The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα  A and B) (Nuclear receptors (NRs) are a family of structurally similar, mostly ligand-regulated, transcription factors that control a diverse set of biological processes, including metabolism, cell proliferation, development, and immunity , 2. SeveA and B) , 6. DespA and B) , 10, 11.A and B) , 13, 14,A and B) , 16, 17,A and B) , 20, 21,A and B) , 24, 25,A and B) , 27, 28.A and B) , 10.Figuvia their protein–protein interaction (PPI) with the hub protein 14-3-3. These 14-3-3 proteins are known to interact, and thereby regulate, hundreds of different, mainly phosphorylated, protein partners. Within the large interactome of 14-3-3 are many well-known disease-related proteins such as the Raf kinases (cancer), LRRK2 (Parkinson’s), and CFTR (cystic fibrosis), which make 14-3-3 proteins highly involved in a larger variety of cellular processes and which makes 14-3-3 protein complexes highly interesting as drug discovery targets (C), thereby reducing E2-dependent transcriptional activity  and analytical ultracentrifugation (AUC). In addition, differential scanning fluorimetry (DSF), fluorescence anisotropy (FA), and hydrogen-deuterium exchange (HDX) experiments determined the role of ERα ligands E2 and 4-OHT on ERα/14-3-3 complex formation and identified 14-3-3 binding to the drug-resistant ERα-Y537S mutant. Finally, we show that the ERα/14-3-3 PPI can be stabilized by the natural product FC-A and that this PPI stabilization can be achieved independently from ERα ligand binding in both wild-type ERα and the ERα-Y537S mutant, thus presenting a potential orthogonal therapeutic strategy for targeting endocrine resistance in breast cancer.Within this work, we aim to enhance our understanding of 14-3-3 binding to ERα A). Since the responsible kinase is not known, native T594 phosphorylation is inaccessible. We thus introduced double-point mutations (F591R/P592R) within the ERα construct, enabling PKA phosphorylation at T594. The two-point mutations make the ERα C-terminal end closely match with the consensus sequence of known PKA substrates (RRXS/TY), thereby increasing the chances of successful phosphorylation of T594 by PKA  , 58. TheFig. 24 by PKA . Fluores4 by PKA .Figure 2E. coli; however, yielded solely in truncated ERα protein (at residues 570 and 572) as apparent from mass spectrometry analysis (The PKA-responsive ERα (ERα(PKA)) construct was expressed and purified from analysis . The truanalysis . Notablyanalysis . Moreoveanalysis  and S9 . Co-expression of 14-3-3ζ and ERα with PKA allowed 14-3-3 to bind in situ to the phosphorylated ERα protein shielding the disordered F domain from proteolytic degradation. A similar co-expression approach was previously applied to successfully identify 14-3-3 binding to Ataxin-1 and to obtain a purified Tau/14-3-3 complex  was expressed together with PKA and strep-tagged 14-3-3ζ in E. coli A. Co-exp complex , 65. Not complex , 67. Yea complex . Therefoman body , and becman body , 51, 69.B). Elution fractions contained both His-tagged ERα and 14-3-3ζ protein, indicating strong binding of 14-3-3ζ to ERα, as 14-3-3 did not contain a His-tag itself. After hydrolysis of the N-terminal His-SUMO tag of ERα, the complex was purified with a streptavidin-column, which again showed co-elution of 14-3-3ζ and ERα (C). The ERα/14-3-3ζ protein complex was finally purified by SEC which resulted in the elution of a uniform peak that contained both proteins (D). Notably, truncation of ERα was not observed after co-expression and -purification with 14-3-3ζ. Quantitative MS experiments showed the approximately equimolar presence of both 14-3-3ζ and ERα within the purified protein complex sample, indicating a 1:1 binding of the two proteins (E).The ERα/14-3-3 protein complex was co-purified using three subsequent chromatography methods. First, a Ni-NTA column was used to select for His-tagged ERα protein B. Elutio and ERα C. The ERproteins . High-reproteins D. Notablproteins  and S13.proteins E.Native PAGE analysis of the purified ERα/14-3-3ζ complex showed a single band protein complex indicating stable complex formation between ERα and 14-3-3ζ without the presence of any major excess of either protein . The addA and B). Analytical SEC results showed a single peak for the individual 14-3-3ζ  and ERα  proteins which eluted similar to monomeric BSA , indicating that both proteins themselves were present as stable homodimers in solution (∼60 kDa), which is in line with reported observations (A)  were used as orthogonal approaches to determine the ERα/14-3-3ζ protein complex size and the stoichiometry of ERα and 14-3-3ζ binding , A and Brvations A 59, 67A and B. ions (A) , 72. Theions (A)  and S13,c(s) validated results observed with SEC. ERα and 14-3-3ζ showed individual peaks with weight-averaged sedimentation coefficients (corrected to 20.0 °C and to the density of water), sw, of 4.0 S and 3.8 S, corresponding to Mw ∼55 kDa and ∼58 kDa, respectively  (B). The ERα/14-3-3ζ complex showed a main peak with a weight-averaged sedimentation coefficient of 6.3 S, corresponding to a Mw ∼124 kDa, thus supporting the 2:2 stoichiometry of the ERα:14-3-3ζ complex (SV-AUC sedimentation coefficient distributions  58 kDa) B. The ER complex , 73. NotC). This result showed that ERα binds to 14-3-3ζ as a stable dimer. The dimeric state of ERα is hypothesized to cooperatively enhance ERα binding affinity to 14-3-3ζ, similar to earlier described multivalent 14-3-3 binders CFTR and LRRK2 . This calculation provided a KD ∼32 ± 6 nM. This affinity is almost 10-fold higher than that of the ERα phosphopeptide binding to 14-3-3, indicating stronger binding of the phosphorylated ERα LBD-F domain protein due to the dimer-to-dimer binding mechanism.Multivalent dimer-to-dimer complex formation was further accessed using a competitive SV-AUC experiment where 14-3-3ζ was added to ERα/14-3-3ζ protein complex to obtain various molar ratios of ERα and 14-3-3ζ. The addition of up to six equivalents of 14-3-3ζ to ERα resulted only in species representing the ERα/14-3-3ζ tetramer and the 14-3-3ζ dimer but did not show any 2:1 14-3-3ζ:ERα complex formation C. This rnd LRRK2 , 75. The complex . Based oD). ERα and 14-3-3ζ alone showed melting temperatures of 45.2 °C ± 0.8 deg. C and 59.0 °C ± 0.2 deg. C, respectively (E and F). ERα phosphorylation at T594 did not influence its melting temperature (E), with ∼2 °C increased thermal stability when compared to the individual protein partners (F). A similar increase in thermal stability for 14-3-3ζ was observed upon the addition of an ERα phosphopeptide  studies D. ERα anectively , E and Fperature . The ERαperature E, with ∼partners F. A simiopeptide . These rHydrogen-deuterium exchange (HDX-MS) assays were performed on the individual proteins 14-3-3ζ and ERα, and the ERα/14-3-3ζ protein complex, to identify which regions within the ERα and 14-3-3ζ protein were involved in protein complex formation . The proapo-ERα and ERα within the ERα/14-3-3ζ complex . Furthermore, DSF studies showed a ∼2 °C enhancement of thermal stability of both ERα-Y537S and 14-3-3ζ upon complex formation, similar to WT ERα (B–D). Interestingly, ERα-Y537S in the absence of 14-3-3 showed melting temperatures of 47.1 °C ± 0.5 deg. C, which is ∼2 °C higher than wildtype ERα, indicating higher thermal stability of ERα when mutated. All data together confirmed ERα-Y537S/14-3-3ζ complex formation, providing an interesting entry point of targeting this mutant via its interaction with 14-3-3.Point mutations in the ERα LBD are known to modulate ERα conformation and transcriptional activity. ERα Y537S is one of the most prevalent somatic mutations in patients with breast cancer, typically acquired after antiestrogen treatment . StructuFig. S22A) (c(s) distribution of the ERα/14-3-3ζ complex in the presence and absence of ligands , where E2 binds in a pocket formed by H3, H6, H8, the beta sheets, and H11 to 12 , which was strongly enhanced upon the addition of E2 (KD = 1.0 ± 0.2 μM) and abolished in the presence of 4-OHT  . Th. ThE andof 4-OHT E and S31 0.1 μM) F and S31apo ERα-Y537S in comparison to WT ERα . These results align with previously published data where the Y537S mutation resulted in a constitutively active conformation of ERα in the absence of agonistic ligands (FA assays with the mutated ERα-Y537S construct showed improved cofactor recruitment of o WT ERα . Apo ERα ligands . Further ligands . Similar ligands .A) and thereby increases the affinity between the binding partners  (B and C). Similarly, FC-A was found to stabilize the ERα-Y537S/14-3-3ζ complex as apparent from the increase 14-3-3ζ melting temperature from 60.2 to 66.3 °C (+6.1 °C)  . FC-A diC and D and E) was not influenced by ERα/14-3-3ζ stabilization by FC-A   D and S24via the formation of a tetrameric complex between an ERα homodimer and a 14-3-3ζ homodimer. Furthermore, the binding of 14-3-3ζ to the disease-relevant Y537S-ERα was confirmed, highlighting the possibility of targeting the ERα/14-3-3ζ PPI as an alternative drug discovery approach for drug-resistant mutants of ERα.NR drug discovery approaches have mainly focused on targeting the NR endogenous ligand binding pocket present within the LBD. Despite great successes using this approach, significant interest has developed in alternative manners to modulate NRs. An orthogonal entry for NR modulation is offered by their PPIs with the 14-3-3 protein. However, the highly relevant molecular understanding of these NR/14-3-3 PPIs is often lacking, while this is necessary to identify new entry points for NR drug discovery. Here we studied the NR ERα and its interaction with 14-3-3 on a molecular level. Co-purification of the intact complex of 14-3-3ζ and the ERα LBD and F domains revealed high-affinity binding between ERα and 14-3-3ζ Both agonist (E2) and antagonist (4-OHT) binding to the ERα LBD did not disrupt 14-3-3 binding to ERα, as apparent from SV-AUC and the native PAGE. Furthermore, natural ligand E2 