The solution combustion synthesis process, in both thin films and bulk Cu:NiOx, is investigated. Thermal analysis studies using TGA and DTA reveal that the Cu:NiOx thin films show a more gradual mass loss while the bulk Cu:NiOx exhibits a distinct combustion process. The thin films can crystallize to Cu:NiOx at an annealing temperature of 300 °C, irrespective of the Acac/Oxidizer ratio, whereas lower annealing temperatures (150 and 200 °C) produce amorphous materials. A detail characterization study of solution combustion synthesized Cu:NiOx, including XPS, UV-Vis, AFM, and Contact angle measurements, is presented. Finally, 50 nm Cu:NiOx thin films are introduced as HTLs within the inverted perovskite solar cell device architecture. The Cu:NiOx HTL annealed at 150 and 200 °C provided PVSCs with limited functionality, whereas efficient triple-cation Cs0.04(MA0.17FA0.83)0.96 Pb(I0.83Br0.17)3-based PVSCs achieved for Cu:NiOx HTLs for annealing temperature of 300 °C.Low temperature solution combustion synthesis emerges as a facile method for the synthesis of functional metal oxides thin films for electronic applications. We study the solution combustion synthesis process of Cu:NiO Thanks to its p-type semiconducting nature, high optical transmittance, enhanced electrical conductivity, and deep-lying valence band (VB) that matches well with the VB of hybrid perovskite photoactive layer, NiOx emerges as an excellent hole transporting layer (HTL) material for inverted perovskite solar cells  as a photoactive layer.The aim of this paper is to identify the Cu:NiOMaterials: Prepatterned glass-ITO substrates (sheet resistance 4Ω/sq) were purchased from Psiotec Ltd., Berkhamsted, UK. All the other chemicals used in this study were purchased from Sigma Aldrich .x solution combustion synthesis: In a typical synthesis, 0.95 mmol Ni(NO3)2.6H2O and 0.05 mmol Cu(NO3)2.3H2O were dissolved in 10 mL 2-methoxyethanol with different concentrations of fuel acetylacetonate to the solution and the mixture was further stirred for 1 h at room temperature. Then, the samples were dried at 80 °C for 5 min and annealed at 150 °C, 200 °C, or 300 °C in air for 1 h. The chemical reaction formula of the solution combustion synthesis is as follows:Cu:NiOx films, the samples were fabricated onto Alumina disk substrates by blade-coating in air. For the AFM, UV-Vis, and contact angle measurements, the Cu:NiOx films were fabricated by doctor blade on quartz substrates.Samples preparations for TGA, AFM, UV-Vis, contact angle analysis: For the thermogravimetric analysis (TGA) of the combustion synthesis behavior of the Cu:NiOx precursors were prepared and blade-coated on ITO substrates as described in the Cu:NiOx solution combustion synthesis section. For the preparations of the triple cation Cs0.04(MA0.17FA0.83)0.96 Pb(I0.83Br0.17)3 perovskite solutions, a previous reported method was used . F. Fx surfresidues c showed i(acac)2 , while te of CuO . Quantitvely see . The hig results . Moreovex films fabricated on quartz substrates by contact angle and UV-Vis spectroscopy. x particles, exhibiting a surface roughness of 0.7 nm  annealed at 300 °C, which is substantially lower (20°); see x surface with moderate wettability, while the films annealed at 300 °C, where the surface is almost free from remnants, show an improved wettability.The contact angle of water was measured on films annealed at 150, 170, and 200 °C, using a fuel to oxidizer ratio of 0 (w/o), 0.1, and 1.5 see , from whx films as HTLs in solar cells, 50 nm thick Cu:NiOx films, synthesized using the previously described conditions, were implemented in inverted perovskite solar cells with structure ITO/Cu:NiOx/perovskite/PC60BM/BCP/Ag and the J-V device characteristics under 1 sun simulated light were recorded.To evaluate the functionality of the different Cu:NiOx films annealed at 150 °C and 200 °C exhibited a very limited functionality. All the devices show low Voc in the range of 0.3 V, and the generated current is below 1 mA/cm2. The device with a 1.5 molar ratio Acac to oxidizer that was annealed at 200 °C shows an almost linear response of the current density to the sweeping voltage, which can be attributed to partially formed metallic Ni, as can be inferred by the corresponding XRD results in x HTLs prepared from precursor solutions containing w/o, 0.1, and 1.5 Acac annealed at 300 °C, delivered higher efficiency. In x HTL where the precursor contained no fuel (w/o Acac) and a 0.1 Acac ratio showed similar PCE values, and the devices which incorporated Cu:NiOx film synthesized with 1.5 Acac/oxidizer ratio showed a reduced Voc and Jsc efficiency, resulting in lower PCE.As it is presented in J–V curves of 0.04(MA0.17FA0.83)0.96 Pb(I0.83Br0.17)3 perovskite formulation infer that the Cu:NiOx oxide’s precursor films that were annealed at temperatures of 150 and 200 °C produce electronic films that cannot function as HTLs for efficient inverted perovskite solar cells. This is ascribed to the incomplete combustion that results in amorphous Cu:NiOx films with remnants. This result is in agreement with a previous report, where amorphous NiOx showed limited functionality as HTL when applied in organic solar cells [x HTL was obtained by annealing at 300 °C for the precursors w/o and with a 0.1 ratio of Acac/oxidizer, whereas, for the 1.5 ratio, metallic Ni are likely to be present within Cu:NiOx films (as indicated within the XRD pattern in x resulted to better PCEs 15.97% (average 14.48%) and 16.58% (average 14.85%), respectively, while, for the 1.5 ratio, the metallic Ni influence delivers lower PCE devices 14.90% (average 13.50%).The experimental results presented within this manuscript using a triple cation Csar cells . On the x films by using different molar ratios  of fuel acetylacetone (Acac) to oxidizer (nitrates) precursors as well as various thermal processing annealing temperatures . Thermogravimetric analysis (TGA and DTA) results showed that the complete combustion process at ~150 °C can occur in bulk analogues. XRD measurements revealed that the corresponding Cu:NiOx films crystallize to NiO phase upon annealing temperature at 300 °C irrespective of Acac concentration, while, for lower annealing temperatures , no crystal phase was observed. XPS, AFM, UV-Vis spectroscopy, and contact angle measurements on the films strongly support the incomplete combustion of the Cu:NiOx thin films for annealing temperatures at 150 °C and 200 °C. XPS measurements of the Cu:NiOx film revealed the presence of a high atomic ratio of remnants for thermal annealing at 200 °C, which are remarkably reduced for films annealed at 300 °C. Surface topography images and thickness measurements via AFM and profilometer showed that the Cu:NiOx films annealed at 300 °C have a lower thickness (~50 nm) and roughness (~0.7 nm) compared to ~80 nm thickness and ~1.5 nm roughness for the Cu:NiOx films annealed at temperatures 150 °C due to remnants in the film. Moreover, Cu:NiOx films annealed at 300 °C have an improved hydrophilicity, showing a contact angle of 20°, while the films annealed at 150 °C and 200 °C show angles of more than 60° due to surface remnants. Regarding the optical absorption measurements, the 300 °C thermally annealed films exhibit a distinct absorption curve ascribed to the formed crystalline Cu:NiOx, while the lower annealing temperature films at 150 °C and 200 °C lack any strong absorption in the range of measured wavelengths due to the amorphous phase; these results are consistent with the paper reported in the XRD findings. To conclude, the presented solution combustion chemistry findings in Cu:ΝiOx thin films are confirmed by applying the various ratios of Acac/Oxidizer and annealing processing temperatures of SCS-based Cu:NiOx HTLs in triple cation-based Cs0.04(MA0.17FA0.83)0.96 Pb(I0.83Br0.17)3 inverted PVSCs. The Cu:ΝiOx HTLs annealed at temperatures 150 °C and 200 °C, irrespective of Acac/Oxidizer ratios, provided limited functionality in the PVSCs due to incomplete an combustion process that resulted in amorphous Cu:NiOx with remnants, as confirmed by the presented XRD and XPS measurements, respectively. The crystalline phase of Cu:NiOx HTLs and efficient inverted PVSCs performance obtained at an annealing temperature of 300 °C irrespective of the Acac/Oxidizer ratio. Following the solution combustion synthesis route that has been investigated within this manuscript, the Cu:NiOx crystalline HTLs annealed at 300 °C, with a 0.1 ratio of Acac/oxidizer resulting in 16.58% PCE for the triple cation-based Cs0.04(MA0.17FA0.83)0.96 Pb(I0.83Br0.17)3 inverted PVSCs.In this work, we examined the solution combustion synthesis of Cu:NiO


The results suggest that such compounds can interact at the same binding site as imatinib, probably sharing a competitive inhibition mechanism. One compound showed the greatest interaction affinity for BCR-Abl-1 in the docking studies.The enzyme tyrosine kinase BCR-Abl-1 is the main molecular target in the treatment of chronic myeloid leukemia and can be competitively inhibited by tyrosine kinase inhibitors such as imatinib. New potential competitive inhibitors were synthesized using the (phenylamino)pyrimidine-pyridine (PAPP) group as a pharmacophoric fragment, and these compounds were biologically evaluated. The synthesis of twelve new compounds was performed in three steps and assisted by microwave irradiation in a 1,3-dipolar cycloaddition to obtain 1,2,3-triazole derivatives substituted on carbon C-4 of the triazole nucleus. All compounds were evaluated for their inhibitory activities against a chronic myeloid leukemia cell line (K562) that expresses the enzyme tyrosine kinase BCR-Abl-1 and against healthy cells (WSS-1) to observe their selectivity. Three compounds showed promising results, with IC Abl1 of chromosome 9 and the BCR gene on chromosome 22  using a riding model. All nonhydrogen atoms were refined anisotropically. Some crystallization and X-ray diffraction experiments for compound 2b were performed. All samples evaluated had low scattering patterns and germinated crystals. This resulted in poor quality data, which limited the quality of the refinement. Structure illustrations were generated using ORTEP-3 for Mercury [www.ccdc.cam.ac.uk/data_request/cif.Single crystal X-ray data for 2, APEX2  and SAIN2, APEX2 , respect2, APEX2 . The str package –44 and w Mercury , and cry Mercury . X-ray c23H24N8O2 (M = 444.50 g/mol): monoclinic, space group P21/c, a = 14.895(3) Å, b = 16.167(3) Å, c = 9.5274(14) Å, β = 92.150(5)°, V = 2292.7(7) Å3, Z = 4, T = 298.0 K, μ(Mo Kα) = 0.087 mm−1, F (000) = 936.0, crystal size = 0.24 × 0.22 × 0.096 mm3, Dcalc = 1.288 g/cm3; of the 21431 reflections measured (3.72° ≤ 2Θ ≤ 50.682°), 4199 were unique  and were used in all calculations. The final R1 was 0.1284 (I >2σ(I)), and wR2 was 0.3858 .C1a,b, and 2a–j) were evaluated for their activity against K562 cells (ATCC® CRL-243TM), a CML cell line, and a WSS-1 healthy cell line (ATCC® CRL-2029TM). The K562 strain was grown in RPMI-1640 medium according to the provider's recommendations [2. WSS-1 cells grew adherently, while cells of the K562 strain grew in suspension. All strains were maintained at 35 °C, at 5% CO2.All novel compounds synthesized (ndations –48. Cult4 (cells per well) in black plates with a 96-well transparent bottom (Greiner Bio-One) for approximately 20 hours to allow for cell growth and adhesion. The compounds obtained were added at concentrations of 1 µM and 10 µM in DMSO and incubated for 46 hours. This assay was performed in triplicate and measured using resazurin (Sigma-Aldrich) at a concentration of 0.1 mg/mL using a FlexStation 3 microplate reader (Molecular Devices).WSS-1 cells were incubated at 5 × 104 cell/mL), the K562 cells were incubated for one hour, and then the compounds dissolved in DMSO (Sigma-Aldrich) were added and further incubated for 47 hours. After this period, resazurin was added at a final concentration per well of 0.01 mg/mL, and the first fluorescence reading was immediately performed  (time zero) using FlexStation 3 microplates (Molecular Devices). After the first reading, the plate was returned to the incubator, and after one hour, the second reading was performed, completing the 48 hours of incubation with the compounds. The reference inhibitor used was IMT at fixed concentrations of 1 μM and 10 μM.After plating  [The Molegro Virtual Docker v6.0 (MVD) program was used to predict the complexes and energies of interaction between the enzyme BCR-Abl 1 and inhibitors e: 3PYY) . The strhttp://www.ks.uiuc.edu/Research/vmd/vmd-1.9.3/).Validation of the molecular docking protocol was performed through the redocking of IMT complexed with the BCR-Abl-1 structure (PDB code: 3PYY), free of water molecules and cofactors, using Molegro Virtual Docker 6.0 (MVD) . Thus, tFile 1Additional experimental and analytical data, and NMR spectra of synthesized compounds.


SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented mouse SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine (NSDP) as a covalent inhibitor of SARM1 that reacted with the cysteines, especially Cys311 in its ARM domain and blocked its NMN-activation, protecting axons from degeneration. The Cryo-EM structure showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering a potential neuroprotective mechanism of dihydropyridines. Axon degeneration (AxD) occurs in most neurodegenerative disorders . SterileIt should be noted that SARM1 is not just a simple NADase activated to deplete the cellular NAD. We have documented that SARM1 is a multifunctional enzyme with properties similar to CD38, a universal signaling enzyme possessing not only NADase activity but also catalyzing both the cyclization of NAD to cyclic ADP-ribose (cADPR) and the exchange of nicotinamide in NADP with nicotinic acid to produce nicotinic acid adenine dinucleotide phosphate (NAADP) . Both cASince SARM1 is important in axon degeneration and potentially other physiological processes as well, we thus aim to design and synthesize probes for visualizing SARM1 activation in live cells and to screen drug library for potent inhibitors.We focused on its base-exchange reaction for designing specific probes for SARM1 and had shown that pyridyl derivatives can readily serve as substrates . We thusO-series), but not the nitrogenated derivatives , nor PC9 with diene as linker. The emission spectra of the reactive O-series showed steady increase as the reaction progressed  . The remThe observed spectral changes showed a linear dependence on NMN, with as low as 10 μM being effective , confirmN. crassa NADase -inducible SARM1  was usedNMNAT1 knockout in HEK293T cells also resulted in increasing PAD6 fluorescence (Endogenous NMN can be increased by ablating NMN-adenylytransferase (NMNAT1)  to activrescence  in a timrescence .Consistent with the in vitro results showing that PC6 is highly selective for SARM1 over CD38 in live cells, cells expressing either wildtype or Type III mutant CD38  did not Sarm1-specific shRNA (Vincristine (VCR)-induced AxD in peripheral neuropathy is a common side effect of chemotherapy  and is tic shRNA . In the ic shRNA , green, ic shRNA , blue. Aic shRNA , temporaic shRNA , which oic shRNA , blue byic shRNA , CTRL.D, KD +VCR) and reduced AxD (A). These results indicate SARM1-activation is a necessary and causal factor, but not a sufficient one for AxD. Other critical factors and downstream events of microtubular dysfunction might contribute to the degeneration.Reducing endogenous SARM1 using shRNA , KD reduuced AxD , furtheruced AxD , red anduced AxD , red, co50 of a reported inhibitor of SARM1, nicotinamide (Nam)  inhibited NADase less than the base-exchange. These remarkable differences underscore the importance of using more than one assay for drug screening (see Discussion).Another prompt application of PC6 is library screening for inhibitors of SARM1. The feasibility was verified by measuring the ICde (Nam) . The meaed value . Next, wed value  and its ed value . Out of ed value , which w50-value of about 150 μM (squares), but its potency increased 75-fold after exposure to UV (50 = 2.36 ± 0.3 μM). Also, fresh NSDP had an HPLC-elution peak at 12.2 min (In the middle sector is nisoldipine (NSDP), a calcium channel blocker having beneficial effects on neurodegenerative diseases. Peculiarly, its inhibition of SARM1 varied widely among batches. Investigations indicated the active compound was not NSDP but its derivative. 12.2 min , but was12.2 min , red. HR12.2 min , inset i12.2 min . The HPL12.2 min , showed The dHNN-induced inhibition of SARM1 was irreversible by washing , strongl50. The IC50 of dHNN in the SARM1-dN-expression cells is around 4 μM, close to the IC50 in vitro. dHNN decreased the cellular cADPR in cells expressing SARM1, but not in those expressing SAM-TIR . dHNN interacts with Glu264, Leu268, Arg307, Phe308, and Ala315  . In 2D-cd Ala315 , green id Ala315 , red towd Ala315 .B, red line).By preventing SARM1 from activation, dHNN also inhibited the VCR-activated cADPR production  in neuroVisualizing the activity of a signaling enzyme in live cells provides clearer understanding of the spatial and temporal aspects of its mechanism and function, a goal sought by many. The PC probes developed here are particularly advantageous. They are cell-permeant, but the SARM1-catalyzed exchange products are not and accumulate in the cytosol, enhancing their detection. The remarkably large red shift of the product fluorescence provides easy visualization away from the interference of autofluorescence.Currently, there are several fluorescent probes for SARM1 activity in use. They are all analogs of NAD and are cell-impermeant, such as εNAD. The fact that NAD is now shown to be an inhibitor of SARM1  makes thScreening library to identify compounds of interest is a straightforward strategy widely used. The case for SARM1 is more complicated, as it is not only a multi-domain protein but also an auto-regulated enzyme catalyzing multiple reactions. Compounds may target the regulatory ARM domain as shown here for dHNN, or the catalytic TIR domain as Nam and the inhibitors reported during the preparation of this manuscript . For SARMuch effort is being invested in targeting SARM1-mediated NAD depletion for therapeutic protection from AxD. Chemical blockers may well be an ideal tool for turning off the NAD depletion. dHNN uncovered in this study is the first compound ever described that can block the activation of SARM1, revealing a druggable allosteric site and can thus usher in a new approach for therapeutic drug development. Another point of interest is that dHNN is a derivative of NSDP. Metabolic conversion of NSDP to dHNN, leading to inhibition of SARM1, may well account for the neural protective effects of NSDP .This study was carried out in strict accordance with animal use protocol approved by Peking University Shenzhen Graduate School Animal Care and Use Committee (#AP0015001). All animals (C57BL6/J), purchased from Guangdong Medical Laboratory Animal Center (China), were handled in accordance with the guidelines of the Committee on the Ethic of Animal Experiments. All surgery was performed after euthanasia and efforts were made to minimize suffering.2.The HEK293 and HEK293T cells were purchased from the American Type Culture Collection and the identity has been authenticated by STR profiling. They have not been contaminated by mycoplasma. The cells were cultured in DMEM supplemented with 10% fetal calf serum and 1% penicillin-streptomycin solution and maintained in a standard humidified tissue culture incubator with 5% CO2PO4, NH4HCO3, and iodoacetic acid were purchased from Sigma-Aldrich. DMEM, Neurobasal Plus Medium, Trypsin-EDTA, penicillin/streptomycin solution, B27 plus, GlutaMax, Laminin, Lipofectamine 2000, ConA-Alex-647, formic acid, acetonitrile were purchased from ThermoFisher. NGF was from Sino Biological. FBS was obtained from PAN Bitotech. Approval drug library (L1000) and Nisoldipine power (Cas # 63675-72-9) were purchased from TargetMol. Dehydro Nisoldipine (Cas #103026-83-1) was obtained from TRC, while dehydronitrosonisoldipine (Cas #87375-91-5) was purchased from Glpbio and TRC. Vincristine was purchased from Selleck. General chemicals for probe synthesis were purchased from Dieckmann, Alfa, Energy, or Sangon Biotech (Shanghai).NAD, NMN, Digitonin, Poly-L-lysine, 5-fluoro-2’-deoxyuridine and uridine, KH2SO4, 15 mL of acetic acid, and 25 mL of anisaldehyde) or KMnO4 . Silica gel (200–300 mesh) was used for flash column chromatography. Nuclear magnetic resonance (NMR) spectra were recorded on either a 300 , 400 , or 500  NMR spectrometer. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet, br = broad. High resolution mass spectra (HRMS) were obtained from a MALDI-TOF mass spectrometer.All air and water-sensitive reactions were carried out with anhydrous solvents in flame-dried flasks under argon atmosphere, unless otherwise specified. All the reagents were obtained commercially and used without further purification, unless otherwise specified. Anhydrous DMF was vacuum distilled from barium oxide, acetonitrile, and dichloromethane was distilled from calcium hydride. Yields refer to isolated yields, unless otherwise specified. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm silica gel plates (60 F-254) that were analyzed by UV light as visualizing method and by staining with anisaldehyde 3 , and triethylamine  in degassed CH3CN (15 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 100°C for 5 hr. The mixture was then diluted with water (20 mL) and aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 3:1) of the residue gave a pale-yellow solid  as the product. PC1: mp = 272–273°C. 1H NMR  δ 8.47 , 7.46 , 7.42–7.31 , 6.88 , 6.59 , 5.51 . 13C NMR  δ 149.8, 145.4, 133.7, 128.5, 123.6, 120.0, 120.0, 113.8. HRMS (+ESI) m/z calcd. for C13H12N2 (M + H)+ 197.1073, found 197.1072.To synthesize PC1, to a stirred solution of 4-vinylpyridine , 4-iodoaniline , P at rt. The resulting mixture was stirred at 70°C for 6 hr. The mixture was then diluted with water (20 mL) and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were washed with water (15 mL ×3), dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 1:20) gave a brown solid  as the product (1). Spectral data of 1 are consistent with those reported in the literature., P(o-tol)3 , and triethylamine  in degassed CH3CN (5 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 100°C for 12 hr. The mixture was then diluted with water (20 mL) and aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 2:1) of the residue gave a pale-orange solid  as the product. PC2: mp = 199–200°C. 1H NMR  δ 8.52 , 7.39 , 7.31 , 7.23 , 6.79 , 6.60 , 3.21 , 1.29 . 13C NMR  δ 149.9, 149.0, 145.5, 133.4, 128.5, 125.2, 121.2, 120.4, 112.6, 38.2, 14.8. HRMS (+ESI) m/z calcd. for C15H16N2 (M + H)+ 224.1386, found 224.1382.To synthesize PC2, to a stirred solution of 4-iodoaniline  in DMF (7.5 mL) was added ethyl bromide  and Naterature. To a sti2CO3  at rt. The resulting mixture was stirred at 70°C for 6 hr. The mixture was then diluted with water (20 mL) and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were washed with water (15 mL ×3), dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. Silica gel flash column chromatography of the residue (ethyl acetate/hexanes = 1: 30) gave a brown oil  as the product (2). Spectral data of 2 is consistent with those reported., 4-vinylpyridine , P(o-tol)3 , and triethylamine  in degassed CH3CN (15 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 100°C for 12 hr. The mixture then was diluted with water (20 mL) and the aqueous phase extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 1:1) of the residue gave a pale-yellow solid  as the product. PC3: mp = 184–185°C. 1H NMR  δ 8.50 , 7.47–7.38 , 7.30 , 7.25–7.19 , 6.74 , 6.66 , 3.47–3.18 , 1.31–1.02 . 