex-peptide hybrids (IPHs) that possess H2N-KK(K)GG peptide sequences  at the 5′-or 4′-position (the para or meta position with respect to the C–Ir bond) of the ligands, 2 and 3 or 4, respectively (59 These IPHs induce cell death in Jurkat cells (T-lymphocyte leukemia)with EC50  valuesof 1.5–16 μM and are less toxic against IMR90 cells , which were used as a normal cellline. In addition, the luminescence emission of IPHs is enhanced indead cells, suggesting that IPHs function as not only PCD inducersin cancer cells but also detectors of dead cells.59 The mechanistic study strongly suggested that 2c, 3b, 3c, and 4 induce paraptoticcell death in Jurkat cells via a mitochondrial Ca2+ overload,the decrease in mitochondrial membrane potential (ΔΨm), and cytoplasmic and mitochondrial vacuolization.58 The findings also suggested the direct transfer of Ca2+ into mitochondria from the ER, an intracellular Ca2+-storingorganelle, during the paraptosis processes.Cyclometalatediridium (Ir) complexes such as ectively 1.50−59 T4, which had the lowestEC50 value among 2–4 against Jurkatcells, focusing on the direct influx of Ca2+ from the ERinto mitochondria. The findings indicate that IPH 4 istransferred to mitochondria,57 where it induces membranefusion between mitochondria and the ER and mediates the direct influxof Ca2+ into mitochondria from the ER, resulting in theinduction of paraptosis. It had been reported that CGP37157 is aninhibitor of the sodium (Na+)/Ca2+ exchanger(mNCX) on the outer membrane of mitochondria61 and that it affects the mitochondrial Ca2+ concentration-2,5-diphenyl-2H-tetrazolium bromide] as a function of time and by microscopic observationsto determine the appropriate conditions for mechanistic studies ofparaptosis (4 (0–25 μM)in 10% FBS /RPMI 1640 medium for 1, 3, 6, 12, and 24 h at 37 °C under 5% CO2, and their EC50 values were determined to be 3.2,2.0, 1.5, 1.2, and 1.7 μM, respectively (4 (5 μM) for 30 min, 1 h, 2 h,and 3 h, and a strong green emission from 4 was observedin the dead cells in 1–3 h (The cytotoxicity of raptosis 1. Jurkatectively 1A. In miin 1–3 h 1Bg–o.4 intoJurkat cells was measured by ICP-MS (inductively coupled plasma-massspectrometry). Jurkat cells were treated with 4 (5 μM)for 30 min, 1 h, 2 h, and 3 h at 37 °C under 5% CO2, washed three times with PBS, and lysed with nitric acid at 4 °Covernight. The lysates were diluted with H2O, and the sampleswere analyzed by ICP-MS. As presented in 4 was increased to 0.64 ± 0.01 fmol/cell after incubation for30 min and to 1.3 ± 0.1 to 1.5 ± 0.4 fmol/cell after 1–3h, indicating that the emission enhancement of 4 in Jurkatcells after 1 h is correlated with the intracellular uptake of 4. On the basis of these results, we decided to perform detailedmechanistic studies of paraptosis after incubation with 4 at 5 μM for 1 h.The time-dependent intracellularuptake of 2+ transport, themaintenance of mitochondrial morphology, and the regulation of ER-mitochondriontethering.74 Therefore, we observed the relationship between mitochondria andthe ER in Jurkat cells by conducting co-staining experiments usingspecific probes, MitoTracker Green (0.5 μM) for mitochondriaand ER-Tracker Red (1 μM) for the ER, by confocal microscopy.As shown in The physiological connection betweenmitochondria and the ER is well-known as mitochondria-associated membranes(MAMs), which function to mediate intracellular signaling pathwaysfor inducing apoptosis and autophagy, Ca2+ from the ER to mitochondria acrossthe MAMs has been extensively studied.84 It has been established that the inositol 1,4,5-triphosphate receptor(IP3R) functions as a Ca2+ channel to releaseCa2+ to MAMs and the cytosol under the control of IP3 and Ca2+75 and thatthe VDAC (voltage-dependent anion channel) on the outer mitochondrialmembrane (OMM) and mitochondrial Ca2+ uniporter (MCU) complexon the inner mitochondrial membrane (IMM) mediate the transfer ofCa2+ from the ER and cytosol to mitochondria.76 The mitochondrial permeability transition pore(mPTP), which is a nonspecific channel located on the IMM, allowsions such as Ca2+ and small molecules produced by mitochondrialmetabolism to pass through the mitochondrial matrix under the controlof mitochondrial Ca2+ overload and/or oxidative stress.78The transfer of Ca2+ channels, including 2-aminophenylborate ,85 ruthenium red ,86 and ER-000444793 (an inhibitor of the mPTP),87 have been reported (the structures of theseinhibitors are shown in Chart S1). Theeffect of these inhibitors on the cytotoxicity by 4 andcelastrol, which had been reported to function as a paraptosis inducer,58 was examinedby microscopic observations and MTT assays . Jurkat cells were incubated in the presence ofthese inhibitors for 1 h and then treated with 4 or celastrolfor 3 or 12 h, respectively. The morphological changes and stronggreen emission from 4 were then observed in dead cells,indicating the negligible inhibitory effects of 2-APB, ER-000444793,and RuRed on the cell death induced by 4 and celastrol.Some inhibitors of Ca+/Ca2+ exchanger (mNCX), which functions to export Ca2+ from the mitochondrial matrix to the cytosol in exchangewith cytosolic Na+, affects the mitochondrial Ca2+ concentration.90 We thereforeexamined the cytotoxicity of CGP37157 (90 against Jurkatcells by means of an MTT assay. Jurkat cells were treated with CGP37157(0–1000 μM) in 10% FBS/RPMI 1640 medium for 1, 3, 6,12, and 24 h at 37 °C under 5% CO2, and the EC50 values were determined to be 74 μM for the 12 h incubationand 55 μM for the 24 h incubation for 0–24 h, and cell death was observed after treatment withCGP37157 for 12–24 h, by staining with propidium iodide (PI)3B. Note 4 (4), and included in Poly 812 resin.The resulting samples were sliced and then observed by TEM. As shownin 4 andcelastrol  paraptosis in HeLa cellsinduced by 8-p-hydroxybenzoyl tovarol,23 and (iii) paraptosis in A2780 cells and SKOV-3cells induced by morusin.22 Therefore,we believe that Jurkat cells undergo vacuolization in the paraptoticprocesses induced by 4 and CGP37157, as well as celastrol.The morphological changes in Jurkat cells induced by CGP37157 3B were s4 1B and co92 necrostatin-1 ,93 and 3-methyladenine94  with respect to the cell death induced by CGP37157. Itwas found that Z-VAD-fmk weakly inhibited the paraptosis induced byCGP37157, while negligible inhibition by other PCD (necroptosis andautophagy) inhibitors was observed , indicating very weak relationships between the cell death inducedby CGP37157 and both necroptosis and autophagy.We then tested several inhibitors of PCD such asZ-VAD-fmk (a broadcaspase inhibitor and an apoptosis inhibitor),2+ overload and loss of mitochondrial membrane potential(ΔΨm) induced by CGP37157were also