Eryngium campestre, obtained from its aerial parts, was investigated by GC-FID, GC/MS, HRMS, NMR and VCD analyses. The main compounds were germacrene D (23.6%), eudesma-4(15)-7-dien-1-β-ol (8.2%) and falcarindiol (9.4%), which are associated with a new uncommon and naturally found 17-membered ring lactone. This 17-membered ring features conjugated acetylenic bonds, named campestrolide (23.0%). The crude extract showed moderate antitrypanosomal (Trypanosoma brucei brucei), antileishmanial (Leishmania mexicana mexicana) and anticancer (cancerous macrophage-like murine cells) activities, and also displayed cytotoxicity,  in similar concentration ranges . Likewise, campestrolide displayed low activity on all tested cells (IC50: 12.5–19.5 µM) except on Trypanosoma, on which it was very active and moderately selective (IC50 = 2.2 µM. SI= 8.9). In conclusion, the new compound that has been described, displaying a singular structure, possesses interesting antitrypanosomal activity that should be further investigated and improved.The chemical composition of a hexanic extract of  Eryngium, the most common of the Apiaceae family, comprises more than 250 species with cosmopolitan distribution in temperate regions of all continents, mainly in Eurasia, North Africa and South America  Nym. ssp. tuberosum [Eryngium species was first reported by Bohlmann and Zdero [E. alpinum, E. coeruleum and E. giganteum. However, this is the first time it has been identified in E. campestre. Falcarindiol has been shown to have anti-inflammatory, antibacterial and anticancer activities, as well as protective effects against hepatotoxicity [Column chromatography was carried out using a gradient of polarity with hexane and diisopropyl ether and then followed by UV detection, producing 75 fractions from the hexane extract of s carota , and Petuberosum . Its idetoxicity .F21: 13 mg, 33 at 96%). GC/MS-EI spectra of 33 exhibited an isotopic cluster at m/z 270 and 271, and a base peak at m/z 115. Other fragment ions were observed at m/z 55, 91, 128 and 145 experimental 293.1513 and (m/z)theoretic 293.1512. error +0.3 ppm), which would correspond to a compound possessing an additional carbon atom and one supplementary unsaturation  as compared to the falcarindiol 34 . The IR spectrum exhibits absorption peaks at 1739 cm−1, 1098 cm−1 and 1225 cm−1, corresponding to C=O and C-O vibrations. This indicates the presence of a cyclic lactone function. Additionally, absorption bands at 2255 cm−1 and 2856–2928 cm−1 indicated the presence of C≡C and CH2 bonds, respectively.Column chromatography using the gradient of solvent diisopropyl ether/hexane (5/95) produces a yellow, odorous, oily compound ( and 145 . The ESI1H- and 13C-NMR spectra (3 groups and the presence of a ketone group (172.98 ppm). Furthermore, the characteristic resonances of a polyacetylene skeleton (constituted by a sequence of two consecutive triple bonds) are identified using the 13C-NMR spectrum in combination with DEPT data .The complete assignment was achieved using the 2D NMR spectra -enantiomer -enantiomer (R)-enantiomer (Z)-17(R)-vinyloxacycloheptadeca-10-en-13,15-diyn-2-one. It should be mentioned that the identified configuration is perfectly consistent with those generally reported in the literature for polyacetylenes derivatives [In the last decade, VCD spectroscopy has become a popular technique for the elucidation of absolute configurations of chiral molecules ,32,33. Tantiomer c, namelyivatives ,35.Khaya ivorensis—a dicotyledone tree from the Meliaceae family [Amphidinium species. Researchers have reported cytotoxic properties for all the described amphidinolides mentioned above [To our knowledge, this uncommon natural macrocyclic lactone that includes one cyclo-1,3-diynes motif has only been reported in one other compound (ivorenolide B), which was extracted from e family ,36. Furte family ,37,38,39ed above .E. campestre and compound 33 were evaluated for their in vitro cytotoxicity and antiprotozoal activities (50 ≈ 4 µg/mL) on all tested cells and hence, without any selectivity for the concerned parasites. The newly identified macrocyclic lactone was strongly active on Trypanosoma (IC50 = 0.6 µg/mL or 2.2 µM ≈ 2 µM), as defined by Beaufay et al. (IC50 ≤ 2 µM) [50 < 1 µg/mL and at least ten-fold higher than on a mammalian cell line. As the calculated campestrolide log P value (log P = 5.56) [Both the crude hexanic extract of tivities . The pla ≤ 2 µM) , with a  ≤ 2 µM) , i.e., a = 5.56)  was clos = 5.56) , we can 50 = 3.4 and 4.8 µM, respectively) but not as selective as those observed for the extract. Another well-known complex polyene macrolide is amphotericin B (IC50 = 0.09 ± 0.06 µM), a reference drug widely used to treat visceral leishmaniasis but still little studied on cutaneaous one and with dose-limiting toxicity [The antileishmanial or anticancer activities were notable  on Trypanosoma brucei brucei compared to HeLa cytotoxicity. The inhibition of the trypanothione reductase was proposed as one target of this very reactive alkylating agent [Trypanosoma brucei rhodesiense, T. cruzi, Plasmodium falciparum and Leishmania donovani axenic and the infected macrophages amastigotes compared on L6 rat skeletal myoblasts (IC50 = 0.1–2.5 µM. SI > 10). The terminal methylene double bond seems to improve activity [Campestrolide can partially explain all the activities observed for the crude extract but it is certainly not the only active component of the mixture, as observed with the other samples containing different percentages of campestrolide (data not shown). Therefore, other components, e.g., the identified polyacetylenes, could influence activities by addition or synergism action. This has already been observed, for example, with the polyphenolic compounds . Indeed,th taxol . Previouth taxol . Falcaring agent . AnotherT. cruzi trypomastigotes at 100 µg/mL. It also inhibited cruzain, an essential T. cruzi cysteine protease, with an IC50 of 22.1 µg/mL [In addition, germacrene D, another major identified compound, possesses some antiparasitic activity as with other non-lactonized sesquiterpenoids and eudesmanolides, with 61% growth inhibition of the .1 µg/mL .E. campestre displayed antileishmanial activity with IC50 values of 36 and 15 µg/mL, respectively, on L. donovani promastigotes. They were inactive on the P. falciparum D6 and W strains [Plasmodium growth inhibition at 4.81 µg/mL [Eryngium species have previously shown interesting antiparasitic activities (IC50 < 20 µg/mL) on T. cruzi [L. donovani, but appeared less active or inactive on Plasmodium falciparum [50 values of 14.33 and 7.84 µM on L. tarentolae promastigotes and L. donovani amastigotes respectively) was isolated from E. foetidum aerial parts [Concerning the reported plant extract, dichloromethane and methanol compounds from the aerial parts of the  strains . However81 µg/mL . Other Elciparum ,53. A daEryngium campestre L. (Apiaceae) were collected in June 2016, from the north-western areas of Algeria. The botanical identification of the plants was performed according to the botanical determination keys summarized in the Flora of Algeria [Aerial parts of  Algeria  and a voThe dried aerial parts (120 g) were powdered and weighed, then extracted with hexane (500 mL) under reflux in a Soxhlet apparatus, at 68 °C. The extract was first filtered on filter paper and then on PTFE filters (0.2 µm). After that, the extract was evaporated to dryness under reduced pressure. This resulted in a yield of 1.05%.E. campestre hexanic extract was carried out using different techniques, such as GC/FID, GC/MS (EI) and 1D and 2D NMR. The identifications were mainly based on comparison of the retention indices (RI) and the MS spectra with those contained in our laboratory library “Arômes”. When spectral data were not present in the “Arômes” library, the RI and the MS data were compared with those from commercial libraries [The identification of individual compounds in the ibraries ,55,56,57® Bonna Agela Technologies, Willington, CT, USA), using an Automatized Combi Flash apparatus  equipped with a fraction collector and monitored by a UV detector. The solvents of elution were n-hexane (A) and diisopropyl ether (B), with a gradients of elution: a , b , c , d , e  and f .We measured 300 mg of the hexanic extract. This was then submitted to chromatography on a silica gel column  by linear interpolation, using the Van den Dool and Kratz (1963) equation with the aid of the software from Perkin-Elmer . The relative percentages of the extract constituents were calculated from the GC peak areas by the normalization procedure, without the application of correction factors.Analyses were carried out using a Perkin-Elmer Autosystem XL GC apparatus , equipped with a dual flame ionization detection (FID) system and fused-silica capillary columns, namely, Rtx-1 (polydimethylsiloxane) and Rtx-wax (polyethyleneglycol) . The oven temperature was programmed from 60 to 230 °C at 2 °C/min and then held isothermally at 230 °C for 35 min and helium was employed as the carrier gas (1 mL/min). The injector and the detector temperatures were maintained at 280 °C and the samples were injected (0.2 µL of pure oil) in the split mode (1:50). The RI of each compound was determined relative to the retention times of a series of Samples were analyzed with a Perkin-Elmer Turbo mass detector (quadrupole) coupled to a Perkin-Elmer Autosystem XL, equipped with fused-silica capillary columns Rtx-1 and Rtx-Wax. The oven temperature was programmed from 60 to 230 °C at 2 °C/min and then held isothermally at 230 °C (35 min) and helium was employed as the carrier gas (1 mL/min). The following chromatographic conditions were employed: the injection volume was 0.2 µL of pure oil; the injector injector temperature was 280 °C; split 1:80; the ion source temperature was 150 °C; the ionization energy was 70 eV; the MS (EI) data were acquired over the mass range 35–350 Da; and the scan rate was 1 s.3  at 300 K using a Bruker Avance DRX 500 NMR spectrometer , operating at 500.13 MHz for 1H and 125.76 MHz for 13C Larmor frequency with a double resonance broadband fluorine observe (BBFO) and 5 mm probehead. 13C-NMR experiments were recorded using the one-pulse excitation pulse sequence (90° excitation pulse), with 1H decoupling during signal acquisition (performed with WALTZ-16). The relaxation delay was set at 2 s. For each analysed sample, depending on the compound concentration, 3 k up to 5 k free induction decays (FID) 64 k complex data points were collected using a spectral width of 30,000 Hz (240 ppm). Chemical shifts (δ in ppm) were reported relative to the residual signal of CDCl3 . Complete 1H and 13C assignments of the requested compounds were obtained using 2D gradient-selected NMR experiments, 1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC and 1H-1H NOESY, for which conventional acquisition parameters were used, as described in the literature [NMR experiments were acquired in CDClterature .