13C NMR  δ 149.9, 148.2, 145.7, 133.4, 128.6, 123.2, 120.4, 120.4, 111.5, 44.5, 12.7. HRMS (+ESI) m/z calcd. for C17H20N2 (M + H)+ 253.1699, found 253.1699.To synthesize PC3, to a stirred solution of 4-iodoaniline  in DMF (15 mL) was added ethyl bromide  and Nareported. To a sti2 , 1-bromo-4-vinylbenzene , P(o-tol)3 , triethylamine  in degassed CH3CN (15 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 100°C for 12 hr. The mixture was then diluted with water (20 mL) and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. Silica flash column chromatography (ethyl acetate/hexanes = 1:30) gave a pale green solid  as the product (3). Spectral data of 3 are consistent with those reported in the literature., 4-vinlypyridine , P(o-tol)3 , and triethylamine  in degassed CH3CN (10 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 100°C for 12 hr. The mixture was then diluted with water (20 mL) and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 3:1) of the residue gave a pale-yellow solid  as the product. PC4: mp = 225-226 °C. 1H NMR  δ 8.54 , 7.54–7.44 , 7.42–7.38 , 7.36 , 7.29 , 7.09 , 6.99 , 6.88 , 6.67 , 3.39 , 1.19 . 13C NMR  δ 150.1, 147.6, 144.9, 139.1, 134.2, 133.1, 129.7, 129.0, 128.6, 128.0, 127.4, 126.3, 124.9, 124.4, 122.9, 120.8, 111.7, 44.4, 12.7. HRMS (+ESI) m/z calcd. for C25H26N2 (M + H)+ 355.2169, found 355.2167.To synthesize PC4, to a stirred solution of terature. To a stio-tol)3 , and triethylamine  in degassed CH3CN (15 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 100°C for 12 hr. The mixture was then diluted with water (20 mL). Upon addition of 5% HCl leads to partial precipitation of the product. The aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 3:1) of the residue gave an off-white solid  as the product. PC5: mp = 281–282. 1H NMR  δ 9.83 , 8.55 , 7.67–7.42 , 7.07 , 6.85 . 13C NMR  δ 158.2, 149.8, 144.8, 133.1, 128.7, 127.2, 122.4, 120.5, 115.7. HRMS (+ESI) m/z calcd. for C13H11NO (M + H)+ 198.0913, found 198.0913.To synthesize PC5, to a stirred solution of 4-vinylpyridine , 4-iodophenol , P(o-tol)3 , and triethylamine  in degassed CH3CN (15 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 100°C for 12 hr. The mixture was then diluted with water (20 mL) and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 3:1) of the residue gave an off-white solid  as the product. PC6: mp = 146–147°C. 1H NMR  δ 8.55 , 7.51–7.42 , 7.33 , 7.27 , 6.97–6.81 , 4.07 , 1.44 . 13C NMR  δ 159.6, 150.1, 145.0, 132.8, 128.7, 128.4, 123.6, 120.6, 114.8, 63.6, 14.8. HRMS (+ESI) m/z calcd. for C15H15NO (M + H)+ 226.1226, found 226.1226.To synthesize PC6, to a stirred solution of 4-vinylpyridine , 1-ethoxy-4-iodobezene , P(2Cl2 (25 mL) was added acetyl chloride  at rt. The resulting mixture was stirred at 0°C for 20 min and then rt for 2 hr. The solution was then diluted with water (20 mL) and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined extracts were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The resulting pale brown oil  was obtained as the product (4) and was used for the next step without any further manipulation. Spectral data of 4 is consistent with those reported in the literature (o-tol)3 , and triethylamine  in degassed CH3CN (15 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 100°C for 6 hr. The mixture was then diluted with water (30 mL) and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 3:1) of the residue gave a white solid  as the product. PC7: mp = 152–153°C. 1H NMR  δ 8.58 , 7.63–7.48 , 7.36 , 7.28 , 7.15–7.09 , 6.97 , 2.32 . 13C NMR  δ 169.4, 150.9, 150.2, 144.5, 133.9, 132.2, 128.1, 126.2, 122.1, 120.9, 21.2. HRMS (+ESI) m/z calcd. for C15H13NO2 (M + H)+ 240.1019, found 240.1018.To synthesize PC7, to a stirred solution of 4-iodophenol  and triethylamine  in CHterature . To a st2CO3  at rt. The resulting mixture was stirred at 70°C for 6 hr. The mixture was then diluted with water (20 mL), and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were washed with water (15 mL ×3), dried over anhydrous Na2SO4, and evaporated under reduced pressure. A white solid was obtained as the product (12). To a stirred solution of the crude product (12), 4-vinylpyridine , P(o-tol)3 , and triethylamine  in degassed CH3CN (30 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 100°C for 5 hr. The mixture was then diluted with water (30 mL) and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 3:1) of the residue gave a pale-yellow solid  as the product. PC8: mp = 114–115°C. 1H NMR  δ 8.59 , 7.80–7.60 , 7.35 , 7.19 , 6.99 , 6.91 , 4.19 , 1.50 . 13C NMR  δ 160.4, 150.0, 143.9, 132.5, 131.8, 130.2, 128.9, 125.7, 120.6, 115.9, 112.3, 102.4, 64.8, 14.3. HRMS (+ESI) m/z calcd. for C16H14N2O (M + H)+ 251.1179, found 251.1178.To synthesize PC8, to a stirred solution of 5-bromo-2-hydroxy-benzonitrile  in DMF (7 mL) was added ethyl bromide , and Ko-tol) , Cs2CO3  and KCl  in DMF (30 mL) under argon was added Pd(OAc)2  quickly. The resulting mixture was stirred at 90°C for 5 hr and then treated with 5% HCl (10 mL) and stirred at rt for 10 min. The mixture was then diluted with water (20 mL) and the aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 1:5) gave a pale-yellow solid  as the product (6). Spectral data of 6 is consistent with those reported in the literature , 4-methylpyridine  in Ac2O (2 cmL) was added NaOAc  at rt. The resulting mixture was heated under reflux for 21 hr. Then the mixture was cooled to rt and diluted with CH2Cl2, washed with H2O, 5% HCl, H2O, and saturated aqueous NaHCO3. The aqueous phase was extracted with ethyl acetate (15 mL ×3). The combined organic extracts were dried over Na2SO4, filtered and evaporated under reduced pressure. Silica gel flash column chromatography (ethyl acetate/hexanes = 3:1) gave a pale-yellow solid  as the product. PC9: mp = 132–133°C. 1H NMR  δ 8.58 , 7.44 , 7.36–7.29 , 7.17 , 6.99–6.74 , 6.57 , 4.11 , 1.48 . 13C NMR  δ 159.0, 149.8, 144.7, 135.4, 133.8, 129.2, 128.2, 127.9, 125.8, 120.3, 114.6, 63.3, 14.6.HRMS (+ESI) m/z calcd. for C17H18NO (M + H)+ 252.1383, found 252.1384.To synthesize PC9, to a stirred solution of 1-ethoxy-4-iodobezene  and 3,3-diethoxyprop-1-ene , P by LR clonase II enzyme according to the manufacturer’s instructions. HEK293F cells overexpressing dtSARM1-dN were constructed by lentivirus infection and selected with 1 μg/mL puromycin. DtSARM1-dN was released by 200 μM digitonin and immunoprecipitated with StrepTactin resin , washed with buffer W  for four times and eluted with 2 mM biotin in buffer W. DtSARM1-dN was used in the experiments on the dHNN-modification, cysteine-to-alanine mutants and Cryo-EM structures.N. crassa NADase were prepared as described previously , respectively, in an Infinite M200 PRO microplate reader (Tecan). For the assays with εNAD，NHD, or NGD as the substrate, 100 μM of each probe was incubated with the enzymes and the kinetics of fluorescence production was measured at λ2PO4 (pH 6.0) and 0.1 M KH2PO4 (pH 6.0) with MeOH (7:3) to elute NMN, cAPPR, ADPR, NAD, and a gradient of ACN from 30 to 70% to elute PAD6 and PC6. The PAD6 fractions were collected and lyophilized for the characterization of absorption and fluorescence spectra.The reactions were prepared by mixing SARM1-IP  with 100 μM NAD, 50 μM PC6, 100 μM NMN, and 0.1 mg/mL BSA in PBS and incubated for 60 min at 37°C. SARM1-IP was removed by centrifugation at 4,500 rpm for 1 min. The cleaned mixtures were applied to a C-18 reverse phase column equipped on an HPLC (Agilent 1260) with a gradient of 0.1 M KHTo analyze PAD6 in cells, the metabolites were extracted from the cells treated with 50 μM PC6 by 0.6 M perchloric acid, followed by the neutralization with Chloroform: Tri-n-octylamine (3:1). The extracts were applied to a C-18 column and PAD6 was eluted with water and acetonitrile by 2% acetonitrile for 8 min, then 30% acetonitrile for 8 min.NMNAT1 were constructed as before  of SARM1 or HEK293T Knocking out s before . Cells, HEK293 cell line carrying doxycycline (Dox)-inducible SARM1 was constructed as previously described . The celMouse DRG culture was performed as described . BrieflyOn div6, the neurons were infected with lentivirus carrying various expression cassettes of genes or shRNAs. Three days later, the cells were treated with 50 μM PC6 in the absence or presence of 200 μM CZ-48 or 50 nM vincristine. The fluorescence images  were captured under a confocal microscope (Nikon A1) with a 60x object. The mean fluorescence intensity was quantified by NIS-Elements AR analysis (Nikon). Axon degeneration was quantified based on axon morphology using ImageJ. The TdTomato fluorescence images were binarized and measured the total axon area (size = 20 infinity pixels) and the degenerated axon  with particle analyzer module of ImageJ. Axon degeneration index was calculated as the ratio of the degeneration axon over total axon area.Sarm1 plasmids were constructed as described previously (Sarm1 (5’- CCGGCTGGTTTCTTACTCTACGAATCTCGAGATTCGTAGAGTAAGAAACCAGTTTTTG-3’) or the scrambled shRNA (5’- CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTG-3’) were inserted to pLKO.1-puro  with EcoRI and AgeI, followed by the replacement of the puromycin resistance gene with a fluorescent protein, TdTomato (GenBank: LC311026.1) with KpnI/BamHI. The lentiviral particles were prepared by transfecting HEK293T cells with the corresponding lentivectors, pMD2.G, and psPAX2 . Axon degeneration was quantified using ImageJ. For each treatment, 60 random grid-squares with 147 × 147 pixels were cropped, binarized and the total axon area (size = 16 infinity pixels) and the degenerated axon  were quantified with the particle analyzer module of ImageJ. Axon degeneration index was calculated as the ratio of the degeneration axon over total axon area.DRG neurons were treated with 50 nM Vincristine or 200 μM CZ-48 for 0, 12, 24, 48 hr on div6. After incubation, DRG was washed with cold PBS and lysed with 0.6 M perchloric acid. The concentration of cADPR was analyzed by the cycling assay, as described previously .Sarm1 sense, 5’-CTTTCTCCAAGGAGGACGAGC-3’, antisense, 5’-CTTGTGTCACTGGCATCCACC-3’; GAPDH sense, 5’- TGGCCTTCCGTGTTCCTAC-3’, antisense, 5’-GAGTTGCTGTTGAAGTCGCA-3’.The total RNAs were extracted from DRG neurons with RNA extraction kit (OMEGA) 48 hr post infection and transcribed with the