induced activating conformational changes of the ERα LBD and subsequent cofactor peptide recruitment, in a similar fashion for ERα in isolation and ERα in complex with 14-3-3ζ. Similarly, synthetic ligand 4-OHT showed antagonistic behavior for ERα alone and ERα in complex with 14-3-3ζ, as observed by reduced cofactor recruitment. Finally, the ERα/14-3-3ζ PPI stabilization by FC-A was shown to be functional and not impeded, nor dependent, on ERα ligand binding in both wild-type ERα and the Y537S mutated protein.Combined, these results inform that 14-3-3ζ binding to ERα, and stabilization of this PPI by FC-A, function independently of conformations, mutations, and liganded state of the ERα LBD. This orthogonality is most likely facilitated by the long and flexible 42-residue F-domain of ERα, accommodating ERα conformations to not affect 14-3-3 binding. Molecular stabilization of the ERα/14-3-3 protein complex with molecular glues like FC-A would therefore be a potential entry point for targeting ERα and its drug-resistant variants. The orthogonality of the molecular events within the ERα would even bode for dual targeting of both the PPI interface and the classical ERα ligand binding pocket. Furthermore, whereas orthosteric drugs such as 4-OHT also show binding to ERβ  and ERRγThe concept of therapeutic targeting of the ERα/14-3-3 PPI could be envisioned to be translatable to other NR/14-3-3 protein complexes. So far, 14-3-3 has been identified as the binding partner of eight NRs, for which 14-3-3 binds to each NR in a unique manner. The NR/14-3-3 interactions form a potential entry point for targeting ‘hard-to-drug’ NRs due to, for example, drug resistance or the absence of an orthosteric pocket in the LBD. A potential example is the Androgen Receptor (AR), an established prostate cancer target. Although prostate cancer is initially often successfully targeted with androgen deprivation therapy or AR antagonists such as enzalutamide, drug- and castration-resistant AR mutants or splice variants are often developed within patients with prostate cancer , 87, 88.P03372) construct was designed which contained three mutations  to introduce a PKA recognition motif for in vitro phosphorylation of T594, and to remove a potential PKA phosphorylation site S305. pCDFDuet-1-His-SUMO-ERaLBD-F-strep was transformed into NiCo21 (DE3) competent cells using heat stock and were plated on LB-agar plates containing 100 μg/ml streptomycin. A single colony was selected and cultured overnight in 50 ml LB medium (100 μg/ml streptomycin) at 37 °C. After overnight incubation, cultures were transferred to 2 l ZYP-5052 medium (100 μg/ml streptomycin) and incubated at 37 °C, 180 rpm until an OD600 of ∼2 was reached. Protein expression was then induced with 0.4 mM isopropyl-β-d-thiogalactoside (IPTG), and cultures were incubated overnight at 18 °C, 180 rpm. Cells were harvested by centrifugation  and resuspended in lysis buffer  glycerol, 2 mM beta-mercaptoethanol (βME) containing cOmplete EDTA-free Protease Inhibitor Cocktail tablets (1 tablet/100 ml lysate) and Benzonase Nuclease . The cell suspension was lysed using an Emulsiflex-C3 homogenizer (Avestin) and the cell lysate was cleared by centrifugation . Protein-containing supernatant was loaded on 2 × 5 ml Ni-NTA columns  at 5 ml/min and 4 °C. Columns were washed with 3 × 5 CV Wash Buffer  glycerol, 2 mM βME) until proteins concentrations dropped below 0.05 mg/ml. His-SUMO-ERαLBD-F-strep was eluted using 2 to 3 CV Elution Buffer  glycerol, 2 mM βME). His-SUMO-ERαLBD-F-strep containing elution fractions were subsequently incubated with SUMO hydrolase (1:100) to remove the His-SUMO tag from ERα during o/n dialysis at 4 °C  glycerol, 2 mM βME). SUMO-cleaved ERαLBD-F-strep was loaded on a 5 ml Streptavidin column  at 3 ml/min. Columns were washed with Buffer W  collecting 2 CV until protein concentrations dropped below 0.05 mg/ml. ERαLBD-F-strep was eluted using 4 CV Buffer BXT . Finally, ERαLBD-F-strep was purified by SEC (Superdex 75pg Hiload 16/600) at 1 ml/min and 4 °C using SEC buffer . Protein-containing fractions were combined and concentrated to >5 mg/ml, aliquoted, and stored at −80 °C. Correct mass and purity of the proteins were confirmed using SDS-PAGE and Q-ToF LC-MS analysis.A His-Sumo-ERαLBD-F-strep  and 500 μM ATP, for 3 h at 4 °C.Phosphorylated ERα (ERα-pT594-strep) was expressed and purified in a similar manner as described earlier for non-phosphorylated ERα-strep. An P63104) construct was transformed into NiCo21 (DE3) competent cells using heat stock and was plated on LB-agar plates containing 100 μg/ml ampicillin. A single colony was selected and cultured overnight in 8 ml LB medium (100 μg/ml ampicillin) at 37 °C. After overnight incubation, cultures were transferred to 500 ml TB medium (100 μg/ml ampicillin) and incubated at 37 °C, 180 rpm until an OD600 of 0.6 to 1.2 was reached. Protein expression was induced with 0.4 mM IPTG, and cultures were incubated overnight at 18 °C, 180 rpm. Cells were harvested by centrifugation  and resuspended in lysis buffer  glycerol, 2 mM βME) containing cOmplete EDTA-free Protease Inhibitor Cocktail tablets (1 tablet/100 ml lysate) and Benzonase Nuclease . The cell suspension was lysed using an Emulsiflex-C3 homogenizer (Avestin), and the cell lysate was cleared by centrifugation . Protein-containing supernatant was loaded on a 5 ml Streptavidin column  at 3 ml/min. Columns were washed with 4x 2CV Buffer W . 14-3-3ζ-strep was eluted using 2 CV Buffer BXT . Finally, 14-3-3ζ-strep was purified by SEC (Superdex 75pg Hiload 16/600) at 1 ml/min and 4 °C using SEC buffer . Protein-containing fractions were combined and concentrated to >5 mg/ml, aliquoted, and stored at −80 °C. Correct mass and purity of the protein were confirmed using SDS-PAGE and Q-ToF LC-MS analysis.A 14-3-3zeta-strep  was co-transformed with full-length 14-3-3zeta  and SUMO-PKA   and resuspended in lysis buffer  glycerol, 2 mM βME) containing cOmplete EDTA-free Protease Inhibitor Cocktail tablets (1 tablet/100 ml lysate) and Benzonase Nuclease . The cell suspension was lysed using an Emulsiflex-C3 homogenizer (Avestin) and the cell lysate was cleared by centrifugation . Protein-containing supernatant was loaded on 2 × 5 ml Ni-NTA columns  at 5 ml/min and 4˚C. Columns were washed with Wash Buffer  glycerol, 2 mM βME) collecting each time 1 to 3 CV fractions until proteins concentrations dropped below 0.05 mg/ml to ensure removal of all excess 14-3-3. 14-3-3ζ/ERα complex was then eluted using 2 to 3 CV Elution Buffer  glycerol, 2 mM βME). The 14-3-3ζ-strep/His-SUMO-ERα complex was subsequently incubated with SUMO hydrolase (1:100) to remove the His-SUMO tag from ERα during overnight dialysis at 4 °C  glycerol, 2 mM βME). SUMO-cleaved 14-3-3ζ-strep/ERα complex was loaded on a 5 ml Streptavidin column  at 3 ml/min. Columns were washed with Buffer W  collecting 2 CV fractions until protein concentrations dropped below 0.05 mg/ml to ensure removal of all excess ERα. 14-3-3ζ/ERα complex was eluted using 8 CV Buffer BXT . Finally, the protein complex was purified by SEC (Superdex 75pg Hiload 16/600) at 1 ml/min and 4 °C using SEC buffer . Protein-containing fractions were combined and concentrated to >10 mg/ml, aliquoted and stored at −80 °C. Correct mass and purity of the proteins was confirmed using SDS-PAGE and Q-ToF LC-MS analysis.His-SUMO-ERαLBD-F  at 20 °C and at 43.000 to 45.000 rev./min rotor speed . All sedimentation profiles were collected by absorbance measurements at 280 nm. The calculated distributions were integrated to establish the weight-average sedimentation coefficients corrected to 20 °C and to the density of water ).Protein samples were dialyzed into 20 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgClDilution series of 14-3-3ζ-strep and ERα-pT594-strep were prepared in MQ (0.1% FA) to final concentrations of 0.025, 0.020, 0.015, 0.010, and 0.005 mg/ml. Furthermore, a 500×, 750× and 1000× dilution of the ERα/14-3-3ζ protein complex were prepared. The final samples (∼100 μl) were transferred to a 200 μl LC-MS vial. UPLC-QToF-MS analysis was performed on a Waters  Acquity I-Class UPLC system coupled to a Waters Xevo G2-XS quadrupole time-of-flight (QToF) mass spectrometer. The devices were controlled by MassLynx Software . Full scan in positive electrospray ionization (ESI+) mode was used as MS acquisition mode with an acquisition range from 150 to 2000 m/z. A 3 μm, 100 × 2.0 mm Polaris 3 C8-A column  was placed inside a column oven at 40 °C and used for chromatographic separation. Flowrate was set at 0.3 ml/min, and a gradient of water containing 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B) was set as follows : 0.0 to 2.0 min (30% to 39% B), 2.0 to 5.0 min (39% B), 5.0 to 7.5 min (39% to 60% B), 7.5 to 8.0 min (60% B), 8.0 to 8.1 min (60% to 30% B) 8.1 to 10.0 min (30% B). Sample injection volume 2 μl. Mass Spectrometry settings were set as follows: capillary voltage: 0.80 kV, cone voltage: 40 V, source offset: 80 V, source temperature: 120 °C, desolvation temperature: 450 °C, cone gas: 10 l/h desolvation gas: 1000 l/h.Data were analyzed using MassLynx software. Chromatograms were background subtracted . The area under the peak was determined using integration in the MassLynx software with a relative area threshold of 10. The obtained area under the curve was then plotted against the protein concentration, after which a linear regression was determined between the five data points. Using the equation of the linear regression, the concentration of 14-3-3 and ERa was determined in each protein complex sample.To perform mass analysis of the individual peaks deconvolution was performed on m/z spectra of each individual peak. After visual inspection of the m/z spectrum, the spectrum was zoomed to the five most abundant peaks from which the mass spectrum was determined using MaxEnt1 . Mass spectra were centered and errors of the deconvolution process were determined.