−1 flow rate , flow of 100 L h−1 and heated at 35 °C). The samples were dissolved and further diluted in methanol  doped with sodium chloride (0.1 mM) prior to the analysis. Accurate mass experiments were performed using reference ions from an internal calibration procedure, with two reference ions formed upon electrospray of a poly (ethylene oxide) (PEO). Data analyses were conducted using MassLynx 4.1 programs, which were provided by Waters .High-resolution mass spectrometry (HRMS) experiments were performed with a Synapt G2 HDMS quadrupole/time-of-flight , equipped with an electrospray source operating in the positive mode, ESI(+). The samples were introduced at a 10 µL min−1) was used to enhance the signal/noise ratio before use of the photoelastic modulator. A transmission cell of 200 μm optical pathlength, equipped with CaF2 windows, was used. The solutions with a concentration of 0.1 mol L−1 were prepared by dissolving the solid samples in CD2Cl2. The VCD spectrum of the enantiomer was measured at room temperature using a sample with an estimated purity of 80%. The baseline of the VCD spectrum was corrected by the subtraction of the VCD spectrum of the solvent. For each individual spectrum, about 16,000 scans were averaged at 4 cm−1 resolution (corresponding to 4 h of measurement time). For the IR absorption spectra, the cell filled with CD2Cl2 served as a reference. The spectra are presented without any smoothing and further data processing.The infrared (IR) and vibrational circular dichroism (VCD) spectra were recorded on a Bruker PMA 50 accessory, coupled to a Vertex 70 Fourier transform infrared spectrometer . A photoelastic modulator  set at l/4 retardation was used to modulate the handedness of the circular polarized light at 50 kHz. Demodulation was performed by a lock-in amplifier . An optical low-pass filter (<1800 cmS)-enantiomer of compound 33 using Gaussian 16 package [2Cl2) effects have been introduced using the implicit solvation model, SMD, which is based on the integral equation formalism of the polarizable continuum model (ief-pcm). The computed harmonic frequencies are generally larger than the fundamentals observed experimentally. They were calibrated using a scaling factor of 0.98. The IR absorption and the VCD spectra were constructed from the calculated dipole and the rotational strengths assuming the Lorentzian band shape with a half-width at a half-maximum of 8 cm−1. The computational method is detailed in the The calculation of the VCD and IR spectra was performed on the ( package . The geoTrypanosoma brucei brucei (Tbb. strain 427) bloodstream forms and Leishmania mexicana mexicana (Lmm. MHOM/BZ/84/BEL46) promastigotes. The tests were performed using [Leishmania and Trypanosoma, respectively). Suramine sodium and pentamidine isethionate salts  were used as a positive control with 3-fold dilutions (0.0046–10 µg/mL) from a stock solution of 2 mg/mL as well as amphotericin B. A maximum of 0.5% DMSO was previously verified to be non-toxic in all the biological assays. The IC50 values were determined using Microsoft Excel and GraphPad Prism 7.0 software , based on a nonlinear regression. The selectivity index was calculated in comparison to the WI38 cytotoxicity to assess the therapeutic properties as antiparasitic or anticancer hits.The crude extract and the newly identified pure compound (91.9%), campestrolide, were evaluated in vitro for their cytotoxicity on two mammalian cell lines: normal human fibroblasts (WI38) and cancerous macrophage-like murine cells. Their antiprotozoal activities were also investigated on ed using ,61 stockEryngium campestre hexane crude extract. This newly described compound presents a particular structure that is composed of a rigid 1,3-dynes motif and a flexible aliphatic part. Moreover, campestrolide possesses relatively strong antitrypanosomal activity but moderate selectivity. Therefore, we assume that campestrolide could be considered for further investigation to improve activity/selectivity. Furthermore, it is partially responsible for the observed antiprotozoal activities and cytotoxicity in the crude extract.A new uncommon macrocyclic lactone named campestrolide, along with other known compounds, was isolated and identified in the


The dispersion of the organic complexes in the copolymer matrix was highly improved, while the transparency of the copolymers was negligibly influenced in comparison with the pristine cross-linked PMMA. In addition, the thermal resistance of the copolymers was enhanced with the complex loading, while their glass transition temperature, cross-linking level, and mechanical properties were to some extent reduced. Yb(TTA)2AAPhen and Nd(TTA)2AAPhen provided the prepared copolymers with selective photothermal effects and shape-memory functions for 980 and 808 nm NIR lights, respectively. Finally, smart optical devices which exhibited localized transparency or diffraction evolution procedures were demonstrated based on the prepared copolymers, owing to the combination of good transparency and selective light wavelength responsivity.Shape-memory polymers (SMPs) selectively induced by near-infrared lights of 980 or 808 nm were synthesized via free radical copolymerization. Methyl methacrylate (MMA) monomer, ethylene glycol dimethylacrylate (EGDMA) as a cross-linker, and organic complexes of Yb(TTA) Remote-controlled actuating of polymeric materials to drive their own movements or to manipulate other substances is one rapidly developing area in advanced intelligent materials ,2,3,4,5.Tsws) of such SMP composites (SMPCs), shape recoveries are triggered. Various substances presenting photothermal effect have been explored as the functional fillers for SMPCs, especially carbon nanomaterials [3Phen and Nd(TTA)3Phen powders in poly[ethylene-ran-(vinyl acetate)] (EVA) offered the shape recovery upon a NIR-NIR irradiation of 980 and 808 nm, respectively.Shape-memory polymer (SMP) is a kind of polymer that has the ability to recover from a temporary shape to its permanent one. Superior to liquid-crystalline polymers ,13,14,15aterials ,32 and naterials ,10. The aterials , visibleaterials ,10, or iaterials ,34. The aterials ,35,36. Iaterials . To dateaterials , UV–NIR aterials , Vis–Visaterials , Vis–NIRaterials ,26, and aterials . The phySMPs can be used to create a series of deformable, programmable, and shape-memory optical devices ,41,42. VHere, acrylic acid (AA) with double bonds was used to prepare reactive Yb and Nd organic complexes. Selectively responsive SMPs to NIR lights were synthesized subsequently among methyl methacrylate (MMA) monomer, ethylene glycol dimethylacrylate (EGDMA), and the reactive organic complexes via in situ copolymerization. The structures and photothermal properties of the functional and reactive complexes were studied, while the properties and the light-induced shape-memory performance of the prepared SMPs were evaluated. Finally, we created temporal structures at the microscale on those SMPs and investigated their feasibility in the field of smart optical devices.3·6H2O  and NdCl3·6H2O  were provided by Funing Rare Earth Industrial Company . Acrylic acid , methyl methacrylate , ethylene glycol dimethylacrylate , 2,2′-azobis(2-methylpropionitrile) , and N,N-dimethylformamide  were obtained from Aladdin, Shanghai, China. All chemicals were used without further purification.The α-thenoyltrifluoroacetone  and 1,10-phenanthroline  were purchased from Sinopharm Chemical Reagent Company . YbCl3·6H2O (10 mmol), NdCl3·6H2O (10 mmol), HTTA (20 mmol), AA (10 mmol) and Phen (10 mmol) were respectively dissolved in 30 mL ethanol. The ethanol solution of YbCl3·6H2O or NdCl3·6H2O was first poured into a three-necked flask at 60 °C in a magnetic stirred water bath. The ethanol solutions of ligands were subsequently added dropwise. The HTTA and AA solutions were added together before the Phen solution. The pH value of the mixture was adjusted to 6–7 using 1 mol·L−1 sodium hydroxide ethanol solution, before the reaction of 6 h. Subsequently, the precipitation was collected using centrifugation , before washing with water and ethanol for three times each and drying in a vacuum oven at 60 °C for 12 h.YbCl2AAPhen  or Nd(TTA)2AAPhen  were first dissolved in DMF (4 g) and sonicated for 30 min. MMA monomer (5 g), EGDMA  and AIBN  were subsequently added to the solution. The whole solution was prepolymerized in a glass vial (20 mL) at 65 °C for 30 min. Subsequently, the prepolymer was injected into a home-made glass container (L × W × D = 70 mm × 20 mm × 1 mm), which was placed in an oven at 65 °C for further copolymerization. After 12 h, the materials were taken out and then heated at 65 °C for 24 h in a vacuum oven. Three different powder loadings  were used and the samples were named as cPMMA, PMMA–Yb5, PMMA–Yb10, PMMA–Nd5, and PMMA–Nd10.Yb(TTA)Two pieces of Nylon fabrics were pressed against both sides of PMMA-Yb10 or PMMA-Nd10, and the sandwich-structured materials were placed between two glass plates and fixed using office clamps. The materials were heated to 150 °C in an oven for 10 min and cooled down gradually to room temperature without manual intervention. The specimens with temporary surface structures were obtained by removing the clamps, glass plates, and Nylon fabrics a. In add2AAPhen and Nd(TTA)2AAPhen as well as the raw materials of AA, HTTA and Phen. The samples were ground with pottassium bromide (KBr) together and pressed into a testing tablet.Fourier Transform Infrared Spectroscopy: Fourier transform infrared spectroscopy  was used to determine the chemical structures of the prepared Yb(TTA)2AAPhen and Nd(TTA)2AAPhen as well as the transmittance spectra of the copolymers were measured via a UV–vis–NIR spectrophotometer , using BaSO4 and air as the references, respectively.Reflectance and Transmittance Measurements: The reflectance spectra of Yb(TTA)2AAPhen and Nd(TTA)2AAPhen as well as the copolymers were evaluated using thermogravimetric analysis  scanning from room temperature to 800 °C at the heating rate of 10 °C·min−1.Thermogravimetric Analysis: In an air flow, the thermal stability of Yb(TTA)−1.Thermal Properties Analysis: Differential scanning calorimetry  was used to determine the thermal properties of the copolymers. The scanning temperature increased from room temperature to 150 °C, and decreased to −75 °C before increasing to 150 °C again. The heating and cooling were conducted under a nitrogen atmosphere at a rate of 10 °C·min2AAPhen and Nd(TTA)2AAPhen powders as well as the copolymers were performed using a Smartlab  thin-film diffractometer employing Cu Kα radiation (λ = 0.15046 nm), with the 2θ angle from 5° to 50° at the scanning rate of 10°·min−1.X-ray diffraction: The structure characterization of Yb(TTA)2AAPhen and Nd(TTA)2AAPhen powders as well as the copolymers were examined using a scanning electron microscope  equipped with a NORAN System 7 EDX detector .Microscopy: The morphology of Yb(TTA)Photothermal Effect: The NIR lights of 980 and 808 nm were generated respectively by a laser diode driver  and a laser driver . The sample temperatures were measured using a hand-held infrared camera . The power density was determined using an optical power/energy meter  equipped with a thermopile detector .m1 was cut and immersed in DMF. After 72 h, the sample was removed from the solvent, while the mass (m2) was immediately measured in the swollen state after cleaning the extra solvent using a piece of tissue. The swollen sample was heated at 60 °C again until completely dry to obtain the final weight (m3). The gel content (G) was given by m3/m1 × 100%. The swelling degree (Q) was calculated using Equation (1). The ρ1 and ρ2 were the specific densities of the swelling solvent of DMF and PMMA.Swelling Experiments: A piece of the sample with the mass of XLD]s in moles per cubic centimeters) of the cross-linked copolymers was determined using the Flory-Rehner equation (Equation (2)) s was also calculated based on the swelling experiment, while the data clearly showed a reduction in cross-linking density 2AAPhen, as expected, generated new peaks located at 975 and 802 nm, respectively, compared to pristine cPMMA. Higher loading increased the peak intensity, i.e., the transmittance at 975 nm decreased from 83.6% for cPMMA to 80.6% and 77.3% for PMMA–Yb5, and PMMA–Yb10, respectively. At the wavelength of 802 nm, cPMMA, PMMA–Nd5, and PMMA–Nd10 exhibited a gradually decreased transmittance from 79.2 to 69.2% and 62.7% as well. Due to such certain absorption peaks, the prepared polymers also presented selective photothermal effects upon the NIR light irradiation of 980 or 808 nm, as shown in −1  highly relied on the suitable light wavelength and power density, which dominated the indirectly generated temperature 2AAPhen enabled the SMPs to recover from temporary shape upon selective NIR light irradiations. It is worth mentioning that since the temperature increase not only depended on the filler content but the light intensity as well, increasing the light power density was expected to trigger the shape recovery of PMMA–Yb5 and PMMA–Nd5 ultimately, which was not further investigated.The light-induced shape recovery ratio 2AAPhen reactivity with methyl methacrylate monomers and ethylene glycol dimethylacrylate. Increasing the contents of organic complexes enhanced the thermal resistance of the copolymers, but caused a reduction in glass transition temperature, cross-link density, and mechanical properties. The transparency, however, was not affected evidently. Further, the discriminated absorptivity of the complexes close to the wavelengths of 980 and 808 nm provided the copolymers with the selective photothermal effect and shape-memory capability. Finally, smart optical devices were created which can switch the localized transparency or the diffraction evolution procedures. Further work should focus on the development of other organic complexes containing different reactive ligands and the in situ synthesis of other NIR light-induced SMPs.Via free radical copolymerization, shape-memory polymers selectively responsive to NIR lights of 980 or 808 nm were synthesized. The usage of acrylic acid containing double bonds offered the prepared Yb(TTA)