examined. Intracellular Ca2+ concentrations weremeasured by flow cytometry using red-emitting Ca2+ probes,Rhod-2  and Rhod-4 (a cytosolicCa2+ probe). Jurkat cells were stained with Rhod-2/AM orRhod-4/AM (at 5 μM) and then treated with CGP37157 (100 μM)or 4 (5 μM) for a given period of incubation, immediatelyafter which the emission intensity of Rhod-2 and Rhod-4 was measuredby flow cytometry. As shown in panels A and C of 4, indicating the inductionof a mitochondrial Ca2+ overload by CGP37157 and 4. On the contrary, a small emission enhancement of Rhod-4was observed in the cytosol in the presence of CGP37157 and 4  was measured by means of DilC1(5) , the emission intensity of which responds to ΔΨm.95 Jurkat cells were treatedwith CGP37157 (100 μM) for 1–12 h, stained with DilC1(5)(5 μM) for 30 min, and observed via confocal microscopy. Thered emission of DilC1(5) was quenched after the treatment with CGP37157for 6 h, as shown in ΔΨm. The decrease in ΔΨm with 4 was also observed by confocal microscopy(4 (5 μM) for 10–30 min, and the decrease in emissionintensity was observed 20 min after the treatment with 4. The change in the emission intensity profiles of DilC1(5) in ΔΨm, triggered by CGP37157  for 10–30min . It should be noted that the emissionfrom 4 in Jurkat cells was very weak  during a 1 h incubation , indicating that green emission in Figure S5). The emission intensity profiles ofthese intracellular probes indicated by white lines in panels d, i,n, s, x, and ac of Figure S7. In panelsa–e, k–o, and u–y of 4, CGP37157, and celastrol,respectively, indicating the “fission state” of mitochondria.The mitochondrial structure was changed from a fragmentated featureto a tubule structure that was distributed around the nucleus afterthe treatment with 4  and CGP37157 , CGP37157 , and celastrol . Interestingly, extensive overlap of MitoTracker Greenand ER-Tracker Red was observed after the treatment with 4  and CGP37157 . In contrast, the distribution (and weak overlap) of mitochondriaand the ER was negligibly changed after the treatment with celastrol, as shown in Figure S7f. Theseresults strongly suggest that 4 and CGP37157 induce membranefusion between mitochondria and the ER and that celastrol does not.The emission intensity profiles of MitoTracker Green andER-TrackerRed in 2+ homeostasis.100 The fusion of the mitochondrial membrane includes the outer membranefusion, which is induced by mitofusin 1 (MFN1) and mitofusin 2 (MFN2),and the inner membrane fusion induced by optic atrophy 1 (OPA1).100 It was reported that MFN1 and MFN2 are localized on the OMM andER membrane and exist as homo- or heterodimers that function to inducethe membrane fusion of mitochondria.102 It has alsobeen reported that these GTPases function to construct mitochondria-ERtethering sites for Ca2+ transport.106 The fission of the mitochondrial membrane is mediated by dynamin-relatedprotein 1 (DRP1), which is assembled on the OMM.110We next investigatedmitochondrial fusion and fission cycle, which is mediated by intracellularguanosine triphosphatases (GTPases) and controls various aspects ofmitochondrial function such as energy metabolism and Ca4 and CGP37157  for 0–3 h, celastrol(1 μM) for 0–24 h, and CGP37157 (100 μM) for 0–24h , and the target proteins were extracted and analyzed byWestern blot analyses. The expression levels of MFN1 and MFN2 wereweakly changed, and DRP1 was somewhat upregulated by 4 at 5 μM for 30 min to 1 h (4 (after incubationfor 1 h) and CGP37157 (after incubation for 12 h) . In contrast,celastrol decreased the expression levels of these proteins in a time-and concentration-dependent manner .The changes in expression levels of MFNs and DRP1in Jurkat cellsby the treatment with 112 and havealso been proposed to be target proteins for cancer therapy.112 Therefore, we tested the effects of GTPaseinhibitors on the cell death induced by 4 and CGP37157 selectively inhibits DRP1.114 CID1067700 has been reported to be a commoninhibitor of GTPase, especially the Ras superfamily, which is importantin the cell progression through the cell cycle, regulation of cellmorphology, and cell invasion and migration116 (the structures of these inhibitors are shown in Chart S3). Jurkat cells were incubated in the presence ofthese inhibitors for 1 h, treated with 4  or CGP37157 , and subjected to MTT assays.As shown in Figure S9, however, these GTPaseinhibitors have a negligible effect on the cytotoxicity by 4 and CGP37157, indicating that the mechanism of 4- andCGP37157-induced paraptosis is unlikely associated with dynamin-relatedendocytosis, DRP1-mediated mitochondrial fission, and the Ras signalingpathway.It has been reported that intracellular GTPases are related tosignal transduction, endocytosis, and protein biosynthesis for celldifferentiation and proliferation4 andCGP37157 were examined by using small interfering RNA (siRNA) forMFN1 and MFN2 to knock down (KD) these proteins. The siRNAs for MFN1(siRNAMFN1) and MFN2 (siRNAMFN2) (5 or 10 nM)were added to Jurkat cells, and then the cells were incubated for48 or 72 h at 37 °C under 5% CO2 to produce MFN1-and/or MFN2-KD Jurkat cells. Western blot analyses suggested thatMFN1 and MFN2 were knocked down by the corresponding siRNA  by 45% and 80%, respectively . The cytotoxicity of 4 and CGP37157 against MFN1-and/or MFN2-KD Jurkat cells was evaluated by MTT assays. The resultspresented in panels B and C of Figure S10 indicate that the knockdown of MFNs weakly inhibited the cell deathinduced by 4 and CGP37157. The effect of a negative controlsiRNA (NCsiRNA) on the expression levels of MFNs and the cell viabilityof Jurkat cells was negligible, implying that MFNs are somewhat relatedto the paraptosis induced by 4 and CGP37157. It shouldbe noted that the treatment with siRNA at 5 nM for 72 h was toxicagainst Jurkat cells in the presence of 4 and CGP37157,although the efficiency of knockdown of these proteins by siRNA couldbe improved by increasing the incubation time, as shown in Figure S11.The roles of MFNs in the cell death induced by Figure S12, a negligible effect of knockdown of MFNs on thecell death induced by celastrol was observed.The cytotoxicity of celastrolagainst MFN1- or MFN2-KD Jurkat cellswas also evaluated by MTT assays. As shown in 4, CGP37157, and celastrolare shown in 4, CGP37157, and celastrol, respectively.On the basis of the aforementionedresults, proposed plausible mechanisms for the development of paraptosisin Jurkat cells induced by IPH 2+ channels on the membrane of the ER andmitochondria such as IP3R, MCU, and mPTP are not majorfactors in the transport of Ca2+ from the ER to mitochondriathat is induced by 4, as confirmed by MTT assays usingspecific channel inhibitors .