−1 using isocratic pump delivering 0.4% formic acid in water. Both protease column and trap column were placed in an icebox. After 3 min, peptides were separated on a C18 reversed-phase column  with a linear gradient 5 to 35% B in 26 min, where solvent A was 2% acetonitrile/0.4% formic acid in water and solvent B 95% acetonitrile/5% water/0.4% formic acid. The analytical column was placed in an icebox. TimsToF Pro mass spectrometer  operating in positive MS/MS mode was used for the detection of peptides. Data were processed by DataAnalysis 5.3 software . MASCOT search engine was used for the identification of peptides using a database containing the sequence of 14-3-3ζ or ERα.100 pmol of 14-3-3ζ-strep or ERα(PKA)-strep was mixed in 1:1 (v/v) ratio with 1 M glycine at pH 2.3 and injected on a mixed Pepsin/Nepenthesin-2 acidic protease column. Generated peptides were trapped and desalted by a Micro trap column (Luna Omega 5 um Polar C18 100 Å Micro Trap 20 × 0.3 mm) for 3 min at a flow rate 200 μl minAll proteins were dialyzed and diluted into 20 mM Hepes, 150 mM NaCl, 10 mM MgCl2, and 0.5 mM TCEP pH7.5 to a final concentration of 20 μM. E2 or DMSO was added to a final concentration of 150 μM. Hydrogen deuterium exchange was initiated by 10-fold dilution of the proteins under different conditions in a deuterated buffer. Fifty microliter aliquots (100 pmol) were taken after 20 s, 2 min, 20 min and 2 h of incubation in deuterated buffer, quenched by 50 μl of 1 M glycine, pH 2.3 and snap frozen in liquid nitrogen. Aliquots were quickly thawed and analyzed using the same system as described above. Peptides were separated by linear gradient 10 to 30% B in 18 min. Mass spectrometer was operated in positive MS mode. Spectra of partially deuterated peptides were processed by Data Analysis 5.3  and by in-house program DeutEx.2, and 0.5 mM TCEP with protein at a final concentration of 2.5 to 5 μM. Ligands were added at a final concentration of 100 μM. All samples were 1:1 diluted into native PAGE loading dye . after which 12 μl of each sample was loaded on a 4 to 20% Mini-PROTEAN TGX Precast Protein Gel (Bio-Rad). Gels ran at 130 V for 2.5 h at 4 °C in running buffer . Gels were washed in MilliQ (20 min), stained with Coomassie Brilliant Blue G-250 (Bio-Rad), and destained in MilliQ until bands were clearly visible. Gels were imaged and analyzed with ImageJ.Samples were prepared in 20 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, 500 μM TCEP) to obtain 40 μl samples containing 5 μM 14-3-3ζ-strep, 5 μM ERα-strep or 10 μM ERα/14-3-3ζ complex, with either 1% DMSO (negative control) or 100 μM ligand . All samples additionally contained 10x ProteoOrange dye  and were heated from 35 to 79 °C at a rate of 0.3 °C per 15 s in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Fluorescence intensity was determined using excitation 575/30 nm and emission 630/40 nm filters. Based on these melting curves, the (negative) first derivative melting curve was obtained, from which the melting temperature TM could be determined. Reported TM values were obtained from three independent experiments from which the average and standard deviations were determined using excel.Proteins were diluted . Wells containing only fluorescein-labeled peptide were used to set as G-factor at 35 mP. All data were analyzed using GraphPad Prism (7.00) for Windows and fitted using a four-parameter logistic model (4PL) to determine apparent binding affinities (KDapp). All results are based on two independent experiments from which the average and standard deviations were calculated to obtain the final values.All FA dilution series were prepared in polystyrene (non-binding) low-volume Corning Black Round Bottom 384-well plates (Corning 4514 or 4511). FA measurements were performed directly after plate preparation, using a Tecan Infinite F500 plate reader at room temperature  Tween20, 0.1% (w/v) BSA. This is done in the presence of DMSO (negative control), 100 μM E2 or 100 μM 4-OHT.ERα(PKA)-strep, ERα(PKA)-pT594-strep, and ERα/14-3-3ζ protein complex (for WT ERα and Y537S mutant) were titrated in a 2-fold dilution series (starting at 40 μM ERα) to 20 nM of fluorescein-labeled SRC1 peptide in 20 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.1% (v/v) Tween20, 0.1% (w/v) BSA. This is done in the presence of a 2-fold dilution series of 3′de-Ac FC-A (0.015–250 μM); each well contained a final concentration of 0.25% DMSO.14-3-3ζ was titrated in a 2-fold dilution series (starting at 300 μM 14-3-3ζ) to 2 nM of fluorescein-labeled peptide , ERα(PKA)-strep) in 20 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2 and 20 μM TCEP) using a final molar stoichiometry of 1:2 or 1:4 protein to peptide. These complexes were used to set up sitting-drop crystallization plates were set up in which each of the four complexation mixtures were combined with 24 crystallization buffers, optimized for 14-3-3σ crystallization , 0.19 M CaCl2, 24 to 29 % (v/v) PEG 400 and 5% (v/v) glycerol). For each combination, a 1:1 mix (both 250 nl) of complexation mixture and crystallization buffer was prepared for crystal growth. Crystals grew within 10 to 14 days at 4 °C. Co-crystals of 14-3-3ζ with ERα peptides (ERα(PKA)-pT594 and ERα(PKA)-pT594-strep) were soaked with 10 mM 3′ deAc-FC-A in crystallization buffer and incubated for 3 days at 4 °C.N-acetylated ERα(PKA)-pT594 and ERα(PKA)-pT594-strep peptides were mixed with 10 mg/ml 14-3-3σΔC protein (truncated after T231 to reduce flexibility) in complexation buffer  with the following settings: 1440 image, 0.25°/image, 100% transmission, and 0.1 s exposure time. Initial data processing was performed at DESY using XDS after which pre-processed data was taken to further scaling steps, molecular replacement, and refinement.Data were processed using the CCP4i2 suite (version 7.1.18). XDS-preprocessed data were scaled using AIMLESS. The data were phased with MolRep, using protein data bank (PDB) entry 4JC3 as a template. A three-dimensional structure of 3′ dAc-FC-A was generated using AceDRG, which was thereafter built in the electron density based on visual inspection Fo-Fc and 2Fo-Fc electron density map. Sequential model building  and refinement were performed with Coot and REFMAC, respectively. Finally, alternating cycles of model improvement  and refinements were performed using Coot and phenix.refine from the Phenix software suite (version 1.20.1-4487). Pymol (version 2.2.3) was used to make the figures in the manuscript. All structures were deposited in the protein data bank (PDB) and obtained IDs: 8C40, 8C42, 8C3Z and 8C43. See 8C40, 8C42, 8C3Z, and 8C43.Crystal structures described in this manuscript have been deposited to the PDB. They have the following PDB codes: This article contains The authors declare the following competing interest(s): L. B. and C. O. are scientific co-founders of Ambagon Therapeutics. C. O. and E. S. are employees of Ambagon Therapeutics.


II atom is in the low-spin state.Molecules of the title complex are centrosymmetric and the Fe—N bond lengths to the N atoms of the porphyrin ring indicate that the Fe II(C44H24Cl4N4)(C5H9N)2] or [FeII(TClPP)(t-BuNC)2] , the metal ion lies on an inversion center and is octahedrally coordinated by the N atoms of the porphyrin ring in the equatorial plane and by carbon atoms of the trans t-BuNC ligands in the axial sites. The Fe—N bond length of 2.0074 (14) Å suggests a low-spin complex (S = 0). The crystal packing of the title compound is sustained by C—H⋯Cl, C—H⋯N and C__H⋯Cg (Cg = the centroid of a pyrrole ring of the TClPP porphyrinate) interactions, leading to a three-dimensional network. The Hirshfeld surface (HS) analysis indicates that 61.4% of the intermolecular interactions are from H⋯H contacts while other contributions are from C⋯H/H⋯C, O⋯H/H⋯O and N⋯H/H⋯N interactions, which comprise 21.3%, 13.3% and 3.6% of the HS, respectively.In the title compound, [Fe These ferrous porphyrin complexes are low-spin 3d6 systems (S = 0). Such FeII models with π-acceptor axial ligands like CN−, CO and isocyanides (R—NC) are also known. Heme-isocyanide derivatives were studied starting from 1951 (t-BuNC)2] compound. In 1984, the Mössbauer-effect data and the IR of the bis(isocyanide) iron(II) porphyrin [FeII(TPP)(PhCO-NC)2] (PhCO-NC = benzoylisocyanide) and the mixed ligand isocyanide-py ligands [FeII(TPP)(PhCO-NC)(py)] were reported (iPrNC)(1-MeIm)] (iPrNC = 2-isocyanopropane and 1-MeIm = 1- methylimidazole) complex. In 2001, spectroscopic investigations of the FeII and FeIIIn-butyl isocyanide complexes P450cam and P450nor were reported (t-BuNC)2] complex where TPBP is the 5,10,15,20-{tetrakis-[para-(benzoyloxy)phenyl]porphyrinate ligand iron(II) complex, [FeII(TClPP)(t-BuNC)2], I.Since the beginning of the 1960s, hexacoordinated iron(II) metalloporphyrins of type  molecule comprises the asymmetric unit of complex I  bond length and the spin state of iron(II) metalloporphyrins. Consequently, in high-spin (S = 2) complexes, the Fe—Np distances are the longest, exemplified by the [Fe(TpivPP)(N3)]− ion complex porphinate, known as the picket-fence porphyrin) where Fe—N = 2.094 (3) Å  complexes, the average Fe—Np bond length is reduced. For example, in the [FeII(TMPP)(amp)2] complex , the Fe—N bond length is 1.988 (2) Å  derivative (I), the Fe—N distance of 2.0074 (14) Å strongly suggests that this species corresponds to an iron(II) low-spin (S = 0) porphyrin. Notably, this value closely resembles those for the related [FeII(TPP)(t-BuNC)2] (t-BuNC)2]  Å, which is very near to those observed in the associated FeII bis(t-BuNC) metalloporphyrins: [FeII(TPP)(t-BuNC)2]  and [FeII(TPBP)(t-BuNC)2] {where TPBP is [4-(benzoyloxy)phenyl]porphyrinate} with FeII—C distances of 1.901 (3) and 1.907 (2) Å, respectively –(t-BuNC) geometry. Specifically, the Fe—C23—N3 and C23—N3—C24 angles measure 165.75 (15) and 163.66 (17)°, respectively, which closely resemble the corresponding angles observed in [FeII(TPP)(t-BuNC)2] (t-BuCN)2]+, these angles exhibit significantly higher values, measuring 174.2 