Rapamycin, an inhibitor of mechanistic Target Of Rapamycin Complex 1 (mTORC1), extends lifespan and shows strong potential for the treatment of age-related diseases. However, rapamycin exerts metabolic and immunological side effects mediated by off-target inhibition of a second mTOR-containing complex, mTOR complex 2. Here, we report the identification of DL001, a FKBP12-dependent rapamycin analog 40x more selective for mTORC1 than rapamycin. DL001 inhibits mTORC1 in cell culture lines and in vivo in C57BL/6J mice, in which DL001 inhibits mTORC1 signaling without impairing glucose homeostasis and with substantially reduced or no side effects on lipid metabolism and the immune system. In cells, DL001 efficiently represses elevated mTORC1 activity and restores normal gene expression to cells lacking a functional tuberous sclerosis complex. Our results demonstrate that highly selective pharmacological inhibition of mTORC1 can be achieved in vivo, and that selective inhibition of mTORC1 significantly reduces the side effects associated with conventional rapalogs. Rapamycin extends lifespan in model organisms by targeting mTORC1, but exerts off-target side effects via inhibition of mTORC2. Here, the authors report the identification of a selective mTORC1 inhibitor, and show that it inhibits mTORC1 activity both in vitro and in vivo, with reduced side effects on glucose homeostasis, lipid metabolism, and the immune system. Excitingly, rapamycin also prevents or delays the onset of age-related diseases in mice, including cancer and Alzheimer’s disease, rejuvenating the aging mouse heart, and ameliorating age-related cognitive decline9. Scientists are now beginning to explore the possibility that rapamycin may be of therapeutic benefit for age-related diseases in dogs and humans12.Age-related diseases, including cardiovascular disease, cancer, neurodegenerative disease, and type 2 diabetes, represent the greatest healthcare challenges in a graying world. Targeting the aging process through dietary or pharmacological interventions has been advanced as way to prevent or delay many age-related diseases simultaneously, extending healthspan. Over the past decade, significant scientific activity has focused on the anti-aging potential of rapamycin, an inhibitor of the serine/threonine protein kinase mTOR (mechanistic Target Of Rapamycin), which can extend the lifespan of organisms including yeast, worms, flies, and mice19. Although short term, low dose, and/or intermittent rapalog-based regimens may partially limit side effects22, and trials of rapamycin in the elderly have begun23, the efficacy of such treatment regimens in treating age-related diseases remains to be determined, particularly as the effects of rapamycin on longevity are dose-dependent25. All three FDA-approved rapalogs—sirolimus, everolimus, and temsirolimus—have similar effects on the glucose metabolism and immune cell profile of mice20. It is clear that there is an urgent need for new molecules that inhibit mTOR signaling with reduced side effects26.Unfortunately, treatment with rapamycin and its analogs  is associated with negative side effects that limit their potential utility as anti-aging therapies. These side effects include immunosuppression, glucose intolerance, an increased risk of type 2 diabetes, and disruption of lipid homeostasis27, functions primarily as a downstream effector of the insulin/IGF-1 signaling pathway, through substrates that include AKT15. Several years ago, while investigating the molecular basis of rapamycin-mediated glucose intolerance, we made the surprising discovery that long-term treatment with rapamycin inhibits not only mTORC1 but also inhibits mTORC2 in vivo in many metabolically active tissues28. This discovery, in combination with extensive results from genetic manipulation of model organisms, has led to the consensus that the beneficial effects of rapamycin on health and longevity are mediated solely by inhibition of mTORC1, while the off-target inhibition of mTORC2 is responsible for many of the negative side effects of rapamycin. Thus, many of the side effects of rapalogs could be avoided by selectively inhibiting mTORC1 signaling14.mTOR is found in two distinct protein complexes. mTORC1, which is acutely sensitive to rapamycin, promotes cell growth, ribosomal biogenesis, and protein translation through substrates that include S6K1 and 4E-BP1, and regulates autophagy via ULK1. mTORC2, originally characterized as rapamycin resistantHere, we report the identification of DL001, a rapamycin analog with much greater selectivity for mTORC1 than rapamycin. We show that DL001 selectively inhibits mTORC1 both in cell culture and in vivo in mice. Critically, we find that unlike rapamycin, DL001 does not disrupt glucose or lipid homeostasis, and has substantially reduced effects on the immune system. Our results demonstrate that highly selective pharmacological inhibition of mTORC1 can be achieved in vivo, and that such a compound minimizes the negative metabolic and immunological impacts associated with rapamycin and other conventional rapalogs.29, recent work investigating the molecular mechanism by which rapamycin disrupts mTORC2 (ref. 30) suggested that rapamycin derivatives with reduced activity against mTORC2 but full potency against mTORC1 might exist. We therefore screened a library of approximately 90 rapamycin analogs in order to identify compounds with reduced activity against mTORC2; for the screens, we primarily utilized PC3 cells , we also identified several compounds with reduced inhibition of mTORC2. We selected DL001  via western blotting, assessing the phosphorylation of the mTORC1 substrate S6K1 and its substrate S6, the phosphorylation of rapamycin-sensitive and -insensitive residues on 4E-BP1 , and the phosphorylation of the mTORC2 substrate AKT S473. While rapamycin and DL001 had very similar effects on mTORC1 activity Fig. , the eff50mTORC2 for DL001, we utilized a commercially available high-sensitivity chemiluminescent assay (AlphaLISA). PC3 cells treated with either vehicle or a range of doses of either rapamycin or DL001 for 24 h were analyzed to determine the phosphorylation of S6 S240/S244 and Akt S473. As shown in Fig. 50 of 63.3 pM, whereas DL001 inhibited mTORC1 with a very similar IC50 of 74.9 pM. In contrast, while rapamycin inhibited mTORC2 activity with an IC50 of 534.9 pM, DL001 inhibited with a significantly greater IC50 of 26,245.4 pM , while upregulating proteins involved in macroautophagy and lysosomal function  of DL001 and rapamycin, and western blotting to determine the phosphorylation of S6 and AKT S473 , rapamycin, and mTOR473 Fig. . We find473 Fig. . Likewis473 Fig. .Fig. 2DL34. We therefore administered DL001 to mice for 20 days, and broadly assessed the effect of DL001 on mTORC1 and mTORC2 signaling in 11 tissues. We observed robust inhibition of mTORC1 signaling by DL001 in 9 of 11 tissues, including liver, gastrocnemius, and heart .We and others have observed that the metabolic effects of rapamycin are apparent in mice after 2–3 weeks of chronic treatmentart Fig. , visceraart Fig. . DL001 dart Fig. , tissuesart Fig. . Strikinart Fig. ), AKT S4−1 DL001 administered every other day for 5 weeks on hepatic mTORC1 signaling. Consistent with our findings above, we confirmed decreased mTORC1 signaling in the liver of both rapamycin and DL001-treated mice as measured via the phosphorylation of S6 S240/S244 . Rapamycin dramatically impacted both the amount of Rictor-associated mTOR and the total amount of mTORC2 in the liver, while DL001 had no effect on mTORC2 integrity or abundance  of DL001; the trough level of rapalogs is clinically monitored in patients subjected to rapalog therapy and is directly linked with rapalog exposure, efficacy, and side effects43. Importantly, the blood levels of both DL001 and rapamycin were substantially higher than levels observed in the blood of mice fed 14 ppm rapamycin24, which as we have previously demonstrated is sufficient to inhibit mTORC2 in vivo44.Our initial hypothesis was that a rapalog that specifically inhibits mTORC1, such as DL001, would enable us to avoid many of the side effects of rapamycin. To test this, we examined the effects of DL001 on glucose metabolism, lipid metabolism, and the immune system. Male C57BL/6J mice, which show a robust metabolic response to rapamycin and to genetic inhibition of hepatic mTORC2 signaling44, rapamycin-treated mice were pyruvate intolerant, indicating a failure to suppress hepatic gluconeogenesis  was conducted Fig. ; as expesis Fig. . Once agsis Fig. . Clinica001 Fig. .Fig. 4Un45. We observed that while rapamycin robustly elevated plasma cholesterol, triglycerides, and free fatty acids, DL001 does not increase blood levels of these lipids  in DL001-treated animals; rapamycin and DL001 both increase the phosphorylation of PKA substrates —are likely a significant barrier to its application for the treatment of chronic age-related diseases. Rapamycin is associated with an increase in viral and fungal infections in humans, short courses of low-dose rapamycin impairs the defense against acute bacterial and viral infections in mice, and chronic treatment with rapamycin impairs adaptive immunity in mice52. As both mTOR complexes play important roles in adaptive immunity, in part by promoting the survival, differentiation, activation, and function of T cells49, we predicted that by more selectively targeting mTORC1, DL001 would have reduced—although not zero—effects on immune cell numbers as compared to rapamycin.In addition to its metabolic side effects, the immunosuppressive effects of rapamycin—mediated in part via inhibition of mTORC2  cell numbers by 40%, and CD3+CD4+ (helper T) cell numbers by 47% 54; rapamycin reduced Tregs by 54%, while DL001 had a significantly smaller effect on Tregs, reducing the numbers by 37%  cell number by 36%, DL0001 had no effect on CD3+CD8+ cell number , a rare genetic disease resulting from a loss of function mutation in 56, and a rapalog (everolimus) has been FDA approved for the treatment of specific symptoms of TSC58. However, suppression of TSC symptoms requires continuous chronic treatment with rapalogs, which can results in significant side effects. Over the course of 4 years, 30% of subjects in one trial of rapalogs for TSC developed hypercholesterolemia;59 in a second study 5 of 18 (27%) participants were hospitalized for pneumonia over the course of 4 years60. One clinical