(1) The Ca4 induces (i) membrane fusion (or tethering) between mitochondriaand the ER via MFNs and related molecules , (ii)direct transport of Ca2+ from the ER to mitochondria, and(iii) a decrease in the mitochondrial membrane potential (ΔΨm), resulting in paraptosis inJurkat cells . We assume that the induction of the membranefusion of mitochondria and the ER by artificial compounds is one ofthe most important findings in this work.(2) Close contact between mitochondria and the ER via mitochondria-associatedmembranes (MAMs) was observed in Jurkat cells before the additionof IPHs , and 9. 50 value of 55 μM for 24 h), as confirmed by microscopic andTEM observations . This scenario is supported by the results showingthat the knockdown of MFNs partially restored the cell viability ofJurkat cells .(3) We found thatCGP37157, a mNCX inhibitor, induces paraptosisin Jurkat cells after treatment for 12–24 h (ECrvations 3 and 4. 4 and CGP37157 weakly changethe expression levels of dynamine-related protein 1 (DRP1), a mitochondrialmembrane fission protein . A negligibleeffect of DRP1 inhibitors on paraptosis was observed, indicating thatDRP1 is scarcely related to 4- and CGP37157-induced paraptosis.(4) Weobserved that 58 the expression levels of MFNs are suppressed andmembrane fusion between mitochondria and the ER was negligible . We previously reported that celastrol increasesCa2+ concentrations in the cytoplasm rather than in mitochondriafor the induction of paraptosis and activates the mitogen-activatedprotein kinase (MAPK) signaling pathway, resulting in autophagy.58 These data imply that different signaling pathways are involvedin paraptosis induced by different drugs.(5) In the presenceof celastrol, which had been reported to bea paraptosis inducer,2–4 are localizedon mitochondria during the earlystages of the cell death process.58 These data, considering that CGP37157 is a mNCX inhibitor,90 strongly suggestthat the main target organelle of these compounds is the mitochondria.On the contrary, several target proteins of celastrol have been identified,120 such as a heat-shock protein 90 (Hsp90)-cell division cycle 37 (Cdc37)complex that controls protein folding118 and IκB kinase (IKK) that regulates gene transcription throughNF-κB activation.119 In addition,it has been proposed that a quinone methide moiety of celastrol functionsas an acceptor of Michael addition  reactions with thethiol residue of the target proteins.120 Although the relationship between these target proteinsof celastrol and paraptotic mechanisms in Jurkat cells remains unclear,it is very likely that these differences result in the different mechanismsin paraptosis induced by 4 and CGP37157 and in paraptosisinduced by celastrol.(6) In our previousstudy, we reported that IPHs such as 2–4 and CGP37157 is associated with membrane fusionbetween mitochondria and the ER.These findings allow us to suggest thatparaptosis can be classifiedinto at least two types. The first is a known type of paraptosis (termedparaptosis I herein) that is induced by celastrol and negligibly involvesmembrane fusion between mitochondria and the ER. The second type ofparaptosis (termed paraptosis II herein) induced by IPHs such as 2+ from the ER tomitochondria. The findings suggest that IPH 4 inducesmembrane fusion (or tethering) between the ER and mitochondria. Wealso found that CGP37157, an inhibitor of a mitochondrial Na+/Ca2+ exchanger (mNCX), induces paraptosis in Jurkat cellsvia intracellular pathways similar to those induced by 4. Importantly, the membrane fusion of the ER and mitochondria bythese two compounds would lead to the direct transport of Ca2+ from the ER to mitochondria. In contrast, celastrol, which had beenknown as a naturally occurring paraptosis inducer, negligibly hassuch a function. To the best our knowledge, this is the first exampleto show the structural fusion of mitochondria with the ER by artificialmolecules and the direct transfer of Ca2+ from the ER tomitochondria to stimulate intracellular pathways for the inductionof programmed cell death.Insummary, we report on the results of a series of more detailedmechanistic studies of paraptotic cell death that is induced by Ircomplex-peptide hybrids (IPHs) that possess basic (cationic) peptides,focusing on the direct transport of Ca2+ overload in the cytoplasm and hardly involves membranefusion between mitochondria and the ER. The second type is a new classof paraptosis induced by 4 and CGP37157 and is termedparaptosis II in this work, which involves mitochondria–ERmembrane fusion and subsequent mitochondrial Ca2+ overload.Because the structures of IPHs and CGP37157 are so different, we donot exclude the possibility that these two molecules activate differenttarget molecules to stimulate intracellular signaling pathways involvedin paraptosis II.The results obtained in this workindicate that paraptosis shouldbe classified into two types. The first is a known type of paraptosisinduced by celastrol, which is termed paraptosis I herein, which involvesCaThe findings reported in this study provideuseful informationnot only for mechanistic studies of PCD such as paraptosis but alsofor the design and synthesis of PCD inducers in cancer cells in thefuture. The design and synthesis of IPHs and other types of peptidehybrids that possess higher anticancer activity and more detailedmechanistic studies are now underway.H-tetrazolium bromide (MTT), Rhod-2/AM, and Rhod-4/AM werepurchased from Dojindo. Z-VAD-fmk was purchased from the Peptide Institute,and necrostatin-1 (Nec-1) was purchased from Enzo Life Science. Propidiumiodide (PI), 3-methyladenine (3-MA), potassium chloride, and bovineserum albumin (BSA) were purchased from Fujifilm Wako Chemicals. CGP37157and DilC1(5)  were purchased from Sigma-Aldrich. Roswell Park Memorial Institute(RPMI) 1640 medium, minimum essential medium (MEM), Dulbecco’smodified Eagle’s medium (DMEM), phosphate-buffered saline (PBS),Tween 20, a 40% (w/v) acrylamide/bisacrylamide solution, tris(hydroxymethyl)aminomethane(Tris), sodium dodecyl sulfate (SDS), ammonium persulfate (APS), glycine,sodium chloride, and RuRed were purchased from Nacalai tesque. Dynasoreand Mdivi-1 were purchased from TCI . Celastroland 2-aminoethoxydiphenylborate (2-APB) were purchased from CaymanChemical Co. ER-000444793 was purchased from MedChemExpress. CID1067700was purchased from Calbiochem. The Pierce BCA Protein Assay Kit waspurchased from Thermo Fisher Scientific Inc. Fetal bovine serum (FBS)was purchased from Capricorn products Inc. The anti-MFN1 antibody,anti-MFN2 antibodies, anti-DRP1 antibody, and siRNA for MFN1 (siRNAMFN1) and MFN2 (siRNAMFN2) were purchased from SantaCruz Biotechnology. The anti-GAPDH, anti-mouse IgG HRP-linked, andanti-rabbit IgG