kit, Transcript II One-step gDNA Removal and cDNA synthesis Supermix from Sangon Biotech. The mRNA level of SARM1 relative to GAPDH was quantified with by Q-RT-PCR using TransStart Tip Green qPCR SuperMix (TransGen Biotech) on CFX Connect Real-Time PCR Detection System (Bio-Rad). The following primer pairs were used: x for the reactions with different drugs, Vmax for the reaction without drugs and Vmin for the reaction without enzyme. The inhibitory rates were calculated by the equation, (Vmax- Vx)/Vmax and plotted using GraphPad Prism 8.0. The standard statistics of the screening were calculated as follows: Z' factor=1-(3*SD(Vmax)+3*SD(Vmin))/(Average(Vmax)-Average(Vmin)), S/N = (Average(Vmax)-Average(Vmin))/SD(Vmin). In the screening of this study, Z’ = 0.69 and S/N = 291.96.For high-throughput screening of the potential inhibitors, 1.5 μg/mL SARM1-dN was pre-incubated with 50 μM drugs  at room temperature for 10 min and the reaction reagents, including 20 μM PC6, 50 μM NAD, and 50 μM NMN were added to start the reaction. Controls including reactions without the drugs, defined as 0% inhibition, and without both the drugs and SARM1-dN, defined as 100% inhibition. The fluorescence  was measured by plate reader  and the initial reaction rates were calculated, V50 measurement, 0.4 μg/mL SARM1-dN was pre-incubated with doses of drugs in vitro for 10 min, and started the reaction by adding 50 μM NAD, 50 μM NMN, and 50 μM PC6. Calculation of IC50 by plotting the initial rate to dose of compounds.For IC2PO4 (pH 6.0) and 0.1 M KH2PO4 (pH 6.0) with MeOH (7:3) to elute NMN, cAPPR, ADPR, NAD, Nam. The amount of ADPR was used to calculate the initial rate. IC50 was calculated by Graphpad Prism 8.0.1 μg/mL of SARM1-dN was pre-incubated with drugs for 15 min at room temperature, and the reactions were started by adding 100 μM NAD and 100 μM NMN. They were stopped by removing the enzyme with MultiScreen Filter Plates (Millipore) after 0, 15, and 30 min incubation at 37°C and the reactants and products were analyzed by a C-18 column  with a gradient of 0.1 M KHThe NSDP powder was dissolved in DMSO and shined with UV at 254 nm for 30 min and analyzed with a C-18 reverse phase column (ZORBAX SB-C18) equipped on a HLPC (Aligent 1260) and eluted with 50% of 0.1%TFA and 50% of 0.1%TFA in 99% ACN. The products after UV treatment were collected and purified by HPLC, as described above. The inhibitory activity of these fractions was determined by PC6 assay after being neutralized with 100 mM Tris (pH7.5), and the main peak was characterized by HRMS .To determine whether dHNN inhibits the activation or enzymatic activity of SARM1 in vitro, SARM1-dN, the autoinhibited form, and SAM-TIR, the constitutively active form, were pre-incubated with different concentrations of dHNN at rt for 10 min, after which the activity was measured with PC6 assay and the inhibition rate was calculated.To test the same effect in cellulo, HEK293 cells overexpressing the inducible SARM1 (iSARM1) or SAM-TIR (iSAM-TIR) were pre-incubated with 20 μM dHNN, or DMSO as controls, for 1.5 hr and then treated with 100 μM CZ-48 or 0.5 μg/mL Dox for the indicated time. The cellular levels of cADPR were measured as described above.2 fragments , the dtSARM1-dN band was sliced, dehydrated with 100% ACN, and the proteins were alkylated by 22.5 mM IAA for 30 min in dark after the reduction by 10 mM DTT at 55°C for 30 min. After overnight in-gel digestion with Trypsin, the peptides were extracted and analyzed with HRMS . The dHNN modifications, determined by Protein Discoverer software (ThermoFisher), were defined as an increase of molecular weight by 370.153 Da, 354.158 Da, 402.143 Da, or 386.148 Da on the cysteine residues characterized in the MSragments . The abu50 of dHNN by PC6 assay in vitro.The cysteine-to-alanine mutants of dtSARM1-dN were amplified by the overlapping PCRs with the primers, listed below, and subcloned into pCDH-EF1-MCS-IRES-neo by Xba I and Not I. To prepare the mutant proteins, HEK293 cells were transfected with the above plasmids by lipofectamine 2000 or Polyethylenimine according to the manufacturer’s instructions, and the proteins were extracted 48–72 hr after transfection and determined the ICGTAGCCCAGGGTCTGGCC GACGCCATCCGC-3’C117A-F: 5’-GCGGATGGCGTCGGCCAGACCCTGGGCTAC-3’C117A-R: 5’-CATTCGGAGGAGACAGCC CAGAGGCTGGTG-3’C199A-F: 5’-CACCAGCCTCTGGGCTGTCTCCTCCGAATG-3’C199A-R: 5’-GCGGTGCTGTATTGGGCACGCCGCACGGAC-3’C215A-F: 5’-GTCCGTGCGGCGTGCCCAATACAGCACCGC-3’C215A-R: 5’-GCGCTGCTGCGCCACGCAGCGCTGGCGCTG-3’C226A-F: 5’-CAGCGCCAGCGCTGCGTGGCGCAGCAGCGC-3’C226A-R: 5’-CTGGCGCTGGGCAACGCAGCGCTGCACGGG-3’C233A-F: 5’-CCCGTGCAGCGCTGCGTTGCCCAGCGCCAG-3’C233A-R: 5’-CTTCGGCTGCACGCCGCACTCGCAGTAGCG-3’C271A-F: 5’-CGCTACTGCGAGTGCGGCGTGCAGCCGAAG-3’C271A-R: 5’-GGCCGCTTCGCCCGCGCC CTGGTGGACGCC-3’C311A-F: 5’-GGCGTCCACCAGGGCGCGGGCGAAGCGGCC-3’C311A-R: 5’-CGCTTGGAGGCGCAGGCAATCGGGGCTTTC-3’C343A-F: 5’-GAAAGCCCCGATTGCCTGCGCCTCCAAGCG-3’C343A-R: 5’-GGGGCTTTCTACCTCGCAGCCGAGGCTGCC-3’C350A-F: 5’-GGCAGCCTCGGCTGCGAGGTAGAAAGCCCC-3’C350A-R: 5’-GGTTTCTCCAAGTACGCAGAGAGCTTCCGG-3’C430A-F: 5’-CCGGAAGCTCTCTGCGTACTTGGAGAAACC-3’C430A-R: 5’-GCCAACTATTCTACGGCC GACCGCAGCAAC-3’C482A-F: 5’-GTTGCTGCGGTCGGCCGTAGAATAGTTGGC-3’C482A-R: 5’-TACGGCCTGGTCAGCGCAGGCCTGGACCGC-3’C508A-F: 5’-GCGGTCCAGGCCTGCGCTGACCAGGCCGTA-3’C508A-R: 5’-CAGCTGCTGGAAGACGCAGGCATCCACCTG-3’C527A-F: 5’-CAGGTGGATGCCTGCGTCTTCCAGCAGCTG-3’C527A-R: 5’-CACTCCCCGCTGCCCGCAACTGGTGGCAAAC-3’C552A-F: 5’-GTTTGCCACCAGTTGCGGGCAGCGGGGAGTG-3’C552A-R: 5’-GGAGCACTGGACAAGGCAATGCAAGACCAT-3’C629A-F: 5’-ATGGTCTTGCATTGCCTTGTCCAGTGCTCC-3’C629A-R: 5’-ATGCAAGACCATGACGCAAAGGATTGGGTG-3’C635A-F: 5’-CACCCAATCCTTTGCGTCATGGTCTTGCAT-3’C635A-R: 5’-GTGACTGCTTTAAGCGCC GGCAAGAACATT-3’C649A-F: 5’-AATGTTCTTGCCGGCGCTTAAAGCAGTCAC-3’ C649A-R: 5’-CAGTCTAGAATGGACTACAAGGATGACGATG-3’ dtSARM1-dN-F: 5’-ATAGCGGCCGCTTAGGTTGGACCCA-3’dtSARM1-dN-R: 5’-Cells were lysed with RIPA buffer . Each sample was loaded onto 10–12% SDS-PAGE gels and the proteins were then transferred to a PVDF membranes. The membranes were blocked with 5% milk and blotted with anti-SARM1 (home-made), with anti-Tubulin (TransGen Biotech) as an internal control. After incubation with HRP-conjugated second antibodies, the signals were developed by ECL (Abvansta), detected, and quantified by a Chemidoc MP system and ImageLab software (Bio-Rad).2. The total dataset consists of 2692 raw movies with a defocus value range of −1.0 to −2.0 μm. Motion correction and CTF parameter estimation were performed with cryoSPARC  before frozen by liquid ethane and stored in liquid nitrogen. The sample without inhibitor was examined at the Cryo-EM center of Chinese University Hong Kong (Shenzhen) on a 300kV Titan Krios (ThermoFisher Scientific) equipped with Gatan K3 direct electron detector under magnification of 105,000x, with the corresponding pixel size of 0.85 Å. The dose rate was set to 17.6 e/pix/s and exposure time was set to 2.5 s to obtain 50 frames, which led to an accumulated dose of 61 electrons per ÅryoSPARC . 2,012,1-/Å2 was set for each exposure and split into 39 frames during data acquisition. The defocus range was set between −0.8 and −2.0 μm. In total, 2890 images were collected. Motion correction and CTF parameter estimation were performed with MotionCor2 and CTFFind4 built within Relion 3.1 (The dHNN-treated sample was examined at the Cryo-EM center of Southern University of Science and Technology on a Titan Krios G3 (ThermoFisher Scientific) with Gatan K2 summit detector with a nominal magnification of 130,000x and corresponding pixel size of 1.076 Å. A total accumulative dose of 50 elion 3.1 . After Clion 3.1 . The parThe previously reported structures of the SARM1 SAM domain (PDB: 6O0S) and TIR domain (PDB: 6O0Q) were used as model templates during initial model building. The initial model of ARM domain was built de novo in Coot . The thrt-test was used to determine statistical significance of differences between means . GraphPad Prism 8.0 was used for data analysis.All experiments contained at least three biological replicates. Data shown in each figure are all means ± SD. The unpaired Student’s  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This is a very nice manuscript describing the development of small molecule probes that can detect the activity of SARM1 in live cells. One of the probe allows the author to monitor the activity SARM1 and discovery a small molecule derived from an hyertension drug as an effective SARM1 inhibitor. SARM1 is an enzyme that can convert NAD+ to ADP-ribose or cyclic ADP-ribose and is implicated in axon degeneration, thus the probe and the inhibitor described in the manuscript could lead to future therapeutic development targeting SARM1 to treat axon degeneration.Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, including Hening Lin as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Michael Marletta as the Senior Editor.Thank you for submitting your article "Permeant Fluorescent Probes Visualize the Activation of SARM1 and Uncover an Anti-neurodegenerative Drug Candidate" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. Since the inhibitors react with multiple cysteines and the mechanism of inactivation is not clear from the MS data, please either propose a reasonable mechanism and/or minimize those aspects of the report and explicitly state that the inhibitor modifies multiple cysteines and is unlikely to be a very useful probe.2. It would be necessary to do a more thorough editing and proof-reading to further improve the language.Reviewer #1:The authors aimed to develop cell-permeable small molecule probes that can monitor the activity of SARM1, an enzyme that hydrolyzes NAD+ and is thought to be important for axon degeneration. They successfully achieved this goal using the base exchange activity of SARM1 to make a donor-π-acceptor type of fluorophore. The best probe described in the manuscript is PC6. A number of experiments were carried to rigorously test that the probe works as expected. PC6 has a number of nice features. It is cell permeable, gives much stronger signal than any other probes known for SARM1, is specific for SARM1 and does not detect the activity of CD38 (another enzyme that has similar activity), and allows detection of endogenous SARM1 activation in neurons.Using this probe PC6, the authors was able to monitor SARM1 activity in neurons treated with vincristine and demonstrated that SARM1 activation precedes axon degeneration and is important but not sufficient for axon degeneration. Most importantly, using this probe to monitor SARM activity, they screened a library of about 2000 drug molecules and discovered that a hypertension drug, nisoldipine, could