trial of a rapalog for TSC had a particularly high incidence of side effects, with 72% of the subjects developing hypercholesterolemia, 66% developing hyperlipidemia, and 22% of subjects developing hyperglycemia; in that trial one subject died as a result of bacterial sepsis13. Thus, TSC represents a disease in which minimizing the side effect of rapamycin may be particularly important.Treatment with rapamycin can suppress many of the effects of TSC in animal models of the diseaseTsc1. We observed that both rapamycin and DL001 effectively suppressed the hyperactive mTORC1 activity of Tsc1−/− MEFs to below wild-type levels  lacking els Fig. . MEFs la001 Fig. . Importa001 Fig. .Fig. 5DL62 is minimal; work in genetic mouse models suggests that inhibition of the rapamycin-resistant functions of mTORC1 may be associated with side effects such as allergic asthma, hypoglycemia, seizures, and the ability to build muscle mass with exercise66. Importantly, DL001 efficiently represses hyperactive mTORC1 in cells lacking a functional TSC. As hyperactive mTORC1 drives much of the pathology of tuberous sclerosis and other mTORopathies67, DL001 and related molecules may be of clinical use in safely treating these disorders.Here, we determined that DL001 is an effective mTORC1-specific rapalog both in cell culture and in vivo, and we demonstrate for the first time that the negative side effects of rapamycin and its analogs, including metabolic disruption and immunosuppression, may be avoided in whole or in part by a compound that specifically targets mTORC1. Importantly, as DL001 is an FKBP12-dependent rapalog, its effects on the rapamycin-resistant functions of mTORC120, the only parameter directly linked to the appearance of adverse events in clinical studies of rapalogs is the trough concentration43. We estimate that the half-life of DL001 is about 60% of that of rapamycin, with a near-trough concentration (Cmin) approximately 25% lower. Importantly, even this reduced near-trough blood level of DL001 is substantially higher than blood levels of rapamycin that we have previously demonstrated to be sufficient to inhibit mTORC2 in vivo44. The minor differences in blood levels of rapamycin and DL001 we have observed are therefore not adequate to explain the in vivo metabolic differences we have observed with respect to mTORC2-mediated side effects, particularly given the potent differences in mTORC2 specificity we observed between rapamycin and DL001 in cell culture. However, we note that the concentration of DL001 or its metabolism in different tissues may contribute to our observation that DL001 appears to more effectively suppress mTORC1 signaling in certain tissues than in others. As rapalogs are distributed extensively in peripheral tissues, including key metabolic tissues, conducting a full analysis of DL001 pharmacokinetics and metabolism in both blood and in specific tissues will be important to help advance our understanding of this molecule.An important limitation of our study is that we have not conducted detailed pharmacokinetic studies to characterize the metabolism and elimination of DL001. While we have previously suggested that rapalogs that are eliminated more rapidly may have reduced (but still significant) side effects as a result of decreased mTORC2 inhibitionImportant work remains to be completed prior to the clinical use of DL001 and related molecules, including pre-clinical studies in disease models, a direct assessment of the effects on immune system function, and a direct comparison of the side effects of DL001 or related molecules with rapalogs such as everolimus, which is FDA-approved to treat specific manifestations of TSC, in both male and female mice. While unanswered questions remain, including the impact of DL001 and related molecules on other rapamycin-associated side effects, the exact molecular mechanisms responsible for the reduced impact of DL001 on mTORC2 activity, and the ability of these molecules to delay age-related diseases and extend healthspan, our results demonstrate for the first time that highly selective pharmacological inhibition of mTORC1 can be achieved in vivo and that this avoids many of the side effects associated with long-term rapamycin treatment.−1) and CD8  were from Tonbo; antibodies to CD25  and FOXP3  were from eBioscience, and antibodies to CD11a , and CD3  were from BD. Protease and phosphatase inhibitor cocktail tablets were from Fisher. Other chemicals were purchased from Sigma unless noted. Glucose measurements were performed using a Bayer Contour blood glucose meter and test strips. Mouse Insulin ELISA kits were purchased from Crystal Chem. Rapamycin was purchased from LC Labs. 2.0 mL Tough Tubes with Caps (13119–500) and 1.4 mM ceramic beads (13113-325), were purchased from Mo-Bio Laboratories, Carlsbad, CA.For western blotting, antibodies to phospho-AKT S473 (4060), AKT (4691), phospho-p70 S6 kinase (9234), p70 S6 kinase (2708), phospho-S6 ribosomal protein (2215 or 5364), S6 ribosomal protein (2217), p-4EBP1 S65 (9451), p-4EBP1 T37/S46 (2855), total 4EBP1 (9452), p-S757 ULK1 (14202), ULK1 (8054), mTOR (2792), RICTOR (2140), HSP90 (4877), β-tubulin (2128S), FKBP51 (12210S), and p-PKA substrate (9621) were from Cell Signaling Technology; β-actin (A2228) was from Sigma; ATGL (SC-365278) was from Santa Cruz; and FKBP12 (ab2918) was from Abcam. All antibodies for western blotting were used at a dilution of 1:1000, except that p-T389 S6K1 was used at a dilution of 1:500 for the experiments shown in Fig. 1H and 13C NMR, 2D Heteronuclear Multiple Bond Coherence (HMBC) and COSY Correlation NMR  ppm 6.44 , 6.39 , 6.26–6.19 , 6.15–6.08 , 5.43 , 5.11 , 5.09 , 4.98 , 4.95–4.92 , 4.88 , 4.42 , 4.26 , 4.04–3.97 , 3.95 , 3.71–3.59 , 3.46–3.40 , 3.34–3.11 , 3.05 , 2.77 , 2.49–2.42 , 2.34 , 2.28–2.16 , 2.13–2.08 , 2.07–1.99 , 1.89–1.75 , 1.74 , 1.69–0.80 , 0.79–0.70 ; 13C NMR : ppm 213.5, 208.4, 199.3, 176.9, 169.7, 167.5, 139.9, 139.8, 138.3, 132.9, 130.9, 127.4, 124.8, 99.5, 82.7, 78.1, 76.5, 74.1, 69.7, 66.6, 55.9, 51.3, 45.7, 44.1, 40.6, 40.2, 39.9, 39.1, 35.8, 35.7, 35.5, 35.2, 33.8, 32.2, 30.8, 30.2, 26.9, 26.7, 25.0, 22.1, 21.0, 16.1, 15.2, 14.1, 13.8, 10.8; LC/MS (m/z): [M]+ calculated for C49H75NO12, 869.5 g mol−1; found 887.7 as [M + NH4]+ and 892.7 as [M + Na] +.The chemical purity and identity of DL001 was determined through Liquid chromatography–mass spectrometry, spectrographic analysis using All animal procedures were performed in complicance with institutional guidelines and all relevant ethical regulations for animal testing and research. Animal studies conducted at the Buck Institute for Research on Aging were approved by the Institutional Animal Care and Use Committee (IACUC) at the Buck Institute for Research on Aging, Novato, CA. Animal studies conducted at WSM VA Hospital were approved by the IACUC of the William S. Middleton Memorial Veterans Hospital, Madison, WI.−1 rapamycin, 13 mg kg−1 DL001, or vehicle every other day for 3 weeks (4 mice/group). Following the last injection, mice were fasted overnight. Mice were injected with insulin (0.75 U kg−1) 15 min prior to dissection and tissues were immediately frozen in liquid nitrogen. Our analysis of mTORC1 and mTORC2 signaling in the tissues of vehicle and rapamycin-treated mice were previously reported30.All animal care and experimental procedures performed at the Buck Institute for Research on Aging were approved by the IACUC at the Buck Institute for Research on Aging. In these experiments, the results of which are described in Fig. −1 rapamycin or 12 mg kg−1 DL001 was administered every other day. Following the last injection, mice were fasted overnight for 16 h. Mice were injected with insulin (0.75 U kg−1) 15 min prior to dissection, and tissues were immediately frozen in liquid nitrogen.Animal studies conducted at WSM VA Hospital were approved by the IACUC of the William S. Middleton Memorial Veterans Hospital. In these experiments, the results of which are described in Figs. g for 15 min at 4 °C. The supernatants were collected and protein concentration was determined using the DC protein assay (Biorad). Equal amounts of protein were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane using the Invitrogen Nu-Page system . Western blot analysis was then performed.Frozen tissues collected at the Buck Institute were homogenized using the Omni TH homogenizer  on ice in RIPA buffer , 50 mM Tris (pH 8.0), Protease inhibitor cocktail (Roche), phosphatase inhibitor 2, 3 (Sigma)), and then centrifuged at 17,000 28. Protein concentration was determined by Bradford (Pierce Biotechnology). Twenty micrograms protein was separated by SDS-PAGE on 8%, 10%, or 16% resolving gels . Proteins were then transferred to PVDF membrane (Millipore). Immunoprecipitation of RICTOR was performed using flash-frozen liver lysed in cold CHAPS buffer68. Following protein lysis and determination of protein concentration, 1 mg of protein lysate in 500 µL (2 µg µL−1) was precleared with 20 µL of protein G agarose beads, and then incubated with 2 µL of RICTOR antibody (1:250) overnight. Following antibody incubation, 25 µL of protein G agarose beads were added and incubated at 4 °C for 3 h. Beads were then washed to removed unbound protein, and boiled in 2× loading buffer prior to SDS page analysis. Imaging was performed using a GE ImageQuant LAS 4000 imaging station. Brain tissue was analyzed by Aeonian Pharmaceuticals using a similar protocol and imaging was performed using a Biorad ChemiDoc MP imager.Frozen tissues collected at UW-Madison, as well as cells and tissues, were lysed in cold RIPA buffer supplemented with phosphatase inhibitor and protease inhibitor cocktail tablets . Tissues for western blotting other than brain were lysed in RIPA buffer using a FastPrep 24  with bead-beating tubes and ceramic beads (Mo-Bio Laboratories), and then centrifugedRaw western blot images are provided in the Source Data file. Quantification of all blots was performed by densitometry using NIH ImageJ software. Approximate molecular weights, based on the expected molecular weight and verified using molecular weight standards run on each gel, are indicated to the right of each immunoblot.l-glutamine  and cultured at 37 °C under an atmosphere of 95% air and 5% CO2. For AlphaLISA experiments, cells were seeded in 96-well plates for 24 h and treated at various concentrations of rapamycin or DL001 (from approx. 