HRP-linked antibodies were purchased from Cell Signaling.The anti-mouse IgG Alexa Fluor 647-linked antibody was purchased fromAbcam. INTERFERin was purchased from Pulyplus. Stock solutions of 4 in PBS, CGP37157, and celastrol in DMSO were stored at 0°C. The results of MTT and BCA assays were confirmed by usinga multilabel counter, Wallac 1420 ARVO (PerkinElmer). Fluorescentimaging studies were conducted using fluorescent microscopes . The intracellularuptake of 4 was measured by ICP-MS .The results of Western blot analyses were analyzed on the ChemiDocMP system (Bio-Rad). Flow cytometric analyses were performed by usinga flow cytometer , and data were analyzedon FlowJo software . TEM images were obtained by usingthe instrument .All reagents and solvents wereof the highest commercial quality and used without further purification,unless otherwise noted. All aqueous solutions were prepared usingdeionized water. MitoTracker Green, MitoTracker Red, ER-Tracker Red,and Opti-modified Eagle’s medium (Opti-MEM) were purchasedfrom Invitrogen. 3--2,5-diphenyl-22 incubator.Jurkat, HeLa S3, and A549 cells wereincubated in RPMI 1640 medium, MEM, and DMEM, respectively, supplementedwith 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycinat 37 °C in a humidified 5% CO4 cells/well)were seeded on a 96-well plate  incell culture medium and incubated overnight at 37 °C under 5%CO2. Jurkat (2.0 × 104 cells/well), HeLaS3, and A549 cells were treated with 4 (0–25 μM)and CGP37157 (0–100 μM) for 1, 3, 6, 12, and 24 h at37 °C under 5% CO2, after which a MTT solution inPBS  was added to each well. After incubation for4 h at 37 °C under 5% CO2, a 10% SDS in 0.01 N HClaqueous solution (100 μL) was used as a formazan lysis solutionand the resulting solutions were incubated overnight under the sameconditions, followed by the measurement of the absorbance at 570 nmusing a multilabel counter, Wallac 1420 ARVO (PerkinElmer).HeLa S3 and A549 cells (2.0 × 104 cells/well) were incubatedin the presence of 2-APB (50 μM), ER-000444793 (10 μM),RuRed (10 μM), Dynasore (10–30 μM), Mdivi-1 (10–30μM), CID1067700 (1–10 μM), Z-VAD-fmk (30 μM),Nec-1 (30 μM), and 3-MA (5 mM) in 10% FBS RPMI medium (50 μL)for 1 h at 37 °C under 5% CO2, and then solutionsof 4 (10 μM) and celastrol (2 μM) in 10%FBS/RPMI 1640 medium (50 μL) were added. The final concentrationsof 4 and celastrol were 5 and 1 μM, respectively.The resulting solutions were incubated at 37 °C under 5% CO2 for 1 and 3 h (4) or 24 h (celastrol), afterwhich a 0.5% MTT solution in PBS (10 μL) was added to each well.After incubation for 4 h at 37 °C under 5% CO2, a10% SDS in 0.01 N HCl aqueous solution (100 μL) was used asa formazan lysis solution and the resulting solutions were incubatedovernight under the same conditions, followed by the measurement ofthe absorbance at 570 nm using a multilabel counter, Wallac 1420 ARVO(PerkinElmer).In a 96-wellplate, Jurkat cells (2.0 × 105 cells) were treatedwith 4 (5 μM), CGP37157 (100 μM), and celastrol(1 μM) in 10% FBS/RPMI 1640 medium (100 μL) at 37 °Cunder 5% CO2 for different periods of incubation time,after which the cells were collected by centrifugation and washedwith PBS. For CGP37157 and celastrol, the cells were treated withpropidium iodide  in PBS for 30 min at37 °C under 5% CO2, washed with PBS, and observedby fluorescence microscopy  using a GreinerCELLview dish (35 mm × 10 mm). Emission images were obtainedby using an FF01 filter (excitation at 377 nm and emission at 520nm) for 4 and a TRITC filter (excitation at 540 nm andemission at 605 nm) for CGP37157 and celastrol.In a1.5 mL Eppendorftube, Jurkat cells (2.0 × 105 cells) were stained with MitoTracker Green(0.5 μM) for 1 h at 37 °C under 5% CO2 and thenER-Tracker Red (1 μM) for 1 h at 37 °C under 5% CO2 in 10% FBS/RPMI 1640 medium (100 μL). After being washedwith PBS, the cells were treated with 4 (5 μM)in 10% FBS/RPMI 1640 medium (100 μL) for 0–30 min at37 °C under 5% CO2. For CGP37157 and celastrol, Jurkatcells were treated with CGP37157 (100 μM) or celastrol (1 μM)in 10% FBS/RPMI 1640 medium (100 μL) for 0–24 h, afterwhich the cells were stained with MitoTracker Green (0.5 μM)for 1 h at 37 °C under 5% CO2 and then ER-TrackerRed (1 μM) for 1 h at 37 °C under 5% CO2 inPBS. The cells were washed with PBS and observed by confocal microscopy using a Greiner CELLview dish (35 mm× 10 mm). Excitation at 473 nm and emission from 485 to 545 nmwere used for MitoTracker Green. Excitation at 559 nm and emissionfrom 570 to 620 nm were used for ER-Tracker Red. The exposure timewas 20 μs/pixel.In a 1.5 mL Eppendorf tube, Jurkat cells(2.0 × 106 cells) were treated with 4 (5 μM) for 0–3 h at 37 °C under 5% CO2 (n = 3). After the cells had been washed threetimes with PBS, HNO3  was added to the cellsand the resulting solutions were incubated overnight at 4 °C.After centrifugation , the supernatantwas transferred to a 15 mL tube, diluted with H2O (4.5mL), and filtered. The number of iridium atoms was measured by ICP-MS.In a 1.5 mL Eppendorf tube, Jurkatcells (1.0 × 106 cells) were incubated in the presenceof 4 (5 μM for 0–3 h or 0–20 μMfor 30 min), CGP37157 (100 μM for 0–24 h or 0–100μM for 12 h), and celastrol (0–10 μM for 24 h or1 μM for 0–24 h) at 37 °C under 5% CO2. After the cells had been washed twice with PBS, the proteins wereextracted by using RIPA buffer  and quantified usinga Pierce BCA Protein Assay Kit (Thermo Scientific). Proteins (7.5μg/well) for MFNs and DRP1 and proteins (5.0 μg/well)for GAPDH were used for sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS–PAGE) (7.5% for MFNs and DRP1 and10% for GAPDH) (Bio-Rad). After SDS–PAGE, the proteins weretransferred to a polyvinylidene fluoride membrane (Merck Millipore)by using a semi dry blotter (Bio-Rad). The membrane was blocked withBlocking One solution  for 30 min at room temperature.After being blocked, the membrane was washed three times with 1×TBST and treated with primary antibodies overnight (at a dilutionof 1/1000 for MFNs and DRP1 or 1/2000 for GAPDH) in a signal enhancerHIKARI-solution A . The membrane was washed threetimes with 1× TBST and treated with the anti-mouse or anti-rabbitIgG HRP-conjugated secondary antibody (at a dilution of 1/4000 forMFNs and DRP1 or 1/10000 for GAPDH) in 1× TBST for 60 min atroom temperature. The protein signal was spotted with a Chemi-LumiOne Ultra solution  using the ChemiDoc MP system (Bio-Rad).In a 1.5 mLEppendorf tube, Jurkatcells (1.0 × 106 cells)were incubated with 4 (5 μM for 3 h), CGP37157(100 μM for 12 h), and celastrol (1 μM for 24 h) in 10%FBS/RPMI 1640 medium at 37 °C under 5% CO2. Aftercentrifugation , the cells were washedtwice with PBS, prefixed with glutaraldehyde (2.5%) at 4 °C for40 min, and then washed