and 173.5° for the average Fe—C—N and C—N—C angles, respectively. The deviations from linearity, represented by the angles 14.3/16.3° for complex I and 11.0/20.9° for [FeII(TPP)(t-BuNC)2]  derivative, where the average deviation values are 5.8/6.5° (t-BuCN)2]+, is consistent with the dominance of the π-backbonding effect in iron(II) derivatives over iron(III) coordination compounds.In the case of the related iron(III) ion complex   ion complexes [FeIII(TPP)(t-BuNC)2]+ (t-BuNC)2]+ /1.12 (2) Å and 1.145 (4)/1.144 (4) Å, respectively.As highlighted in the IR spectroscopy section, the Fet-BuNC) derivative and the shortest C—N bond length of an iron(III)–bis(t-BuNC) metalloporphyrin is small (0.042 Å).It is certainly true that the iron(II) derivatives display longer C—N distances. Nevertheless, the difference between the longest C—N bond length of an iron(II)–bis(t-BuNC)2] complexes are linked to each other via weak non-classical C—H⋯Cl, C—H⋯N hydrogen bonds and C—H⋯Cg intermolecular interactions where Cg is the centroid of a pyrrole ring. It may be noted that Cl1 acts as acceptor for three different C—H groups.Within the crystal structure of I Figs. 2, the  where Porph is a porphyrin and L is a N-donor, O-donor, S-donor or C-donor axial ligand gave 25 hits where L is a neutral N-donor axial ligand, ten hits for neutral O-donor axial ligands, three S-donor neutral S-donor axial ligands and four neutral C-donor axial ligands. In fact, for the latter type of neutral axial ligands, it is the tert-butyl isocyanide corresponding to [FeII(TPP)(t-BuNC)2] (t-BuNC)2] (OOEP = octaethyloxophlorinato) (t-BuNC)2] phenyl]porphyrinate]).A search in the Cambridge Structural Database (t-Bu-NC)2] (I)  of the methyl groups of the t-BuNC axial ligand occurs at 2883 cm−1. The strong band at 996 cm−1 is attributed to the bonding vibration δ(CCH) of the porphyrin core for which a value around 1000 cm−1 is characteristic of a metalled porphyrin while a δ(CCH) value around 960 cm−1 is specific of a free base porphyrin.The FT–IR spectrum of [FeI) Fig. 4 was obtat-BuNC metalloporphyrins are displaced by at least 60 cm−1 to higher frequency compared to those of ferrous t-BuNC porphyrin complexes. Thus for the [FeIII(TPP)(t-BuNC)2]+ ion complex (t-BuNC)2] compound  species (I), exhibits two bands attributed to the ν(C≡N) frequency value of the t-BuNC axial ligand with the weak one at 2202 cm−1 and the main strong IR band is shown at 2126 cm−1. This indicates clearly that complex I is iron(II) metalloporphyrin.It has been found that the values of the C≡N stretching frequency for ferric I was obtained in chloroform using a WinASPECT PLUS scanning spectrophotometer (t-BuNC)2] and [FeII(TBPPP)(t-BuNC)2], which are 432 nm and 437 nm, respectively  ferric metalloporphyrins, the λmax of the Soret band value is blue shifted compared to those of the ferrous bis(t-BuNC) porphyrin complexes, e.g., for [FeIII(TPP)(t-BuNC)2]+  analysis performed with 7.para-chlorophenyl)porphyrin (H2TClPP) and [FeIII(TClPP)(SO3CF3)] were prepared as described in the literature (SO3CF3)]  in dichloromethane (35 ml) was added an excess of tert-butyl isocyanide (t-BuNC) . The reaction mixture was stirred at room temperature for 3 h. Crystals of the title complex were obtained by diffusion of the n-hexane and dichloromethane solutions. Elemental analysis calculated (%) for C54H42Cl4FeN6: C 66.68, H 4.35, N 8.64; found: C 66.81, H 4.41, N 8.78,The starting materials 5,10,15,20-tetra(8.Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023008083/hb8075sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023008083/hb8075Isup2.hklStructure factors: contains datablock(s) I. DOI: 975663CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We show that CD1d is expressed by the majority of patient MM, (myelo)monocytic AML, and CLL cells and that the bsTCE triggers type 1 NKT and Vγ9Vδ2-T cell-mediated antitumor activity against these patient tumor cells and improves survival in in vivo AML, MM, and T-ALL mouse models. Evaluation of a surrogate CD1d-γδ bsTCE in NHPs shows Vγ9Vδ2-T cell engagement and excellent tolerability. Based on these results, CD1d-Vδ2 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML.Bispecific T cell engagers (bsTCEs) hold great promise for cancer treatment but face challenges due to the induction of cytokine release syndrome (CRS), on-target off-tumor toxicity, and the engagement of immunosuppressive regulatory T cells that limit efficacy. The development of Vγ9Vδ2-T cell engagers may overcome these challenges by combining high therapeutic efficacy with limited toxicity. By linking a CD1d-specific single-domain antibody (VHH) to a Vδ2-TCR-specific VHH, we create a bsTCE with trispecific properties, which engages not only Vγ9Vδ2-T cells but also type 1 NKT cells to CD1d  •CD1d is expressed on the majority of patient MM, (myelo)monocytic AML, and CLL cells•A CD1d-Vδ2 bsTCE triggers dual activation of type 1 NKT and Vγ9Vδ2-T cells•CD1d-Vδ2 bsTCE-activated type 1 NKT and Vγ9Vδ2-T cells exert robust antitumor activity•A surrogate fully cross-reactive CD1d-γδ bsTCE is well tolerated in NHP studies Bispecific T cell engagers (bsTCEs) show anticancer therapeutic activity but face challenges due to toxicity and regulatory T cell engagement, limiting efficacy. Lameris et al. demonstrate that selective engagement of Vγ9Vδ2-T and type 1 NKT cells using a CD1d-Vδ2 bsTCE results in robust antitumor activity and is well tolerated in NHPs. By using a functional and structural approach, we showed that VHH1D12 simultaneously contacts CD1d and the type 1 NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity, which translates directly into antitumor activity and has the potential to overcome at least some of the clinically encountered limitations of glycolipid Ag-based therapeutic approaches. Indeed, a superior effect of VHH1D12 over glycolipid Ag on type 1 NKT cell-mediated antitumor activity was noted using patient-derived tumor samples.+ tumor cells, resulting in strong activity in in vivo models and ex vivo against patient multiple myeloma (MM), monocytic acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL) cells. Exploratory toxicology studies with a fully cross-reactive (surrogate) CD1d-γδ bsTCE in non-human primates (NHPs) demonstrated good tolerability, no clinical, chemistry, or hematology abnormalities or organ toxicity, and only low levels of IL-6.Here, we generated a CD1d-Vδ2 bsTCE that can trigger robust activation of both type 1 NKT and Vγ9Vδ2-T cells toward CD1d50) of the CD1d-Vδ2 bsTCE for CD1d and Vγ9Vδ2-TCR was in the low-nanomolar range and equivalent to that of the individual monospecific VHHs (+ (but not CD1d−) tumor cell lines with low-nanomolar and low-picomolar EC50s, respectively (50s were observed in co-cultures of CD1d+ tumor cells and mixed (1:1) type 1 NKT/Vγ9Vδ2-T cells , and interferon (IFN)-γ, whereas Vγ9Vδ2-T cells mainly secreted TNF and IFN-γ  of type 1 NKT/Vγ9Vδ2-T cells  B and S2C-T cells C. MoreovImportantly, in 7-day co-cultures of healthy donor-derived PBMCs  and tumor cell lines, AML growth was controlled and MM growth further reduced in the presence of the CD1d-Vδ2 bsTCE even at low to very low E:T ratios A. ParticCollectively, these data demonstrate that CD1d-Vδ2 bsTCEs trigger dual engagement of type 1 NKT and Vγ9Vδ2-T cells, which results in their expansion and potent and selective CD1d-restricted antitumor activity.,,,,,Pichia pastoris yeast strain selected for large-scale production). The humanized CD1d-Vδ2 (hu-)bsTCE had an immunogenicity risk in the range of humanized antibodies. When tested against sera from healthy human donors, the hu-bsTCE showed a low incidence and level of binding to pre-existing human anti-VHH antibodies  was identical between humanized and wild-type (WT) CD1d-Vδ2 bsTCE .tibodies A. Bindinδ2 bsTCE E.Figure In conclusion, CD1d-Vδ2 bsTCE humanization was successful and reduced the predicted immunogenicity risk without compromising functionality.,,,,Next we assessed the antitumor effects of CD1d-Vδ2 hu-bsTCE using patient-derived tumor samples of (myelo)monocytic and B cell lineage origin, as these generally express CD1d.h1  and Th2 type cytokines ; for Vγ9Vδ2-T cells predominantly Th1 type cytokines   thereof, CD1d-Vδ2 hu-bsTCE induced robust type 1 NKT and/or Vγ9Vδ2-T cell degranulation and cytokine production; for type 1 NKT cells, both Tme IL-2) B–S4D. Nome IL-2) C–5E. Theme IL-2) C–5E. Altme IL-2) E–S4G. Inme IL-2) E. Since  IL-2) ,,H, indica IL-2) ,,F.Overall, these data demonstrate that CD1d is expressed by the majority of patient-derived MM, (myelo)monocytic AML, and CLL cells and that CD1d-Vδ2 hu-bsTCE strongly enhanced type 1 NKT and Vγ9Vδ2-T cell activation, resulting in robust cytotoxic responses toward these cells. In CLL, type 1 NKT (but not Vγ9Vδ2-T) reactivity depended on the presence of (exogenous) agonistic lipid Ags in CD1d.in vivo antitumor efficacy, we established disseminated AML and MM models via intravenous (i.v.) injection of MOLM-13 and MM.1s.CD1d in immunodeficient NOD scid gamma (NSG) mice. In the aggressively growing AML model, i.v. administration of mixed (1:1) human type 1 NKT/Vγ9Vδ2-T cells at day 0 and 7 had a modest effect on survival, which was significantly prolonged in combination with twice-weekly i.v. administration of CD1d-Vδ2 bsTCE   A. In thectively) B. A strictively) B. The onctively) A and S5Bin vivo antitumor efficacy of the humanized CD1d-Vδ2 bsTCE using whole PBMCs admixed with CCRF-CEM cells  in a s.c. T-ALL model in NSG mice. In the absence of PBMCs, twice-weekly i.p. treatment with CD1d-Vδ2 hu-bsTCE (2 mg kg−1) did not inhibit tumor growth, whereas admixed PBMCs (in absence of bsTCE) slightly reduced tumor growth (−1 and 0.2 mg kg−1). Again, treatment with CD1d-Vδ2 hu-bsTCE (2 mg kg−1) alone had no effect, and PBMCs alone or combined with control gp120-Vδ2 bsTCE administration slightly reduced tumor growth, resulting in a minor increase in survival   (We next assessed the r growth C and S5Cr growth C and S5Cctively) D. Clear mg kg−1) D and S5Din vivo hematologic malignancy models using either expanded type 1 NKT and Vγ9Vδ2-T cells or PBMCs as effector cells and demonstrate antitumor efficacy with intermittent dosing.Overall, these data show that CD1d-Vδ2 bsTCE improves survival in multiple ,Macaca mulatta and/or Macaca fascicularis  CD1d or Vγ9Vδ2-T cells  and pharmacodynamic (PD) parameters in NHP, which have a pAg-reactive Vγ9Vδ2-T cell population highly homologous to that of human.