The current paper contrasts the extensive in vitro, in vivo, and ex vivo assessments of the radiotracers [18F]FE@SNAP and [11C]SNAP-7941 and provides comprehensive information about their biological and physicochemical properties. Furthermore, it examines their suitability for first-in-man imaging studies.The melanin-concentrating hormone receptor 1 (MCHR1) has become an important pharmacological target, since it may be involved in various diseases, such as diabetes, insulin resistance, and obesity. Hence, a suitable positron emission tomography radiotracer for the 18F]FE@SNAP and [11C]SNAP-7941 were conducted on adherent Chines hamster ovary (CHO-K1) cells stably expressing the human MCHR1 and MCHR2. Small animal imaging studies on mice and rats were performed under displacement and baseline conditions, as well as after pretreatment with the P-glycoprotein/breast cancer resistant protein inhibitor tariquidar. After the imaging studies, detailed analyses of the ex vivo biodistribution were performed. Ex vivo metabolism was determined in rat blood and brain and analyzed at various time points using a quantitative radio-HPLC assay.Kinetic real-time cell-binding studies with [11C]SNAP-7941 demonstrates high uptake on CHO-K1-hMCHR1 cells, whereas no uptake was detected for the CHO-K1-hMCHR2 cells. In contrast, [18F]FE@SNAP evinced binding to CHO-K1-hMCHR1 and CHO-K1-hMCHR2 cells. Imaging studies with [18F]FE@SNAP and [11C]SNAP-7941 showed an increased brain uptake after tariquidar pretreatment in mice, as well as in rats, and exhibited a significant difference between the time-activity curves of the baseline and blocking groups. Biodistribution of both tracers demonstrated a decreased uptake after displacement. [11C]SNAP-7941 revealed a high metabolic stability in rats, whereas [18F]FE@SNAP was rapidly metabolized.[11C]SNAP-7941 underlines the decisive superiority over [18F]FE@SNAP.Both radiotracers demonstrate appropriate imaging properties for the MCHR1. However, the pronounced metabolic stability as well as superior selectivity and affinity of [ A PET tracer would also facilitate preclinical to clinical translation. The specific MCHR1 antagonist SNAP-7941 ((+)-methyl(4S)-3-{[(3-{4-[3-(acetylamino)phenyl]-1piperidinyl}propyl)amino]carbonyl}-4--6-(methoxymethyl)-2-oxo-1,2,3,4-tetra-hydro-5-pyrimidenecarboxylate hydrochloride) [11C]SNAP-7941 , a cyclic 19 amino acid long polypeptide, is primarily produced by neurons in the lateral hypothalamic area (LHA), the incerto-hypothalamic area (IHy), and zona incerta (ZI) , colonichloride)  served ain vitro, in vivo, and ex vivo assessments of [11C]SNAP-7941 and [18F]FE@SNAP to determine the superior radioligand with respect to its biological and physicochemical properties. A comprehensive illustration of the applied in vitro, in vivo, and ex vivo experiments is shown in Fig. The current paper contrasts novel and previously attained ments of 1CSNAP-79The racemic mixture of SNAP-7941 ((±)-SNAP-7941), the fluoroethylated analog (±)-(2-fluoroethyl)-3-{[(3-{4-[3-(acetylamino)phenyl]-1-piperidinyl}propyl)amin]carbonyl}-4--6-(methoxymethyl)-2-oxo-1,2,3,4-tetra-hydro-5-pyrimidenecarboxylate (FE@SNAP), as well as the precursor compounds (±)-3-{[(3-{4-[3-(acetylamino)phenyl]-1piperidinyl}propyl)amino]carbonyl}-4--6-(methoxymethyl)-2-oxo-1,2,3,4-tetra-hydro-5-pyrimidenecarboxylate acid (SNAP-acid), and (±)-2-(tosyloxy)ethyl-3-{[(3-{4-[3-(acetylamino)phenyl]-1piperidinyl}propyl)amino]carbonyl}-4--6-(methoxymethyl)-2-oxo-1,2,3,4-tetra-hydro-5-pyrimidenecarboxylate acid (Tos@SNAP) were provided by the Department of Pharmaceutical Chemistry and the Department Organic Chemistry of the University of Vienna  . Tariqui11C]SNAP-7941, the radiolabeled analog of (±)-SNAP-7941, was performed in a fully automated synthesizer  as previously reported [18F]FE@SNAP was performed in a microfluidic device  as described elsewhere [11C]SNAP-7941 and [18F]FE@SNAP were determined by analytical high-performance liquid chromatography (HPLC) .The radiosynthesis of SNAP-7941 and [18F]FE@SNAP as previously reported SNAP-7941 and [18F]FE@SNAP. Binding to the seeded cells was ensured by adding different concentrations of the radiotracer (0.05–1000 nM). Association-time curves were monitored in real-time until the binding equilibrium was achieved. The observed rate constant of the association reaction (kobs) was determined using non-linear regression curve fitting algorithms implemented in GraphPad Prism 6.0 , as previously reported SNAP-7941) or 20 min ([18F]FE@SNAP) after the radiotracer application via the lateral tail vein. To investigate a potential binding to the P-glycoprotein (P-gp) and/or breast cancer resistance protein (BCRP) brain efflux transporter system, rats as well as mice were pretreated either with the P-gp/BCRP inhibitor TQD  or the respective solvent ). Mice received the P-gp/BCRP inhibitor 30 min and rats 60 min before the radiotracer application. Experiments were initiated with a 7-min cone beam attenuation CT (CBCT) of the brain  using a small animal cone beam computed tomography (CBCT) scanner . Subsequently, the animals were positioned in the Siemens Inveon microPET scanner . The radiotracers were injected intravenously, and dynamic PET imaging was performed for 45 min (rats) in case of [11C]SNAP-7941  and 30 min (mice)—60 min (rats) for [18F]FE@SNAP .Anesthetized rats and mice were immobilized in a multimodal animal carrier unit . The body temperature was preserved at 37 °C throughout the whole experiment. Rats received either [11C]SNAP-7941: 1 × 3 s, 3 × 2 s, 1 × 6 s, 1 × 15 s, 1 × 35 s, 1 × 145 s, 1 × 270 s, 1 × 285 s, 1 × 165 s, 3 × 30 s, 1 × 120 s, 1 × 240 s, 1 × 420 s, 1 × 900 s and for [18F]FE@SNAP: 1 × 2 s, 1 × 3 s, 1 × 5 s, 3 × 10 s, 1 × 20 s, 1 × 60 s, 1 × 120 s, 4 × 240 s, 4 × 600 s. PET image reconstruction were performed using an OSEM 3D/OP-MAP with scatter correction and a ramp filter (matrix size 128 × 128). Image data were normalized and corrected for random events, dead time, and radioactive decay. A calibration factor was applied to convert the activity information into absolute concentration units. Multimodal image registration and data post-processing was performed using the biomedical image analysis software PMOD 3.8  and the Inveon Research Workplace . Volumes of interest (VOIs) were outlined on the CT images and transferred to the PET data set. Time-activity curves (TACs) were calculated, normalized to dose and weight and expressed as standardized uptake values .Image reconstruction of the CT raw data was performed with a Feldkamp algorithm using a ramp filter followed by standard rat beam-hardening correction and noise reduction . All CT image data was calibrated to Hounsfield units (HU). PET list mode data were sorted into three-dimensional sinograms according to the following frame sequences, ).After the imaging studies, the animals were sacrificed by decapitation; blood and tissues were removed and collected in tubes, weighed, and subjected to radioactivity measurements in a Gamma Counter  of acetonitrile and 0.9 % saline solution using an ULTRA-TURRAX® . Subsequently, blood and brain were centrifuged , and the obtained supernatant was analyzed by an analytical HPLC equipped with a radioactivity detector (radio-HPLC) , mobile phase: /acetonitrile 70/30 v/v; flow: 1 ml/min, λ = 254 nm). The ratio between metabolite and intact radiotracer was calculated using quantitative HPLC analysis.For the analysis of potential radiometabolites, blood samples (50–80 μl) from rats were withdrawn at 10 and 45 min for SNAP-7941 (kobs = 0.0629 ± 0.0020 min−1) on CHO-K1-hMCHR1 cells showed no significant differences (P = 0.9). High statistical significance was observed for the accumulation of both radiotracers on the native CHO-K1 cells compared to the CHO-K1-hMCHR1 cells  compared to [11C]SNAP-7941. No significant difference in the binding pattern was determined for [11C]SNAP-7941 on the native CHO-K1 cells compared to the CHO-K1-hMCHR2 cells, which indicates negligible accumulation to these two cell lines  and 77 ± 0.2 % (n = 3) for FE@SNAP.The PPB analysis of (±)-SNAP-7941 resulted in 80 ± 0.9 % . Furthermore, both radiotracers showed an increased brain uptake for the TQD blocking group in rats. Brain uptake was increased 2.45-times for [18F]FE@SNAP .[18F]FE@SNAP and [11C]SNAP-7941, for the brown adipose tissue (BAT), brain, and lung. Corresponding TACs are depicted in Fig. P = 0.3117). Differences in the binding profiles before and after displacement with 15 mg/kg (±)-SNAP-7941 in all other regions of interest were not statistically significant, for both [18F]FE@SNAP and [11C]SNAP-7941.TACs of selected regions were analyzed before and after displacement with 15 mg/kg (±)-SNAP-7941. A clear difference in the binding pattern was observed for both tracers, [18F]FE@SNAP (blue); [11C]SNAP-7941 (green); displacement (red)). A detailed statistical analysis was performed for selected target regions -SNAP-7941  and fewer preparation steps. Moreover, its radiosynthesis is faster (40 min) compared to [18F]FE@SNAP (100 min), which has to be synthesized via a microfluidic device SNAP-7941 evinced selective binding to the CHO-K1-hMCHR1 cells, which is in good agreement with previously published data [18F]FE@SNAP additionally exhibited accumulation to the CHO-K1-hMCHR2 cells  and the experimental setup (membranes vs. living cells). In this context, it has to be highlighted that experiments on living cells, as performed in the present study, enhance the understanding of the complex interplay between the radiotracer and the dedicated biological target [Ki = 3.91 ± 0.74 nM) compared to FE@SNAP (Ki = 9.98 ± 1.12 nM) [11C]SNAP-7941 exhibits an improved target affinity and superior selectivity.Since MCHR2 is not expressed in rodents, additional kinetic real-time cell-binding studies were performed to substantiate the target selectivity. In this context, both radiotracers demonstrated a specific accumulation profile on the CHO-K1-hMCHR1 cells and negligible accumulation on the native CHO-K1 cells  . Based oin vivo pharmacology, it has been demonstrated in preceding [11C]SNAP-7941 is a P-gp/BCRP substrate, as confirmed in small animal imaging studies of rat brains  for [11C]SNAP-7941 compared to [18F]FE@SNAP (logD = 3.83 ± 0.1). In previous studies, plasma free fractions of 12.6 ± 0.2 % were reported for [18F]FE@SNAP and of 20.96 ± 1 % for [11C]SNAP-7941 using an ultrafiltration method with human pooled plasma FE@SNAP compared to [11C]SNAP-7941 results from the formation of radiometabolites  supported by the findings from the ex vivo metabolism studies in rat whole blood -SNAP-7941 could be detected for the lung -SNAP-7941 for both radiotracers -SNAP-7941, [11C]SNAP-7941 revealed a more pronounced difference. However, a significant displacement in the colon was only observed for [11C]SNAP-7941  contrasts the higher suitability of SNAP-7941 evinced a high metabolic stability in rat whole blood, whereas [18F]FE@SNAP was rapidly metabolized (Fig. in vitro as well as in vivo studies. A detailed analysis of the brain metabolism demonstrated an extensive degradation of [18F]FE@SNAP, while [11C]SNAP-7941 remained metabolically stable SNAP-7941 clearly demonstrated superior imaging properties due to its higher selectivity, affinity, and metabolic stability. Based on the combined data, we recommend [11C]SNAP-7941 as the tracer of choice for the imaging of MCHR1.Even though both tracers exhibit suitable properties for the imaging of the MCHR1, FE@SNAP and [11C]SNAP-7941 demonstrate appropriate imaging properties for the MCHR1. Yet, some physiological processes influenced by the MCH system, as for instance its contribution to BAT stimulation, remain unclear and demand further elucidation.The MCHR1 has become an interesting pharmacological target for clinical medicine, as well as for biomedical research, since it may be involved in a plethora of lifestyle diseases. In this context, the availability of a suitable PET radiotracer is a crucial step for the quantitative 11C]SNAP-7941 reveal the decisive superiority over [18F]FE@SNAP. Since humans express both the MCHR1 and MCHR2, tracer selectivity is essential for prospective first-in-man imaging studies. Therefore, [11C]SNAP-7941 is the ideal candidate for initial clinical trials, addressing the imaging of endocrinological and psychiatric disorders.However, the pronounced metabolic stability as well as superior affinity and selectivity of [