inhibits SARM1. Surprisingly, further studies showed that a derivative of nisoldipine, dehydronitrosonisoldipine , is actually the inhibitor of SARM1. They then carried nice mechanistic studies (including mass spectrometry and cryo-EM structures) showing that dHNN inhibits SARM1 by covalently modify Cys311 residue in the ARM domain. The dHNN binding site is similar to the previously established NAD+ inhibitory site.Overall, the probe is novel with many useful features, the study is rigorous and rather complete, and the conclusion is well supported. I believe the study will be important for the field and will be well received by the field.Reviewer #2:The manuscript by Li et al. describes the development of styrylpyridines as cell permeant fluorescent sensors of SARM1 activity. This work is significant because SARM1 activity is increased during neuron damage and SARM1 knockout mice are protected from neuronal degeneration caused by a variety of physical and chemical insults. Thus, SARM1 is a key player in neuronal degeneration and a novel therapeutic target. SARM1 is an NAD+ hydrolase that cleaves NAD+ to form nicotinamide and ADP ribose  via a reactive oxocarbenium intermediate. Notably, this intermediate can either react with water (hydrolysis), the adenosine ring (cyclization to cADPR), or with a pyridine containing molecule in a 'base-exchange reaction'. The styrylpyridines described by Li et al. exploit this base-exchange reaction; the styrylpyridines react with the intermediate to form a fluorescent product. Notably, the best probe (PC6) can be used to monitor SARM1 activity in vitro and in cells. Upon validating the utility of PC6, the authors use this compound to perform a high throughput screen of the Approved Drug Library (L1000) from TargetMol and identify nisoldipine as a hit. Further studies revealed that a minor metabolite, dehydronitrosonisoldipine (dHNN), is the true inhibitor, acting with single digit μM potency. The authors provide structural and proteomic data suggesting that dHNN inhibits SARM1 activity via the covalent modification of C311 which stabilizes the enzyme in the autoinhibited state.Key strengths of the manuscript include the probe design and the authors demonstration that they can be used to monitor SARM1 activity in vitro in an HTS format and in cells. The identification of C311 as potential reactive cysteine that could be targeted for drug development is an important and significant insight.Key weaknesses include the fact that dHNN is a highly reactive molecule and the authors note that it modifies multiple sites on the protein (they mentioned 8 but MS2 spectra for only 5 are provided). As such, the compound appears to be a non-specific alkylator that will have limited utility as a SARM1 inhibitor. Additionally, no information is provided on the proteome-wide selectivity of the compound. An additional key weakness is the lack of any mechanistic insights into how the adducts are generated.Moreover, it is not clear how the proposed sulphonamide and thiohydroxylamine adducts are formed. From the images presented, it is unclear whether there is sufficient 'density' in the cryoEM maps to accurately predict the sites of modification. Finally, the authors do not show whether the conversion of PC6 to PAD6 is stable or if PAD6 can also be hydrolyzed to form ADPR. Essential revisions:1. Since the inhibitors react with multiple cysteines and the mechanism of inactivation is not clear from the MS data, please either propose a reasonable mechanism and/or minimize those aspects of the report and explicitly state that the inhibitor modifies multiple cysteines and is unlikely to be a very useful probe.Although dHNN may react with multiple cysteines, our results indicate the primary one is Cys311. Nevertheless, we have added the qualification that other Cys may contribute to the inhibition . The evidence for Cys311 being dominant includes quantification of the intensity of the modified peptides and normalizing with that of the corresponding total peptides, with or without modification, showing that the modification is mainly on Cys311 . The dominant role of Cys311 is also confirmed by our mutagenesis and structural studies. Furthermore, quantification of cysteine-containing peptides of other proteins showed no dHNN modification. So, we conclude that dHNN shows significant specificity to the Cys311 of SARM1. The inhibitory mechanism of dHHN we propose is like that of NAD, prohibiting the conformational change of the ARM domain by acting on the allosteric site that includes Cys311 to inhibit the activation of SARM1.2. It would be necessary to do a more thorough editing and proof-reading to further improve the language.We have carefully edited and improved our manuscript throughout.Reviewer #2:The manuscript by Li et al. describes the development of styrylpyridines as cell permeant fluorescent sensors of SARM1 activity. This work is significant because SARM1 activity is increased during neuron damage and SARM1 knockout mice are protected from neuronal degeneration caused by a variety of physical and chemical insults. Thus, SARM1 is a key player in neuronal degeneration and a novel therapeutic target. SARM1 is an NAD+ hydrolase that cleaves NAD+ to form nicotinamide and ADP ribose  via a reactive oxocarbenium intermediate. Notably, this intermediate can either react with water (hydrolysis), the adenosine ring (cyclization to cADPR), or with a pyridine containing molecule in a 'base-exchange reaction'. The styrylpyridines described by Li et al. exploit this base-exchange reaction; the styrylpyridines react with the intermediate to form a fluorescent product. Notably, the best probe (PC6) can be used to monitor SARM1 activity in vitro and in cells. Upon validating the utility of PC6, the authors use this compound to perform a high throughput screen of the Approved Drug Library (L1000) from TargetMol and identify nisoldipine as a hit. Further studies revealed that a minor metabolite, dehydronitrosonisoldipine (dHNN), is the true inhibitor, acting with single digit μM potency. The authors provide structural and proteomic data suggesting that dHNN inhibits SARM1 activity via the covalent modification of C311 which stabilizes the enzyme in the autoinhibited state.Key strengths of the manuscript include the probe design and the authors demonstration that they can be used to monitor SARM1 activity in vitro in an HTS format and in cells. The identification of C311 as potential reactive cysteine that could be targeted for drug development is an important and significant insight.Key weaknesses include the fact that dHNN is a highly reactive molecule and the authors note that it modifies multiple sites on the protein (they mentioned 8 but MS2 spectra for only 5 are provided). As such, the compound appears to be a non-specific alkylator that will have limited utility as a SARM1 inhibitor. Additionally, no information is provided on the proteome-wide selectivity of the compound.Although dHNN may react with cysteines in general, our results indicate it does target specifically Cys311. Quantification of cysteine-containing peptides of other proteins showed no dHNN modification. So, we conclude that dHNN shows significant specificity to the Cys311 of SARM1. Some other SH-reactive agents we tested show little inhibition on SARM1. The evidence for Cys311 being dominant includes quantification of the intensity of the modified peptides and normalizing with that of the corresponding total peptides, with or without modification, showing that the modification is mainly on Cys311 . The dominant role of Cys311 is also confirmed by our mutagenesis and structural studies. Our result strongly suggested that the C311 is a druggable site for designing allosteric inhibitors against SARM1 activation.dHNN is effective in inhibiting SARM1 activation and AxD at low μM range, making it a useful inhibitor. Considering that the neuroprotective effect of NSDP, an approved drug, may well be due to dHNN, labeling it as inhibitor of SARM1 serves focus more attentions.Revision has been made in Discussion.An additional key weakness is the lack of any mechanistic insights into how the adducts are generated. Moreover, it is not clear how the proposed sulphonamide and thiohydroxylamine adducts are formed.Please refer to Figure 1 in the paper :14794.), which has been cited in our manuscript.From the images presented, it is unclear whether there is sufficient 'density' in the cryoEM maps to accurately predict the sites of modification.Please refer to Figure 5F, in which we show the close-up view of dHNN in the ARM domain. dHNN (purple) linked to the residue C311 and formed the hydrophobic interactions with surrounding residues E264, L268, R307, F308, and A315. The extra electron densities near the residue C311 fit the shape of dHNN and were shown as grey mesh.Finally, the authors do not show whether the conversion of PC6 to PAD6 is stable or if PAD6 can also be hydrolyzed to form ADPR.PAD6 is stable and cannot be hydrolyzed by the activated SARM1, as shown in


Ambrosia artemisiifolia L. and Artemisia annua L. from Korea. For extraction of volatile compounds, headspace-solid phase micro extraction (HS-SPME) and simultaneous distillation extraction (SDE) were applied and analyzed by gas chromatography/mass spectrometry (GC/MS). From the results, SDE extraction was found to give the highest concentration of volatile compounds with an average concentration of 1,237.79 mg/kg for A. annua L. leaves compared to 1,122.73 mg/kg by HS-SPME technique. A total of 116 volatile organic compounds were identified, including 76 similar volatile organic compounds detected by both the methods of extraction in leaves of subject species at varying concentrations. Among these 33 volatile organic compounds were reported for the first time from the subject plant species. Thus the present research findings extend the characterization of volatile organic compounds from leaves of A. annua L. and A. artemisiifolia L. species and reported some distinguishing compounds which may be used for their discrimination.This study was designed to analyze the volatile organic compounds in the leaves of  Ambrosia artemisiifolia L. (common ragweed) leaves are highly allergenic pollens, causing great agricultural losses  is a medicinal herb; native to South East Asia, including South Korea, China, and India  is a quick, solvent less, and simple method for analysis of volatile organic compounds Holt, . This teA. artemisiifolia L. leaves instead of A. annua L. leaves. So, it was very important to understand the volatile organic compounds characteristics of leaves of both the subject