twice with PBS. Osmium tetroxide (1%) wasused for postfixation, and the solution was incubated at 4 °Cfor 30 min. The cells were washed with PBS, included in an agarosegel, and dehydrated by using 50–100% and anhydrous EtOH. Thecells were embedded into Poly 812 resin (Nisshin EM Co. Ltd.) at 60°C for 3 days. The resin was sliced with a glass knife (150 nmthickness) on an ultramicrotome , and the samples werestained with EM stainer (Nisshin EM Co. Ltd.) and observed on TEMinstrument  with electron irradiation at 100 kV.In a1.5 mL Eppendorf tube, Jurkat cells (3.0 × 105 cells) were incubated in the presence of Rhod-2/AM  or Rhod-4/AM  in 10% FBS/RPMI1640 medium for 30 min at 37 °C under 5% CO2 and thentreated with 4  or CGP37157 in 10% FBS/RPMI 1640 medium (100 μL)for 30 min at 37 °C under 5% CO2. Immediately afterthe treatment, 10% FBS/RPMI 1640 medium (300 μL) was added tothe cells and the samples were analyzed on a flow cytometer . The data were analyzed on FlowJo software .In a 1.5 mL Eppendorf tube, Jurkat cells (2.0 × 105 cells) were stained with DilC1(5) in 10% FBS/RPMI 1640 medium for 30 min at 37°C under 5% CO2, followed by the treatment with 4  in the medium for 0–60min at 37 °C under 5% CO2. For CGP37157, Jurkat cells(2.0 × 105 cells) were treated with CGP37157  in 10% FBS/RPMI 1640 medium for 0–12h at 37 °C under 5% CO2, followed by the treatmentwith DilC1(5)  in PBS. The cells were washedwith PBS and observed by confocal microscopy using a Greiner CELLview dish (35 mm × 10 mm). Excitation at405 nm and emission from 470 to 520 nm were used for 4. Excitation at 635 nm and emission from 650 to 750 nm were usedfor DilC1(5). The exposure time was 20 μs/pixel.In a 1.5 mL Eppendorf tube, Jurkatcells (2.0 × 106 cells) were stained with MitoTrackerRed  or ER-Tracker Red  in 10%FBS/RPMI 1640 medium at 37 °C under 5% CO2. Afterbeing washed with PBS, the cells were fixed with 4% paraformaldehydein PBS (500 μL) for 10 min at 37 °C under 5% CO2, washed with PBS, and permeabilized by using 0.1% Tween 20 in PBS(200 μL) for 15 min at room temperature. After the cells hadbeen blocked with 2% BSA in 1× PBST (200 μL) for 1 h atroom temperature and washed with 1× PBST, the cells were treatedan anti-MFN1 or an anti-MFN2 antibody (1:100 dilution in 1× PBST)at 4 °C overnight. The cells were washed with 1× PBST andtreated with anti-mouse IgG H&L (Alexa Fluor 647) (1:100 dilutionin 1× PBST) for 1 h at room temperature. After being washed with1× PBST, the cells were observed by confocal microscopy  using a Greiner CELLview dish (35 mm × 10 mm).Excitation at 559 nm and emission from 570 to 620 nm were used forMitoTracker Red and ER-Tracker Red. Excitation at 635 nm and emissionfrom 650 to 750 nm were used for the detection of MFN1 and MFN2. Theexposure time was 20 μs/pixel.In a 1.5 mL Eppendorf tube, Jurkatcells  in 10% FBS/RPMI 1640 medium that had beenseeded on a 12-well plate and incubated overnight at 37 °C under5% CO2 ,and the resulting solution was incubated for 48 or 72 h at 37 °Cunder 5% CO2. The cells were collected by centrifugation, and the expression levels of MFNs wereevaluated by Western blot analysis as described above. The cytotoxicityof 4 (3 or 5 μM), CGP37157 (50 or 100 μM),and celastrol (1 μM) against MFNs-KD Jurkat cells was evaluatedby MTT assays as described above.To a solution of transfection reagent  in OPTI-MEM (0.3 mL) in a 1.5 mL Eppendorf tube was addedsiRNA  in Ht test. Data are presented as means ± the standarddeviation of three independent experiments, and a P of <0.05 was considered to indicate a statistically significantdifference.Statistical analyses of MTT assayswere performed by using Graphpad Prism 9 software with the Student’s


The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of the spike, and therefore lose neutralization efficacy against emerging SARS-CoV-2 variants, which frequently mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralize antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both NGS and epitope mapping of a monoclonal antibody with a known binding site. The most prominent epitope captured represented parts of the fusion peptide (FP) of the spike. It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell; the serine protease TMPRSS2 cleaves the spike within this sequence. Blocking this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence have been rather rare among SARS-CoV-2 variants so far, this may provide an advantage in the fight against future virus variants. The novel beta-coronavirus SARS-CoV-2 was described in late 2019 and is responsible for the current public health crisis of global concern ,4. To taIn total, 20 samples were obtained in Germany and Northern Italy between February 2020 and January 2021 from non-vaccinated patients that had been hospitalized at intensive care units (ICU). Sampling was performed according to the Declaration of Helsinki. Plasma samples were taken between March 2020 and February 2021. Approval for the serum samples was given from the ethical committee of the Technische Universität Braunschweig  and from the Institutional Review Board of Policlinico San Matteo (protocol number P_20200029440).E. coli SS320  were used for transformation in a pre-chilled 0.1 cm electroporation cuvette using a micropulser  with 1.8 kV for at least 5 ms. Immediately after pulsing, 1 mL of pre-warmed recovery medium (Lucigen) was added and cells were recovered for one hour at 37 °C, 650 rpm. Next, 10 µL of transformed E. coli suspension were used for a dilution series ranging from 10−4 to 10−6 in 2xYT medium and plated them on 2xYT agar plates supplemented with 100 µg/mL Ampicillin and 0.1 M glucose (2xYT-GA). The next day, the obtained colonies were counted and the maximum library diversity was determined. The remaining 990 µL recovered E. coli were plated onto a 15 × 15 cm 2xYT-GA plate. After overnight cultivation, the clones were collected by applying 25 mL 2xYT-GA medium onto the plates and incubation for 15–30 min on a plate rocker. The suspension was harvested with a L-spatula and 1 mL aliquots were saved as glycerol stocks (750 µL medium and 250 µL 80% glycerol solution) by freezing them in liquid nitrogen and storing them at −80 °C.The SARS-CoV-2 encoding sequences were separately amplified from seven genome fragments in pUC57 vectors via PCR using individual primers for each fragment. DNA of each fragment was purified using the Macherey-Nagel PCR purification Kit according to the manufacturer’s instructions. PCR products were sonicated for 30 s on/30 s off for 70 cycles . The obtained fragmented DNA was blunt-end polished , purified with the NucleoSpin Gel and PCR Clean-up Kit  and cloned into pHORF3 with T4 DNA Ligase (NEB) overnight at 16 °C followed by a heat-inactivation at 70 °C for 10 min . LigatioE. coli cells that contained a pORF3 vector were analyzed with PCR according to the method described by Kügler et al. [r et al. . Briefly11 cfu of Hyperphage. The culture incubated at 37 °C without shaking for 30 min and at 37 °C with shaking for 30 min at 250 rpm. Cells were harvested by centrifugation at 3220× g for 10 min and resuspended in 2xYT-KA medium for overnight phage production at 30 °C, 210 rpm. The next day, the culture was centrifuged for 30 min at 8000 rpm . The supernatant was harvested and 1/5 (v/v) of 20% PEG (v/v) in 2.5 M NaCl were added. The suspension was incubated overnight on ice in a 4 °C environment to precipitate phage. Next, the suspension was centrifuged for 1 h at 11,000 rpm. The supernatant was discarded and the remaining pellet was resuspended in 10 mL phage dilution buffer (PDB). The phage suspension was added to 1/5 of the final volume of 20% PEG (v/v) in 2.5 M NaCl and incubated overnight on ice. Then the suspension was centrifuged for 30 min at 20,000 rpm and the supernatant was discarded. The phage pellet was diluted in 1 mL PDB and added to a 2 mL Eppendorf tube. Finally, the cells were centrifuged 2× at 18,000× g and filtered in a 2 mL vial using a 0.45 µm filter. The libraries were stored at 4 °C. Libraries were titrated in a dilution series ranging from 10−8 to 10−12 and 10 µL of each dilution was used to infect 50 µL E. coli XL-1 Blue MRF’ cells (OD600 = 0.5). The infected bacteria were plated out on 2xYT-GA plates. To enrich SARS-CoV-2 related protein fragments, the library was packaged using Hyperphage  that lacks the gene encoding pIII . To do sE. coli XL1-Blue (MRF’) cells at an OD600 = 0.4–0.6. The infected bacterial culture was inoculated overnight at 37 °C, 250 rpm and used for plasmid preparation with the Macherey-Nagel Easy Pure kit according to the manufacturer’s instructions. The obtained plasmids were used for PCR amplification of the sequence of interest. Here, it was important to introduce barcode sequences via primers for NGS analysis at GENEWIZ, according to the GENEWIZ Amplicon-seq guidelines. The PCR products were purified and analyzed on a 1% agarose gel. Resulting FASTQ files were mapped to the Wuhan reference genome (Genbank No.: MT326090.1) using BWA-MEM with the UGENE workflow “Processing of raw DNA-seq paired-end reads”. The resulting BAM file was further processed using samtools [For Next-Generation-Sequencing (NGS), the libraries were used to re-infect 5 mL of samtools . The cov11 phage/well) and incubated overnight at 4 °C. In parallel, a 100-fold excess (diversity/library titer) of each library was pre-incubated for one hour at RT in panningblock solution (1% (w/v) BSA and 1% (w/v) milk powder in PBS + 0.05% (v/v) Tween20 on an unrelated human IgG antibody and on the anti-human Fc specific antibody MC002-M , used for capturing of the sera. Serum antibodies dissolved in panningblock were captured by the anti-human Fc specific antibody MC002-M for two hours at RT. After washing, the pre-incubated libraries were applied and incubated two hours at RT. Unbound phage were washed away and 150 µL/well trypsin (10 µg/mL in PBS) was applied to each well for 30 min at 37 °C for elution. Then, 100 µL of eluted phage were used for infection of 5 mL E. coli TG1 cells at an OD600~0.5 in a 24-well MTP. The plates were incubated 30 min without and then 30 min with shaking at 450 rpm at 37 °C in a shaker . Medium was changed by centrifuging the plate 10 min at 2500× g (Eppendorf 5810R) and drying the plate briefly on a paper towel. Afterwards, 5 mL of 2xYT medium supplemented with 100 µg/mL Ampicillin and 0.1 M glucose (2xYT-GA) were added to the pellet and incubated 30 min at 37 °C, 450 rpm. The suspension was infected with 17 µL Hyperphage (3 × 1012 cfu/mL) and incubated 30 min at 37 °C without and afterwards 30 min at 37 °C with shaking at 450 rpm. Plates were centrifuged as before and 5 mL 2xYT-A + 70 µg/mL Kanamycin (2xYT-KA) were added to the pellet. Phage production was carried out overnight at 30 °C, 450 rpm. For the next round of panning, 100 µL produced phage were added to 50 µL panningblock solution and applied to the pre-incubation steps. After the second panning round, 1:10, 1:100 and 1:1000 dilutions of the eluted phage were prepared in 2xYT medium. From each dilution, 10 µL were used to infect 50 µL E. coli XL1-Blue MRF’ (OD600~0.5). The infected bacteria were plated on 2xYT-GA agar plates. Randomly selected clones were analyzed as indicated above and alignments were done with UGENE (version 34). The panning procedure was adapted from Zantow et al. . Brieflyv/v) glycerol. The next day, 10 µL were transferred from each well to 180 µL 2xYT-GA in a new 96-well MTP. The MTP was incubated two hours at 37 °C, 800 rpm before it was infected with 10 µL/well of Hyperphage (3 × 1012 cfu/mL). After incubation for 30 min at 37 °C with and without shaking at 800 rpm, the plate was centrifuged at 3220× g for 10 min. The pellets were resuspended in 2xYT-KA medium and phage particles were produced overnight at 30 °C, 800 rpm. For epitope mapping of monoclonal scFv-Fc antibodies, a single panning round was performed.For each panning approach, a 96-well MTP was filled with 150 µL 2xYT-GA medium per well after the third panning round. Individual clones were picked from the 2xYT-GA agar plates and inoculated in the 96-well MTP overnight at 37 °C, 800 rpm. This plate served as a master-plate and was stored at −80 °C in 20% (v/v)) and blocked with 350 µL/well of 2% (w/v) M-PBST. Overnight produced phage particles were captured and COVID-19 patient sera were diluted 1:100 in 2% M-PBST supplemented with 1010 cfu Hyperphage and 1:10 (v/v) E. coli cell lysate and applied to the captured phage particles. To detect bound serum antibodies, an anti-human IgG polyclonal antibody conjugated with HRP  was applied at a dilution of 1: 70,000 and incubated two hours at RT. The plates were washed three times with Milli-Q + Tween20 and TMB substrate was applied for 20 min to each well. After stopping the reaction with 1 N sulfuric acid the absorption at 450 nm and 620 nm was measured. In the assays using monoclonal antibodies, clones were considered positive with a ΔOD450-620 nm signal higher than 1.3.For screening ELISA experiments, 96-well MTP  were coated with 25 ng/well of a mouse anti-pVIII antibody  and incubated overnight at 4 °C. Wells were washed three times with Milli-Q + Tween20 (0.1% (w/v) in TBS) and incubated on the slides over night at 4 °C. Bound serum antibodies were stained with Alexa Fluor® 647 conjugated, isotype specific secondary antibodies . Data analysis was performed by visual inspection.Microarray assays were carried