Most γδ-T-cells become activated via a major histocompatibility complex (MHC)-independent pathway by the interaction of their receptor, Natural Killer Group 2 Member D (NKG2D) with the tumor-specific NKG2D ligands, including MHC class I-related chain A/B (MICA/B) and UL16-binding proteins (ULBPs), to kill tumor cells. However, despite their potent antitumor effects, the treatment protocols specifically targeting ovarian tumors require further improvements. Ovarian cancer is one of the most lethal and challenging female malignancies worldwide because of delayed diagnoses and resistance to traditional chemotherapy. In this study, we successfully enriched and expanded γδ-T-cells up to ~78% from peripheral blood mononuclear cells (PBMCs) with mostly the Vγ9Vδ2-T-cell subtype in the circulation. We showed that expanded γδ-T-cells alone exerted significant cytotoxic activities towards specific epithelial-type OVCAR3 and HTB75 cells, whereas the combination of γδ-T cells and pamidronate (PAM), a kind of aminobisphosphonates (NBPs), showed significantly enhanced cytotoxic activities towards all types of ovarian cancer cells in vitro. Furthermore, in tumor xenografts of immunodeficient NSG mice, γδ-T-cells not only suppressed tumor growth but also completely eradicated preexisting tumors with an initial size of ~5 mm. Thus, we concluded that γδ-T-cells alone possess dramatic cytotoxic activities towards epithelial ovarian cancers both in vitro and in vivo. These results strongly support the potential of clinical immunotherapeutic application of γδ-T-cells to treat this serious female malignancy. Epithelial ovarian cancer (EOC), the most common and lethal type of ovarian cancer, causes 140,000 deaths annually worldwide [+ T-cells expresses the γ/δ T-cell receptor (TCR) which account for <10% of T-cells in the thymus, spleen, lymph nodes, and peripheral blood; but they are predominant T-cell types in certain epithelial tissues [In primates, a minor portion of CD3 tissues . Most ci tissues . Moreove tissues , and int tissues . However tissues .These γδ-T-cells retain a combination of innate and adaptive immune cell characteristics, rendering them appropriate for immunotherapy ,9. FurthSome clinical studies using Vγ9Vδ2 T-cells cytotoxicity towards various tumors are currently in progress ,16,17; hn > 20). The same expanded cells were then separately stained using Vγ9Vδ2 T-cell markers and showed complete correlation to the percentage increase of expanded pan-γδ-T cells over time, indicating that the expanded pan γδ-T-cells, see PBMCs from healthy donors were isolated and activated with PAM plus IL-2 to enrich γδ-T-cells during the culture period, see n = 12–20) using a high-grade serous ovarian carcinoma (HGSC) [n > 12; **** p < 0.0001 for HTB75 and OVCAR3) and 60% , respectively, compared to the activated αβ T-cell-treated, naive CD3 T-cell-treated and PAM-treated only controls, see n > 12; * p = 0.03 ~ **** p < 0.0001, In order to investigate whether these expanded Vγ9Vδ2 T-cells retained cytotoxic activities towards ovarian cancer cells, cytotoxicity experiments were performed multiple times (a (HGSC)  with epi+T cells) along with the replacement of old medium at 24-h with the E/T ratio of 1, and 20 caused a slight decrease in cell index (CI) of control cells, which was probably related to stress. The addition of E/T ratios at 1 and 20 of γδ-T cells alone resulted in an abrupt decrease in impedance and the CI values were significantly lower in two epithelial-type cells  compared to each of the control cells, see +T cells, see A real-time cell monitoring system, xCelligence System , was usep < 0.001, whereas SKOV3 showed significantly increased levels of both MICA/B and ULBPs following treatment with PAM compared to the non-treated cells, respectively, see p < 0.05 and ** p < 0.01, respectively.Mechanistically, as γδ-T-cells also express the NKG2D receptor, we examined the relative contribution of NKG2D during the γδ-T-cell expansion period on days 0, 6, and 10. Around 90% of expanded γδ-T-cells expressed NKG2D, see To further investigate how γδ-T-cells’ contact cytotoxicity acts towards EOC OVCAR3 cells, we set up co-culture cytotoxicity experiments with E/T ratios of 0, 1, 10, and 20 on chamber slides. After 24 h, cells were mounted for CD3 immunohistochemical (IHC) staining, see 7) s.c. implanted into mice grew to form discernible tumor masses (~5 mm in diameter) for two weeks before initiating immunotherapy. To test the antitumor efficacy, we divided the experimental mice into three groups (n = 8/group) which received either a combination of PAM+Vγ9Vδ2 T-cells, Vγ9Vδ2 T-cells alone, or no treatment. The PAM was intravenous (i.v.) injected (50 µg/kg) on day 14 in xenograft NSG mice. After 24 h, expanded human Vγ9Vδ2 T-cells (106) were i.v. injected into mice. In mice treated with schedule 2, OVCAR3 cells (107) were s.c. implanted in mice and allowed to form tumor masses (~8 mm in diameter) for four weeks before initiating immunotherapy. Subsequently, systemic administration of PAM+Vγ9Vδ2 T-cells or Vγ9Vδ2 T-cells alone was repeated weekly for four cycles. Strikingly, mice treated with schedule 1, the growth of OVCAR3 tumors was completely eliminated in mice receiving either systemic PAM+Vγ9Vδ2 T-cells or Vγ9Vδ2 T-cells alone, compared to the control mice group (Ctrl) treated with 1x PBS . Apart from this, the experimental mice survived without significant body weight loss or physical disability before the experimental endpoint (tumor mass > 800 mm3 in control group). Furthermore, the anti-CD3 IHC analysis revealed the presence of infiltrating human Vγ9Vδ2 T-cells in tumor sections of NSG mice that had received Vγ9Vδ2 T-cells, see Accordingly, the average tumor size, see p = 0.035 ~ **** p < 0.0001; Tumor-infiltrating γδ-T-cells are composed of Vδ1 and Vδ2 subtypes, which were reported to react to tumors but not to healthy cells. Among these, Vγ9Vδ2 T-cells, a unique subtype in human peripheral blood that showed strong cytotoxicity against cancer cells, including ovarian cancer, may serve as a potent candidate for cancer immunotherapy ,23. HereUpon contacting with tumor cells, γδ-T-cells extended their pseudopodia and burrowed into target cells as demonstrated by SEM. Also, reactive tumor cells formed spherical protrusions on tumor cell surfaces . Blebbing, a display of plasmalemmal protrusions, is most commonly associated with cell injury and apoptosis ,31. The An improvement of adoptive γδ-T-cell transfer approaches might be to combine them with NBP treatments, which can sensitize tumor cells to γδ-T-cell recognition. This combined approach was supported by a recent phase I trial in which renal carcinoma patients showed a complete response and their disease pathology was rapidly stabilized . AlthougIn summary, our study provides strong preclinical evidence for a powerful immunotherapy against EOCs using adoptive Vγ9Vδ2 T-cell transfer. We showed that the EOC, a susceptible tumor type in ovarian cancer, can be effectively treated by γδ-T cells. Importantly, our results indicated that such an approach not only prevented tumor expansion but also completely eradicated preexisting tumors when the tumor size was relatively small in early tumor growth. Furthermore, bisphosphates such as PAM treatment can potentially enhance the therapeutic efficacy by γδ-T-cells transfusion. However, it should be noted that its impact varies with individual tumors, which is tightly associated with the induction of NKG2D ligands. Therefore, for the γδ-T-cells therapeutic applications, it might be beneficial to examine the expression levels of NKG2D ligands in patients’ tumors prior to the choice of whether to combine with PAM treatment or not. In addition, we also evaluated, in detail, the efficacy of targeted cytotoxicity and morphology changes of the tumors and elucidated the mechanisms of tumor eradication. However, although both the size and weight of larger size tumors were significantly reduced by the administration of γδ-T-cells, the complete elimination of large tumors was not achieved. This was probably due to the hypoxic environment within large tumors that might enhance resistance to the infiltrating γδ-T-cell-mediated immune attack. Although the OVCAR3 xenograft model lacked activated αβ T-cells infusion control, which then requires further experimental confirmation, our in vitro cytotoxicity data were precisely indicative of the in vivo results and thereby supported the unique function of γδ T-cells mediated cytotoxicity. A further detailed mechanistic study is required to improve this therapeutic approach.The Taipei Medical University-Joint Institutional Review Board (TMU-JIRB) approved the study . Informed consent was obtained from each individual healthy volunteer. The research including biohazards, biological agents, toxins, materials or reagents follows the standard biosafety regulations and is reviewed and approved by the Institute’s “Environmental Protection and Biological Safety Committee”  before the project started.2 at a density of 1.5 × 106 cells/mL in RPMI 1640 . On the day of isolation, IL-2 , and pamidronate  were added. Additional medium and cytokines were added every 2–3 days over a total culture period of 14 days.Human PBMCs obtained from different healthy individual volunteers were isolated by gradient separation with Ficoll-Paque Plus , and were cultured in 37 °C with 5% CO® NxT Acoustic Focusing cytometer and analyzed using CellQuest Pro  software version 2.7.873.0.Expanded Vγ9Vδ2 T-cells were purified by positive selection using a human anti-TCR γ/δ T-cell MicroBeads kit , according to the manufacturer’s instructions. The following monoclonal antibodies (mAbs) were obtained from BD Bioscience : FITC-mouse anti-human CD3, PE-mouse anti-human TCRγδ, FITC-mouse anti-human TCRαβ, PE-mouse anti-human Vγ9-TCR, FITC-mouse anti-human Vδ2-TCR, PE-Cy7-mouse anti-human CD314 (NKG2D), PE-mouse anti-human ULBP3, PE-mouse anti-human ULBP2/5/6, APC-mouse anti-human MICA/B, and their IgG controls. After expansion, Vγ9Vδ2 T-cells and tumor cell lines were confirmed by flow cytometry using a FACSCalibur and/or an Attune2 incubation. All cell lines were tested for species