-T cells A. We the-T cells A. Since -T cells A and S6Bar range B, demons−1) study. As expected, and related to their small size and absence of an Fc domain, a single escalating dose of either bsTCE showed a short plasma half-life , rapid plasma clearance, and large apparent volume of distribution , the binding intensity of the CD1d-Vγ9 bsTCE to peripheral blood Vγ9-T cells was lower compared with that observed with the control Vγ9 bsTCE, although binding remained detectable throughout the study at both the 0.3- and 1.0-mg kge levels A. While e levels . Body wee levels . PostmorOverall, these data show that the fully NHP cross-reactive CD1d-Vγ9 bsTCE has a rapid plasma clearance but prolonged on-target binding, triggering clear Vγ9-T cell engagement, and was well tolerated in NHP.,,,,,h1 cytokine responses is deemed important for downstream effector cell activation and has been linked to an improved response in several immunotherapy studies.,,CD1d is a (glyco)lipid Ag-presenting molecule frequently expressed by malignant cells of myelomonocytic and B cell lineage origin and some solid malignancies , and may promote tumor growth via the presentation of (low-affinity) lipid Ags causing immunosuppressive cytokine release by type 1 NKT cells and engagement of protumoral diverse NKT cells.h1 cytokine response and robust tumor cell lysis was also observed using patient-derived tumor samples. Of interest was the observation that the CD1d-Vδ2 bsTCE only triggered limited type 1 NKT cell activation in co-culture with patient-derived CLL cells. As CD1d expression in CLL appears to increase in more advanced stages of the disease,Expression of CD1d on tumor cells was confirmed in the majority of MM, (myelo)monocytic AML, and CLL patients, and the CD1d-Vδ2 bsTCE-mediated engagement of type 1 NKT and Vγ9Vδ2-T cells resulting in a T50s observed in in vitro experiments, and prolonged binding of the cross-reactive bsTCE to peripheral blood Vγ9Vδ2-T cells despite a short plasma half-life as observed in the NHP study, these data indicate an extended functional half-life and are supportive of an intermittent-dosing approach of the CD1d-Vδ2 bsTCE in humans.Low-MW/low-affinity bsTCEs, such as blinatumomab, require continuous infusion to be effective.,,,−1. Importantly, blood plasma analysis showed low levels of IL-6 release only after the first and highest dose, indicating a low risk for CRS. Furthermore, no depletion of CD1d+ B cells and monocytes was observed. This is in agreement with our in vitro results showing preferential lysis of malignant cells while B cells and monocytes were relatively spared, implicating a low risk for on-target off-tumor toxicity.Common adverse events of bsTCE and CAR-T cell therapies include CRS and ICANS that are potentially life threatening and frequently mandate dose adjustment and/or immunosuppressive measures.ClinicalTrials.gov identifier: NCT04887259).Collectively, we have generated a first-in-class, off-the-shelf, humanized CD1d-Vδ2 bsTCE that selectively engages both type 1 NKT and Vγ9Vδ2-T cells to trigger a potent antitumor response to CD1d-expressing tumor cells. Considering the expression of CD1d in CLL, MM, and AML, and the favorable tolerability profile of the surrogate engager in NHPs, the CD1d-Vδ2 hu-bsTCE was termed LAVA-051 and selected for evaluation in a first-in-human clinical phase 1/2a study in patients with CLL, MM, or AML that are refractory to prior therapy  or transduced with the lentiviral plasmid FUW-mCherry/luc,,54 was grown in RPMI-1640 supplemented as above. The epithelial cell line HeLa (WT and transfected with CD1d) was a gift from dr. M. Kronenberg and grown in DMEM supplemented as above. The epithelial cell line A-431 was obtained from the ATCC and grown in DMEM supplemented as above. Cell lines were confirmed to be Mycoplasma-free. Authentication of CCRF-CEM and MM.1s transfectants (BaseClear and Eurofins) showed no signs of contamination or misidentification.The T-ALL cell line CCRF-CEM was obtained from the ATCC and grown in RPMI-1640 supplemented with 10% (v/v) fetal calf serum (FCS), 0.05 mM β-mercaptoethanol (β-ME), and 100 IU mLPBMC from healthy donors were isolated from venous blood by density gradient centrifugation, obtained from Sanquin blood supply under written informed consent (the Netherlands) or commercially acquired . Bone marrow mononuclear cells (BMMC) from patients with MM ) or patients with AML  were isolated from bone marrow samples by density gradient centrifugation, obtained after approval by the institutional review board  and written informed consent from the patients or commercially acquired . PBMC from CLL patients ) were isolated from venous blood by density gradient centrifugation, obtained after approval by the institutional review board  and written informed consent from the patients.+ MM cells, respectively, into NOD scid gamma (NSG) mice (Charles River). For the AML model, female 8-9-week-old NSG mice were i.v. injected with 0.25×106 MOLM-13 cells via the tail vein (day 0). One hour later and on day 7 PBS or a mixture of 0.5×107 human type 1 NKT and 0.5×107 human Vγ9Vδ2-T cells were i.v. injected. Mice were twice weekly i.v. injected with CD1d-Vδ2 bsTCE . Mice were euthanized when pre-set human endpoints were reached. All mice were maintained in a specific pathogen-free facility (12h light/12h dark cycle) and supplied with autoclaved bedding and ad libitum water and food. The AML in vivo study was performed by Oncodesign, France, and approved by the animal Care and Use Committee of Oncodesign (Oncomet) agreed by French authorities (CNREEA agreement number 91).Disseminated AML and MM models were established by i.v. transfer of AML and CD1d6 MM.1s.CD1d cells via the tail vein (day 0). On day 7, 14, and 21 PBS or a mixture of 0.5×107 human type 1 NKT and 0.5 × 107 Vγ9Vδ2-T cells with or without CD1d-Vδ2 bsTCE (100 μg per mouse) were i.v. injected. Mice were twice weekly i.p. injected with PBS or CD1d-Vδ2 bsTCE (100 μg per mouse). Peripheral blood type 1 NKT (mCD45−hCD45+CD3+TRAV-10+) and Vγ9Vδ2-T cell frequencies (mCD45−hCD45+CD3+TRGV9+(TRDV2+)) were determined over time by flow cytometric bead-based cell counting. Mice were euthanized when pre-set human endpoints were reached. Blood in the one mouse, treated with type 1 NKT and Vγ9Vδ2-T cells plus CD1d-Vδ2 bsTCE, that was found dead could not be reliably analyzed due to clotting and was excluded from the analyses. The study was terminated 90 days post tumor engraftment. All mice were maintained as described above. Animal experiments were carried out in compliance with Dutch/European ethical guidelines and approved by the Dutch Central Authority for Scientific Procedures on Animals (permission number AVD114002016402).For the MM model, female 16-26-week-old NSG mice (Charles River) were irradiated with 2 Gy 24h prior to i.v. injection of 2.5 × 107 CCRF-CEM cells alone or mixed with 0.5–1.0×107 (as indicated) healthy donor PBMC (day 0) in female 5-9-week old NSG mice . Mice were i.p. injected with PBS or CD1d-Vδ2 hu-bsTCE (indicated dose and frequency). Tumor volumes were measured twice a week in two dimensions using a caliper, and the tumor volume (V) calculated using the formula: V = (W2×L)/2, where L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). In the dose escalating part, mice were euthanized when the tumor volume of individual mice reached >3000 mm3, mean tumor volume of the groups reached >2000 mm3 or when pre-set human endpoints were reached. In the dosing interval study, mice were euthanized when the tumor volume of individual mice reached >2000 mm3 or when pre-set human endpoints were reached. All mice were maintained as described above. The T-ALL in vivo study was performed by Crown Bioscience, China, in compliance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and approved by the Institutional Animal Care and Use Committee (IACUC) of Crown Bioscience.A s.c. T-ALL model was established via s.c. inoculation of 1 × 10M. fascicularis, at Crown Bioscience, Beijing, China. The study was divided in two phases. Phase 1: NHP received a single dose CD1d-Vγ9 bsTCE or control Vγ9 bsTCE  to determine PK values. Venous blood samples were collected pre-dose (day −14 and −7), 0, 0.5, 1, 2, 4, 6, and 8h, and day 1, 2, 3, 4, 5 and 6 post-infusion to determine PK values and/or flow cytometry analyses. Free plasma concentrations of the bsTCEs, measured using a free drug assay  by ABL immunology , were used to estimate PK parameters (PK Solver software version 2.055). Peripheral blood Vγ9-T cell percentages (CD3+TRGV9+(RαL+)), CD69 expression, and binding of CD1d-Vγ9 bsTCE or control Vγ9 bsTCE to Vγ9Vδ2-T cells were determined over time by flow cytometry. Binding to CD1d+ cells could not reliably be detected since the used RαL antibody could not bind VHH1D22 when bound to CD1d.PK and PD of CD1d-Vγ9 bsTCE or control Vγ9-bsTCE were studied in 12 female −1, 0.3 mg kg−1 or 1 mg kg−1 via a 30-min i.v. infusion, n = 1 animal per group). Venous blood samples were collected pre-dose (day −14 and −7), 0.5, 2, 4, 8 and 24h after the first and last dose and 2 and 24h after the remaining doses. Hematological parameters (complete blood cell count), coagulation , chemistry  and cytokines (U-PLEX Biomarker Group 1 (NHP) Assays; IL-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, TNF, IFN-γ and CCL2) were determined over time. Axillary lymph nodes were surgically collected under general anesthesia on day −14 and 24h after the final i.v. dose and processed to single cell suspensions. 