species selected for current study and to find out the distinguishing marker compounds. From literature search it was come to know that HS-SPME and SDE techniques were not used to isolate volatile organic compounds from A. artemisiifolia L. leaves and also there was no comparative study found on the volatile organic compounds of A. artemisiifolia L. leaves and A. annua L. leaves. In the current analysis, therefore both HS-SPME and SDE techniques coupled to GC/MS analysis were applied to establish the comparative results of volatile organic compounds profile of A. artemisiifolia L. leaves and A. annua L. leaves. It was also attempted to report distinguishing marker volatile organic compounds which may be used for their identification.Some wild toxic plants are morphologically very similar to important medicinal plants. When such wild toxic plants are accidentally taken, instead of medicinal plants for different purposes, they cause many problems. According to Kim and Jang , in SoutA. artemisiifolia L. leaves and A. annua L. leaves were collected in triplicate from Korea native plants growers farm association corporation, Seongnam-si, Republic of Korea These two leave varieties were thoroughly washed with clean water, followed by rinsing with distilled water. Then, using a blender  the leaves were crushed, transferred to polythene bags, properly labelled and used for volatile compounds extraction and GCMS analysis.Figures and taxonomy of the sampled plants are shown in Fig. n-pentane and diethyl ether), a wire spiral packed double distilling apparatus  was used. Anhydrous sodium sulphate was burned overnight at 650 °C in furnace  and then used for dehydration of organic solvent.All chemicals and reagents used in this study were purchased from Sigma-Aldrich Co.  and Thermo Fisher Scientific . The water purification system  was used to prepare ultra-pure water. For the re-distillation of the organic solvents  and then injected 50.0 µL (100 ppm in n-pentane) n-butyl benzene as an internal standard. After that, the headspace glass vial was hold on for 30 min in equilibrium temperature at 70 °C and then SPME fibre  was exposed for 5 min to absorb volatile organic compounds. Finally, the temperature of the injector was maintained at 250 °C, and the fibre was kept in GC/MS objector for 5 min to analyse the volatile organic compounds.Around 2.0 g of each homogenized sample of 2SO4. Finally, the vigreux column was used to concentrate the extract volume to 1.5 mL. After that, it was again concentrated to 0.5 mL under N2 gas mild flush. Finally, the concentrated extract was injected into the GC/MS system for determination of volatile organic compounds  apparatus with 100 mL redistilled n-pentane:diethyl ether  mixture. The experiment was maintained under normal atmospheric pressure for 3 h  with the EI (electron impact) mode. The ionization voltage was 70 eV, and temperatures of the injector and of ion source were maintained at 250 °C and 220 °C, respectively. The volatile organic compounds were isolated by GC column DB-5  and the mass spectra scanned were from 41 to 500 m/z. The GC/MS oven temperature was set as follows; 40 °C  raised to 220 °C at 2 °C/min and then to 280 °C at 10 °C/min . Helium gas at flow rate of 1.0 mL/min was used as the carrier gas. The sample injector volume was 1.0 µL and the split ratio was 1:20  mixture was used as an external reference .As = Amount of sample (g).Pc = Peak area (component in sample).The volatile organic compounds were calculated in triplicate, and the data was evaluated using the Software Version 20  of Statistical Package for Social Sciences (SPSS). The final results were reported as mean ± standard deviation (mg/kg).A. artemisiifolia L. leaves and A. annua L. leaves via HS-SPME and SDE techniques were as displayed in Fig. The GC/MS chromatograms of volatile compounds of various extracts from A. artemisiifolia L. leaves and A. annua L. leaves ranged from 651.53 mg/kg to 1,122.73 mg/kg and 566.1 mg/kg to 1,237.79 mg/kg through HS-SPME and SDE techniques, respectively.In the current analysis, leaves of both plants were extracted for volatile compounds by HS-SPME and SDE. The extracts were analyzed by GC/MS, which identified more compounds compared to already reported studies , 6 oxygenated sesquiterpenes , 8 oxygenated monoterpenes , and 7 monoterpene hydrocarbons  , followed by γ-muurolene , spathulenol , and δ-cadinene . A. artemisiifolia L. leaves gave 6 compounds including phenyl acetaldehyde, (E)-β-ocimene, artemisia alcohol, germacerene B, palustrol, and α-humuleneepoxide II by HS-SPME which were not detected by SDE extraction , 7 ketones , 7 alcohols , 5 aldehydes , 3 ethers , and 2 esters . As per Table β-selinene , β-caryophyllene , alloaromadendrene epoxide , phytone , and aromandendrene . The HS-SPME method found 6 compounds such as cuminaldehyde, benzyl pentanoate, α-acoradiene, alloaromadendrene epoxide, 4-cadinen-7-ol, and phytone, which were not detected by SDE analysis , 5 oxygenated sesquiterpenes , 6 oxygenated monoterpenes , and 37 monoterpene hydrocarbons  , valencene , borneol , and β-caryophyllene . A. artemisiifolia L. leaves were found to have 12 volatile organic compounds such as sulcatone, benzene acetaldehyde, linalool, β-bourbonene, β-cubebene, neryl acetone, β-sesqui sabinene, junenol, and α-guaiol which were reported by SDE and not by HS-SPME technique , 22 hydrocarbons , 11 aldehydes , 7 ketones , 3 ethers , 1 ester . The highest proportion was of terpene group compounds  which included 23 oxygenated monoterpenes, 11 sesquiterpene hydrocarbons, 2 oxygenated sesquiterpenes, and 11 monoterpene hydrocarbons having 40.17%, 493.56 mg/kg; 27.80%, 346.09 mg/kg; 20.50%, 254.96 mg/kg and 3.55%, 44.23 mg/kg, respectively , (E)-pinocarveol , and β-selinene . In the current study, 14 volatile organic compounds were found only by SDE compared to HS-SPME method. Both extractions of A. artemisiifolia L. leaves identified compounds such as γ-terpinene, phenethyl alcohol, α-campholenal, camphene hydrate, (Z)-chrysanthenol, p-cymen-8-ol, α-terpineol, (Z)-carveol, indole, 2-methoxy-4-vinylphenol, eugenol, (Z)-jasmone, and (Z)-nerolidol  is used to extract volatile organic compounds with short time and to extract more volatile monoterpenes. Simultaneous distilled extraction (SDE) is capable of extracting higher amount of volatile organic compounds and requires longer extraction time at high temperature. Both techniques have been used for volatile compounds in the past around the world  identified in the leaves of both species applying the 2 extraction methods. From Table A. artemisiifolia L. leaves whereas oxygenated sesquiterpenes  were detected in higher concentrations in A. annua L. leaves by HS-SPME technique. Both HS-SPME and SDE methods extracted more terpene hydrocarbons from A. artemisiifolia L. leaves and oxygenated terpene compounds from A. anuua L. leaves.In the current study, 116 volatile organic compounds were reported, with 11 compounds  by SDE technique whereas the lowest value was observed in the same leaves (566.1 mg/kg) by HS-SPME extraction. A total of 116 volatile components were identified, extracted by SDE compared to 71 volatile organic compounds by HS-SPME technique, while 33 volatile organic compounds were detected for the first time compared to published literature on A. artemisiifolia L. leaves and A. annua L. leaves. The common major volatile organic compounds (caryophyllene oxide and champhor) were detected in leaves of both samples. The identified marker compounds in A. artemisiifolia L were γ-muurolene and spathulenol while β-selinene and caryophyllene were reported in A. annua L. leaves. Thus, the present findings extend the characterization of volatile organic compounds of A. annua L. leaves and A. artemisiifolia L. leaves and reported some distinguishing compounds which may be used for their characterization.In conclusion, this study determined the volatile organic compounds from


Natural products frequently have unique physiological activities and new action mechanisms due to their structural diversity and novelty, and are an important source for innovative drugs and lead compounds. We present herein that natural product santamarine targeted thioredoxin reductase (TrxR) to weaken its antioxidative function in cells, accompanied by accumulation of high levels of reactive oxygen species (ROS), and finally induced a new mechanism of tumor cell oxidative stress-mediated apoptosis. TrxR knockdown or overexpression cell lines were employed to further evaluate the cytotoxicity of santamarine regulated by TrxR, demonstrated that TrxR played a key role in the physiological effect of santamarine on cells. Santamarine targeting TrxR reveals its previously unrecognized mechanism of antitumor and provides a basis for the further development of santamarine as a potential cancer therapeutic agent. Small molecule cancer therapeutics have perpetually played a crucial role in controlling tumor proliferation and progression, yet the primary challenge remains to discriminatively eradicate cancer cells with little or no toxicity to normal counterparts . TherefoThe mammalian thioredoxin reductase (TrxR) enzymes are a vital redox regulator in cells and normally overexpress in tumor tissues to maintain tumor phenotypes and metastasis . Physiolvia regulating the redox state of cancer cells . NADPH was obtained from Roche . The Annexin V-FITC and PI apoptosis assay kit was purchased from Zoman Biotech . DMEM, puromycin, DMSO, G418, DCFH-DA, DHE, DTT, Hoechst 33342, Ac-DEVD-pNA, insulin, and CHAPS were products of Sigma-Aldrich . FBS was a product of Sijiqing . DTNB was a product of J&K Scientific . All other reagents used were of analytical purity. The recombinant rat TrxR1 (WT TrxR1) was provided by Professor Jianqiang Xu . The Escherichia coli (E. coli) Trx was produced as described by our previous reference -2,5-diphenyl-2H-tetrazolium bromide), streptomycin, and penicillin were obtained from Amresco . BSA, PMSF, and Naeference . The plad States . The estterature .2 atmosphere, and DMEM medium. HeLa-shNT and HeLa-shTrxR1 cells were cultured under standard conditions and supplemented with 1 μg mL−1 puromycin. HEK-IRES and HEK-TrxR1 cells were cultured under standard culture conditions, and 0.4 mg mL−1 G418 and 0.1 μM sodium selenite were supplemented for each cell subculture.Unless mentioned otherwise, the experimental cell lines HeLa, HepG2, HL-60, MDA-MB-231, BEAS-2B, and L02 were purchased from the Shanghai Institute of Biochemistry and Cell Biology. These cell lines were cultured under standard culture conditions of 37°C incubator, 5% COInula racemosa Hook. f. The purity analysis of santamarine was performed on Waters pump 1,525 and PDA 2998 series HPLC systems with reversed-phase C18  chromatographic columns at room temperature. The chemical characterization spectrum and HPLC purity analysis diagram of santamarine were shown in the Supporting Information  δ: 6.05 , 5.39 , 5.33 , 3.93 , 3.65 , 1.82 , 0.86 . 