identification, mycoplasma detection and authentication confirmed via short tandem repeat (STR) profile analysis at ATCC.The ovarian cancer cell lines, HTB75, OVCAR3, SKOV3, TOV112D, A2780 and a melanoma cell line A375 were purchased from American Type Culture Collection  and grown in Dulbecco’s modified Eagle medium  supplemented with 10% FBS at 37 °C with 5% CO4 cells/well) were plated in triplicate wells. After 24 h, one of the NBPs, pamidronate (PAM), at the indicated titration ratios of expanded Vγ9Vδ2 T-cells, activated αβ T-cells and naïve CD3 T-cells at approximate effector/tumor (E/T) ratios were added at different treatment time slots. Where indicated, γδ effector cells were pre-incubated for 30 min with 10μg/mL anti-NKG2D mAb  before the addition of tumor target cells. MTT values were evaluated using absorbance at 570 nm on an ELISA reader . The viability of the cells was calculated as the percentage of MTT reduction.MTT  cytotoxicity assays were performed in 96-well plates. Tumor cells . Cells were grown overnight and impedance was measured every hour, prior to treatments, as described . Cell imn = 4 mice/group): Ctrl (1x PBS), Vγ9Vδ2 T-cells , and PAM plus Vγ9Vδ2 T-cells (50 μg/kg PAM i.v. 1 day before administering Vγ9Vδ2 T-cells (106 cells/mouse) via an i.v. injection).All mice were kept according to National Institutes of Health guidelines for animal care and the guidelines of the Genomics Research Center, Academia Sinica  and maintained in a specific pathogen-free (SPF) facility. The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee . NOD.Cg-PrkdcscidIl2rgtmlWjl/SzJ (NSG) mice were used at 6–10 weeks of age. Mice were randomly assigned to three treatment groups , and further drying samples in an oven for 24 h. Fixed samples were sputter-coated with gold and imaged using SEM.After co-culture for 24 h at an E/T ratio of 20, attached cells were imaged using scanning electron microscopy  to compare the morphology of OVCAR3 cells on culture transwells (control group). SEM imaging was performed after fixing cells with 2% paraformaldehyde + 2.5% glutaraldehyde , dehydrating samples with graded ethanol . The statistical significance of the difference between means was determined using a two-tailed Student’s


As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP–SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using  R6K4 ‘heavy’ and R0K0 ‘light’ (DC Biosciences))-Dialysed calf serum (DC Biosciences)-SDS-polyacrylamide gel -NuPAGE LDS Sample Buffer (Invitrogen)-NuPAGE Sample Reducing Agent (Invitrogen)-NuPAGE MOPS SDS Running Buffer (Invitrogen)-NuPAGE Antioxidant (Invitrogen)-GelCode Blue Safe protein stain (Thermo Fisher Scientific)-Razor blade-50 mM Ammonium bicarbonate (ABC) (Sigma)-Acetonitrile (Thermo Fisher Scientific)-10 mM DTT (Sigma)-55 mM Iodoacetamide (IAA) (Sigma)-20 μg Trypsin glass vial (Thermo Fisher Scientific)-0.1% Trifluoroacetic acid (TFA) (Sigma)-10% TFA (Sigma)-Trypsin buffer , 200 μl 50 mM ABC  and 700 μl water. Add 3 μl of trypsin solution to 227 μl acetonitrile/ABC solution to make Trypsin buffer).-Stage tips C18 (Sigma Supelco)-Methanol (Fisher Scientific)-Mass spectrometer2.2-Primers for your RNA of interest, including a forward primer that contains the T7 promoter sequence TAATACGACTCACTATAGG followed by 20 nt of sense DNA template-High-fidelity PCR kit -PCR machine-T7 RNA polymerase -rNTPs 20 mM (Roche)-RNaseOUT (Invitrogen)-TURBO DNase (Ambion)-3 M Sodium acetate (NaOAc) (Fisher Scientific)-100% Ethanol (EtOH) (Fisher Scientific)-70% EtOH-Urea loading buffer (ULB) -10% polyacrylamide gel  containing 7 M Urea (Fisher Scientific)-1X Tris/Borate/EDTA (TBE) buffer , and bring the final volume to 1 L using deionized water)-Stains All (Sigma-Aldrich)-RNA extraction solution (0.3 M NaOAc pH 5.2 (Fisher Scientific), 0.5 mM EDTA (Fisher Scientific), 0.1% w/v SDS (Fisher Scientific))2.3-In vitro-transcribed and purified RNA (500 pmol)-Adipic acid dihydrazide-Agarose (Sigma)-3 M NaOAc pH 5 (Fisher Scientific)-0.1 M NaOAc pH 5-0.1 M Sodium (meta) periodate (Sigma)-100% EtOH-70% EtOH-4 M KCl (Fisher Scientific)-2 M KCl-Roeder D buffer  glycerol (Fisher Scientific), 0.2 mM EDTA (Fisher Scientific), 100 mM Tris pH 8.0 (Invitrogen), 0.5 mM DTT (Thermo Fisher Scientific), 0.2 mM PMSF (Sigma))-2 (VWR)100 mM MgCl-0.5 M Sodium creatine-phosphate dibasic tetrahydrate (CIP) (Sigma)-100 mM ATP (Sigma)-RNaseOUT (Invitrogen)-2 (Fisher Scientific), 1 mM DTT, 0.2 mM PMSF (Thermo Fisher Scientific))Buffer G , 1 mM EDTA, 1% TritonX-100 (Fisher Scientific), 10% glycerol, 1.5 mM MgCl-NuPAGE LDS Sample Buffer (Invitrogen)-NuPAGE Sample Reducing Agent (Invitrogen)-SDS-polyacrylamide gel -NuPAGE MOPS SDS Running Buffer (Invitrogen)-NuPAGE Antioxidant (Invitrogen)-GelCode Blue Safe protein stain (Thermo Fisher Scientific)33.1By combining RNA pull-down with SILAC in vitro transcription or chemical synthesis), performing the RNA pull-down assay, and undertaking mass spectrometry analysis.The method involves producing cell extracts derived from SILAC-labelled cells, obtaining RNA (by 3.23.2.113C-labelled arginine and 2D-labelled lysine (R6K4 (DC Biosciences)) as the heavy SILAC medium, and control DMEM containing unlabelled arginine and lysine (R0K0 (DC Biosciences)) as the light SILAC medium.Grow cells in SILAC media, ‘heavy’ and ‘light’, supplemented with dialysed calf serum, for six passages. We use DMEM medium containing 3.2.23.2.2.1-Electrophorese the protein extract (diluted in 1X NuPAGE LDS Sample Buffer and 1X NuPAGE Sample Reducing Agent) into an SDS-polyacrylamide gel using 1X NuPAGE MOPS SDS Running Buffer supplemented with 1:1000 NuPAGE Antioxidant. Stain the gel with GelCode Blue.-Using a razor blade, remove an approximately 0.5-cm piece of gel from the centre of the protein lane and cut into small pieces that are approximately 1 mm × 1 mm.-Transfer the gel pieces to a microfuge tube with 200 μl 50 mM ABC and incubate at room temperature (RT) for 5 min. Remove liquid, add 200 μl acetonitrile, and incubate at RT for 5 min. Repeat the ABC and acetonitrile washes until the solution is no longer blue.-Add enough 10 mM DTT to cover the pieces, using 20–50 μl or more, and leave at 37 °C for 30 min.-Remove the liquid, and add 200 μl acetonitrile for 5 min.-Turn off the light. Remove the acetonitrile, add 55 mM IAA to cover the gel pieces, and incubate at RT for 20 min in the dark. Remove the IAA, add 50 mM ABC for 5 min, and then acetonitrile for 5 min.-On ice, prepare Trypsin buffer.-Add Trypsin buffer to just cover the gel pieces, and incubate on ice for 15 min. As the gel absorbs the buffer, add more Trypsin buffer to slightly above the gel pieces, and incubate at 37 °C for 30 min. Add enough acetonitrile/ABC solution to slightly above gel pieces (to make sure the gel pieces do not dry out), and leave at 37 °C overnight.-Add 1:1 vol of 0.1% TFA to samples (e.g. 100 μl), and leave 20 min. Check that the pH is 1–3, or adjust by adding 10% TFA .-Activate a stage tip C18 by pushing or spinning 20 μl methanol through the tip. Next, condition the tip by pushing or spinning 40 μl 0.1% TFA through the tip.-Load the sample onto the tip by spinning. Wash by pushing or spinning 60 μl 0.1% TFA through the tip.-Maintain the stage tip at −20 °C until ready to load onto a mass spectrometer.3.2.2.2-Following digestion and peptide purification, perform an LC-MS/MS analysis using an orbitrap mass spectrometer or a similar machine. Determine the efficiency of heavy label incorporation into peptides by manually examining randomly selected peptides from the raw file. Since arginine and lysine may have different labelling efficiencies, this needs to be done separately for arginine- and lysine-containing peptides. Arginine-to-proline conversion should also be examined.3.33.3.1-Design primers that encompass your RNA of interest, appending a T7 promoter sequence (TAATACGACTCACTATAGG) to the 5′ end of the forward primer. Amplify the DNA using high-fidelity PCR, and confirm the size of the PCR product by electrophoresis in an agarose gel.3.3.2-Mix the following in a tube. We use NxGenT7 RNA Polymerase.-Incubate at 37 °C for 1.5 h.-Add 1–2 μl TURBO DNase, and incubate at 37 °C for 10 min.-Add 1/10 vol of 3 M NaOAc and 3 × volume of 100% EtOH, and precipitate on dry ice for 30 min or at −20 °C overnight.-Precipitate by spinning at 4 °C and full speed for 20 min. Wash the pellet with 70% EtOH, spinning 3 min at full speed, and re-suspend in 40 μl water and 40 μl ULB.-Boil at 90 °C for 2 min, and subsequently cool for 2 min on ice.-Split the sample into two, and electrophorese each half in separate wells of a pre-run, warm 10% polyacrylamide/urea gel using 1X TBE as a running buffer.-Stain with Stains All, and cut out the bands.-Add 300 μl RNA extraction solution, and leave on the bench overnight.-Next day, transfer the supernatant to a new tube, and add 900 μl 100% EtOH. Incubate at −20 °C over-night, or on dry ice for 1 h.-Spin at full speed for 20 min to precipitate the RNA, and wash the resulting RNA pellet with 70% EtOH.3.43.4.1-Prepare the following mix in a total volume of 200 μl:-Wrap all tubes in foil to protect from light. Leave rotating on a wheel at RT for 1 h.-Precipitate the RNA by adding 15 μl 3 M NaOAc and 600 μl 100% EtOH, and leave on dry ice for 10–30 min. Spin at 4 °C and full speed for 20 min. Wash with 1 ml 70% EtOH, and spin at 4 °C and full speed for 3 min. Re-suspend the RNA in 500 μl 100 mM NaOAc pH 5.-Transfer 250 μl Adipic acid dihydrazide-Agarose resin (50%) per reaction to a 15 ml tube using a cut pipette tip, and wash 3 times with 10 ml 100 mM NaOAc, spinning 300 rpm for 3 min after each wash. Re-suspend the final pellet in enough 100 mM NaOAc to have a mix of 50% beads/liquid. You will need 200 μl mix per reaction. Add 200 μl beads to the 500 μl periodate-treated RNA, and leave rocking at 4 °C overnight.-Remove the foil. Add 700 μl 4 M KCl to the mix, and rock at RT for 30 min. Pellet the resin at 3000 rpm for 3 min, discarding the supernatant. Wash the resin twice with 1 ml 2 M KCl, and 3 times with Roeder D buffer, spinning at 300 rpm for 2 min after each wash.3.4.2-Incubate at 37 °C for 30 min with rocking at 400 rpm. Pellet by spinning at 1000 rpm for 3 min. Save the supernatant as a loading control.Add to the pelleted resins:3.4.3-Work on ice. Wash reactions three times with 1 ml Buffer G, spinning at 4 °C and 1000 rpm for 2 min. Combine the “light” and “heavy” samples during the last wash.3.4.4-2O, 15 μl NuPAGE LDS Sample Buffer, and 6 μl NuPAGE Sample Reducing Agent.To the beads add: 39 μl H-Boil at 70 °C for 10 min while shaking. Spin at RT and full speed for one min, and load 30 μl.-Electrophorese the sample into an SDS-polyacrylamide gel using 1X NuPAGE MOPS SDS Running Buffer supplemented with 1:1000 NuPAGE Antioxidant, so that the dye migrates 1 cm into the gel. Stain the gel with GelCode Blue Safe protein stain.3.5-Cut out the band, proceed with in-gel digestion as described in Section 4We developed the RP–SMS method to identify proteins that bind to the let-7a-1 pre-miRNA (pre-let-7a-1). Pre-let-7a-1 is known to be regulated by LIN28A We incorporated R6K4 into HeLa cells constitutively expressing LIN28A, and confirmed the SILAC incorporation . ProteinNotably, a H/L ratio of 1 in the RNA pull-down compared to the beads-only control signifies that there is no enrichment of protein binding to RNA. H/L ratios of ≤0.5 indicate either preferential cellular protein binding to the beads-only control or protein contaminants that derive from the environment . H/L ratios of ≥2 indicate specific protein binding to the RNA.H/L ratios revealed that 81 cellular proteins were enriched at least two-fold (H/L ratio ≥2) in the pre-let-7a-1 pull-down compared to the beads-only control , among wTo validate the specificity of our RNA pull-down assay, we introduced a well-known LIN28A-binding site (GGAG) into the terminal loop of pre-miR-16, which does not bind LIN28A A [27], [in vitro processing assays and/or in vivo miRNA processing analysis using extracts from wild-type, candidate factor-depleted, or candidate factor-overexpressing cells.As the next step towards identifying miRNA processing factors, one could take all significant hits from the RP–SMS experiment and perform loss-of-function (RNAi and/or CRISPR/Cas9) or gain-of-function (over-expression) experiments. This should be followed by 5The regulation of RNA processing by RBPs is at the functional heart of all cells. RBPs mediate and control gene expression by regulating virtually all steps of RNA metabolism Many methods to identify protein binding to specific RNAs have recently been established