out according to the method described by SK-Hotop et al. . Brieflyw/v) BSA dissolved in PBS-T. Next, 1 µg of biotinylated peptide per well was captured for one hour at RT and plates were washed three times as described before. Sera were titrated in 2% BSA in PBS-T ranging from the dilution factor of 1:300 to 1:3 × 10−8 and applied to a 384-well MTP. Unbound serum antibodies were washed away three times with Milli-Q + Tween20. To detect bound serum antibodies, we applied an anti-human IgG polyclonal antibody conjugated with HRP  and incubated for one hour at RT. The plates were washed three times with Milli-Q + Tween20 and TMB substrate was applied for 20 min to each well and analyzed as described before. Data analysis and graphical plots were carried out using OriginPro (version 2018).Biotinylated peptides were obtained from Peps4LS . Costar 384-well MTP were coated with 200 ng/well Streptavidin overnight at 4 °C. The following day, all wells were washed three times with Milli-Q + Tween20 and blocked for one hour at RT with 2%  upon packaging with Hyperphage, phages display viral protein fragments of different sizes on their surface ,40. The To determine whether the generated SARS-CoV-2 genomic ORFeome library could be used to identify antibody epitope, we tested the non-neutralizing monoclonal antibody STE73-6C10 that was shown to recognize a peptide sequence within the S1 subunit . The ORFFive COVID-19 patient sera from Northern Italy and Germany were panned against the generated ORFeome library to determine the mimotopes of the antibodies generated during humoral response. Prior to ORFeome display, antibody titers against spike and RBD were determined . PatientIn this study, we identified an immunogenic polypeptide located within the spike protein of SARS-CoV-2 which is prominently recognized by COVID-19 patient sera. This peptide was found in four out of five serum samples from COVID-19 ICU patients by ORFeome phage display. The minimal epitope region (MER) encodes the 16 amino acid (aa) sequence (811-PSKRSFIEDLLFNKVT-828) and refers to the N-terminus of the fusion peptide (FP) of spike, which is involved in viral cell entry after the spike protein is cleaved by the host protease TMPRSS2 ,43. ThisIn our study, we show that the identification of this epitope is possible using ORFeome phage display of a whole-genome SARS-CoV-2 library. The library quality determines whether immunogenic epitopes can be found within serum samples or not. To this end, our library was analyzed by NGS; the results indicated that the majority of the genome sequence was covered by the library, despite some accessory proteins being only marginally represented. For additional control, we showed that monoclonal antibody STE73-6C10  binds itNewly emerging SARS-CoV-2 variants can escape the immune response to earlier variants by introducing mutations in the RBD ,24. TargWe showed that ORFeome phage display could identify this epitope in a fast and effective manner. The sequences may be useful for the development of therapeutics  which are more likely to be active against future variants, based on the observation that the FP sequence has remained conserved so far among SARS-CoV-2 circulating variants.


This new cryptand-like system acts as an effective ligand and allows for complexation of BF2 (boron difluoride) subunits. Three BODIPY arrays, containing two, three, and four BF2 subunits, namely 4·2BF2, 4·3BF2 and 4·4BF2, could thus be isolated from the reaction of 4 with BF3·Et2O in the presence of triethylamine at 110 °C, albeit in relatively low yield. As prepared, bicycle 4 is characterized by a rigid C2 symmetric structure as inferred from VT NMR spectroscopic analyses. In contrast, the three BODIPY-like arrays produced as the result of BF2 complexation are conformationally flexible and unsymmetric in nature as deduced from similar analyses. All four products, namely 4, 4·2BF2, 4·3BF2 and 4·4BF2, were characterized by means of single crystal X-ray diffraction analyses. Tetramer 4·4BF2 gives rise to a higher extinction coefficient (by 2.5 times) relative to the bis- and tris-BODIPY arrays 4·2BF2 and 4·3BF2. This was taken as evidence for stronger excitonic coupling in the case of 4·4BF2. All three BODIPY-like arrays proved nearly non-fluorescent, as expected given their conformationally mobile nature. The efficiency of reactive oxygen species (ROS) generation was also determined for the new BODIPY arrays of this study.A fully conjugated cryptand-like bicyclic porphyrinoid ligand 4, incorporating three carbazole linkages and four dipyrrin moieties, was prepared  A cryptand-like bicyclic porphyrinoid was obtained in preference over the monocyclic porphyrinoid by controlling the reaction stoichiometry and condensation conditions. The cryptand-like species supports formation of multiple 3D BODIPY-like arrays. The strong exciton coupling22,24 expected for systems containing more than one BODIPY-like subunit has made such systems of interest in the context of specific applications, including light-harvesting, fluorescence-based sensor development, and photodynamic therapy (PDT).27–33 Recently, Werz and co-workers prepared a series of benzene-fused oligo-BODIPYs containing up to 31 rings that exhibited intriguing NIR-absorbing and redox properties.34 Early on several macrocyclic BODIPY arrays incorporating 1,4- and 1,3-phenylene moieties were reported by Nabeshima and co-workers, which were shown to act as hosts for cationic guests.35,36 Our own group reported two giant calix[8]- and calix[16]phyrin-derived BF2 arrays with strong NIR absorption features.22 However, to the best of our knowledge, there are no reports concerning covalently linked three-dimensional conjugated BODIPY arrays. Accordingly, their photophysical properties remain unexplored.Recently, covalent arrangements of BODIPYs  metallocage structure,38 might allow construction of a 3D porphyrinoid containing co-planar dipyrrin units suitable for BF2 complexation. The present study was undertaken as a test of this hypothesis. In fact, both a figure-of-eight monocyclic porphyrinoid 3 and a fully conjugated cryptand-like bicyclic porphyrinoid 4 could be formed via a controllable MacDonald-type condensation using such a precursor   followed by oxidation with DDQ. The crude reaction mixture was subjected to alumina column chromatography using 50% CH1H NMR spectrum of 4 recorded in THF-d8 at room temperature was characterized by the presence of well resolved signals consistent with a symmetric system  is consistent with strong intramolecular hydrogen bonding interactions between the dipyrromethene units.The c system . Three sO Fig. S2. The pyrO Fig. S2 and S7†.1H–1H COSY and 2D ROESY analyses ), 7.80 ), 7.70 ), 7.60 ), 7.46 ), 7.39), 7.37 ), 7.14 ), 6.99 ), and meso –CH protons signal at 6.97 ppm ), respectively. These peak assignments were made with the aid of via the slow diffusion of methanol into a THF solution. The resulting X-ray structure confirmed that in the solid state 4 possess a cryptand-like shape with C2 molecular symmetry  are small . Such binding behaviour would be expected to inhibit molecular motion.The  Fig. S26. Similar2, 4·3BF2 and 4·4BF2. All three systems are bicyclic and bear structural analogy to 4. For instance, the bis-BODIPY analogue, 4·2BF2, is pseudo-D2-symmetric with the two BF2 units bound to the meso-substituted dipyrrin moieties in a diagonal arrangement. The unbound dipyrrin units are engaged in intramolecular hydrogen bonding interactions characterized by bond distances and angles of 2.108 Å and 123.57° and 2.257 Å and 122.23° for N5–H5⋯N4 and N10–H10⋯N9, respectively (9N2B planes indicated as A and B) is 10.475 Å. Five methanol molecules, solvents of crystallization, are observed in each unit  to the carbazole units, while the final methanol molecule does not appear to interact directly with 4·2BF2.Further insights into the molecular structure of the BODIPY arrays came from single-crystals X-ray structural analyses. ectively . The cen Fig. S37. Two of 2 BODIPY a shorter centroid-to-centroid distance between the adjacent chromophore units (defined by C9N2B planes) of 4.94 Å (A and B) was observed  of 4.942 and 4.983 Å were found . Such a finding is consistent with the free rotation inferred from the solution phase NMR spectral studies  with an extinction coefficient, ε = 2.27 × 105 M−1cm−1, which is ca. 2.5-fold larger than those for the corresponding bis- and tris-BODIPY arrays. In the case of 4·4BF2 , a high energy absorption feature is seen at 400 nm. These spectral features are taken as evidence of interchromophore exciton coupling between the two pair of orthogonally-aligned BODIPY subunits present in 4·4BF2.28,40The absorbance and luminescent properties of ligand 4 and corresponding BODIPY arrays 4·2BFluene cf.. As the 41 Across the board, these TD-DFT studies revealed a good correspondence between the observed and predicted spectral features  calculations at the CAM-B3LYP/6-311G level.ϕFL) were determined to be 0.03%, 0.14%, and 0.25% for 4·2BF2, 4·3BF2, and 4·4BF2, respectively. This lack of appreciable fluorescence in the case of the BODIPY arrays is ascribed to their conformational mobility as revealed in the VT NMR spectral studies discussed above. Such a molecular property would be expected to result in an enhanced rate of nonradiative decay.42 When the polarity of the solvent increased  slight changes in the absorption features were seen for 4·2BF2, 4·3BF2, and 4·4BF2, whereas an apparent decrease in the fluorescence intensity was observed . In toluene, the absolute fluorescence quantum yields  and 2.6 ns (85%) for 4·3BF2, and 1.86 ns (21%) and 4.01 ns (79%) for 4·4BF2, respectively. The biphasic decay pattern is consistent with the presence of two excited-state components that likely reflect the inherent molecular flexibility. Such findings provide support for the inference that all three 3D BODIPY arrays preserve their linear, orthogonally-aligned features in the photoexcited state.45,46The fluorescence lifetimes of the BODIPY arrays were determined in toluene by means of time-correlated single photo counting (TCSPC) with photo-excitation at 450 nm Fig. S48. The flu47,48 we investigated their singlet oxygen generation efficiency under conditions of green light (ca. 520 nm) irradiation. Here, 1,3-diphenylisobenzofuran (DPBF) and Rose Bengal (RB) were used as the singlet oxygen scavenger and reference compound,33 respectively. A time-dependent decrease in the absorbance of DPBF (λmax = 414 nm) was seen upon irradiation of the BODIPY arrays in CH2Cl2 solution, whereas no detectable decrease was seen in the dark  and 4·3BF2 . This was taken as evidence of a facilitated ISC process in the case of 4·4BF2.Inspired by previously reported orthogonally-aligned BODIPY dimers or oligomers that produced reactive oxygen species (ROS) upon photoexcitation as the result of inferred spin–orbit charge transfer intersystem crossing (SOCT-ISC),2, the second reduction step is well separated from the first by 510 mV. This exceeds the splitting seen for 4·2BF2 (300 mV) and 4·3BF2 (240 mV). The larger splitting seen for 4·4BF2 is consistent with the high degree of electronic interaction between the BODIPY units and the relatively stronger exciton coupling inferred on the basis of the absorption spectral studies discussed above. We thus conclude that the structure, as well as degree of internal interaction, can be modified by conversion of 4 to the corresponding BF2-bound forms.The redox properties of ligand 4 and its corresponding BODIPY arrays were also examined by cyclic voltammetry (CV) and differential pulse voltammetry (DPV)  than its bis- and tris-BF2 congeners, 4·2BF2 and 4·3BF2. Consistent with their conformationally dynamic features, all three BODIPY arrays proved only weakly fluorescent. However, evidence of intra-subunit coupling was inferred in at least the case of 4·4BF2. This leads us to suggest that further studies of 3D cryptand-like porphyrinoids is warranted both as potential receptors and as optical materials. Work along these lines is ongoing in our laboratories.In summary, we have optimized the synthesis of a bicyclic porphyrinoid 4 relative to a competing figure-of-eight monocyclic porphyrinoid product 3. This was done by controlling the stoichiometry and the acid-catalysed reaction conditions. A single-crystal X-ray diffraction based structural analyses revealed that ligand 4 interacts with THF and CH2, 4·3BF3 and 4·4BF2 have been deposited at the CCDC under accession numbers 2110148 and 2150674–2150676, respectively, and can be obtained from the Cambridge Crystallographic Data Centre (CCDC). Data supporting this report, including cyclic voltammetry, singlet oxygen measurement, NMR and MS spectra, absorption and emission spectra, DFT calculation details and the calculated Cartesian coordinates of various complexes, have been uploaded as part of the ESI.†Crystallographic data for 4, 4·2BFProject conceptualization and research supervision: J. L. S., T. S. and C. L.; synthesis, characterization, NMR, spectroscopy, CV and DPV studies: W. N. and L. Y.; single crystal generation and data analysis: W. N.; theoretical calculations: C. L.; writing–original draft: W. N.; writing–review and editing: J. L. S., C. L. and W. N. All authors proofread, commented on, and approved the final version of this manuscript.There are no conflicts to declare.SC-013-D2SC01635D-s001SC-013-D2SC01635D-s002