24h after the 7th infusion the animals were euthanized for autopsy. Peripheral blood and lymph node Vγ9-T cell frequencies and binding of CD1d-Vγ9 bsTCE or control Vγ9 bsTCE (CD3+TRGV9+(RαL+)), and T cell (CD3+), B cell (CD3−CD20+) and monocyte (CD3−CD14+) frequencies were determined over time by flow cytometry. Absolute numbers were calculated as follows: [target population ml−1 blood] = ([counts in target population gate (FACS)]/[Counts in leukocyte gate (FACS)]) x [white blood cell count ml−1].Phase 2 (conducted in sequel of phase 1): NHP received 7 daily i.v. doses of CD1d-Vγ9 bsTCE or control Vγ9 bsTCE  and all procedures related to housing, handling, caring and treatment of the animals were performed in compliance with the guidelines approved by the AAALAC.All antibodies were used at empirically determined dilution factors. Staining was performed at 4°C in PBS supplemented with 0.1% BSA and 0.02% sodium azide unless otherwise stated. Analyses and cell sorting were performed using a FACS Canto II or FACS LSRFortessa (BD Biosciences). Data analysis was performed using Kaluza (Beckman Coulter), FCAP array and FlowJo (BD Biosciences).,4Ser(G4S)-linker (VHH1D12-VHH5C8 (further referred to as CD1d-Vδ2 bsTCE)) and purified, tagless, and endotoxin free CD1d-Vδ2 bsTCE was produced by UPE, the Netherlands. N-terminal positioning of VHH1D12 had a minimal functional advantage compared to C-terminal positioning in triggering type 1 NKT cell degranulation and was equally effective in triggering Vγ9Vδ2-T cell degranulation (data not shown). Control gp120-Vδ2 bsTCE (anti-gp120 VHHL8CJ3 which binds to glycoprotein (gp)120 of HIV-1)4S-linker to the N-terminus of anti-Vδ2 TCR VHH (clone VHH5C8)) and produced by UPE.Human CD1d-specific and human Vγ9-and Vδ2-TCR-specific VHH were generated and screened as previously described.in vitro binding experiments of monovalent VHH or bsTCE, ∼1×105 cells  or indicated concentrations of the constructs for 30–45 min at 4°C, followed by extensive washing and incubation for 30 min at 4°C with GαL-FITC, RαL-VHH iFluor488 or RαL-VHH iFluor647, washed and analyzed by flow cytometry. The relative percentage of binding was calculated by dividing delta median fluorescence (MF) [condition] (MF condition minus MF 0 nM) by delta MF [1000 nM] (MF 1000 nM minus MF 0 nM) multiplied by 100. In case delta MF 0–1000 nM was ∼0, binding was set to 0% for all conditions.For −1 sodium penicillin, 100 μg mL−1 streptomycin sulfate, 2.0 mM L-glutamine, 1000 U ml−1 GM-CSF and 20 ng mL−1 rhIL-4 for 5–7 days and subsequently matured with 100 ng mL−1 lipopolysaccharide (LPS) in the presence of 100 ng mL−1 α-GalCer for 48–72h. Type 1 NKT cells were purified from PBMC from healthy donors by magnetic bead sorting using anti-type 1 NKT microbeads. Purified type 1 NKT cells were stimulated weekly with irradiated (50 Gy) α-GalCer loaded mature moDC in Yssel’s medium−1 sodium penicillin, 100 μg mL−1 streptomycin sulfate, 2.0 mM L-glutamine, 10 IU mL−1 rhIL-7, 10 ng mL−1 rhIL-15.Primary human type 1 NKT cells were generated as described previously.−1 sodium penicillin, 100 μg mL−1 streptomycin sulfate, 2.0 mM L-glutamine, 10 IU mL−1 rhIL-7, 10 ng mL−1 rhIL-15 and 50 ng mL−1 phytohaemagglutinin (PHA).Primary human Vγ9Vδ2-T cells were generated and expanded as described previously.+TRBV25-1+ and TRGV9+TRDV2+, respectively.Expanded type 1 NKT and Vγ9Vδ2-T cells selected for experiments were >90% (>97% for mouse experiments) TRAV105 target cells  whether or not labeled with PBSE (CCRF-CEM only) were co-cultured with medium (negative control), VHH1D12 or bsTCE at the indicated concentration, and with 5×104 type 1 NKT cells, 5×104 Vγ9Vδ2-T cells or a mixture of 2.5×104 type 1 NKT and 2.5×104 Vγ9Vδ2-T cells for 4h in the presence of PE-labelled anti-CD107a, after which type 1 NKT ((PBSE−)CD3+CD45+TRGV9−TRAV-10+-tetramer+)) and Vγ9Vδ2-T cells ((PBSE−)CD3+CD45+TRGV9+TRAV-10-((TRDV2+)-tetramer−/+)) were analyzed for CD107a expression. Supernatants from 24h co-cultures of 1 × 105 MM.1s.CD1d and 5×104 type 1 NKT, Vγ9Vδ2-T cells or a 1:1 mixture thereof (ratio 1:1) with medium (negative control) or CD1d-Vδ2 bsTCE (50 nM) were analyzed for cytokine production using a cytometric bead array (CBA).To evaluate induction of degranulation of type 1 NKT and Vγ9Vδ2-T cells, 1×105 cells were co-cultured overnight ±1 × 105 C1R.WT or C1R.CD1d cells and CD1d-Vδ2 bsTCE (1 μM), vehicle control (DMSO), sulfatide (25 μg mL−1) or a combination thereof. CD3+CD19− cells were analyzed for CD69 expression.For the stimulation assays of the CD1d(sulfatide)-restricted TCR DP10.7 JRT3 transfectant, 1×10+(TRDV1+)7-AAD- cells was assessed using flow cytometry.CD1d(sulfatide) tetramer-PE was generated as described previously.+ T cells was determined .For assessment of the capacity of CD1d-Vδ2 bsTCE to interfere with CD1d-sulfatide reactivity of diverse NKT cells in PBMC, healthy donor PBMC were enriched for CD1d(sulfatide)-reactive diverse NKT cells by magnetic bead sorting using CD1d(sulfatide) tetramer-PE in combination with anti-PE IgG microbeads , MOLM-13, NOMO-1 cells) were incubated with 5×104 type 1 NKT, Vγ9Vδ2-T cells or a 1:1 mixture thereof with medium (negative control) or bsTCE at indicated concentration. After 16h incubation, cells were stained with an antibody cocktail, washed in annexin-V buffer and briefly incubated with annexin-V FITC, 7-AAD and flow cytometric counting beads, for subsequent flow cytometry analysis. Absolute number of living target cells (PBSE+ (CCRF-CEM cells only) CD3-/dim(CD7+)(CD33+)TRAV10−TRGV9−annexin V− 7-AAD-) was determined according to manufacturer’s instructions. Specific lysis of target cells was calculated as follows; 100 - ((absolute cell number [condition]/absolute cell number [target cells only]) x 100).The ability of human type 1 NKT and Vγ9Vδ2-T cells to lyse target cells was determined by flow cytometry. 1×10-(CD45−/+)TRAV-10−TRGV9-7-AAD-), type 1 NKT (CD3+(CD45+)TRAV-10+TRGV9-7-AAD-) and Vγ9Vδ2-T cells (CD3+(CD45+)TRAV-10−TRGV9+7-AAD-) were quantified by flow cytometric counting beads. Tumor growth and effector cell expansion factor was calculated by dividing the cell count after co-culture by the starting cell count at day 0.To examine tumor growth control, and type 1 NKT and Vγ9Vδ2-T cell expansion, MM.1s.CD1d or MOLM-13 cells were incubated with medium (negative control) or CD1d-Vδ2 bsTCE (50 nM) and cultured for up to 7 days in the presence of human healthy donor derived type 1 NKT, Vγ9Vδ2-T cells or a 2:3 mixture thereof (E:T 1:10). Living target cells  and with medium (negative control) or CD1d-Vδ2 bsTCE (50 nM or concentration range) (and PE-labelled anti-CD107a for 16h culture only) and cultured up to 7 days.To examine B cell and monocyte lysis, tumor growth control, and type 1 NKT and Vγ9Vδ2-T cell degranulation and expansion in PBMC, 1×10−CD19+CD33−CD45+7-AAD-), monocytes (CD3−CD14+CD19−CD33+CD45+7-AAD-), and MM.1s.CD1d (CD3−CD38+CD45-7-AAD-), and CD107a expression on type 1 NKT -tetramer+7-AAD-) and Vγ9Vδ2-T cells -tetramer−/+7-AAD-) were quantified/determined by flow cytometry counting beads after 16h. Specific lysis was calculated as described above. To determine tumor growth and effector cell expansion, living tumor cells  and living type 1 NKT (CD3+(CD33−)CD45+TRGV9−TRDV2−TRAV10+CD1d-tetramer+7-AAD-) and Vγ9Vδ2-T cells (CD3+(CD33−)CD45+TRGV9+(TRDV2+)TRAV10−CD1d-tetramer−/+7-AAD-) were quantified by flow cytometric counting beads. Expansion factor was calculated by dividing the cell count after co-culture by the starting cell count.Living target cells (B cells (CD34 target cells (monocytes (>65% CD14+ after isolation), THP-1 cells (stained with cell trace violet (CTV)) were incubated with 5×104 Vγ9Vδ2-T cells (>75% of CD3+ cells after isolation) with medium (negative control) or 1 nM CD1d-Vδ2 bsTCE. After 24h incubation, cells were stained with an antibody cocktail, washed and briefly incubated with 7-AAD, for subsequent flow cytometry analysis. The percentage of 7-AAD+ monocytes (CD3−CD14+TRGV9-) and THP-1 cells (CD3−TRGV9−CTV+) was determined.To determine the ability of freshly isolated (untouched) Vγ9Vδ2-T cells to lyse either THP-1 tumor cells or freshly isolated (untouched) monocytes, Vγ9Vδ2-T cells and monocytes were isolated from healthy donor PBMC by magnetic bead sorting using a human γδ-T cell isolation kit and a pan monocyte isolation kit, respectively. 5×10In silico humanization was performed by Lonza (Lonza Biologics) via selection of human antibody germline sequences similar to monovalent VHH , identification of VHH critical residues, and complementarity-determining region (CDR) grafting and substitution of mismatched residues between parental and acceptor framework regions, similar as described previously.4S-linker to Vδ2 TCR  and expressed in P. pastoris . VHH1D12.Q1E P. pastoris strain (data not shown). HLA-DRB1-binding score of the monovalent binding arms of the CD1d-Vδ2 bsTCE (i.e. CD1d VHH1D12 and Vδ2-TCR VHH5C8) was compared to the HLA-DRB1-binding score of therapeutic human IGHV.125) and purified. I125-labeled bsTCE (∼0.5 ng per test) was subsequently incubated with 1 μL healthy donor human serum (1:50 diluted in 0.3% PBS-BSA) and possible IgG-VHH complexes were then captured onto protein-G coated Sepharose beads. After overnight incubation and extensive washing, radioactivity of sepharose-beads was measured by a gamma counter. Specificity of the test was confirmed by inhibition of the signal by adding an excess of unlabeled bsTCE . Percentage of binding was calculated by dividing the number of disintegrations measured on the sepharose-bound radiolabeled bsTCE in the experimental condition by the number of disintegrations found in the total input of radiolabeled bsTCE. The cut-off point for positivity was calculated on the mean percentage binding  plus 1.645 SD.The frequency of pre-existing human anti-VHH antibodies in serum was determined via an Ag binding test by Sanquin Blood Supply foundation. CD1d-Vδ2 bsTCE and CD1d-Vδ2 hu-bsTCE were labeled with radio-active iodine (I−CD19−/+CD38+CD45-/dimCD56−/+(CD81−/+)(CD117−/+)CD138+), AML (CD3−CD33−/+CD34−/+CD45dim/+CD56−/+CD117−/+HLA-DRdim/+) and CLL cells (CD3−CD5+CD19+). Geometric MF index (MFI) was calculated by dividing MF [stained tumor cells] by MF [unstained tumor cells or CD1d− population]. T cell percentage (CD3+CD45+) of mononuclear cells, type 1 NKT percentage -tetramer+/TRBV25-1+) of CD3+ cells and Vγ9Vδ2-T percentage (CD3+CD45+TRGV9+TRDV2+) of CD3+ cells was determined using multicolor flow cytometry. For autologous and allogeneic type 1 NKT and Vγ9Vδ2-T cell degranulation, and cytotoxicity toward patient MM, monocytic AML and CLL cells, 1×105 thawed BMMC (MM and AML) or PBMC  or thapsigargin  pre-incubation) were cultured with medium (negative control) or CD1d-Vδ2 hu-bsTCE (50 nM) in the presence or absence of 5×104 type 1 NKT, Vγ9Vδ2-T cells or a 1:1 mixture thereof for 16h in the presence of PE-labelled anti-CD107a, after which type 1 NKT (7-AAD−CD3+ (CD19−)(CD33−)CD45+(CD138-)TRGV9−TRAV-10+CD1d-tetramer+) and Vγ9Vδ2-T cells (7-AAD−CD3+(CD19−)(CD33−)CD45+(CD138-)TRGV9+TRAV-10-((TRDV2+)-tetramer−/+)) were analyzed for CD107a expression. Live MM (7-AAD−CD3−(CD19−)CD45dim/−CD38+(CD56−/+)CD138+), monocytic AML (7-AAD−CD3−CD33dim/+(CD34+)CD45dim/+(CD56+CD117+)HLA-DRdim/+) and CLL cells (7-AAD−CD3−CD5+CD19+CD45+) were quantified by flow cytometry counting beads. Specific lysis was calculated as described above. Supernatants were collected and analyzed for cytokine production by CBA.Using multicolor flow cytometry, CD1d geometric median fluorescence (MF) was determined on MM (CD36 human PBMC (positive control), M. fascicularis  PBMC or M. mulatta PBMC  were subsequently incubated with indicated VHH  and antibody cocktail . CD11b+ CD20− TRGV9- pan-γδ- cells and CD11b− CD20− TRGV9+ pan-γδ+/dim were analyzed by flow cytometry for CD1d VHH and Vγ9Vδ2-TCR VHH binding respectively as determined by streptavidin-APC fluorescence.To examine cross-reactivity of CD1d-Vδ2 bsTCE toward NHP CD1d and Vγ9Vδ2-TCR, monovalent VHHs ) and Vγ9-T cells (CD3+CD11b-streptavidin-APC+). Relative percentage of binding was calculated as described above. To evaluate induction of degranulation of Vγ9-T cells, thawed human and NHP primate PBMC (0.25–1×105 per condition) were incubated with rhIL-2 (50 IU/mL) for 24h to recover followed by incubation with or without CD1d-Vδ2 bsTCE, CD1d-Vγ9 bsTCE or control Vγ9 bsTCE (concentration range) for an additional 24h in the presence of PE-labelled anti-CD107a, after which Vγ9-T cells (CD3+ CD11b− TRGV9+) were analyzed for CD107a expression.To be able to assess safety, PK and PD parameters in NHP, a surrogate bispecific molecule was generated using sequence information from an NHP cross-reactive mouse antibody  directed to the Vγ9 chain of the TCR, coupled to the cross-reactive CD1d specific VHH1D22  and produced by UPE. Bispecific molecule CD1d VHH1D12-scFv7A5 (control Vγ9 bsTCE), which lacks cross-reactivity toward NHP CD1d, was generated as a control and produced by UPE. Binding of the various bispecific molecules to CD1d and Vγ9Vδ2-TCR was analyzed at indicated concentrations by flow cytometry as described above. For binding to monocytes and Vγ9-T cells in PBMC (human or NHP), 0.25–1×10in vivo experiments was determined by using a two-sided, two-sample equal-variance t-test (assuming a population mean difference of 0.25 with an s.d. of 0.15 and with a significance level (α) of 0.050 with >80% power) and previous experience. Sample size for the AML and T-ALL in vitro experiments was estimated based on previous experience with these tumor cell lines. Mice were randomly assigned to treatment groups. No statistical method was used to predetermine sample size for the in vitro experiments. For in vitro experiments each n represents an independent experiment with, when applicable, primary type 1 NKT and Vγ9Vδ2-T cell lines, and PBMC obtained from individual healthy donors. For experiments with patient MM and AML BMMC and CLL PBMC each n represents an individual patient. For in vivo experiments each n represents an individual animal. No data were excluded from the analyses. The experiments were not randomized and the investigators were not blinded to allocation during the experiments and outcome assessment.Sample size for the MM 50) was calculated using non-linear regression . To determine the relationship between CD1d-expression and cell lysis, linear regression was used to fit the line through the data points, determine goodness of fit (R2) and calculate the p value. Survival data was analyzed with Kaplan–Meier survival curves and the log rank test  was used to calculate statistically significant differences. Statistical details can be found in the figure legend. Statistical analysis was performed using Prism v.9.1.0 (GraphPad Software).For data with one variable and two groups, a two-tailed paired/unpaired t-test was used to calculate the p value. For data with one variable and multiple groups, a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test to calculate the multiplicity-adjusted p value was used. For data with two variables, a two-way ANOVA with either Tukey’s or Šídák’s multiple comparisons test to calculate the multiplicity-adjusted p value was used. Dose–response curves, half maximal effective concentration (EC


These data suggest that AC5 inhibition with vidarabine might be a new therapeutic approach for the treatment of cardiovascular disease associated with occlusal disharmony.We recently reported a positive relationship between occlusal disharmony and cardiovascular disease via activation of β-adrenergic signaling in mice. Furthermore, inhibition of type 5 adenylyl cyclase (AC5), a major cardiac subtype in adults, protects the heart against oxidative stress. Here, we examined the role of AC5 in the development of occlusal-disharmony-induced cardiovascular disease in bite-opening (BO) mice, prepared by cementing a suitable appliance onto the mandibular incisor. We first examined the effects of BO treatment on cardiac function in mice treated or not treated for 2 weeks with vidarabine, which we previously identified as an inhibitor of cardiac AC. Cardiac function was significantly decreased in the BO group compared to the control group, but vidarabine ameliorated the dysfunction. Cardiac fibrosis, myocyte apoptosis and myocyte oxidative DNA damage were significantly increased in the BO group, but vidarabine blocked these changes. The BO-induced cardiac dysfunction was associated with increased phospholamban phosphorylation at threonine-17 and serine-16, as well as increased activation of the CaThe online version contains supplementary material available at 10.1186/s12576-022-00826-4. Oral health decrease with age, and this is a major risk factor for many diseases, including cardiovascular disease . In addiAdenylyl cyclase (AC) is the target enzyme of β-adrenergic receptor (β-AR) signaling stimulation. At least 9 isoforms are known, 7 of which are expressed in the heart; the type 5 isoform (AC5) is a major adult cardiac isoform, while type 6 is a major fetal isoform  and we hWe previously examined the effects of occlusal disharmony on cardiac remodeling (fibrosis and myocyte apoptosis), cardiac function and susceptibility to atrial fibrillation in bite-opening mice (BO), in which a 0.7-mm BO was introduced by cementing a suitable appliance onto the mandibular incisor. We found that BO-induced cardiac dysfunction and susceptibility to atrial fibrillation are ameliorated by co-treatment with propranolol, a non-selective β-blocker , 16. TheHowever, β-blockers have several critical side effects. The inhibition of sympathetic signaling reduces cardiac function , 18, andHowever, the role of AC5 in the occlusal-disharmony-induced cardiac deterioration remains poorly understood. In this study, therefore, we examined the effects of AC5 inhibition with vidarabine on cardiac function, cardiac fibrosis, myocyte apoptosis, and oxidative DNA damage induced by occlusal disharmony in mice Fig. : Fig. S1All experiments were performed on male 12-week-old C57BL/6 mice obtained from CLEA Japan . Mice were group-housed at 23 °C under a 12–12 light/dark cycle with lights on at 8:00 AM in accordance with the standard conditions for mouse studies by our group , 22, 23.n = 5 each)   , midazolam (0.4 mg/ml), and butorphanol (0.5 mg/ml) , 16, 24.n = 6, BO: n = 6, V: n = 7, BO + V: n = 6) were anesthetized via a mask with isoflurane vapor (1.0–1.5% v/v) titrated to maintain the lightest anesthesia possible, and echocardiographic measurements were performed by means of ultrasonography  at 14 days after BO treatment [Mice  [https://imagej.nih.gov/ij/download.html) to evaluate the percentage of blue area in the Masson-trichrome sections [Among several quantitative methods available to determine interstitial fibrotic regions , 28, 29,: n = 4) , 22, 23.sections , 22, 23.n = 4, BO: n = 5, V: n = 5, BO + V: n = 4) over a microscopic field of 20 x, then averaged and expressed as the ratio of TUNEL-positive nuclei (%) [Apoptosis was determined by terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-deoxyuridine (TUNEL) staining using an Apoptosis in situ Detection Kit . TUNEL-positive nuclei per field of view were manually counted in six sections of each of the four groups (Control: clei (%) , 30. By n = 10, BO: n = 8, V: n = 6, BO + V: n = 8) was homogenized in a Polytron  in ice-cold RIPA buffer , 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) without addition of inhibitors [g for 10 min at 4 °C. The supernatant was collected and the protein concentration was measured using a DC protein assay kit . Equal amounts of protein (5 μg) were subjected to 12.5% SDS–polyacrylamide gel electrophoresis and blotted onto 0.2 mm PVDF membrane .Cardiac muscle excised from mice   under our standard conditions [2O2 in TBS-T before the anti-8-OHdG antibody treatment. The sections were incubated with anti-8-OHdG antibody  overnight at 4ºC in a humidified