8 fM to 10 μM) for 24 h using two methods: for examination of mTORC1 signaling, the assay was performed in the continuous presence of serum; for examination of mTORC2 signaling, cells were (1) treated with the compounds for 8 h in growth media containing 10% serum; (2) the media was then replaced by serum-free media containing the respective drug concentration and incubated for an additional 16 h; and (3) immediately prior to cell lysis, cells were treated with 12% serum for 15 min Cells were harvested by lysis in the buffer supplied with the AlphaLISA kit. mTORC1 inhibition was then determined using the AlphaLISA SureFire kit for Phospho-S6 Ribosomal Protein (Ser240/244) . mTORC2 inhibition was determined by the AlphaLISA SureFire kit for AKT 1/2/3 (S473) . Cells from plates were lysed using 50 μL of the lysis buffer and incubated for 10 min at room temperature while shaking. Four microliters of cell lysates were then analyzed for 2 h at room temperature according to the manufacturer’s protocol. The donor mix was then added and the resulting solution was incubated for 2 h at room temperature. AlphaLISA signal was read on a Fusion-Alpha FP HT (Perkin Elmer). Percent inhibition was calculated by comparison to the highest inhibition value obtained in the response–concentration curve. IC50s were calculated using Prism software. All IC50 experiments were conducted in triplicates with rapamycin and vehicle controls.PC3 cells were maintained in F12K media  supplemented with additional 10% fetal bovine serum (FBS) , 1% Penicillin/Streptomycin , and 2 mM FKBP12 or FKBP51 or a nonspecific control were thawed from Kennedy laboratory stocks30 and reselected in puromycin prior to experimentation. PC3 and HepG2 cells were cultured in DMEM  containing 10% FBS  and 1% penicillin–streptomycin . AML12 cells were cultured in DMEM/F-12 media  with 10% FBS and 1% penicillin–streptomycin.PC3 (CRL-1435), AML12 (CRL-2254), and HepG2 (HB-8065) cells were purchased from the American Type Culture Collection (ATCC). PC3 cells transduced with lentivirus expressing shRNA against Tsc1, and paired Tsc1+/+ cells, were the kind gift of Dr. David Kwiatkowski, and were grown in DMEM containing 10% FBS. For signaling and gene expression experiments, cells were cultured for 24 h in serum-free DMEM containing the specified concentration of rapamycin or DL001, or an equal volume of DMSO (vehicle). For gene expression, cells were harvested and RNA was extracted, cDNA was synthesized, and qPCR was performed69. Mouse qPCR primer sequences for the genes examined have been utilized previously61 and are listed here: Pdk1: F: ggcggctttgtgatttgtat, R: acctgaatcgggggataaac; Pfkp: F: aggagggcaaaggagtgttt, R: ttggcagaaatcttggttcc; Mvk: F: gggacgatgtcttccttgaa, R: gaacttggtcagcctgcttc; Sc5d: F: ccaaatggctggattcatct, R: gtccacagggtgaaaagcat; Ascl3: F: ggggctggaacaattacaga, R: atagccaccttcctcccagt; Scd1: F: ctgacctgaaagccgagaag, R: gcgttgagcaccagagtgta.Immortalized mouse embryonic fibroblasts lacking −1 glucose or 2 g kg−1 pyruvate. For glucose and pyruvate tolerance tests, small blood samples were taken from a tail vein nick at time intervals and read using a Bayer Contour blood glucose meter and test strips. For determination of fasting glucose and insulin, blood glucose levels were read using a glucometer and then 50 μL of blood was collected into a EDTA tube immediately prior to and 15 min following glucose administration. Insulin levels were determined using a Mouse Insulin ELISA kit .Mice were fasted overnight for 16 h and then injected with either 1 g kgg for 10 min, cells were resuspended in the same buffer, and recentrifuged. The splenocytes were suspended in PBS and 0.1% BSA with Ca2+ and Mg2+, and incubated with approximately 100 units of DNAase (Sigma). Splenocytes were then filtered through a 40 μM filter and red blood cells were lysed. Splenocytes were then centrifuged, suspended in PBS with 0.1% BSA, and brought to the UWCCC Flow Cytometry Lab for immunostaining and flow cytometry on a BD LSRII . Data were collected using BD FACSDiva, Version 8.0 and analyzed with FlowJo X, Version 10.0.7r2 . An example of the gating strategy used to analyze the flow cytometry data is shown in Supplementary Fig. Splenocytes were prepared following a procedure from Life Technologies. Briefly, spleens were collected in a buffer containing PBS, 0.1% bovine serum albumin (BSA), and 0.6% Na-citrate, and then macerated through a 70 μM filter using a syringe plunger. Following centrifugation at 3002, 50 mM NaF, 50 mM b-glyceradelhyde phosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate) with addition of protease and phosphatase inhibitor (Thermo Fisher Scientific). Cells were then sonicated and supernatant was collected following centrifugation. Samples were then prepared for quantitative proteomic analysis by the Proteomics Facility of the University of Wisconsin Biotechnology Center. Briefly, peptides from each of the 4 Vehicle, 3 Rapamycin-, and 3 DL001-treated samples were digested with trypsin, the peptides were derivatized separately with isobaric mass tags , and the samples were pooled. Following offline fractionation, four high-pH fractions were analyzed using shallow, 4-h gradients by nanoflow HPLC on an Orbitrap Elite (ThermoFisher Scientific). Database searching and quantitation was performed using Proteome Discoverer 2.2 (ThermoFisher Scientific). Peptides with significant differences between treatment groups were identified using the limma R package70 and the resultant p values were then FDR-adjusted using q values. Differentially affected KEGG and Reactome categories were identified by analyzing significantly altered proteins (q < 0.05) using gProfiler .PC3 cells were grown in 10% DMEM with FBS and treated with 100 nM rapamycin or 100 nM DL001 for 24 h. Cells were washed with cold PBS and harvested in 8 M Urea buffer .Statistical analysis was conducted using Prism 7 (GraphPad Software). Significance was tested by a two-sided Student's Further information on research design is available in the Supplementary InformationReporting SummarySource Data


FXN binds the NFS1-ISD11-ACP-ISCU complex (SDAU), to activate the desulfurase activity and Fe-S cluster biosynthesis. In the absence of FXN, the NFS1-ISD11-ACP (SDA) complex was reportedly inhibited by binding of recombinant ISCU. Recent studies also reported a substitution at position Met141 on the yeast ISCU orthologue Isu, to Ile, Leu, Val, or Cys, could bypass the requirement of FXN for Fe-S cluster biosynthesis and cell viability. Here, we show that recombinant human ISCU binds zinc(II) ion, as previously demonstrated with the E. coli orthologue IscU. Surprisingly, the relative proportion between zinc-bound and zinc-depleted forms varies among purification batches. Importantly the presence of zinc in ISCU impacts SDAU desulfurase activity. Indeed, removal of zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SDA alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SDA. Taking into consideration the inhibition of desulfurase activity by zinc-bound ISCU, we characterized wild type ISCU and the M140I, M140L, and M140V variants under both zinc-bound and zinc-depleted conditions, and did not observe significant differences in the biochemical and biophysical properties between wild-type and variants. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SDA complex. This study therefore identifies an important regulatory role for zinc-bound ISCU in modulation of the human Fe-S assembly system in vitro and reports no ‘FXN bypass’ effect on mutations at position Met140 in human ISCU. Furthermore, this study also calls for caution in interpreting studies involving recombinant ISCU by taking into consideration the influence of the bound zinc(II) ion on SDAU complex activity.Human  •Recombinantly expressed human ISCU varies in amount of bound zinc between batches.•Zinc stabilizes ISCU, but inhibits NFS1 desulfurase activity.•A frataxin bypass in the human Fe-S complex for ISCU variants was not observed. The methylene blue assay was then used in the presence of buffer containing a serial dilution of zinc concentrations and plotting [inhibitor] vs. response.Sulfide production, due to cysteine desulfurase enzyme activity, was measured using the methylene blue colorimetric assay as described previously ,38. The 2.3E. coli ACP, and will hereafter be referred to as the NFS1-ISD11-ACP-ISCU (SDAU) complex.Reconstitution of the recombinant NFS1-ISD11-ISCU complex was mediated by co-expression of all three proteins in a poly-cistronic fashion, where only ISD11 is His-tagged. Expression and affinity chromatography were carried out as described above for single proteins. Complex-containing fractions eluted from affinity step were pooled, and analytical gel filtration was performed using a Dionex Ultimate ™ 3000 system. The Sepax SRT SEC-300 7.8 × 300 mm column was pre-equilibrated in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol and 2 mM TCEP, and run at 0.5 mL/min. Complex formation was confirmed by TCA-precipitation followed by SDS-PAGE analysis. Recombinant NFS1-ISD11-ISCU complex co-purified with 2.42. Thermal unfolding was carried out using a linear thermal ramp  and unfolding midpoint  was determined from changes in the emission wavelengths of tryptophan and tyrosine fluorescence at 330 and 350 nm.Miniaturized DSF (nanoDSF) was performed in 10 μL capillaries using the Prometheus device (NanoTemper Technologies) that uses excitation at 280 nm to measure emission from tryptophan and tyrosine residues at two wavelengths: 330 (non-polar environment emission) and 350 nm (polar environment emission). Each capillary consists of ISCU protein at 0.8 mg/mL in buffer containing 50 mM HEPES pH 7.5, 200 mM NaCl and 10 mM DTT, supplemented with either 300 μM EDTA or 100 μM ZnCl2.52 and then buffer exchanged into 10 mM Tris pH 7.5 (the pH was adjusted with phosphoric acid) and 50 mM NaF before acquiring data. Data points were collected with a resolution of 0.2 nm, an integration time of 1 s, and a slit width of 1 nm. Each spectrum shown is the result of nine averaged consecutive scans, from which buffer scans were subtracted.Circular dichroism (CD) spectra were recorded on a J-815 spectropolarimeter (JASCO) at 20 °C with a scan speed of 100 nm/min using 0.1 cm pathlength quartz cells from Starna Scientific UK. The concentration of ISCU protein was at 0.1 mg/mL in buffer which consisted of 50 mM HEPES pH 7.5, 200 mM NaCl, 5 mM DTT and either 100 μM EDTA or 30 μM ZnCl2.62 or 300 μM EDTA. 50 μL of 1 mg/mL of biotinylated ISCU was diluted to 650 μL and loaded to the streptavin coated sensors. The concentration for SDA used ranged from 10 μM to 1.56 nM. Measurements were performed using a 300 s association step followed by a 300 s dissociation step on a black 384-well plate with tilted bottom (ForteBio). The baseline was stabilized for 30 s prior to association and signal from the reference sensors was subtracted. A plot of response vs. [NFS1] was used for Kd determination using one site-specific binding fit in GraphPad Prism (GraphPad Software).BLI experiments were performed on a 16-channel ForteBio Octet RED384 instrument at 25 °C, in buffer containing 50 mM HEPES pH 7.5, 200 mM NaCl, 5% Glycerol, 2 mM TCEP, 5 mM DTT, 0.5 mg/mL BSA, which is further supplemented with either 100 μM ZnCl33.1M140I, ISCUM140L and ISCUM140V) in order to compare their biochemical and biophysical properties.In frataxin-deleted yeast, a substitution on the scaffold protein Isu at position Met141 to either Cys, Ile, Leu, or Val, corrected the loss-of-frataxin phenotypes and rescued cell viability ,34. In tWT 14604 Da), and the other with an additional mass of 64 Da (14668 Da), which suggests a subset of ISCU is zinc-bound form with one zinc(II) ion per ISCU monomer   . The met3.22 (50 μM) (2-containing buffer). The addition of ISCU to NFS1-ISD11-ACP (SDAU) increased the desulfurase activity by ∼1.5 fold when assayed in EDTA-containing buffer. To our surprise, when SDAU was assayed in ZnCl2-containing buffer, desulfurase activity was almost completely abolished. The activity of SDA alone, without ISCU, was not affected by increasing ZnCl2 concentration (2-containing buffer (30-fold increase from SDAU) , which generates and transfers the inorganic sulfur to ISCU, in a reaction activated by FXN protein ,39. We t (50 μM) A. SDA dintration , indicatom SDAU) .C138A can still be inhibited in the presence of zinc (C69A-C95A. Therefore Cys138 is not involved in zinc(II) ion coordination. Altogether, our results strongly indicate that the inhibition of SDA desulfurase activity by ISCU in vitro, an observation previously reported by various groups [The Cys138 residue on ISCU has been shown to be the persulfide acceptor from NFS1 . We test of zinc B. We furs groups ,39,43, i2 concentrations, prior to assaying for desulfurase activity  > ISCUM140L (i.e. SDAUM140LF complex) > ISCUM140V (i.e. SDAUM140VF complex) > ISCUM140I (i.e. SDUM140IF complex)  .4E. coli IscU was previously shown to exist in these two interconvertible conformations that can be influenced by interaction with zinc [2Z7E) [3LVM) [2KQK) [1WFZ, 1RP9P, 1XJS, 1SU0) [E. coli apo-IscU (PDB: 2L4X) undergoing a helix-to-coil rearrangement in the helix containing the sulfur acceptor residue Cys106  [In this study we set out to characterize recombinant human ISCU wild-type and variant