13C NMR  δ: 75.3, 32.9 , 121.4 , 133.5 , 51.2 , 81.7 , 51.1 , 21.3 , 34.4 , 41.0 , 139.1 , 171.0 , 117.0 , 11.2 , 23.5 .Santamarine , white p3–1 × 104/well) were added with the specified concentrations of the experimental drug and 0.1% DMSO, and grown in triplicate in 96-well plates at 37°C for 24 or 48 h. Cells were exposed to the specified periods, MTT reagent  was then added and continued to incubate at 37°C for 4 h. Then 100 μL extraction buffer  was employed to dissolve formazan crystals. Cell viability was calculated by reading the absorbance at 570 nm.HeLa, HepG2, HL-60, MDA-MB-231, BEAS-2B, and L02 cells  was incubated with 0, 5, 10, 25, or 50 μM santamarine in a 96-well plate at room temperature for 30, 45, or 60 min. The final incubation volume was 50 μL. The TE buffer  containing 200 μM NADPH and 2 mM DTNB was then added, and the absorbance was recorded at 412 nm during the initial 3 min . The relAs we mentioned previously , the cry5) were seeded in a 6-well plate, and cultured overnight. Subsequently, the HeLa cells were exposed to 0, 10, 25, or 50 μM santamarine and further cultured for 8 h, and then treated with TRFS-green  for 4 h at 37°C in dark. The images were captured on a fluorescent microscope.According to our previously established TRFS-green-based live-cell imaging assay , HeLa ceHeLa cells were exposed to 0, 10, 25, or 50 μM santamarine for 24 h, and harvested. The RIPA buffer and the Bradford procedure were employed to extract and quantify the total cellular proteins. Intracellular TrxR activity was measured according to our published procedures .5) were plated into a 12-well plate and were cultured overnight. HeLa cells were exposed to 0, 10, 25, or 50 μM santamarine for indicated times, and subsequently incubated with DCFH-DA or DHE  for 30 min at 37°C in dark with a method as described previously  assay for cellular total thiols. After treating HeLa cells with santamarine, collect the cells and lyse them with RIPA buffer for 30 min on ice, vortexing occasionally. The total thiol level was determined by DTNB titration according to the method we previously described .5) were seeded in a 12-well plate, and cultured overnight. Subsequently, the HeLa cells were exposed to 0, 10, 25, or 50 μM santamarine for 24 h. Hoechst 33342 (5 μg mL−1) was added and continued to incubate for 30 min. The stained nuclei were photographed under a fluorescence microscope.The morphological changes of apoptotic cells were detected by the dye Hoechst 33342 as described previously . BrieflyHeLa cells were exposed to 0, 10, 25, or 50 μM santamarine for 24 h, and lysed with RIPA buffer. Bradford procedure was employed to quantify the total cellular protein contents. The total proteins (120 μg) from extracts were incubated with the protease activity assay mixtures  for 2 h at 37°C. The caspase 3 activity was assessed by detecting the absorbance at 405 nm as described previously .5) were seeded in a 6-well plate, and cultured overnight. Then, the HL-60 cells were exposed to 0, 10, 25, or 50 μM santamarine for 24 h. The cells were harvested and washed with PBS. According to Annexin V-FITC/PI dual staining apoptosis detection kit, the apoptotic cells after santamarine action were determined and analyzed by flow cytometry with Cell Quest software .Annexin V-FITC/PI staining was used to detect the number of apoptotic cells . HeLa cep < 0.05 was regarded as a statistically significant standard.The Students t-test is used to evaluate the statistical difference between the two sets of data, and a one-way ANOVA is used for multiple groups. All experimental values are expressed as the mean ± S.E. of three independent experiments. The value of Asteraceae family  imaging was firstly used to detect the activity of TrxR in cells treated with santamarine . As the TrxR gene knockdown and overexpression cell lines, further confirming the correlation between TrxR inhibition and cytotoxicity induced by santamarine. As shown in shTrxR1, a cell line that established stable knockdown of TrxR1 gene by our lab previously developed (shNT cells). We also compared the sensitivity of our previously acquired HEK cells stably overexpressing TrxR1 gene (HEK-TrxR1) and cells stably transfected with the vector (HEK-IRES) to santamarine treatment   . The higTAT3 (−) , 5. SantTAT3 (−) . The cytTAT3 (−) . Our resi.e., evading apoptosis, due to the intricate interaction of genetic aberrations and signal pathways (One of the hallmarks of cancer is the inherent or acquired circumvention of apoptosis, pathways . Evadingpathways . Therefopathways , thus enpathways . Santamapathways . The limpathways . The prepathways .In conclusion, we unveiled TrxR as a novel target of santamarine, and unraveled that santamarine represses TrxR to cause the disorder of cell redox homeostasis, which is beneficial to the mechanism of oxidative stress-mediated apoptosis in HeLa cells. Santamarine targeting TrxR reveals its previously yet-unrecognized mechanism of antitumor and provides a basis for the further development of santamarine as a potential cancer therapeutic agent.


Artemisia vulgaris L. produces a wide range of valuable secondary metabolites. The aim of the present study is to determine the effects of various concentrations of farnesyl diphosphate (FDP) on β-caryophyllene content in both callus and hairy root (HR) cultures regeneration from leaf explants of A. vulgaris L. Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid , α-naphthaleneacetic acid , and FDP (1 and 3 μM) was used for callus induction and HR regeneration from leaf explants of A. vulgaris L. In this study, precursor-treated  callus displayed the highest biomass fresh weight (FW)/dry weight (DW): 46/25 g, followed by NAA 10.7 μM + FDP 3 μM with FW/DW: 50/28 g. Two different Agrobacterium rhizogenes strains (A4 and R1000) were evaluated for HR induction. The biomass of HRs induced using half-strength MS + B5 vitamins with 3 μM FDP was FW/DW: 40/20 g and FW/DW: 41/19 g, respectively. To determine β-caryophyllene accumulation, we have isolated the essential oil from FDP-treated calli and HRs and quantified β-caryophyllene using gas chromatography–mass spectrometry (GC–MS). The highest production of β-caryophyllene was noticed in HR cultures induced using A4 and R1000 strains on half-strength MS medium containing 3 μM FDP, which produced 2.92 and 2.80 mg/ml β-caryophyllene, respectively. The optimized protocol can be used commercially by scaling up the production of a β-caryophyllene compound in a short span of time. Artemisia vulgaris L. is an important medicinal herb that belongs to the family Asteraceae. These plants are found in several temperate countries in Europe, Asia, Northern Africa, Alaska, and North America. A. vulgaris L. has been widely used as a traditional medicine for treating a number of diseases, such as neonatal jaundice  has an anti-proliferative property against tumor cell lines. They also found that β-caryophyllene caused a strong induction of apoptosis accompanied by DNA ladder formation and caspase-3 catalytic activity in tumor cell lines. However, the plant sesquiterpenes biosynthesis pathway is considered as an isopentenyl diphosphate (IPP), which is the universal biological precursor of all isoprenoids (basic C5 isoprene unit). It can be obtained through either mevalonate (MVA) or 2-methylerythritol 4-phosphate (MEP) pathways  cultures and to isolate the essential oil from both calli and HRs in order to understand the metabolic profile of β-caryophyllene using gas chromatography–mass spectrometry (GC–MS).Caryophyllene is present in nfection . Gertschpathways . Moreovepathways . There apathways . There wpathways . IPP inspathways . Isopentynthesis . The objA. vulgaris L. were collected from Johnny’s Selected Seeds, Winslow, ME, United States. The botanical identity of A. vulgaris was confirmed by comparing them with respect to the reference standards at Johnny’s Selected Seeds. After that, 35-day leaf explants excised from old seedlings were inoculated into Murashige and Skoog (MS) medium (Seeds of ) medium  suppleme) medium .Agrobacterium rhizogenes (A4 and R1000) transferred to the medium were cultured overnight at 25°C in an orbital shaker at 180 rpm. The bacterial cells were harvested by centrifugation at 5,000 rpm for 15 min at 25°C. The pellet-containing bacterial cells were re-suspended in liquid MS medium, and the final optical density (OD) was adjusted to 0.6.From the overnight glycerol stock, 500 μl was taken and transferred to the Luria–Bertani (LB) medium, and the same was subcultured thrice to obtain active culture. The Gram-negative soil bacteria A. rhizogenes infection medium, and incubated for 10 min at 25°C. After infection, the explants were dried on a sterile filter paper and placed on semisolid 1/2 MS medium supplemented with 3% sucrose and supplemented separately with 50, 100, and 150 μM of acetosyringone . AS was filter-sterilized by using a 0.2-μm syringe filter 109  and added to the cooled autoclaved medium. After cocultivation, explants were washed with liquid 1/2 MS containing 500 mg/L cefotaxime, followed by sterile distilled water. The explants were then inoculated on 1/2 MS medium supplemented with 500 mg/L cefotaxime and kept under dark condition for HR induction. HRs of more than 1–2 cm length were excised from the explants and transferred to 30 ml 1/2 MS, 1/2 MS + B5 vitamins, 1/2 MS + FDP 1 μM, 1/2 MS + FDP 3 μM, 1/2 MS + B5 vitamins + FDP 1 μM, and 1/2 MS + B5 vitamins + FDP 3 μM supplemented with 3% sucrose with 500 mg/L cefotaxime until the residual bacteria are killed completely. The experiment was run thrice with 25 explants for each experiment.In aseptic condition, leaf explants derived from 45-day-old plants were wounded with a sterile scalpel, transferred into the 5 vitamins, 1/2 MS + FDP 1 μM, 1/2 MS + FDP 3 μM, 1/2 MS + B5 vitamins + FDP 1 μM, and 1/2 MS + B5 vitamins + FDP 3 μM. The growth index (GI) of all the four lines was measured initially and inoculated with 20 mg of FW bacteria-free HRs (3 weeks old). It was transferred into a 30 ml liquid medium in a 250 ml conical flask and maintained on a rotary shaker at 120 rpm in a dark room for 12 weeks. The in vitro grown HRs were subcultured in a fresh medium for 6 weeks to stimulate further growth of the hairs. Each treatment