chamber, then incubated with 0.3% H2O2 in 0.3% horse serum for 1 h at room temperature to inactivate endogenous peroxidase, rinsed with TBS-T, incubated with anti-mouse IgG in M.O.M. Diluent, and processed with an ABC kit . The ratio of 8-OHdG nuclei with oxidative DNA damage (stained dark brown) per total cell number was evaluated.Oxidative DNA damage in the myocardium was evaluated by immunostaining for 8-hydroxy-2’-deoxyguanosine (8-OHdG) using the Vector M.O.M Immunodetection system . Comparisons were performed using two-way repeated-measures ANOVA followed by the Bonferroni post hoc test Fig. a–f, 6a–dThe BW in the control and vidarabine groups showed no significant change during the experimental period Fig. c. HoweveWe monitored the daily consumption of pellet food and water per mouse, measured as an average of the animals in each cage, during the 2-week experimental period. Consumption levels of food .We examined the effect of BO with/without vidarabine on heart size in terms of cardiac muscle mass per tibial length ratio Fig. d and theThese data suggest that BO did not induce cardiac hypertrophy or lung edema during the experimental period.n = 4) vs. BO (n = 5); 0.9 ± 0.4% vs. 2.5 ± 0.8%, P < 0.01 vs. Control) in accordance with our previous finding [n = 5) vs. BO + vidarabine (n = 4); 2.5 ± 0.8% vs. 1.1 ± 0.5%, P < 0.001 vs. BO).We examined cardiac fibrosis in BO mice with/without vidarabine by means of Masson-trichrome staining Fig. a, b. BO  finding . Vidarabn = 5) vs. BO (n = 4); 1.0 ± 0.4 vs. 2.5 ± 0.9 arbitrary unit (A.U.), P < 0.05 vs. Control), and the increase was significantly suppressed by vidarabine (BO (n = 4) vs. BO + vidarabine (n = 4); 2.5 ± 0.9 vs. 0.7 ± 0.5 A.U., P < 0.01 vs. BO)  vs. BO (n = 5); 1.6 ± 1.3% vs. 4.2 ± 1.7%, P < 0.05 vs. Control) in accordance with our previous findings [n = 5) had no effects on the number of TUNEL-positive cardiac myocytes, but it blocked the BO-induced increase of TUNEL-positive cardiac myocytes (BO (n = 5) vs. BO + vidarabine (n = 4); 4.2 ± 1.7% vs. 1.3 ± 0.7%, P < 0.05 vs. Control).We also examined cardiac myocyte apoptosis in BO mice with/without vidarabine by means of TUNEL staining Fig. a, b. BO findings . VidarabWe also evaluated cardiac myocyte apoptosis by examining the ratio of Bax protein, an accelerator of apoptosis, and BCL-2 protein, a regulator of apoptosis, in the heart Fig. c.n = 4) vs. BO (n = 4); 1.0 ± 0.6% vs. 5.9 ± 2.6 A.U., P < 0.05 vs. Control). Vidarabine alone (n = 5) had no effects on the ratio of Bax/BCL-2, but it blocked the BO-induced increase in the Bax/BCL-2 ratio (BO (n = 4) vs. BO + vidarabine (n = 5); 5.9 ± 2.6 vs. 1.3 ± 1.0 A.U., P < 0.05 vs. Control)  vs. BO (n = 6); 68 ± 1.1% vs. 61 ± 3.0%, P < 0.001 vs. Control; %FS: Control (n = 6) vs. BO (n = 6); 33 ± 0.8% vs. 28 ± 1.9%, P < 0.001 vs. Control). Vidarabine alone (n = 7) had no effect on EF or %FS, but blocked the BO-induced decrease of EF and %FS (EF: BO (n = 6) vs. BO + vidarabine (n = 6); 61 ± 3.0% vs. 66 ± 1.4%, P < 0.01 vs. Control; %FS: BO (n = 6) vs. BO + vidarabine (n = 6); 28 ± 1.9% vs. 31 ± 1.0%, P < 0.01 vs. Control).We performed echocardiography to evaluate cardiac function in terms of left ventricular cardiac function (EF) and fractional shortening (%FS) Table . Both paThese data suggest that BO treatment decreases cardiac function at least in part through the activation of AC5.We evaluated oxidative stress in the myocardium by means of 8-OHdG immunostaining Fig. a, b and 2O2 in TBS-T for 1 h at room temperature before the anti-8-OHdG antibody treatment and confirmed that the 8-OHdG staining procedure could clearly discriminate 8-OHdG-positive and non-positive nuclei /without (negative control) 0.3% Hn = 4) vs. BO (n = 5); 1.1 ± 0.6% vs. 3.0 ± 1.4%, P < 0.05 vs. Control), and the increase was suppressed by vidarabine (BO (n = 5) vs. BO + vidarabine (n = 4); 3.0 ± 1.4% vs. 1.1 ± 0.4%, P < 0.05 vs. Control).The ratio of 8-OHdG-positive/total cells was significantly increased in the BO group  vs. BO (n = 5); 1.0 ± 0.1 vs. 2.7 ± 1.0 A.U., P < 0.01 vs. Control), and again the increase was suppressed by vidarabine (BO (n = 5) vs. BO + vidarabine (n = 4); 2.7 ± 1.0 vs. 1.1 ± 0.4 A.U., P < 0.05 vs. Control).The amount of oxidized proteins, measured using the OxiSelectThese results, together with the data shown in Figs. We first examined the effects of BO treatment on AC5 expression with/without vidarabine, since AC5 was previously found to be increased by chronic catecholamine stress . HoweverReactive oxygen species (ROS) are produced through a number of pathways, including NOX and XO, and may be involved in various physiological and pathological processes in the heart, including fibrosis, apoptosis and heart failure –37.n = 10) vs. BO (n = 8); 1.0 ± 0.2 vs. 1.6 ± 0.4 A.U., P < 0.001 vs. Control). This increase was significantly inhibited by vidarabine (BO (n = 8) vs. BO + vidarabine (n = 8); 1.6 ± 0.4 vs. 1.1 ± 0.2 A.U., P < 0.01 vs. BO)   Fig. b. We alsFig. n =  each (Fin = 8) vs. BO (n = 5); 1.0 ± 0.2 vs. 2.1 ± 0.7 A.U., P < 0.001 vs. Control). This increase was significantly inhibited by vidarabine (BO (n = 5) vs. BO + vidarabine (n = 7); 2.1 ± 0.7 vs. 1.1 ± 0.4 A.U., P < 0.01 vs. BO)  vs. BO (n = 5); 1.0 ± 0.3 vs. 2.8 ± 1.7 A.U., P < 0.05 vs. Control). This increase was significantly inhibited by vidarabine (BO (n = 5) vs. BO + vidarabine (n = 5); 2.8 ± 1.7 vs. 1.1 ± 0.4 A.U., P < 0.05 vs. Control)  signaling pathway, leading to cardiac dysfunction . We ther. BO n = ; 1.0 ± 0These data suggest that overproduction of ROS derived from BO-induced upregulation of NOX4 and XO activates the p38 MAPK signaling pathway, which might lead to cardiac remodeling and dysfunction.n = 4) vs. BO (n = 4); 1.0 ± 0.3 vs. 2.6 ± 1.4, P < 0.05 vs. Control). This increase was significantly inhibited by vidarabine (BO (n = 4) vs. BO + vidarabine (n = 5); 2.6 ± 1.4 vs. 0.5 ± 0.2, P < 0.01 vs. BO)  vs. BO (n = 4); 1.0 ± 0.3 vs. 5.4 ± 2.3 A.U., P < 0.001 vs. Control)  vs. BO + vidarabine (n = 4); 5.4 ± 2.3 vs. 1.7 ± 0.5 A.U., P < 0.01 vs. BO)  Fig. a. Again,BO) Fig. a.These data suggest that activation of AC5 might contribute, at least in part, to the upregulation of NOX4 and XO.n = 4) vs. BO (n = 5); 1.0 ± 0.2 vs. 1.9 ± 0.2 A.U., P < 0.001 vs. Control). The increase was significantly attenuated by vidarabine (BO (n = 5) vs. BO + vidarabine (n = 4); 1.9 ± 0.2 vs. 1.2 ± 0.3, P < 0.01 vs. BO)  Fig. b.These data suggest that BO treatment might activate NOX4/RIP3/CaMKII signaling via activation of AC5.2+ leakage mediated by PLN phosphorylation contributes considerably to the pathogenesis of cardiac remodeling and dysfunction via ROS derived from NOX4 [The elevation of diastolic sarcoplasmic reticulum Carom NOX4 , 46. We n = 4) vs. BO (n = 5); 1.0 ± 0.2 vs. 2.6 ± 1.3 A.U., P < 0.05 vs. Control)  vs. BO + vidarabine (n = 5); 2.6 ± 1.3% vs. 0.7 ± 0.2 A.U., P < 0.01 vs. BO)  was significantly increased in cardiac muscle of BO mice (Control (n = 4) vs. BO (n = 5); 1.0 ± 0.5 vs. 2.2 ± 0.8 A.U., P < 0.05 vs. Control). Again, the increase was significantly attenuated by vidarabine (BO (n = 5) vs. BO + vidarabine (n = 5); 2.2 ± 0.8 vs. 0.8 ± 0.1 A.U., P < 0.01 vs. BO)  was also significantly increased in cardiac muscle of BO mice  and drinking water (b) throughout the 2-week experimental period. C: Control, B: BO, V: Vidarabine, BO + V: BO + Vidarabine. Fig. S3. Representative full-length western blotting of Fig. 2c. The amounts of activated α-SMA (left panel) and GAPDH (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S4. Representative full-length western blotting of Fig. 3c. The amounts of Bax (left panel) and BCL-2 (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped parts of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S5. Representative images of negative (left) and positive (right) controls of 8-OHdG immunostaining. Fig. S6. Representative full-length western blotting of Fig. 4c. The amounts of activated oxidized protein (left panel) and GAPDH (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot. C: control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S7. Representative full-length western blotting of Fig. 5a. The amounts of AC5 (left panel) and GAPDH (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S8. Representative full-length western blotting of Fig. 5b. The amounts of NOX4 (left panel) and GAPDH (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S9. Representative full-length western blotting of Fig. 5c. The amounts of NOX2 (left panel) and GAPDH (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S10. Representative full-length western blotting of Fig. 5d. The amounts of XO (left panel) and GAPDH (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S11. Representative full-length western blotting of Fig. 5e. The amounts of phosphorylated p38 at Thr-180/Tyr-182 (left panel) and total p38 (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S12. Representative full-length western blotting of Fig. 5f. The amounts of phosphorylated ASK-1 at Thr-845 (left panel) and total ASK-1 (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S13. Representative full-length western blotting of Fig. 6a. The amounts of RIP3 (left panel) and GAPDH (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S14. Representative full-length western blotting of Fig. 6b. The amounts of phosphorylated CaMKII at Thr-286 (left panel) and total CaMKII (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S15. Representative full-length western blotting of Fig. 6c. The amounts of phosphorylated PLB at Thr-17 (left panel) and total PLB (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine. Fig. S16. Representative full-length western blotting of Fig. 6d. The amounts of phosphorylated PLB at Ser-16 (left panel) and total PLB (right panel) are shown. The box outlined in black in each panel, indicated by the arrow, corresponds to the cropped part of the blot shown in the main article. C: Control, B: BO, V: Vidarabine, BOV: BO + Vidarabine.