proteins for their functional interaction with SDA and effect on NFS1 activity. Our biophysical characterization of human ISCU is consistent with the existence of two conformational states, one being structured and one disordered around the Fe-S cluster binding site . In factith zinc ,25,26. Sc [2Z7E) , in comp) [3LVM) ,29, carr) [2KQK) , or, mosS, 1SU0) ,31. The  Cys138) .in vitro [Previous publications have shown that ISCU, in the absence of frataxin, inhibits the NFS1 desulfurase activity in vitro ,39,45. Oin vitro , reveal in vitro . This woin vitro .E. coli, a phenomenon that may have occurred during sample preparation for previous in vitro studies of recombinant E coli IscU and human ISCU [The zinc(II) ion observed in our ISCU samples is likely derived from recombinant expression in man ISCU ,39,45. Sman ISCU ,39,45 shman ISCU , with thman ISCU . Therefoin vitro [E. coli, were essentially indistinguishable from wild-type with regards to zinc binding, secondary structure, thermostability, and complex formation. Importantly, under both zinc-replete and zinc-depleted buffer conditions tested, there is negligible desulfurase activity for the frataxin-free SDAU complex with either ISCU wild-type or Met140 variants. The SDAU complexes containing ISCU variants retained the ability to be activated by frataxin under both buffer conditions tested but observed a trend in desulfurase activity with the elongated and less branched side chains being most active  ion, can directly influence NFS1 activity. Uncontrolled variability in zinc content and its impact on reported complex activities between studies, and even between batches of protein from within one study, may be an important factor that contributed to the discrepancy between studies. In summary, the complexity of the multi-component Fe-S assembly system and the level of zinc(II) ion co-purified with ISCU need to be taken into account in future experimental design when exploring human SDAU/SDAUF complex activity.Our inability to observe a FXN bypass effect with the human ISCU suppressor mutants may be due to differences between the yeast and human proteins, or due to a number of factors including assays and reagents, and potentially variable levels of zinc(II) ion that were not controlled by other studies ,38. For


Tityus serrulatus scorpion venom (TsV) is composed of diverse molecules, including toxins that induce a catecholamine storm and mediate classical symptoms of scorpion envenomation. However, the same toxins promote an intense inflammatory response coordinated by innate immune cells, such as macrophages, contributing significantly to the lung edema and mortality caused by TsV injection. Macrophages sense TsV via innate immune receptors, including TLR2, TLR4, and CD14 that promote inflammation and mortality via PGE2/cAMP/PKA/NF-κB/IL-1β axis. The scavenger receptor CD36 also recognizes TsV, but in contrast to the other receptors, it drives the production of leukotriene B4 (LTB4). This lipid mediator operates via BLT1 receptor to reduce cAMP production and consequently IL-1β release, which results in resistance to fatal outcomes of experimental scorpion envenomation. EP80317 is an hexapeptide that serves as a ligand for CD36 and features protective effects under conditions such as atherosclerosis and vascular inflammation. In this study, we evaluated the effects of EP80317 treatment during experimental scorpion envenomation. EP80317 treatment suppressed mouse peritoneal macrophage production of IL-1β, IL-6, tumor necrosis factor (TNF-α), CCL3, and PGE2in vitro. EP80317 treatment also boosted the production of LTB4 and IL-10 in response to TsV. Importantly, EP80317 restrained lung inflammation and mortality caused by TsV in vivo. Taken together, these data indicate a strong therapeutic potential of EP80317 as a supportive treatment to control inflammation induced by scorpion envenomation.Over 1 million cases of scorpion stings are estimated every year, whereas current treatment is limited to antivenom serum combined with supportive therapy.  Tityus serrulatus contribute significantly for this scenario, whose venom (TsV) is composed by a cocktail of bioactive compounds (2 (PGE2) and leukotriene B4 (LTB4)  . The cAMP-dependent protein kinase A (PKA) is activated and phosphorylates the nuclear factor-κB (NF-κB), potentiating IL-1β release  , 2013. M (IL-1β) , 2018b.  release . IL-1R s release . In star release . This einomation , 2018b. nomation .Recommended therapy is limited to antivenom serum, and depending on the severity, the serum is combined with supportive treatment such as prazosin, ionotropic agents, atropine, vasodilators, and benzodiazepines . HoweverT. serrulatus scorpions were maintained at the vivarium of the Ribeirão Preto Medical School, University of São Paulo. TsV was obtained by electrical stimulation, lyophilized, weighted, and stored at −80°C. Before experiments, TsV was diluted in PBS, filtered, and tested for LPS contamination as described . Six- to eight-week-old C57BL/6 female or male mice were weighed before experiments under all conditions. Animals were injected with vehicle (PBS) or a lethal  or excessive  dose of TsV . Followi5 cells/well) were cultured at 37°C, 5% CO2 for 2 h. After this period, PMs were incubated with EP80317 (100 nM) or vehicle (PBS) for 2 h, followed by stimulation with TsV (50 μg/ml) for 24 h at 37°C, 5% CO2. Culture supernatants were collected and stored at −20°C for further analysis.Resident peritoneal macrophages (PMs) were isolated from naïve C57BL/6 mice by injecting 3 ml of PBS into the abdominal cavity and massaged for 1 min. Peritoneal fluid then collected using a syringe with a needle inserted into the inguinal region as described previously . PMs , after lipid extraction and purification from cell culture supernatants or lung homogenates using Sep-Pak C18 cartridges , as described previously (2−) was measured as an indicator of NO production by obtaining a standard curve using serial NaNO2 dilutions, as described , and IL-10 using enzyme-linked immunosorbent assay (ELISA) kits . Quantifications of LTBeviously . Total pescribed . cAMP waescribed . Cell cuescribed .p values <0.05 were considered significant.Data were analyzed with GraphPad Prism 5.0 software , using one-way ANOVA followed by Bonferroni’s multi-comparison test or log-rank test. Adjusted via CD36  and treated with vehicle or EP80317  at 0.5 and 2 h after the experimental envenomation. TsV injection in mice causes significant perturbations in the lung . This suggests that EP80317 might restrain the production of inflammatory mediators by suppressing NF-κB activity. As expected, exposure to EP80317 significantly reduced NF-κB phosphorylation induced by TsV stimulation  via COX-2 pathway in macrophages  and carried out in accordance with the ethical principles for animal research adopted by the Sociedade Brasileira de Ciência em Animais de Laboratório.KZ designed and performed the experiments. KZ and LG analyzed the data and wrote the manuscript. EA and KB provided the scorpion venom. EA, KB, SM, and HO discussed the data. SM and HO provided the EP80317. LF conceived and supervised the study. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


At effective PDT concentrations, the predominant type of induced cell death was apoptosis. Overall, our results show that the PEGylated derivatives presented have significant potential as novel PSs with substantially augmented phototoxicity for application in the PDT of cervical, prostate, pancreatic and breast cancer.Purpurin 18 derivatives with a polyethylene glycol (PEG) linker were synthesized as novel photosensitizers (PSs) with the goal of using them in photodynamic therapy (PDT) for cancer. These compounds, derived from a second-generation PS, exhibit absorption at long wavelengths; considerable singlet oxygen generation and, in contrast to purpurin 18, have higher hydrophilicity due to decreased logP. Together, these properties make them potentially ideal PSs. To verify this, we screened the developed compounds for cell uptake, intracellular localization, antitumor activity and induced cell death type. All of the tested compounds were taken up into cancer cells of various origin and localized in organelles known to be important PDT targets, specifically, mitochondria and the endoplasmic reticulum. The incorporation of a zinc ion and PEGylation significantly enhanced the photosensitizing efficacy, decreasing IC Chlorins are natural photosensitive chlorophyll derivatives containing twenty π electrons in the aromatic ring. Various modified substructures derived from their basic core have been discovered within the plant kingdom ,2,3,4. ODuring the PDT treatment of cancer, ubiquitous oxygen in the triplet state turns into highly reactive singlet oxygen  that triChlorins possess optimal properties for use in PDT but are rather hydrophobic and, thus, aggregate in aqueous media, limiting their application. Consequently, various chemical modifications of chlorin-based PSs have been investigated with the aim of improving their physico-chemical characteristics: core metalation ; PEGylat1, 1  increased in direct dependence to concentration.To verify the mechanism of cell death induced by the tested PSs, MCF-7 cells were treated with compounds ing with ,62, but ll cells . At 5 µMo et al. . At lowe1 rose to 52% as the concentration rose to 1 µM  triggered apoptosis in 61% and 68% of cells, respectively. 1–4 (1 µM).More interestingly, after photoactivation, the proportion of apoptotic cells among the MCF-7 cells treated with compound  to 1 µM . Under t1 (light dose of 1 J·cm−2) predominantly underwent apoptosis, while at higher concentrations (>2 µM) necrosis was prevalent. Tsai et al. showed that not only the concentration and the structure of a PS influences cell death type but also the light dose applied; for instance, at a light dose of 8 J·cm−2, 5-aminolevulic acid  induced the apoptosis of MCF-7 cells while a doubled light dose induced necrosis propanoato(2-)}zinc36{3-−; To a solution of compound Tert-butyl{15-heptacosa-1(24),2(7),6(27),9,11(26),12,14,16,18,20-decaen-23-yl-κ4N8,N24,N25,N26]-13-oxo-3,6,9-trioxa-12-azapentadecan-1-yl}carbamatato(2-)]zinc+; To a solution of compound N-(2-{2-[2-(2-Aminoethoxy)ethoxy]ethoxy}ethyl)-3-heptacosa-1(24),2(7),6(27),9,11(26),12,14,16,18,20-decaen-23-yl-κ4N8,N24,N25,N26]propanamidato(2-)]zinc+, 823.27637 [M+Na]+; Purpurin derivative 1–4 in DMEM + FBS was evaluated using 9,10-anthracenediyl-bis(methylene)dimalonic acid  as a compound reactive with singlet oxygen. A photosensitizer Rose Bengal  was used as a reference. A stock solution of RB was prepared in water. Stock solutions of compounds 1–4 were prepared by dissolving a solid in DMSO. Working solutions were prepared by diluting the stock solutions with air-saturated solvent which was either phosphate buffered saline  or DMEM + FBS. Concentrations of the working solutions of RB and compounds 1–4 were 1.34 × 10−5, 3.19 × 10−4, 3.34 × 10−4, 2.99 × 10−4 and 3.12 × 10−4 M, respectively. A stock solution of AB was prepared by dissolving solid substance in DMSO (4.87 × 10−3 M). Two different amounts of RB and compounds 1–4 (25 and 50 µL of stock solution) and 15 µL of AB stock solution were mixed with 1 mL of solvent in a plastic cuvette . Two replicates of the same PS concentration were prepared. The first was kept in the dark, the second was illuminated. Absorption spectra of both solutions (illuminated and kept in the dark) were collected against the recorded baseline  after 0, 10, 20, 30 and 40 min All experiments were done in duplicates.Singlet oxygen production by compounds −2. Relative spectral emission intensity of the illuminationg source was measured using spectrofluorometer Fluoromax 2 . The lamp illuminated a plate made of barium sulfate positioned on front surface accessory of the spectrofluorometer and its spectrum was recorded as reported by Pavlíčková et al. [A 150 W halogen lamp with an edge-pass filter (Panchromar filter (58 mm), VEB Glastechnik Lommatzsch, Lommatzch, Germany) that transmitted light at wavelengths longer than 500 nm was used for illumination of solutions. The fluency rate at the cuvette was 5 mW·cmEmission correction function supplied by manufacturer was applied to the measured spectra. The same correction factor was used to correct spectra of quinine sulfate solution in sulfuric acid and results corresponded well with standard spectra by Velapoldi and Tønnesen .A in solutions was estimated, and then, relative exposure IA(t) for solution illuminated for time t was calculated asA is absorbance of a tested compound at wavelength λ and time τ from the beginning of illumination. Wavelength integration limits were λ1 = 450 nm and λ2 = 800 nm, and trapezoidal rule for integration was used for absorbance in intervals between spectral measurements. Chemical photodynamical efficiency γx of a photosensitizer was evaluated supposing single exponential dependence of relative AB concentration on light exposureData were evaluated using MS Excel 2010  with a procedure described in detail by Pavlíčková et al.  