consisted of three replicates, and each replicate contained 25 explants per treatment. The GI was determined by using the following formula . The amplified products were analyzed by electrophoresing 1.4% (w/v) of agarose gel along with 100 bp DNA marker  staining with ethidium bromide.Integration of the T-DNA, which is responsible for HR formation, was confirmed by polymerase chain reaction (PCR) analysis using  primers . Genomic primers . The fol2SO4). The oil was then filtered and stored at 4°C for further experiments.Calli and HRs (10 g each) were subjected to hydrodistillation for 4 h at 60°C using a Clevenger-type apparatus. The obtained essential oil was dried over anhydrous sodium sulfate  equipped with an AOC-20i autosampler. One microliter of the essential oil was injected, and the chromatographic separation of β-caryophyllene was achieved with HP5-MS column . Helium with a purity of 99.999% was utilized as the carrier gas at a flow rate of 10 ml/min.The extracted sample was cleaned by passing it through a silica gel column (glass column) containing glass wool, silica gel 60 , and anhydrous Nar2 = 0.9999). Scanning interval and SIM sampling rate were 0.5 and 0.2 s, respectively.The injector port, interface, and ion source temperatures were set at 260, 300, and 230°C, respectively. The programmed oven temperature was as follows: the initial temperature was set at 120°C for 1 min, then raised to 270°C at 6°C/min and finally to 320°C at 7°C/min, and held for 10 min. A solvent delay of 6 min was set to protect the filament from oxidation. The mass spectrometer was operated in electron impact (EI) mode at an ionization potential of 70 eV and at an emission current up to 60 mA. The quantification was performed in selected ion monitoring (SIM) mode by monitoring mass ions for each β-caryophyllene. For quantification, four different calibrators  were used for linearity check. It showed good correlation coefficient , as well as the number of explants responding for HR initiation growth rate. The statistical analyses were performed by ANOVA using SPSS. The differences between means were tested for significance by Duncan’s multiple range test at p = 0.05.The evaluation of β-caryophyllene from various callus and HR samples of A. vulgaris L.  elicitor treatment of Silybum marianum callus cell culture medium  followed by  fresh and dry weight was exhibited  cultures of Catharanthus roseus with the highest biomass production of FW 10 g and DW 6 g  and NAA (5–16 μM) with FDP as a precursor (1 and 3 μM) individually, as per a previous study by garis L. , b. The egerrima . MS mediegerrima . Sujathae medium . In recegulators . Arya anxhibited . Then frxhibited . Similar culture . This stia annua . Quercet culture . Similar1.8 mg/g . A simild DW 6 g . PhenyleDW 0.4 g . In earlDW 0.4 g . The preDW 0.4 g .A. rhizogenes required a different time and duration for the root induction from leaf explants of A. vulgaris L. (4 and R1000) strains after 10–15 days of culture , the HR culture attained a maximum and minimum of a 30-fold and a 65-fold increase of the initial weight of 200 mg (strain), and the HR culture attained a maximum and minimum of a 25-fold and a 55-fold increase in the initial weight of 163 mg (R1000) within 12 weeks on 1/2 MS + FDP 1 μM and 1/2 MS + FDP 3 μM, respectively. After 6 weeks, there was no significant difference in the GI% in another precursor-treated HRs. However, after 12 weeks of culture, GI% was greater in the GI of A. vulgaris L. HRs with a strain at 6 and 12 weeks in the rotary shaking system. At 6 weeks GI of A4 HR culture, GI% was found without precursor treatment in 1/2 MS (42.10%) and 1/2 MS + B5 vitamins (32.55%) and with precursor treatment in 1/2 MS + FDP 3 μM (70.85%) and 1/2 MS + B5 vitamins + FDP 3 μM (60.23%). After 12 weeks, the GI of A4 HR culture without precursor treatment was observed as 1/2 MS (27.33%) and 1/2 MS + B5 vitamins (30.33%), and with precursor treatment, it was observed with the highest response in 1/2 MS + FDP 3 μM (55.00%) and 1/2 MS + B5 vitamins + FDP 3 μM (77.33%), respectively  and 1/2 MS + B5 vitamins (27.44%), and with precursor treatment, it was observed in 1/2 MS + FDP 3 μM (45.11%) and 1/2 MS + B5 vitamins + FDP 3 μM (61.00%). After 12 weeks, the GI of R1000 HR culture without precursor treatment was observed in 1/2 MS (27.33%) and 1/2 MS + B5 vitamins (30.31%), and with precursor treatment, it was observed in 1/2 MS + FDP 3 μM (55.00%) and 1/2 MS + B5 vitamins + FDP 3 μM (77.33%) , MS, and 1/2 MS, after 30 days showed 3-fold increases in growth rate , 1/2 MS + B5 vitamins (26 g), 1/2 MS + FDP 3 μM (39 g), and 1/2 MS + B5 vitamins + FDP 3 μM (40 g) and DW 1/2 MS (12 g), 1/2 MS + B5 vitamins (14 g), 1/2 MS + FDP 3 μM (17 g), and 1/2 MS + B5 vitamins + FDP 3 μM (20 g). The R1000 HRs showed high production with increased FW and DW on different media: FW 1/2 MS (21 g), 1/2 MS + B5 vitamins (22 g), 1/2 MS + FDP 3 μM (39 g), and 1/2 MS + B5 vitamins + FDP 3 μM (41 g) and DW 1/2 MS (10 g), 1/2 MS + B5 vitamins (11 g), 1/2 MS + FDP 3 μM (19 g), and 1/2 MS + B5 vitamins + FDP 3 μM (19 g)  in 7 days. Anthocyanin content peaked around 10.2% DW with yeast elicitor at 1% (v/v). Simultaneously, the maximum production of OA and ursolic acid was 19 and 27 mg/L, respectively, and a 46 and 24% increase in cultures administered with a 2% (v/v) yeast elicitor  , 6C,D. Iwth rate . SimilarL. plant . In anot culture . In Genttivation . The esticrantha  and Gentrophylla . InconsiM (19 g) . The pre 50 days . The larsinthium  and Swer chirata . Inoguch g of DW . Perillaelicitor .rolB gene fragment (780 bp) and rolC gene fragment (540 bp) was confirmed by using gene-specific primers, and successful transformation was confirmed by the presence of rolB and rolC gene fragments. The results of PCR study indicated that the rolB and rolC gene fragments were incorporated into the A. vulgaris L. HR cultures. It indicates that the presence of the incorporation of the T-DNA into the genome of A. vulgaris L. HRs has been successful  genes harbored by the Ri plasmid of this bacterium are integrated into the host plant genome, causing HRs. rol genes are a reflection to affect the growth and development of transformed roots and induce secondary metabolite synthesis by turning on the mediated transcription defense genes  was determined to be utilized. GC–MS of β-caryophyllene compound retention time peak was observed at 7.34 s. In the present study, extracts of callus samples were analyzed through GC–MS by the scan mode method, and the highest β-caryophyllene compound was observed in without precursor-treated callus 2,4D 13.5 μM (1.61 mg/ml) and precursor-treated callus 2,4D 13.5 μM + FDP 3 μM (2.38 mg/ml), followed by NAA 16.1 μM (2.28 mg/ml)  and NAA (2 mg/L), along with amino acids, such as valine and cysteine (12.5 mg/L), resulted in higher content of artemisinin compound  were used for callus induction from MS media along with various growth regulators , and the final quantitative estimation of essential oils was observed at the highest amount of oil content at a 9-week-old callus culture  . These r8 mg/ml) , where ecompound . Enhance culture . Similar culture . Various culture . A simil culture . In this4 induced HR, the highest β-caryophyllene compound in HR cultures without precursor treatment was 2.44 mg/ml, whereas precursor-treated HR showed 2.92 mg/ml (1000 induced HR cultures without precursor treatment was 1/2 MS (2.16 mg/ml) and precursor treatment with 1/2 MS + FDP 3 μM (2.80 mg/ml)  (in vitro secondary metabolites engineering strategies for increasing β-caryophyllene content (17%) that was found in P. minor roots  from A. annua was confirmed in the genome of the Cyanobacterium synechocystis species by means of gas chromatography with flame-ionization detection (GC-FID) and GC–MS analyses  . The obsCC13333) . FDP preor roots , Micromeruticosa , Copaife species , Litsea a cubeba , Ailanthltissima , Rosmarinalis L. , and Polum minus . Recentlanalyses .Valeriana officinalis HRs for the enhancement of β-caryophyllene compound by recombinant and elicited methods  and precursor treatments (cyclooxygenase inhibitor); the highest vincamine compound was observed at 17%  for yeast extract + Ag+ + MeJA, and elicitation metabolite profiling was analyzed by ultra-performance liquid chromatography (UPLC) , 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), and synthase from squalene (SQS) to use a library of cDNA made from hairy A. annua roots. Previously, overexpression of farnesyl pyrophosphate synthase in A. annua leads to high artemisinin aggregation levels by converting IPP and DMADP into diphenylphosphinic acid pentafluorophenyl ester (FDPP) (A. annua 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (AaHDR) involved in enhanced artemisinin production via improved artemisinin biosynthetic pathway precursor development through the MEP pathway were being reported in A. annua. Tryptophan alkaloid compound enhanced through precursor feeding in HR culture has been identified  gene co-transformation system was developed in  methods . The attcultures . The resd at 17% . Enhancey (UPLC) . The useoduction . A solub defense . Recentlr (FDPP) . Cloningentified . The effelidonii . Robins elidonii , 1991 alications  that areications . In thisications .in vitro precursor treatment for enhancement of β-caryophyllene content by using callus and HR culture of A. vulgaris L. Although the biosynthesis pathway of secondary metabolites has not been well explored, the terpenoid biosynthesis pathway is well known for the production of β-caryophyllene via FDP. In this study, we aimed to enhance the output by using FDP as a precursor, and our findings proved that by using precursor-treated cultures, we can improve β-caryophyllene production. In conclusion, β-caryophyllene enhancement was successfully done with precursor-treated HR culture of A. vulgaris L. after 12 weeks in combined treatment culture of 1/2 MS + FDP 3 μM . These findings could encourage and provide support to synthesize new active natural bioactive compounds as an alternative to synthetic repellent and insecticides.This study, to the best of our knowledge, is the first attempt in reporting The original contributions presented in the study are included in the article/SB and BR conceived the idea presented. SB developed the study, performed the experimental work, and wrote the main manuscript. RB developed the GC–MS methodology and performed the analysis. AM and DS analyzed the data interpretation. SS and GP revised the manuscript contents. Finally, all authors have read and approved the 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.