The molecule is not planar, the phenyl and pyridazine rings are twisted with respect to each other, making a dihedral angle of 29.96 (2)° and the dichlorophenyl ring is nearly perpendicular to the pyridazine ring, with a dihedral angle of 82.38 (11)°. In the crystal, pairs of N—H⋯O hydrogen bonds link the molecules to form inversion dimers with an R22(8) ring motif. The dimers are linked by C—H⋯O interactions, forming layers parallel to the bc plane. The intermolecular interactions were investigated using Hirshfeld surface analysis and two-dimensional fingerprint plots, and the molecular electrostatic potential surface was also analysed. The Hirshfeld surface analysis of the title compound suggests that the most significant contributions to the crystal packing are by H⋯H (31.4%), Cl⋯H/H⋯Cl (19.9%) and C⋯H/H⋯C (19%) contacts.The asymmetric unit of the title compound, C The fingerprint plot in Fig. 8b shows that the H⋯H contacts clearly make the most significant contribution to the Hirshfeld surface (31.4%). In addition, Cl⋯H/H⋯Cl, C⋯H/H⋯C, O⋯H/H⋯O and N⋯H/H⋯N contacts contribute 19.9%, 19%, 9.3% and 6.7%, respectively, to the Hirshfeld surface. In particular, the O⋯H/H⋯O contacts indicate the presence of intermolecular N—H⋯O and C—H⋯O interactions. Much weaker Cl⋯C/C⋯Cl (6.1%) and C⋯C (3.7%) contacts also occur.Fig. 8A view of the molecular electrostatic potential, in the range −0.0500 to 0.0500 a.u. using the 6-31G basis set with DFT method, for the title compound is shown in Fig. 9H)-one and  of 2,6-dichlorobenzaldehyde in 30 ml of ethanol, sodium hydroxide 10%  was added. The solvent evaporated under vacuum, the residue was purified through silica gel column chromatography using hexane/ethyl acetate (7:3 v/v). Single crystals were obtained by slow evaporation at room temperature.To a solution  of 6-phenyl-4,5-dihydropyridazin-3(2Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019005139/dx2016sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019005139/dx2016Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019005139/dx2016Isup3.cmlSupporting information file. DOI: 1896404CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


These supramolecular chains are further extended by weak C—H⋯Cl, C—H⋯O and C—O⋯π intermolecular interactions forming a 3D supramolecular network.2-Oxo-1,3-thiazolidin-4-iminium cations interact with a chloride anion  3H5N2OS+·CI−, the asymmetric unit consists of one independent 2-oxo-1,3-thiazolidin-4-iminium cation and one independent chloride anion. The cation interacts with a chloride anion via N—H⋯Cl hydrogen bonds forming a supramolecular chain along [010]. These supramolecular chains are further extended by weak C—H⋯Cl and C—H⋯O interactions, forming a two-dimensional network parallel to (001). The crystal structure is further stabilized by weak C—O⋯π interactions, supporting a three-dimensional architecture. The structure was previously determined by Ananthamurthy & Murthy  but has been redetermined with higher precision to allow the hydrogen-bonding patterns and supramolecular interactions to be investigated.In the redetermination of the title compound, C We report herein the molecular structure and supramolecular architecture of the title salt, C3H5N2SO+CI−, (I)Pbca and determine the cell parameters , these were not accurate enough to examine the hydrogen-bonding patterns and supramolecular interactions that are described here.Thiourea and its derivatives are an important group of organic compounds because of their diverse application in fields such as medicine, agriculture, coordination, and analytical chemistry via the N2—H4⋯Cl hydrogen bond  Fig. 4. The sup1) Fig. 4. All of 1) Fig. 4.dl-2-amino-2-thiazoline-4-carboxylic acid trihydrate -N,N-diphenylthiourea  and chloro acetic acid (37 mg) were mixed in a 1:1 molar ratio. The resulting solution was warmed over a water bath for half an hour and then kept at room temperature for crystallization. After a week, light-yellow prismatic crystals suitable for single-crystal X-ray analysis were obtained.Uiso(H) = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019003189/jj2207sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019003189/jj2207Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019003189/jj2207Isup3.cmlSupporting information file. DOI: 1901297CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


It adopts the  12H21NO7, (I), is conformationally unstable; the predominant form present in its solution is the β-pyranose form (74.3%), followed by the β- and α-furanoses , α-pyranose (3.4%), and traces of the acyclic carbohydrate tautomer. In the crystalline state, the carbohydrate part of (I) adopts the 2C5 β-pyranose conformation, and the amino acid portion exists as a zwitterion, with the side chain cyclopentane ring assuming the E9 envelope conformation. All heteroatoms are involved in hydrogen bonding that forms a system of antiparallel infinite chains of fused R33(6) and R33(8) rings. The molecule features extensive intramolecular hydrogen bonding, which is uniquely multicentered and involves the carboxylate, ammonium and carbohydrate hydroxy groups. In contrast, the contribution of intermolecular O⋯H/H⋯O contacts to the Hirshfeld surface is relatively low (38.4%), as compared to structures of other d-fructose-amino acids. The 1H NMR data suggest a slow rotation around the C1—C2 bond in (I), indicating that the intramolecular heteroatom contacts survive in aqueous solution of the molecule as well.The title compound, C Although the normal physiological functions of d-fructosamines are not understood, a number of bacterial, fungal, and mammalian carbohydrate-processing enzymes , a structural analog of the galectin inhibitor d-fructose-l-leucine , with an emphasis on the hydrogen-bonding patterns in the structure.As a part of our search for efficient blockers of galectins-1, −3 and −4, we have prepared I), crystallizes in the monoclinic space group P21, with two equivalent molecules per unit cell. The molecule may be considered as a conjugate of a carbohydrate, 1-amino-1-deoxy-d-fructose, and an amino acid, 1-aminocyclopentane-1-carboxylic acid, which are joined through the common amino group. The β-d-pyranose ring of the carbohydrate portion exists in the 2C5 or 1C(D) chair conformation, with puckering parameters Q = 0.5763 Å, θ = 172.71°, and φ = 248.80°. These parameters correspond to a conformation with the lowest energy possible for fructose , the β-d-pyranose anomer dominates the tautomeric equilibrium  or Cs—Cγ-exo conformation, with puckering parameters Q = 0.4220 Å, φ = 254.94°, and pseudorotational parameters  Å, short enough for this heteroatom contact to qualify as a strong hydrogen bond. Then the central motif of the intramolecular hydrogen-bonded structure can be described in terms of a compact ring S22(4) pattern represented by the four-atom O7⋯H1A⋯O1—H1O⋯O7 cycle. In the 1H NMR spectrum of (I), two protons, H1C and H1D, which are attached to C1, produce two distinct signals at 3.332 and 3.199 ppm, with J1C,1D = −12.8 Hz  oxygen atoms are involved in intramolecular hydrogen bonding Table 2. At the 1O Fig. 1. AlthougHz Fig. 3. The nonI) features infinite chains of antiparallel hydrogen bonds running along the a-axis direction  and R33(8), and a small finite chain D22(4). Alternatively, the fused rings pattern can be described in terms of two chains, C22(4) and C33(8). The ammonium proton H1A is involved in a rare five-centered hydrogen bond, involving three weakly directional intramolecular contacts with O1, O5, and O7  and one intermolecular, shorter (2.00 Å distance) bond with O3. The carboxyl atom O7 is also involved in an unusual multicenter hydrogen bond, by coordinating four surrounding protons at reasonably short distances, 1.97–2.40 Å  is provided by non- or low-polar H⋯H interactions -1′-aminocyclopentane-1′-carboxylic acid or d-fructose-cycloleucine; thus the compound appears to be novel. Since the conformational instability of the d-fructosamine moiety determines the chemical reactivities and biological activities of d-fructosamine derivatives  with solved structures of other d-fructose-amino acids. The most closely related structures are d-fructose-2-aminoisobutyric acid , has also been observed in CCDC 1583254 , features the cyclic motif of intramolecular multicentered hydrogen bonding , more than in any other d-fructose-amino acid structure known to date. Such effect of the ‘internalization’ of hydrogen bonding in (I) is also revealed in a comparative analysis of the fingerprint plots . The relative abundance of these contacts in structures of d-fructose-amino acids decreases with an increase in the number of intramolecular hydrogen bonds; this trend is clearly revealed by the data presented in Table 4I) is comparable to the respective bond-length differences noted in other fructose-amino acid structures, including CCDC 1583254 .Searches of SciFinder 2018 and the s Table 4. Howeverng Fig. 1, which its Fig. 7 that arets Fig. 7 and delid-glucose , and sodium acetate  were dissolved in 100 mL of a methanol/glycerol (3:1) mixture and refluxed for 3 h. The reaction progress was monitored by TLC on silica. The reaction mixture was diluted with 900 mL of water and passed through a column charged with 80 mL of Amberlite IRN-77 (H+-form). The target compound was then eluted with 0.2 M pyridine, and fractions containing pure (I) were pooled and evaporated. The residue was redissolved in 100 mL of water, decolorized with 0.5 g of charcoal and evaporated to a syrup. The latter was dissolved in 30 mL of ethanol and made nearly cloudy with the dropwise addition of acetone. Crystallization occurred within a week at room temperature. Yield 3.4 g .Cycloleucine , 13C NMR spectrum in D2O: 179.82 (C12); 98.34 (C2); 76.30 (C7); 72.33 (C4); 72.20 (C3); 71.80 (C5); 66.66 (C6); 52.89 (C1); 37.44, 37.40 ; 28.00, 27.98 . See Table 11H NMR spectrum: 4.035 ; 4.017 ; 3.896 ; 3.774 ; 3.771 ; 3.332 ; 3.199 ; 2.220 ; 1.955 ; 1.83 ; J1C,1D = −12.8; J3,4 = 9.8; J4,5 = 3.4; J5,6A = 1.3; J6A,6B = −12.9.Major β-pyranose tautomer peaks (ppm) in Uiso(H) = 1.2Ueq (methine or methylene). As a result of the unrealistic value obtained for the Flack absolute structure parameter [−0.4 (4) for 1097 quotients; Parsons et al., 2013R,3S,4R,5R) was assigned on the basis of the known configuration for the starting compound d-glucose  I. DOI: 10.1107/S2056989019009253/eb2020Isup2.hklStructure factors: contains datablock(s) I. DOI: 1583255CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The bile acid-sensing transcription factor farnesoid X receptor (FXR) regulates multiple metabolic processes. Modulation of FXR is desired to overcome several metabolic pathologies but pharmacological administration of full FXR agonists has been plagued by mechanism-based side effects. We have developed a modulator that partially activates FXR in vitro and in mice. Here we report the elucidation of the molecular mechanism that drives partial FXR activation by crystallography- and NMR-based structural biology. Natural and synthetic FXR agonists stabilize formation of an extended helix α11 and the α11-α12 loop upon binding. This strengthens a network of hydrogen bonds, repositions helix α12 and enables co-activator recruitment. Partial agonism in contrast is conferred by a kink in helix α11 that destabilizes the α11-α12 loop, a critical determinant for helix α12 orientation. Thereby, the synthetic partial agonist induces conformational states, capable of recruiting both co-repressors and co-activators leading to an equilibrium of co-activator and co-repressor binding. The ligand-activated transcription factor farnesoid X receptor (FXR) acts as a cellular sensor for bile acids and is of interest as a drug target. Here the authors employ X-ray crystallography and NMR to characterize the molecular determinants of FXR agonists, antagonists and a partial agonist that drive FXR activation and antagonism. As a member of the nuclear receptor (NR) superfamily it translates ligand signals into changes in gene expression and affects a variety of genes involved in bile acid, glucose and lipid metabolism. For this central biological role, FXR has gained considerable attention as drug target for severe liver disorders4 and metabolic diseases5. The first in class FXR agonist obeticholic acid (OCA)6 has revealed very promising effects of pharmacological FXR activation in non-alcoholic steatohepatitis (NASH), in primary biliary cholangitis (PBC) and in diabetes. OCA has recently been approved by the FDA for PBC treatment and is currently investigated in late stage clinical development for NASH8 despite its adverse effects that appear partly arising from FXR over-activation. Elevated cholesterol levels have been reported from clinical trials9 with OCA that can be correlated with the finding that complete FXR activation blocks bile acid biosynthesis and hinders metabolic cholesterol degradation as consequence. Importantly, conversion to bile acids accounts for approximately one third of cholesterol elimination. Partial FXR activation, therefore, appears as a valuable strategy to avoid mechanism-based side effects of FXR targeting and recently, promising preclinical and phase 1 clinical data of a partial FXR agonist have been reported10. Partial agonists induce activation of the NR with reduced activation efficacy compared to agonists which translates to modest modulation of FXR-regulated gene expression11. However, the molecular mode of action by which partial agonism is established and its structural determinants in terms of ligand binding and conformational changes in the FXR ligand binding domain (LBD) have remained elusive.The ligand-activated transcription factor farnesoid X receptor 12.NRs share a common architecture of an N-terminal ligand-independent activation domain followed by a DNA-binding domain (DBD), a flexible hinge region and a ligand-binding domain composed of twelve α-helices (α1-α12). Especially the C-terminal α12, the so-called activation function 2 (AF-2) of the LBD, has been identified as crucial for NR activation since stabilisation of α12 and concomitant binding to the core of the LBD provides a surface for co-activator binding13. Later studies (reviewed in14) have used various techniques to revise this model and have suggested that the terminal LBD region is dynamic in apo-state and that α12 is only formed upon agonist binding. This contradicted the model of two (active and inactive) positions of a permanently stable α12 and already indicates that more than a single active conformation of NR-LBDs may exist.Previous studies have already evaluated general molecular mechanisms of NR activation. Observations in early co-crystal structures of NR-LBDs revealing differences in the position of α12 for liganded and unliganded (apo) states, have established the common model of a mouse-trap mechanism for NR activation where α12 was thought to be positioned away from the core LBD in apo-state. Agonist binding would then alter its position to be bound to the core LBDAccording to current understanding, agonistic NR ligands promote stabilisation of α12 and binding to the core LBD to induce co-activator recruitment while antagonists stabilise an inactive state with unordered AF-2 of the transcription factor that binds to co-repressors. However, this commonly accepted mechanism of α12 stabilisation and subsequent co-activator recruitment upon agonist binding explains FXR agonism and antagonism fairly well while it does not offer a sound concept concerning partial FXR agonism, so far.15, and differential effects of FXR agonists GW4064, CDCA and fexaramine on FXR regulated gene expression16 already indicated several years ago, that FXR activation is more complex than just switched on or off. Later studies revealed that FXR ligands can differ in their recruitment profiles of various co-activators18, a characteristic that has also been observed for other NRs such as peroxisome proliferator-activated receptors20. Moreover, ivermectin18 and structural analogues21 were identified as specific FXR modulators that despite recruiting the NR co-repressor NCoR-2 to the FXR-LBD induced FXR-mediated reporter activity and gene expression, and recent results also point to an allosteric mechanism22 of FXR activation by small molecules. However, the molecular basis of the differential modes of FXR modulation has not been deciphered.Differential binding modes within the FXR ligand binding site of FXR agonists OCA and MFA-11 . In vitro characterisation of the previously reported FXR modulator ivermectin revealed a partial agonistic/antagonistic profile, as well. Evaluation of 1 in a hybrid Gal4-FXR reporter gene assay confirmed its partial FXR agonism/antagonism , a single oral dose of 1 (10 mg/kg) resulted in a favourable pharmacokinetic profile with high bioavailability and a half-life of 2.1 h confirming suitability of 1 for in vivo studies  of FXR regulated genes relative to the endogenous FXR-agonist CDCA (2) was observed  or intestinal (HT-29) cells were incubated with ved Fig. . Accordived Fig. . Togethe2 (6HL1) and with partial agonist 1 (4QE8). The FXR-LBD complex containing 1 crystallised as a dimer. Both ligand-bound FXR-LBDs are composed of 12 α-helices with bound α-helical NCoA-2740-2752 peptide and adopt the typical three-layer α-helical sandwich arrangement that resembles most NR structures28. The 1.65 Å high-resolution FXR/CDCA (2) structure comprises residues E244-Q472. Notably, the loop region linking α4/5 and α6 (K339-P341) is invisible suggesting flexibility and dynamics of this loop induced by ligand binding. The 2.6 Å FXR/1 structure comprises the identical range as FXR/CDCA and also lacks the loop region between α4/5 and α6 (P341-S342). Additionally, the electron density of the loop region connecting α11 and α12  is ambiguous or invisible, due to induced flexibility by binding of 1. The temperature factors .As first step towards understanding the molecular determinants of FXR partial agonism, we solved the co-crystal structures Fig.  of the F2 6HL and with2  or GW4064 (PDB-ID: 3DCT30). Positions and orientations of all α-helices are very well conserved independently of the bound ligand and expectedly, all ligands occupy a similar binding site. Minor shifts of the N-terminal part of α7 and the C-terminal part of α11 result from different ligand sizes and binding modes. Additionally, there are apparent differences in loop (L) regions connecting α1-α2 (L:α1-α2), α5-α6 (L:α5-α6), α6-α7 (L:α6-α7) and α11-α12 (L:α11-α12) which are differently orientated indicating that residues of these regions are involved in or affected by compound binding. Binding of CDCA (2) disturbs L:α5-α6 folding and binding of 1 destabilises L:α5-α6 as well as L:α11-α12.The structures solved here containing 2), OCA and GW4064 occupy the same binding pocket but show differences in their residue specific interaction modes. CDCA (2) is bound via conserved sidechain interactions involving R331, S332, Y361, Y369, H447 and non-bonded contacts  are similarly stabilised, while synthetic ligands such as GW4064 and 1 are differently bound. GW4064 binding is mainly mediated by 15 non-bonded interactions, two polar contacts of the carboxyl group with R331 and one with the backbone of M265. Partial agonist 1 is exclusively bound by 15 direct non-bonded interactions and one water mediated H-bond to R331  as well as partial agonists 1 and ivermectin on FXR mutants W454Y and W454A. The 1H/15N-HSQC spectra of the fully labeled W454Y and W454A mutant FXR-LBDs indicated that the protein fold is not affected by the mutations and that the mutations are structurally silent  was inactive on both W454 mutants at 50 µM. This may result from a less hydrophobic environment when the highly lipophilic tryptophan residue is replaced by alanine or tyrosine. For partial agonists 1 and ivermectin, mutation of W454 to Y caused increased activation efficacy and for 1 also markedly enhanced potency. Mutation to A had a similar effect for partial agonist 1 but rendered ivermectin inactive. The lower EC50 values of 1 on the mutants may result from reduced energetic penalty compared to the large movement of W454 in wild-type FXR and its exposure to the solvent.To probe the importance of residue W454 of FXR in mediating partial activation, we studied the activity of FXR agonists GW4064 and CDCA  containing ivermectin completely lacks α12 and L:α11-α12. Additionally, α11 is shortened and the structure comprises no co-activator but a co-repressor peptide , α12 is affected which in turn modifies the binding mode of the co-activator. In the agonistic conformation, the co-activator is bound by two conserved ionic interactions forming a charge clamp with K303 (α3) and E467 (α12) stabilising the dipolar NCoA-2 α-helix . Hence, the surface for co-activator binding is crucially dependent on the position of α12. The agonistic structure of CDCA comprises the α12 helix and the entire loop connecting α11 and α12 Fig.  whereas ide Fig. .Fig. 3FX2), GW4064 and fexaramine15 bind close to helix 4 whereas the binding position of partial agonist 1 protrudes towards L:α11-α12 compared to agonistic ligands and ivermectin is shifted towards the Ω-loop causing partial destabilisation of the FXR-LBD thereby suggesting that it may adopt a “combined” binding mode similar to agonist and partial