Briefly,RB in PBS and DMEM+FBS, respectively, as a standard  usin+), PC-3 (prostate carcinoma), U-2 OS (osteosarcoma), MIA PaCa-2 (pancreatic adenocarcinoma), HeLa  and HaCaT (keratinocytes). Unless otherwise specified, the cells were cultured in DMEM medium GlutaMAX  supplemented with 10% FBS . Cells were maintained at exponential phase of growth under standard physiological conditions at 37 °C in humidified atmosphere with 5% CO2.In our study, we used the following human cell lines: MCF-7 (breast carcinoma), LNCaP  dishes for live-cell imaging  and left to adhere for 16 h. Then, the cells were washed with PBS and incubated with compounds 1–4  dissolved in complete cell cultivation medium without phenol red at 37 °C for 3 and 24 h. After that, the cells were washed twice with PBS and the medium was exchanged for phenol-red free DMEM. Stock solutions of compounds 1–4 were prepared in dimethylsulfoxide (DMSO) fresh before the experiments. The final concentration of the vehicle (DMSO) in cell culture medium did not exceed 0.02%.The number of 1 × 101–4, the cells were seeded and treated with the tested compounds as described in 4 particles per cell). All the markers used from stock solutions as supplied by the manufacturer .In order to determine the intracellular localization of compounds 2 atmosphere. The images were acquired by an inverse fluorescence microscope Olympus IX-81 operated by xCellence System  using a high-stability 150 W xenon arc burner and EM-CCD camera C9100-02 . Living cells were analyzed under physiological conditions  by a 60× oil immersion objective  with the numerical aperture of 1.4. All images were deconvolved using xCellence 2D deconvolution module and background-corrected.The intracellular localization of purpurin 18 derivatives was studied by real-time live-cell fluorescence microscopy at 37 °C and in 5% CO5 PC-3 cells was seeded in DMEM+FBS onto glass-bottom MatTek dishes . After 16 h, compounds 1–4 at 1 µM concentration were added to the cells in DMEM with 10% FBS and incubated for another 24 h. Then, the medium was removed, the cells were washed with PBS, which was replaced with FluoroBrite DMEM media  and subjected to fluorescence microscopy. The fluorescence emission intensity was measured in cells in at least 10 view fields at 600× magnification. The images were taken at the same exposition time (600 ms) and light intensity (100%). Then, the data were evaluated using ImageJ 1.52a software by an equation:To calculate corrected total cell fluorescence (CTCF), the number of 1 × 10CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings).1–4 was evaluated in vitro by WST-1 viability assay  similarly as in Rimpelová et al. [−2). The second half of the samples was kept in the dark. Next, 24 h after illumination, the cell culture medium was removed and 100 µL of fresh phenol red-free DMEM with 4 µL of WST-1 was added. After 2-h incubation, the absorbance of formed formazan was measured spectrophotometrically at 450 nm (the reference wavelength of 650 nm) using UV–Vis spectrometer (Tecan). The absorbance is directly proportional to the oxidoreductase activity, and thus to the number of metabolically active cells. Cells treated only with cell culture medium and cells treated with a vehicle (DMSO) served as controls. All samples were tested in quadruplicates. The IC50 values were determined (GraphPad Prism 6) as the concentration necessary to kill 50% of cells.Photo- and dark toxicity of compounds á et al. . The WST1–4 (0.1–5 µM) for 24 h. Then, the cells were illuminated for 13 min by a 150-W halogen lamp with an edge-pass filter transmitting wavelengths longer than 500 nm . The cells were incubated at standard cultivation conditions for next 24 h. Afterwards, the cells were harvested by trypsinization, washed in cold PBS and resuspended in annexin-binding buffer followed by labeling with Alexa Fluor® 488 Annexin V and propidium iodide according to the manufacturer’s protocol  as described in Kirakci et al. [Evaluation of the proportion of dead cells was adapted from Vermes et al.  MCF-7 cei et al.  and Ruml1–4 (ligands) were created by CORINA Classic  software. Other modifications of ligands and proteins were done by Maestro  software. To minimize the ligand energy and addition of missing hydrogen atoms, a function “ligand preparation” with the force field OPLS3e was used. The structure of HSA protein with the code of 1N5U was obtained from Protein Data Bank  database. From the structure of HAS 1N5U, molecules of myristic acid, protoporphyrin IX and water were removed. In order to define the binding pocket, we chose four amino acids  present in the binding pocket of HSA and using the function “receptor grid generator”, a small and big box was created with the edge length depending on the docked ligand  [For compounds zerland) ,90.1). Compared to their parent compound, both derivatives (compounds 3 and 4) exhibited improved accumulation in all of the tested cell lines . Live-cell fluorescence microscopy showed that compounds 3 and 4 localized predominantly in the endoplasmic reticulum and mitochondria, which are desired targets for PDT; moreover, compound 4 also localized in lysosomes. Upon illumination, both compounds efficiently generated singlet oxygen production. This corresponded with their good photodynamic activity at nanomolar to micromolar concentrations in all tested cell lines, with the strongest effect detected for compounds 3 and 4 in the LNCaP and HeLa cell lines, respectively. At submicromolar concentrations, the photoactivated compounds 1–4 prevalently induced apoptosis with negligible necrosis. The most efficient apoptosis inducers (61% to 68% of cells in apoptosis) were compounds 3 and 4. In terms of photodynamic therapy, we believe that our PEGylated derivatives have the ability to outperform their parent photosensitizer purpurin 18, application of which is limited by its aggregation. Furthermore, their enhanced water solubility can overcome the high hydrophobicity and, thus, limited bioavailability associated with photosensitizers, such as chlorin, currently used in photodynamic therapy.We have designed, synthesized and presented the biological activity of two PEGylated derivatives of purpurin 18 (compound


Triple-negative breast cancers are particularly aggressive. In vitro cultures are one of the major pathways for developing anticancer strategies. The effectiveness and reproducibility of the drug screenings depend largely on the homogeneity of culture media. In order to optimize the predictive responses of triple-negative breast cancer 3D cell culture models, these works were focused on the development of SUM1315 and MDA-MB-231 cell lines in OptiPASS medium, a new serum-free formulation (BIOPASS). In monolayer cell culture, OptiPASS medium was more suitable for MDA-MB-231 than SUM1315 cell line but maintained cell phenotype and allowed sufficient proliferation. For spheroids produced in OptiPASS, the size monitoring showed a 1.3 and 1.5-fold increase for MDA-MB-231 and SUM1315 cell lines, respectively and viability/mortality profiles were maintained. Spheroids drug sensitivity thresholds were also improved allowing quicker high throughput drug screenings. These results showed the suitability of OptiPASS for 2D and 3D cell cultures of these two triple-negative breast cancer cell lines, with reproducibility of spheroid formation superior to 98%. This opens the way to the common use of this synthetic medium in future preclinical breast cancer research studies. Triple-negative breast cancers (TNBC) are particularly aggressive and of poor prognosis ,2. It isThis discipline is using more and more three-dimensional (3D) cell culture models, as they reproduce more precisely the tumor microenvironment. Indeed, 3D cell cultures produced by liquid overlay or scaffold techniques, allow the formation of pseudo-microtissues also called spheroids or organoids ,14. ThesFor this research, two liquid-overlay 3D cell culture types already developed by our team for drug screening (i) the proliferative MDA-MB-231 spheroid model and (ii) the non-proliferative SUM1315 spheroid model were used. This modeling highlighted that the choice of a proliferative or non-proliferative 3D model seems to be decisive for drug efficacy’s prediction, according to drug’s mechanism of action, i.e., cytotoxic or cytostatic . In this2. Cell culture was processed in 75 cm2 culture flasks  with 15 mL of the appropriate culture medium. For culture of MDA-MB-231 line, the reference medium was the RPMI 1640 medium  supplemented with fetal calf serum  and gentamycin . For the SUM1315 cell line, the reference medium was the Ham’s F-12 medium (Thermo Fisher Scientific), supplemented with 5% FCS, 10 mM HEPES buffer (SIGMA), insulin  and epidermal growth factor . These two respective media were labeled “reference” in experiments with both cell lines. For experiments with OptiPASS medium (BIOPASS), the latter was supplemented with gentamycin (20 μg/mL) and was labeled “OptiPASS” in our experiments. Cells were only cultured before passage number 30 to prevent cell line drift.The triple-negative breast cancer cell lines (TNBC) MDA-MB-231 (ATCC HTB-26) and SUM1315  were maintained in a humidified incubator at 37 °C under 5% CO4 cells per cm2 in 24-well microplates or 6-well microplates  in 2 or 3 mL of culture medium, respectively. For cultures with OptiPASS medium, prior to seeding, wells were coated for 1 h under agitation with 2% Geltrex LDEV-free  diluted in medium.For both cell lines, cells were seeded at least in triplicates for each culture condition, at concentrations of 2 × 10cellular analysis” algorithm of Gen 5.03 software, with the following settings: threshold value = 1200, background = light, min object size = 15 µm, max object size = 50 µm. The proliferation ratio was calculated by dividing the cell number of each well at day X by the number of cells one day after seeding (day one).The cellular carpet of each microplate wells was imaged daily with the Cytation3MV plate reader  coupled to Gen 5.03 software until cells have reached the confluence (day seven). The number of living-adherent cells was then calculated for each culture condition by the “Then, when cells were at confluence, the supernatant containing floating cells (living and dead) was collected. Adherent cells were trypsinized and also collected, allowing the determination of “adherent”, “floating” and “dead” cells proportion of each cell culture condition, by a trypan blue exclusion test.ex/em 570/584 nm) by the metabolically active cells. For this, the cellular carpet of each condition was rinsed in hot D-PBS (37 °C) and incubated with 100 µL of 60 µM resazurin . The quantity of resorufin formed was monitored with Cytation3MV plate reader and presented in the graphs as the metabolic activity of cells ± s.d (standard deviation).Cell metabolic activity was determined by the resazurin test after one, three, five and seven days of culture (confluence) in reference or OptiPASS medium. It is a colorimetric assay involving the reduction of resazurin (non-fluorescent) into resorufin  was realized prior seeding. Twenty-four hours after seeding, cells were fixed for 20 min with a solution of 4% paraformaldehyde  and permeabilized in ethanol 70° at −20 °C for 5 min. Cells were incubated for one hour with a saturation solution (0.1/1% v/w Triton/BSA in PBS) and then for one hour with the primary antibody  or the isotype control . Then, cells were incubated with the anti-mouse secondary antibody  for one hour, and then with 10 µg/mL Hoechst 33258 (Sigma) for nuclear counterstaining. Finally, cells were dried, mounted with ProlongDiamond (Thermo Fisher Scientific) and observed with Cytation3MV plate reader (Dapi and Cy5 cube fluorescent filter cubes). The vimentin expression per cell was quantified from the immunofluorescent staining images obtained with both cell lines and culture conditions. For this, the fluorescent intensity of vimentin was normalized on the cellular positive area of each image (Cytation3MV).SUM1315 and MDA-MB-231 cells cultured with reference or OptiPASS medium were seeded in Ibitreat microplates (IBIDI) at concentrations of 5 × 103D cell culture protocol with TNBC cell lines was processed according to Dubois et al. . Brieflycellular analysis” algorithm of the software with the following settings: threshold value = 10000, background = light, min object size = 180 µm, max object size = 1000 µm.Spheroids formed with the two culture conditions were imaged daily (M = 4X) from day one (day of effective spheroid formation) to day 14, with Cytation3MV coupled to Gen 5.03 software. The size of each spheroid was measured with the “Spheroid viability was monitored after seven and fourteen days of culture  by the live/dead kit , labeling viable cells in green  and dead cells in red . For this, spheroids were harvested and deposited into six-well microplates. Spheroids were rinsed twice with D-PBS (37 °C) and incubated with a solution of 2 µM calcein-AM and 4 µM etdh-1 in D-PBS for 45 min (protected from light), according to supplier’s instructions. Spheroids were then transferred into eight-well uncoated micro slides  and imaged on Cytation3MV plate reader with green fluorescent protein cube (GFP) and Propidium Iodide (PI) fluorescence cube loaded on the device (M = 4X).Spheroids formed in reference medium or OptiPASS medium were treated with 1 µM of epirubicin , for five days. The size of control and 1 µM-treated spheroids was monitored with Cytation3MV for five days, as previously described. The percentage of treated spheroids cell viability at day five was determined with resazurin test, by the ratio of resorufin formed in treated spheroids on control spheroids. These values were then normalized on the percentage of spheroids treated size on control size, at day five. Finally, the viability assay live/dead was performed on treated and control spheroids at day five. The quantification of ethidium homodimer-1 was realized on epirubicin-treated spheroids by the ratio of Etdh1 intensity in treated spheroids normalized Etdh1 intensity in control spheroids (Cytation3MV).n-independent experiments. Every experiment was performed at least in triplicate and then statistically compared using a two-sided student’s t-test. Data comparison was considered statistically significant when the p-value was p < 0.05 (*). Lower p-values were noted in figures as follows: p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****) and p < 0.00001 (*****). Non-significant results were noted as “ns”.Results were expressed as means ± standard deviation (s.d.) or standard error (s.e.m) of In this study, the performances of the serum-free medium OptiPASS were first determined in 2D cell culture on two TNBC cell lines, MDA-MB-231, and SUM1315. For this, cells were cultured with their reference medium  or OptiPASS medium (coated with 2% Geltrex), until confluence. In order to characterize cell behavior, several parameters, such as (i) cell adherence, (ii) cell proliferation, (iii) cell metabolic activity, and (iv) cell morphology, were analyzed in both cell culture media. Additionally, the mesenchymal phenotype status of both cell lines was also determined in each culture medium by vimentin immunostaining .p = 0.71, two-sided student’s t-test), to 3.4 ± 0.8 and 3.0 ± 0.2 at day seven (p = 0.05) for reference medium and OptiPASS medium, respectively. The cell proliferation time course seemed to be similar in reference and OptiPASS medium. In parallel, cell metabolic activity was monitored for the seven days of culture by the resazurin test. It was 0.066 ± 0.007 and 0.052 ± 0.005 at day one (p = 0.0003), 0.181 ± 0.039 and 0.084 ± 0.006 at day three (p = 0.0005) and increased to 0.335 ± 0.078 and 0.366 ± 0.056 at day seven (p = 0.40), for reference and OptiPASS medium, respectively . These results demonstrated that for all analyzed parameters, i.e., cell proliferation rates, cell metabolic activity and the proportion of attached cells/floating/dead cells, similar cell culture performances were detected for OptiPASS and reference media, with MDA-MB-231 cell line.For MDA-MB-231 cell line, the cell proliferation rate was first studied in both culture media by a cell counting experiment using the Cytation3MV plate reader. For this, the ratio of adherent cells at day X on adherent cells at day one (one day after seeding) was calculated a. The prectively b. Then, ectively c. The prectively d. Finallectively e,f showe−6 compared to reference) and 1.9 ± 0.3 at day seven (p = 10−12 compared to reference), in OptiPASS medium (−9) at day three. Then, it increased at day seven for both cell culture media with 0.148 ± 0.019 and 0.067 ± 0.014 (p = 10−6), for reference and OptiPASS, respectively   f. For SU = 0.79) c. Thus, OptiPASS medium performances were then compared to reference medium for the development of 3D cell cultures with MDA-MB-231 and SUM1315 triple-negative breast cancer cell lines. For these experiments, cell lines previously cultured in monolayer with reference or OptiPASS medium were harvested and cultured in 3D condition according to Dubois et al.  with the−17) at day eight for reference and OptiPASS medium, respectively (−13), for reference and OptiPASS medium, respectively  and increased to 856 ± 117 µm and 1137 ± 120 µm  and to 585 ± 82 µm and 961 ± 81 µm at day 14 (p = 10−44) in reference and OptiPASS medium, respectively  and increased to 476 ± 51 µm and 711 ± 77 µm at day eight  (−34). In contrast, epirubicin-treated spheroids size increased slightly to day three with 542 ± 25 µm  and remained stable until day five (p = 0.84). Thereby, for the five days of 1 µM epirubicin treatment, spheroids from both cell culture medium conditions showed significantly decreased sizes compared to their respective untreated controls. This highlights the cytostatic activity of epirubicin in these both cell culture conditions. Then, the percentage of spheroid viability  was determined after five days of treatment with epirubicin  to 659 ± 11 µm  a. After irubicin c, by theirubicin c. Finallirubicin e. The reirubicin f, demonsirubicin f, reveal−14). In parallel, epirubicin treated spheroids size increased until day three with 263 ± 7 µm , but remained stable until day five . Thus, with this cell line, a significant difference in spheroid size between untreated and treated conditions was detected at day five, only when spheroids were cultured in OptiPASS medium (p = 0.0001). This showed the cytostatic action of epirubicin on SUM1315 spheroids only with OptiPASS medium. Then, the analysis of spheroid viability after five days of treatment showed similar percentages of the viability of 84 ± 8% and 91 ± 10% (p = 0.23), for reference treated spheroids and OptiPASS treated spheroids, respectively  b. Similaectively d. Finallectively f and Etdectively h showed Preclinical studies aim to screen new drugs or new treatment strategies. These consist of several steps, including in vitro cell culture modeling. For this, the culture medium plays a crucial role for the metabolism and the growth of cells in culture. As a result, the stability and homogeneity of culture medium components are essential factors for ensuring the proper cell response to drugs in in vitro screenings ,21,22. O®/Matrigel® are produced by EHS controlled cells, extracted and purified by an industrial process [−12) and (iii) cell metabolic activity  were detected in reference medium compared to OptiPASS medium, respectively. Additionally, SUM1315 cells mostly presented an ovoid shape in bright field microscopy. These results suggest that cells cultured with OptiPASS medium may partly not adhere to the Geltrex coating or may detach. This may cause the decreased values of cell proliferation rates and metabolic activity for this cell line, as the cells may have detached prematurely. SUM1315 mesenchymal phenotype was still maintained in OptiPASS medium as depicted with the vimentin staining [Firstly, OptiPASS medium performances were determined in monolayer cell culture by the analysis of cell adherence, cell proliferation, and cell metabolic activity. Then the expression of the mesenchymal phenotype marker vimentin was determined. Indeed, vimentin is the most commonly used marker for the determination of a phenotype change from epithelial to mesenchymal for various models, including breast cancers ,23,24,25 process . Thus, t process . With MDstaining . BesidesSince a few decades, new cell cultures models in three-dimensions (3D cell cultures) have been dramatically developing, as they reproduce more closely tumor’s microenvironment ,29, and In order to use 3D cell cultures produced in OptiPASS medium for therapeutic drug screenings, spheroid sensitivity to epirubicin was determined in OptiPASS and compared to the reference medium. Spheroids were treated with epirubicin, a strong intercalating agent used in conventional chemotherapeutics protocols, presenting high cytostatic (slowed or stopped proliferation rate) and cytotoxic (decrease in viability and/or presence of dead cells) activities. For MDA-MB-231 spheroids treated with 1 µM epirubicin in reference medium, a 2.2-fold decrease in size, as well as a decrease in viability (67%), were detected compared to controls. Similarly, MDA-MB-231 spheroids cultured in OptiPASS medium presented a 1.6-fold size decrease after treatment associated with lowered viability . Therefore, for this cell line, the cytostatic and cytotoxic actions of epirubicin were detected whatever the culture condition (reference or OptiPASS). In contrast, for SUM1315 spheroids cultured in reference medium, no difference in spheroid size was noted after the treatment, but only a viability drop (84% viability). Conversely, SUM1315 spheroids cultured in OptiPASS presented a 1.2-fold size spheroid decrease with slowed viability . These results showed that the culture with OptiPASS medium allowed to detect the epirubicin cytostatic and cytotoxic actions on both cell lines.All these results demonstrated the added-value of OptiPASS synthetic medium, for the TNBC 3D cell cultures development. Indeed, cell proliferation, cell viability and cell drug sensibility in MDA-MB-231 and SUM1315 spheroids models were clearly optimized. These results also highlighted the key role of the culture medium in the optimization of culture models’ predictive response within the drug screening pipeline and opened the way to the common use of this medium in preclinical breast cancer research studies. Other experiments aiming to determine OptiPASS performances with other cancer cell lines are currently underway.