agonist. Of note, the FXR-LBD structure containing ivermectin is complexed with a fragment of nuclear receptor co-repressor 1 (NCoR-1) and, therefore, has to be considered as antagonistic conformation of the FXR-LBD although ivermectin biologically behaves as partial agonist on FXR. This observation, however, suggests that both the ligand and the co-regulator peptide affect FXR-LBD conformation.Superposition of FXR-LBD structures Fig.  reveals 1 results in disruption of five of these H-bond interactions by shifting of α12 towards the co-activator peptide and thereby inducing an upward movement of NCoA-2  and several non-bonded contacts. However, binding of α atoms of the two residues forming the charge clamp  and co-repressor  peptides each containing one 13C/15N-labeled leucine . The 1H-15N-HSQC of the quaternary mixture of FXR-LBD, guggulsterone, co-activator and co-repressor peptide, revealed no co-repressor signals while the Cβ signals of the co-activator were fully present without detectable shifts. This observation confirmed the assumption that the FXR-LBD in presence of an antagonist is fully complexed with co-repressor and that the co-activator is not bound.Next, we applied the FXR antagonist guggulsteroneAccording to our NMR experiments, unliganded FXR-LBD in solution recruits the co-repressor peptide, which is partly released upon agonist binding. The equilibrium of bound and free co-repressor in presence of a full agonistic ligand but in absence of co-activator slightly differs for distinct agonists but in all cases shifts to strong release of the co-repressor upon addition of co-activator peptide. FXR antagonist guggulsterone, in contrast, stabilised the interaction of the FXR-LBD with the co-repressor which is not released even in presence of co-activator. Hence, the NMR experiments allow a clear differentiation of agonistic and antagonistic ligands.1 in this setting  and line broadening which confirms the binding of both peptides. Thus, binding of partial agonists to the FXR-LBD induces a conformation that is capable of binding co-activator and co-repressor. This FXR-LBD conformation having affinity to co-activator as well as co-repressor explains the pharmacological behaviour of partial FXR agonists and, altogether, this indicates that FXR activity is not either switched on or off but can be tuned to several stages in between.Knowing these effects on co-repressor and co-activator binding caused by FXR agonists and antagonists we then analysed the behaviour of partial agonists ivermectin and ing Fig. . Their aIn addition to explaining the structural and mechanistic basis of partial FXR activation, our results suggest that not only ligand binding affects the conformation of the FXR-LBD but that co-activator binding has even stronger influence. According to current understanding, agonist binding to FXR causes conformational changes in the FXR-LBD that lead to stabilisation of α12 at the core domain which is a mandatory prerequisite to co-activator binding. However, this would indicate that co-activator binding has less stabilising effects on the FXR-LBD than ligand binding. To resolve this anthology, we studied the conformation and position of α12 in the unliganded FXR-LBD to figure out whether it follows the canonical model for NRs of a mouse trap.1H/15N-HSQC spectra of 13C/15N-labeled FXR-LBD and a 13C/15N-labeled FXR-LBD mutant lacking α12  indicating strong preference for co-activator binding. FXR antagonist guggulsterone, in contrast, fully stabilised the complex of the FXR-LBD with the co-repressor and no co-activator was bound to the FXR-LBD (ratio 15:85). In accordance with the other experiments in solution, partial agonists 1 and ivermectin adopted a state between agonists and antagonist displaying free co-repressor/free co-activator ratios of 30:70 and 25:75, respectively.Going more into detail we subsequently focused on labeled co-activator and repressor in NMR and studied their behaviour upon agonist, partial agonist or antagonist binding to the FXR-LBD. Unliganded FXR-LBD robustly recruited the co-repressor which could be observed by disappearance of co-repressor signals upon addition of the LBD. While agonists induced significant displacement of the co-repressor, full release only happened in presence of the co-activator peptide leading to entire reappearance of the co-repressor Cβ signals while no Cβ signal of the co-activator was present. Congruently, antagonist guggulsterone caused the opposite by stabilising the interaction of LBD and co-repressor which was hardly released even in presence of the co-activator. In this case, only the Cβ signal of the co-activator was present in the NMR spectra. Studying the partial agonists 1 causes a shifted position of α12, which in turn affects position and strength of binding of the co-activator.The results from co-crystal structure investigations and NMR experiments in solution together draw a sound mechanism for FXR activation that also explains partial agonism: The unliganded FXR-LBD is bound to co-repressor in solution. An agonistic ligand destabilises the interaction of FXR-LBD and co-repressor which leads to partial dissociation of the co-repressor from the NR. In presence of a co-activator, the co-repressor is then completely released, and the co-activator is bound to the FXR-LBD. An antagonist, in contrast, stabilises the interaction between FXR-LBD and co-repressor. Therefore, the co-repressor is not even released in presence of a co-activator, which can consequently not bind to the NR. A partial agonist behaves like an agonist by weakening the FXR-LBD-co-repressor interaction but in contrast to a full agonist it induces a conformation of the FXR-LBD that has affinity to the co-activator and the co-repressor. This is congruent with our observation that outward movement of W454 and destabilisation of the AF-2 loop in the co-crystal structure of the FXR-LBD in complex with partial agonist 10 helix in the loop region between α3 and α4 (L:α3-α4) thereby stabilising the FXR-LBD and promoting co-activator binding  and CDCA (6HL1), which suggest involvement of Trp454 in binding of CDCA but not GW4064 , the mixture was cooled to room temperature and filtered. The solvent was then partly evaporated under reduced pressure until crystallisation of the product started. The crude product was filtered off and recrystallised twice from hexane/ethyl acetate/acetic acid to obtain the title compound as colourless solid . 1H NMR  δ = 10.62 , 7.95−7.91 , 7.91–7.86 , 7.80 , 7.42 , 7.14 , 7.03  ppm. 13C NMR  δ = 167.42, 167.30, 143.70, 132.94, 132.46, 130.63, 129.73, 129.64, 126.24, 125.96, 120.35, 120.10 ppm. C14H12N2O3. MS (ESI-): m/z 255.0 .4-(2-Aminobenzamido)benzoic acid: 4-Aminobenzoic acid  was dissolved in ethanol  and heated to reflux. Then, isatoic anhydride  was added to the solution with the immediate formation of COtert-Butylbenzamido)benzamido)benzoic acid (1): 4-(2-Aminobenzamido)benzoic acid  was dissolved in THF  and pyridine  was added. After a clear solution had formed, 4-tert-butylbenzoyl chloride  was added drop-wise. The reaction mixture was kept stirring at room temperature and the reaction progress was monitored by TLC. After 4 h, the reaction mixture was diluted with ethyl acetate, washed three times with 10% hydrochloric acid and dried over Na2SO4. Further purification was performed by column chromatography on silica using hexane/ethyl acetate with 2% acetic acid as mobile phase to obtain the title compound as colourless solid . 1H NMR  δ = 12.80 , 11.43 , 10.79 , 8.42 , 7.97–7.94 , 7.92 , 7.87 , 7.85 , 7.63 , 7.59 , 7.31 , 1.31  ppm. 13C NMR  δ = 168.17, 167.39, 165.11, 155.46, 143.25, 139.03, 132.85, 132.20, 130.67, 129.62, 127.43, 126.43, 126.18, 123.77, 123.68, 122.02, 120.57, 35.21, 31.34 ppm. C25H24N2O4. MS (ESI-): m/z 415.9 ; HRMS (ESI+): calculated for C25H24N2O4Na: m/z 439.16283, found: m/z 439.16256 ([M + Na]+). Combustion analysis: measured : C 71.72 (72.10), H 5.78 (5.81), N 6.77 (6.73). 1H- and 13C-NMR of 1 are shown in Supplementary Fig. 4- was used as a reporter plasmid and contains a shortened construct of the promotor of the bile salt export protein (BSEP) cloned into the SacI/NheI cleavage site in front of the luciferase gene35. pRL-SV40 (Promega) served as a control for normalisation of transfection efficiency and cell growth. Assay procedure: HeLa cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Leibniz-Institut, Braunschweig, Germany) were grown in DMEM high glucose supplemented with 10% fetal calf serum (FCS), sodium pyruvate (1 mM), penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 °C and 5% CO2. 24 h before transfection, HeLa cells were seeded in 96-well plates with a density of 8000 cells per well. 3.5 h before transfection, medium was changed to DMEM high glucose, supplemented with sodium pyruvate (1 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and 0.5% charcoal-stripped FCS. Transient transfection of HeLa cells with BSEP-pGL3, pRL-SV40 and the expression plasmids pcDNA3-hFXR and pSG5-hRXR was carried out using calcium phosphate transfection method. Sixteen hours after transfection, medium was changed to DMEM high glucose, supplemented with sodium pyruvate (1 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and 0.5% charcoal-stripped FCS. 24 h after transfection, medium was changed to DMEM without phenol red, supplemented with sodium pyruvate (1 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mM) and 0.5% charcoal-stripped FCS, now additionally containing 0.1% DMSO and the respective test compound or 0.1% DMSO alone as untreated control. Each sample/concentration was tested in triplicate wells and each experiment was repeated independently at least three times. Following 24 h incubation with the test compounds, cells were assayed for luciferase activity using Dual-Glo™ Luciferase Assay System (Promega) according to the manufacturer’s protocol. Luminescence was measured with a Tecan Infinite M200 luminometer . Normalisation of transfection efficiency and cell growth was done by division of firefly luciferase data by renilla luciferase data multiplied by 1000 resulting in relative light units (RLU). Fold activation was obtained by dividing the mean RLU of the tested compound at a respective concentration by the mean RLU of untreated control. Relative activation was obtained by dividing the fold activation of the tested compound at a respective concentration by the fold activation of FXR full agonist GW4064 at 3 µM. EC50 and standard deviation values were calculated with the mean relative activation values of at least three independent experiments set up in triplicates by SigmaPlot 12.5  using a four parameter logistic regression. The assay was validated with FXR agonists OCA  and GW4064 36. FXR mutants were prepared by designing an appropriate primer with the single amino acid mutation and following the standard QuickChange® site-directed mutagenesis protocol. Assay procedure was performed as described for the wild type.FXR transactivation assay: Plasmids: pcDNA3-hFXR2. HT29 cells were grown in McCoy’s 5 A medium, supplemented with 10% FCS, penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 °C and 5% CO2. Cells were seeded in six-well plates (2 × 106 per well) in the same medium. Twenty four after seeding, medium was changed to MEM supplemented with 1% charcoal-stripped FCS (CS-FCS), penicillin (100 U/mL), streptomycin (100 µg/mL) and L-glutamine (2 mM). After additional 24 h, medium was again changed now additionally containing the test compounds in DMSO or DMSO alone . Cells were incubated with the test compounds for 6, 18 or 24 h, harvested, washed with cold PBS and then directly used for RNA extraction. RNA extraction and cDNA synthesis: Two micrograms of total RNA extracted from above described cells with the E. Z. A. Total RNA Mini Kit  were reversely transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s protocol. q-RT PCR: FXR target gene expression was evaluated by quantitative PCR analysis with a StepOnePlusTM System (Life Technologies) using PowerSYBRGreen  and the primers (300 nM each) listed in Supplementary Table n ≥ 3): BSEP: CDCA (50 µM): 624 ± 33%; 1 (5 µM): 205 ± 2%. SHP: CDCA (50 µM): 537 ± 98%; 1 (5 µM): 262 ± 83%. CYP7A1: CDCA (50 µM): 33 ± 7%; 1 (5 µM): 61 ± 3%. OSTα: CDCA (50 µM): 813 ± 159%; 1 (5 µM): 297 ± 34%. IBABP: CDCA (50 µM): 306 ± 17%; 1 (5 µM): 137 ± 10%. FGF19: CDCA (50 µM): 221 ± 18%; 1 (5 µM): 164 ± 8%.FXR target gene quantification (qRT-PCR): Cell culture: HepG2 cells (DSMZ) were grown in DMEM high glucose, supplemented with 10% fetal calf serum (FCS), sodium pyruvate , penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 °C and 5% CO37, pFA-CMV-hPPARγ-LBD37, pFA-CMV-hPPARδ-LBD37, pFA-CMV-hLXRα-LBD38, pFA-CMV-hLXRβ-LBD38, pFA-CMV-hRXRα-LBD39, pFA-CMV-hRXRβ-LBD39, pFA-CMV-hRXRγ-LBD39, pFA-CMV-hRARα-LBD39, pFA-CMV-hRARβ-LBD39, pFA-CMV-hRARγ-LBD39, pFA-CMV-hVDR-LBD39, pFA-CMV-hCAR-LBD39 and pFA-CMV-hPXR-LBD39 coding for the hinge region and ligand binding domain (LBD) of the canonical isoform of the respective nuclear receptor have been reported previously. pFR-Luc  was used as reporter plasmid and pRL-SV40 (Promega) for normalisation of transfection efficiency and cell growth. Assay procedure: HEK293T cells (DSMZ) were grown in DMEM high glucose, supplemented with 10% FCS, sodium pyruvate (1 mM), penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C and 5% CO2. The day before transfection, HEK293T cells were seeded in 96-well plates (2.5 × 104 cells/well). Before transfection, medium was changed to Opti-MEM without supplements. Transient transfection was carried out using Lipofectamine LTX reagent  according to the manufacturer’s protocol with pFR-Luc, pRL-SV40 (Promega) and the respective pFA-CMV-hNR-LBD. 5 h after transfection, medium was changed to Opti-MEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), now additionally containing 0.1% DMSO and the respective test compound or 0.1% DMSO alone as untreated control. Each concentration was tested in duplicates and each experiment was repeated independently three times. Following overnight (12–14 h) incubation with the test compounds, cells were assayed for luciferase activity using Dual-Glo™ Luciferase Assay System (Promega) according to the manufacturer’s protocol. Luminescence was measured with an Infinite M200 luminometer (Tecan) or a Tecan Spark 10 M luminometer (Tecan). Normalisation of transfection efficiency and cell growth was done by division of firefly luciferase data by renilla luciferase data and multiplying the value by 1000 resulting in relative light units (RLU). Fold activation was obtained by dividing the mean RLU of a test compound at a respective concentration by the mean RLU of untreated control (0.1% DMSO). Reference agonists  were included in every individual assay as positive control to monitor performance and all hybrid assays were validated with the above-mentioned reference agonists which yielded EC50 values in agreement with literature.Hybrid reporter gene assays: Plasmids: The Gal4-fusion receptor plasmids pFA-CMV-hPPARα-LBD1 on TGR5 was evaluated by measuring the level of cAMP using a HTR-FRET assay. In brief, NCI-H716 cells were cultured in DMEM supplemented with 10%FBS, using 96-well plates coated with Matrigel . After 24 h, cells were stimulated with test compound 1 or lithocholic acid as positive control for 60 min at 37 °C in OptiMEM with 1 mM IBMX . The level of intracellular cAMP was assessed using the Lance kit  according to the manufacturer’s protocol. Each experiment was repeated independently three times in duplicates.TGR5 assay: The activity of 4 cells/well). After 24 h medium was changed to DMEM containing penicillin (100 U/mL), streptomycin (100 μg/mL) and 1% FCS, and cells were incubated with the respective compounds for 48 h. As positive control, TRITON X-100 (2%) was added 1 h before measurement. After incubation, supernatant of each well was transferred into a fresh plate and LDH substrate/reagent was added. After 20 min incubation absorption at measurement (490 nm) and reference (690 nm) wavelength was determined with a TECAN infinite 200 (Tecan). All experiments were performed in triplicates in four independent repeats. Results  = 1): 10 µM: 0.93 ± 0.04; 30 µM: 1.01 ± 0.08; 60 µM: 0.81 ± 0.06; 100 µM: 1.37 ± 0.02; TRITON X-100: 4.22 ± 0.12.Cytotoxicity assay: LDH assay  was performed according to manufacturer’s instructions. In brief, HepG2 cells (DSMZ) were seeded in DMEM containing 1% SP, 1% PS and 10% FCS in 96-well plates  was preincubated at 37 °C in 432 μL of phosphate buffer  together with a 50 μL NADPH regenerating system . After 5 min, the reaction was started by the addition of 13 μL of microsome mix from the liver of Sprague−Dawley rats  in a shaking water bath at 37 °C. The reaction was stopped by addition of 250 μL of ice-cold methanol at 0, 15, 30, and 60 min. The samples were diluted with 250 μL of DMSO and centrifuged at 10000 g for 5 min at 4 °C. The supernatants were analysed, and the test compound was quantified by HPLC: mobile phase, MeOH 83%/H2O 17%/formic acid 0.1%; flow-rate, 1 mL/min; stationary phase, MultoHigh Phenyl phase, 5 μm, 250 × 4 ; precolumn, phenyl, 5 μm, 20 × 4 (CS-Chromatography); detection wavelength, 330 and 254 nm; injection volume, 50 μL. Control samples were performed to check the stability of the compound in the reaction mixture: first control was without NADPH, which is needed for the enzymatic activity of the microsomes, second control was with inactivated microsomes (incubated for 20 min at 90 °C), third control was without test compound (to determine the baseline). The amounts of the test compound were quantified by external calibration. The metabolism experiments showed the following results : 0 min: 96 ± 1%; 15 min: 84 ± 3%; 30 min: 77 ± 2%; 60 min: 63 ± 2%40.Metabolism Assay: The solubilised test compound 1 in water containing 1% HPMC/Tween 80 (99:1), three animals received 10 mg/kg body weight of FXR agonist CDCA in the vehicle and three animals received the vehicle (water containing 1% HPMC/Tween 80 (99:1)). All animals behaved normal throughout the study and showed no adverse effects.Animals and compound application: 15 male C57BL/6JRj mice  were used in the present study. The animals were housed in a temperature-controlled room (20–24 °C) and maintained in a 12 h light/12 h dark cycle. Food and water were available ad libitum. The in-life phase was performed by the contract research organization Pharmacelsus . All experimental procedures were approved by and conducted in accordance with the regulations of the local Animal Welfare authorities . Nine animals received a single oral dose of 10 mg/kg body weight of FXR partial agonist 1, blood from three restrained and conscious mice was obtained from the lateral tail vein. At another three time points  was obtained by retro-orbital puncture leading to three plasma samples per time point. For liver collection, mice receiving 1 sacrificed at the last time point and mice receiving CDCA or vehicle were sacrificed 480 min after application by cervical dislocation and the complete liver was immediately snap-frozen and stored at −80 °C until further evaluation. 1 was quantified from blood samples by HPLC-MS with a flow rate of 600 µl/min on a Kinetex Phenyl-Hexyl, 2.6 µm, 50 × 2.1 mm  analytical column with a pre-column . Gradient elution with water and 0.1% formic acid as aqueous phase (A) and acetonitrile with 0.1% formic acid as organic phase (B) was used: % B (t (min)), 0(0–0.1)–97(0.4–1.7)–0(1.8–3.0). Full scan mass spectra were acquired in the positive ion mode using syringe pump infusion to identify the protonated quasi-molecular ions [M + H]+. Pharmacokinetic analysis was performed applying a non-compartment model using the Kinetica 5.0 software . Key pharmacokinetic parameters are shown in Supplementary Table Sample collection and pharmacokinetic analysis: At three time points  in a 50 mL Falcon tube. Every tissue was rinsed with PBS buffer containing 10% FCS, penicillin (100 U/mL) and streptomycin (100 μg/mL), and pressed through the cell strainer until 5 mL cell suspension had been collected. The samples were centrifuged at 1200 rpm for 10 min at 4 °C. The supernatant was discarded, and the pellets were washed with 5 mL cold PBS buffer and again centrifuged at 1200 rpm for 10 min at 4 °C. After discarding the supernatant, the cell pellets were re-suspended in 1 mL PBS and total RNA was extracted using the E. Z. A. Total RNA Kit I (Omega Bio-tek) following the Animal Tissue Protocol. The extracted RNA was used for qRT-PCR and equally treated as described for mRNA quantification from HepG2 cells (see above). PCR primers for the murine genes are shown in Supplementary Table −ΔCt method. Results : BSEP: vehicle: 0.030 ± 0.011; CDCA (10 mg/kg): 0.248 ± 0.035; 1 (10 mg/kg): 0.129 ± 0.018. CYP7A1: vehicle: 0.0453 ± 0.0200; CDCA (10 mg/kg): 0.0067 ± 0.0005; 1 (10 mg/kg): 0.0176 ± 0.0014.Quantification of FXR target gene mRNA from mouse livers: Hepatocyte isolation of mouse liver tissue for RT-qPCR: To homogenise the liver samples, one third of each liver was placed on one FalconConstruct: FXR ligand binding domain  and the C-terminally (amino acids 244–442) truncated constructs were cloned in pET (ampicillin resistance) with an N-terminal hexahistidine tag and a Tev cleavage site between FXR-LBD and His-tag. DNA sequence (insert):GAACTGACCCCAGATCAACAGACTCTTCTACATTTTATTATGGATTCATATAACAAACAGAGGATGCCTCAGGAAATAACAAATAAAATTTTAAAAGAAGAATTCAGTGCAGAAGAAAATTTTCTCATTTTGACGGAAATGGCAACCAATCATGTACAGGTTCTTGTAGAATTCACAAAAAAGCTACCAGGATTTCAGACTTTGGACCATGAAGACCAGATTGCTTTGCTGAAAGGGTCTGCGGTTGAAGCTATGTTCCTTCGTTCAGCTGAGATTTTCAATAAGAAACTTCCGTCTGGGCATTCTGACCTATTGGAAGAAAGAATTCGAAATAGTGGTATCTCTGATGAATATATAACACCTATGTTTAGTTTTTATAAAAGTATTGGGGAACTGAAAATGACTCAAGAGGAGTATGCTCTGCTTACAGCAATTGTTATCCTGTCTCCAGATAGACAATACATAAAGGATAGAGAGGCAGTAGAGAAGCTTCAGGAGCCACTTCTTGATGTGCTACAAAAGTTGTGTAAGATTCACCAGCCTGAAAATCCTCAACACTTTGCCTGTCTCCTGGGTCGCCTGACTGAATTACGGACATTCAATCATCACCACGCTGAGATGCTGATGTCATGGAGAGTAAACGACCACAAGTTTACCCCACTTCTCTGTGAAATCTGGGACGTGCAGTGAProtein sequence:ELTPDQQTLLHFIMDSYNKQRMPQEITNKILKEEFSAEENFLILTEMATNHVQVLVEFTKKLPGFQTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPSGHSDLLEERIRNSGISDEYITPMFSFYKSIGELKMTQEEYALLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKIHQPENPQHFACLLGRLTELRTFNHHHAEMLMSWRVNDHKFTPLLCEIWDVQE. coli BL21(DE3) gold cells (Invitrogen) in TB medium containing ampicillin in shaking flasks. Cells were initially grown at 37 °C and then continuously cooled to reach 18 °C when OD600 reaches 1. Expression was then induced with IPTG , cells were grown overnight and then harvested. Pellets were stored at −80 °C or directly used for purification.FXR-LBD was expressed in Protein purification: To the pellet of 1 L culture (~16 g), 60 mL lysis buffer  were added. Cells were disrupted by sonication on ice and then centrifuged at 12,000 rpm for 30 min. The cleared supernatant was treated with imidazole  and incubated with 10 mL NiNTA. After 1 h incubation, protein was washed with additional lysis buffer containing 20 mM imidazole before product was eluted with elution buffer . Elute was treated with 500 U Tev and dialysed against 50 mM Tris pH 8.3, 10% glycerol, 100 mM NaCl, 10 mM DTT overnight. The sample was then concentrated to 10 mL and filtrated over Superdex 200 26/60  in 50 mM Tris pH 8.3, 500 mM NaCl, 10 mM DTT, 10 % glycerol. Pure fractions with FXR-LBD monomer were collected and dialysed against 10 mM Tris pH 8.3, 100 mM NaCl, 5 mM DTT at 4 °C overnight to yield 20–50 mg FXR-LBD of >99% (SDS-PAGE) purity per liter cell culture. MS(ESI): calculated: 27181.2 Da; found: 27184.0 Da. Functionality was tested by ITC with GW4064. Mutants were prepared by designing an appropriate primer with the single amino acid mutation and following the standard QuickChange® site-directed mutagenesis protocol. Expression and purification procedure was performed as described for the wild type.Co-activator  and co-repressor  were synthesised with free amino and carboxy terminus using solid phase peptide synthesis on an ABI 433 A peptide synthesizer (Thermo Fisher). The resulting product was dissolved in water and further purified by reversed phase HPLC with C18 column material, followed by determination of purity and identity of the product by NMR and ESI-MS.2O. The spectra were processed and analysed using Topspin 3.5 (Bruker Biospin). For acquiring the 1H, 13C/15N-HSQC spectra of the FXR –peptide (NCoA or NCoR) complex, a ratio of 1:1  was used for the measurements. The direct interaction of ligands  and their influence on the binding of the co-activator/co-repressor binding was monitored by measuring 1H/13C-HSQC spectra of the 13C-15N-Arg/Leu selectively labeled peptides (ratio: Protein:peptide:ligand was 50:50:250 µM) in the apo and in complex with protein. Samples were measured in a 3 mm NMR-tube with a sample volume of 0.2 mL. FXR was at a concentration of 50 µM and the peptide and ligands were added to a final concentration of 50 µM  and 250 µM (from a stock solution of 100 mM in d6-DMSO), respectively. (Note: most ligands were not completely soluble. The insoluble precipitate of the excess ligand was spun down and the clear sample solution was then used for NMR measurements).NMR experiments were performed at 298 K, on Bruker 800 MHz spectrometers equipped with cryogenic triple-resonance probes. Typically, NMR samples contained 0.2 ml of up to 50/100 µM protein in 50 mM Tris pH 8.3, 500 mM NaCl, 2 mM TCEP, 1 mM TMSP, 5% D1 or 2). The complex was concentrated up to 375 µM and incubated for an additional hour with 750 µM in-house synthesised Nuclear receptor co-activator 2 peptide (NCoA-2740-752). The fresh samples were crystallised at 4 °C as hanging drop in 50 mM Ca(CH3COO)2 pH 7.5, 10 mM DTT, 25-30% PEG 3350 (CDCA) or 300 mM MgCl2, 100 mM Bis-Tris pH 7, 15-20% PEG 3350 (DM175). FXR/CDCA crystals appeared within short period, were soaked in 20% ethylene glycol and flash-frozen in liquid nitrogen. Initial FXR/DM175 crystals diffracted weak and were improved by micro seeding. Diffraction data have been collected on I03 MX at the Diamond Light Source  and BL14.2 operated by the Joint Berlin MX-Laboratory at the BESSY II electron storage ring 41. Data was processed using autoPROC42 or XDSAPP43. Structures were determined by molecular replacement utilising pdb 3FLI as model structure and refined using PHENIX44.FXR244-472 (75 µM) was incubated on ice for 1 h with 150 µM Iigands  was used to search for the structures pertaining to the LBD using the code IPR000536. The resulting PDB codes were read to extract the amino acid sequence from each chain and multiple sequence alignments were performed using Kalign. The residues Lys (α3) and Glu (α12) are widely conserved and typically form a salt bridge in the ligand-bound state of the LBD. D1 indicates the distance between Cα of both residues and was extracted for each of the chains within the PDB. In total there were 578 structures with peptide (co-activator/co-repressor) bound and 740 structures without peptide. The D1 distances extracted from these structures were plotted against the PDB.Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


The title compound comprises a neutral molecule, its zwitterionic tautomer whereby the N-bound proton of the central ring is now resident on the pendant ring, and a water molecule of crystallization. Conventional hydrogen bonding leads to supramolecular layers in the crystal. 14H14N4O·H2O, comprises a neutral molecule containing a central pyrazol-3-one ring flanked by an N-bound phenyl group and a C-bound 5-methyl-1H-pyrazol-3-yl group (at positions adjacent to the carbonyl substituent), its zwitterionic tautomer, whereby the N-bound proton of the central ring is now resident on the pendant ring, and a water molecule of crystallization. Besides systematic variations in geometric parameters, the two independent organic molecules have broadly similar conformations, as seen in the dihedral angle between the five-membered rings [9.72 (9)° for the neutral molecule and 3.32 (9)° for the zwitterionic tautomer] and in the dihedral angles between the central and pendant five-membered rings . In the crystal, pyrazolyl-N—H⋯O(carbonyl) and pyrazolium-N—H⋯N(pyrazolyl) hydrogen bonds between the independent organic molecules give rise to non-symmetric nine-membered {⋯HNNH⋯NC3O} and {⋯HNN⋯HNC3O} synthons, which differ in the positions of the N-bound H atoms. These aggregates are connected into a supramolecular layer in the bc plane by water-O—H⋯N(pyrazolide), water-O—H⋯O(carbonyl) and pyrazolyl-N—H⋯O(water) hydrogen bonding. The layers are linked into a three-dimensional architecture by methyl-C—H⋯π(phenyl) interactions. The different interactions, in particular the weaker contacts, formed by the organic molecules are clearly evident in the calculated Hirshfeld surfaces, and the calculated electrostatic potentials differentiate the tautomers.The title compound, 2C Applications in this context include their possible utilization as cardiovascular drugs -4-methoxyaniline -2-phenyl-2,3-dihydro-1H-pyrazol-3-one, its zwitterionic tautomer 5-methyl-4-(5-methyl-1H-pyrazol-2-ium-3-yl)-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-1-ide and a molecule of water. Evidence of tautomerism is twofold. Firstly, in the nature of the hydrogen-bonding interactions operating in the crystal; see Supramolecular features. Secondly, in small but systematic variations in key geometric parameters, Table 1syn with the pendent ring imine-N atom in the neutral molecule and is designated the NH-Z form The differences in geometric parameters characterizing the two independent organic molecules in (I)3O} and {⋯HNN⋯HNC3O} synthons, differing only in the relative placement of one of the ring-N-bound H atoms, Fig. 1bc plane by hydrogen bonding involving the water molecule, which functions as a donor to the pyrazolide-N6 and carbonyl-O1 atoms and as an acceptor from a pyrazolyl-N2 atom, Fig. 3a. Further consolidation of the layers is provided by π–π interactions with the shortest of these involving the N1-pyrazolyl and phenyl(C9–C14)i rings . As shown in Fig. 3b, the points of contact between layers involve methyl-C8—H⋯π interactions with the symmetry-related phenyl (C23–C28) ring, Table 2The molecular packing of (I)Crystal Explorer dnorm for the neutral tautomer in Fig. 4a and b and for the zwitterionic tautomer in Fig. 4c. In addition to these, a short intramolecular H⋯H contact between symmetry-related pyrazolyl-H4N and H7N atoms  and pyrazolyl-N2—H⋯O1W(water) are also viewed as blue and red regions in Fig. 5e and f.The donors and acceptors of the hydrogen bonds summarized in Table 2s Table 3 is viewednorm range, see Fig. 6a. The presence of faint-red spots near pyrazole-N2, C2, C17, C21 and phenyl-C13, C14 atoms in Fig. 6B and phenyl-C28 atoms, and the spots near the methyl-H18A and phenyl-C14, H14 atoms in Fig. 6A and H8B atoms as the donors and the phenyl (C23–C28) ring as the acceptor in the C—H⋯π contacts are also confirmed from the Hirshfeld surface mapped with the shape-index property through blue and red regions, respectively, in Fig. 7The short interatomic contacts characterizing weak intermolecular interactions in the crystal of (I)a and those delineated into H⋯H, O⋯H/H⋯O, N⋯H/H⋯N and C⋯H/H⋯C, C⋯C and C⋯N/N⋯C contacts for the overall structure are illustrated in Fig. 8b–g and the percentage contributions from the different interatomic contacts to the Hirshfeld surfaces of the neutral tautomer, zwitterion and the overall structure are summarized in Table 4The overall two dimensional fingerprint plot in Fig. 8b, the short contact involving the methyl-H18A and phenyl-H14 atoms, Table 3de + di ∼2.1 Å while the points corresponding to the H13⋯H18A contact are merged within the plot. The involvement of the water molecule in the N—H⋯O hydrogen bond results in a pair of long spikes at de + di ∼1.8 Å in the fingerprint plot delineated into O⋯H/H⋯O contacts, Fig. 8c; these encompass the pair of spikes corresponding to the O—H⋯O hydrogen bond involving carbonyl-O1 atom. The percentage contribution from these contacts to the Hirshfeld surface of the overall structure is less than the individual tautomers e with the shortest C⋯H contact represented as the pair of peaks at de + di ∼2.7 Å, Table 3f and g, arise from the presence of inter-layer π–π contacts between pyrazolyl and phenyl rings whereas the other short C⋯C contacts summarized in Table 3i.e. methyl-C8⋯C22(methyl). The small contribution from C⋯O/O⋯C contacts appears to have a negligible effect on the molecular packing.The fingerprint plot delineated into N⋯H/H⋯N contacts in Fig. 8et al., 1995et al., 1995et al., 2017carbonyl—C—C—Nexternal ring torsion angle of 57.1 (3)°. In two other derivatives, a similar situation pertains. In the derivative where the original phenyl ring of the neutral molecule in (I)et al., 2011et al., 2010carbonyl—C—C—Nexternal ring torsion angles of −57 (1) and 56 (1)° are found for the two crystallographically independent molecules comprising the asymmetric unit.There are no direct precedents for the neutral molecule found in (I)4-Acetoacetyl-3-methyl-1-phenyl-2-pyrazolin-5-one 1  and hydrazin hydrazine (1 ml) were refluxed in a mixture of ethanol (50 ml) and acetic acid (50 ml) for 2 h. The reaction mixture was allowed to stand at room temperature. The precipitate was filtered and recrystallized from ethanol solution as fine needles, M.p. 409–410 K. Yield: 70%.Uiso(H) set to 1.2–1.5Ueq(C). The O- and N-bound H atoms were refined with distance restraints of 0.84±0.01 and 0.86±0.01 Å, respectively, and with Uiso(H) = 1.5Ueq(O) and 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 610.1107/S2056989019004389/hb7813sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019004389/hb7813Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019004389/hb7813Isup3.cmlSupporting information file. DOI: 1906841CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: