The fluorescent-based organic sensors are not suitable for Cu2+ detection due to their short penetration depth caused by the UV/visible excitation source. Therefore, we have demonstrated a highly sensitive and selective near-infrared (NIR) excitable poly(acrylic acid) (PAA) coated upconversion nanoparticles (UCNPs) based sensor for Cu2+ detection. We construct the PAA modified NaF4@NaF4:Tm@NaGdF4 core-shell-shell structured UCNPs based sensor via a co-precipitation route. The upconversion emission intensity of the PAA-UCNPs decreases linearly with the increase in the Cu2+ concentration from 0.125 to 3.125 μM due to the copper carboxylate complex formation between Cu2+ and PAA-UCNPs. The calculated detection limit of the PAA-UCNPs based sensor is 0.1 μM. The PAA-UCNPs based sensor is very sensitive and selective toward detecting the Cu2+ ions, even when the Cu2+ co-exist with other metal ions. The EDTA addition has significantly reversed the upconversion emission quenching by forming the EDTA-Cu2+ complex based on their greater affinity toward the Cu2+. Therefore, the PAA-UCNPs based sensor can be a promising candidate for Cu2+ detection because of their higher sensitivity and selectivity under 980 nm NIR excitation.Detection of the Cu To detect the trace elements, both essential  and non-essentials (P and S), developing highly selective and sensitive sensors is a crucial, useful and challenging task in the fields of medical, environment, and biology , being the third most essential and abundant micronutrient, exists in very low concentration and participates in various organism's physiological processes   is around 100–150 μg/dL (15.7–23.6 μM) and 1.3 ppm (~20 μM), respectively  in adults  and a multidentate ligand. The reaction between the probe with Cu2+ produces a stable Cu2+ complex that quenches the fluorophore's emission. This method detects the Cu2+ up to 2 ppb  doped UCNPs exhibit several intra 4f−4f transitions producing strong tunable emissions of different colors with longer lifetimes by changing the size and morphology of the UCNPs. Also, the UCNPs can produce tunable multi-color emissions via controlling the dopants concentrations. Low energy NIR light excitation provides a high sensitivity detection system with negligible auto-fluorescence and light scattering. In this regards, different research groups have developed UCNPs based sensors for the detection of organic and inorganic entities such as toluene/phenol , which convert the near Infrared (NIR) light  into visible light, have been become a promising candidate for the bio detection application. Compared with the dyes and quantum dots, the UCNPs exhibit several benefits: sizeable anti-Stokes shift, low toxicity, high chemical/photostability, lower photodamage, and deeper light-penetration depth  and Yb3+ concentration can reach up to ~100% in hexagonal NaYF4:Yb3+/Tm3+@NaYF4 core/shell nanocrystals after overcoming concentration quenching of Yb3+  in organic solvents (oleic acid (OA)/oleylamine/1-octadecene). Besides, the bare UCNPs induces the quenching effect due to the surface defects and vibrational deactivation from solvents  and 2-amino-3′,6′-bis(ethylamino)-2′,7′-dimethyl-3′,9a′-dihydrospiro-3-one (a rhodamine derivative) based on energy transfer process between UCNPs and rhodamine derivative. The rhodamine derivative was excited by the UCNPs, and detected Cu2+ in the 2–14 μM range with good selectivity and high chemo stability. The core-shell technology also helps to improve the sensitivity of Cu2+ detection. Zhang Y. et al.  (PAA) functionalized NaF4@NaF4:Tm@NaGdF4 core-shell-shell structured UCNPs, where the UCNPs work as the energy donor and Cu2+ ions as the acceptor. The reversible process of Cu2+ detection comprising the fluorescence quenching and recovery upon adding Cu2+ ions and EDTA, respectively, is schematically illustrated in 4@NaF4:Tm@NaGdF4 UCNPs, composed of NaYb0.5Nd0.5F4 core, NaYb0.5Gd0.49Tm0.01F4 first shell and NaGdF4 second shell were prepared by using co-precipitation method. At first, the OA ligand, attached during the preparation, was removed, followed by functionalization with PAA to form PAA-UCNPs for realizing efficient Cu2+ detection (Process I and II). With Cu2+ (Process III), the carboxylate group of PAA-UCNPs can bind selectively to the Cu2+ ions owing to the electrostatic interaction between Cu2+ and carboxylate anion of PAA, forming copper carboxylate complex to quench the UCNPs fluorescence. Upon 980 nm NIR light excitation, in the presence of Cu2+, the emission intensity of the UCNPs was quenched linearly as the Cu2+ concentration increased from 0.125 to 3.125 μM with the detection limit of 0.1 μM. The upconversion emission intensity of the UCNPs was recovered up to 90% after further addition of EDTA (Process IV). Besides, the current UCNPs based nano-platform showed high sensitivity and selectivity of Cu2+ ions over the range of other metal ions .In this study, we construct a new upconversion sensor for Cu3CO2)3 · xH2O, 99.9%; Yb(CH3CO2)3 · 4H2O, 99.9%; Gd(CH3CO2)3 · xH2O, 99.9%, Tm(CH3CO2)3 · xH2O, 99.9%; NaOH, >98%; NH4F, >98%; 1-octadecene, 90%; oleic acid, 90%; poly(acrylic acid) (PAA), MW ≈ 1800; HCl, >98%, were purchased from Sigma-Aldrich. C10H14N2O8Na2 · 2H2O (EDTA) (>98%) were purchased from Sangon Biotech, China. Ethanol (99.5%), cyclohexane (99.5%), diethylene glycol (DEG) (>99%) were purchased from Macklin, China. The solutions of metal cations were performed from their corresponding salts of analytic grade or molecular biology grade, such as LiCl, NaCl, KCl, MgCl2·6H2O, CaCl2·4H2O, AlCl3·6H2O, BaCl2, ZnCl2, MnCl2·4H2O, CoCl2·6H2O, NiCl2·6H2O, and CuCl2·3H2O. The water used in this work is ultrapure water (18.2 MΩ cm) deionized by the Milli-Q system.Following chemicals with appropriate purities were used as received without further purification. Nd3 (0.2 M) and Yb(CH3CO2)3 (0.2 M) was mixed with 4 mL of oleic acid and 6 mL of 1-octadecene solution in 50-mL flask. The mixture was heated at 120°C for 30 min, then heated at 170°C for 40 min to form the lanthanide-oleate complexes and then cooled down to room temperature. Thereafter, mixed methanol solution of NH4F (1.5 mmol) and NaOH (1 mmol) was added to the above mixture and heated at 45°C for 30 min. After methanol evaporation at 110°C, the solution temperature was raised to 295°C and maintained for 1.5 h under argon. Post 295°C heating, the solution was cooled to room temperature and then the resulting nanoparticles were washed with ethanol and cyclohexane for several times and re-dispersed in 6.5 mL of cyclohexane.The core UCNPs were synthesized by using a modified co-precipitation method, as reported elsewhere 3 , 4 mL oleic acid and 6 mL 1-octadecene to a 50 mL flask, and heated at 120°C for 30 min, then heated at 170°C for 40 min to form the lanthanide-oleate complexes, and then cooled down to room temperature. The preformed core nanoparticles dispersed in 3 mL of cyclohexane with methanol solution of NH4F (1.5 mmol) and NaOH (1 mmol) were added and heated to 45°C for 30 min. After methanol evaporation, the reactant was heated at 295°C for 1.5 h under argon atmosphere followed by cooling to room temperature. The resulting nanoparticles were washed several times with ethanol and cyclohexane, and re-dispersed in 4 mL of cyclohexane. The procedures were repeated for the preparation of core-shell-shell nanoparticles.In a typical procedure to the shell growth of NaYbThe oleate capped UCNPs (denoted as OA-UCNPs) dispersed in 2 mL of cyclohexane were precipitated by ethanol and dispersed again in 2 mL of HCl solution (0.1 M). The mixture was then sonicated at 35°C for 1 h to remove the OA ligands. After the reaction, the uncapped UCNPs were collected via centrifugation for 0.5 h , followed by washing twice with deionized water and re-dispersed in deionized water .Typically, 30 mg of poly acrylic acid (PAA) was added to the deionized water (9 mL) with NaOH (0.1 M in deionized water) to adjust the pH at 8 under robust stirring at room temperature, followed by the addition of uncapped UCNPs (1 mL) dropwise, and the mixture was stirred for another 2 h. This solution was then mixed with DEG (10 mL) and heated at 105°C for 1 h under stirring for water evaporation. The resultant solution was then heated at 160°C for 2 h in a 20 mL Teflon-lined autoclave. Next, the PAA capped UCNPs (denoted as PAA-UCNPs) were collected through centrifugation at 15,000 rpm for 0.5 h. PAA-UCNPs were washed several times with deionized water and ethanol, re-dispersed in deionized water , and stored at 4°C in fridge.−1 at a resolution of 1 cm−1 using a Nicolet 6700 FT-IR Spectrometer . Ultraviolet-visible (UV–Vis) spectra were measured on SHIMADZU UV2700 spectrophotometer. Upconversion luminescence spectra were recorded by FluoroMax-4 fluorescence spectrometer  at the same condition, except using an external 980 nm CW laser at 3W or 808 nm laser at 2 W . The photoluminescence intensity was obtained by integration the upconversion emission spectra in wavelengths range of 330–850 nm. Thermogravimetric analysis (TGA) was performed using Netzsch DTA-TG STA449F5 instrument under N2 atmosphere at a heating rate of 10° per minute.To investigate the morphology/size of UCNPs, the samples were characterized by a HT7700 transmission electron microscope (TEM) with the working voltage of 100 KV . The crystal phase of UCNPs was identified by DY735 X-ray diffractometer (XRD)  with 2θ range from 10 to 80° at a scanning rate of 5° per minute, with Cu Kα irradiation (λ = 1.5406 Å). Fourier transform infrared (FT-IR) spectra of UCNPs using KBr pellet technique were recorded between 400 and 4,000 cm−3 M were prepared in deionized water using the corresponding metal salts and diluted in deionized water at concentrations of 5 × 10−4 M and 5 × 10−6 M, respectively. The PAA-UCNPs dispersion with concentrations of 0.25 and 0.025 M were prepared in deionized water. The PAA dispersion with a concentration of 5 × 10−3 M was prepared in deionized water. The EDTA stock solution with a concentration of 5 × 10−3 M was prepared in deionized water.The metal ion stock solutions with a concentration of 5 × 102+ solution (5 × 10−4 M) and diluted to 4 mL in deionized water. In the UV-Vis experiment of 2+ (5 μM) and PAA (40 μM) chelating with Cu2+ (40 μM) were prepared in deionized water by diluting corresponding bulk solutions. The mixture was filled in a quartz cell with an optical path of 1.0 cm to achieve an absorption spectrum. For the upconversion emission experiment with 980 nm laser excitation, 40 μL PAA-UCNPs dispersion (0.025 M) was added to a 4 mL bottle, then the Cu2+ solution (5 × 10−6 M) was added by means of a micro-pipette and diluted to 4 mL. The mixture was filled in a quartz cell with an optical path of 1.0 cm for testing. For the upconversion emission experiment with 808 nm laser excitation, 100 μL PAA-UCNPs dispersion (0.25 M) was added to a 3 mL bottle, then the Cu2+ solution (5 × 10−6 M) was added by means of a micro-pipette and diluted to 3 mL. The mixture was filled in a quartz cell with an optical path of 1.0 cm for testing. For upconversion emission spectra of the selective experiment with 980 nm laser excitation, 40 μL PAA-UCNPs dispersion (0.025 M) was added to a 4 mL bottle, then the metal ion solution , was added by means of a micro-pipette and diluted to 4 mL. The mixture was filled in a quartz cell with an optical path of 1.0 cm for testing. For upconversion emission spectra of the reversibility experiment, EDTA was added dropwise to the solution of PAA-UCNPs whose fluorescence has been quenched by copper ions. After mixed well, filled it into a quartz cell with a 1.0 cm optical path for testing. After recovery experiment through addition of EDTA, the PAA-UCNPs were collected via centrifugation, followed by washing several times with deionized water and ethanol, re-dispersed in deionized water, and stored at 4°C in fridge for another Cu2+ sensing test.In the UV-Vis experiment of 2+ was determined using the following equation  of the UCNPs. The FT-IR spectra of the OA-capped, un-capped, and PAA-capped UCNPs measured in the range of 500–4,000 cm−1 are presented in 2+ ion. The FT-IR spectrum of the as-prepared OA-capped UCNPs exhibits five prominent peaks at 1,463, 1,558, 2,851, 2,921, and 3,443 cm−1. The presence of the doublets at  and  attributes to the [symmetric (νs) and asymmetric (νas)] stretching vibrations of the carboxylic group (–COO) and methylene group (–CH2), respectively, which indicates the presence of OA ligand on the surface of the UCNPs  group , and CO2 (νO2C) groups of PAA ligand, respectively , respectively. The intensity of the doublet caused by the CH2 (νH2C) group decreases significantly in comparison to the OA capped UCNPs. Thus, we conclude that the capping of the PAA group onto the surface of the UCNPs is successful. This is consistent with the TGA results. The TGA plots of PAA-UCNPs and un-capped UCNPs are shown in 0.5Nd0.5F4 and core-shell structure with the composition of NaYb0.5Nd0.5F4@NaYb0.5Gd0.49Tm0.01F4 nanoparticles, respectively. The core and core-shell particles exhibit the particle size of 18 ± 0.5 and 25 ± 2.1 nm, respectively. Thus, the thickness is ~7 nm and ~2 nm for the inner shell and outmost shell layer, respectively.The structure and morphology characteristics of the as-prepared NaYb2+ and PAA-UCNPs. The UV-Vis spectrum F4:Tm@NaGdF4 , and NaYbF4@ NaF4:Tm@NaGdF4 UCNPs (excited at 980 nm) UCNPs. The emission spectra displayed several sharp characteristics peaks of Tm3+ at 344, 360, 450, 474, 645, 695, 723, and 801 nm, which can be attributed to the 1I6 → 3F4, 1D2 → 3H6, 1D2 → 3F4, 1G4 → 3H6, 1G4 → 3F4, 3F3 → 3H6  transition shows significant higher emission intensity than the 1G4 → 3H6 (474 nm). The higher emission intensity of the 3H4 → 3H6 transition originates due to the depletion of 3H4 level by surface quenching, which will supress the population of 1G4 by subsequent excited state absorption or energy transfer (ET) F4:Tm@NaGdF4, the emission intensity of the peaks below the 460 nm (UV and blue emissions) has increased with decrease in the 474 nm (1G4 → 3H6) emission peak, which agrees with the result as reported by Wen et al.  under 980 nm excitation within 0.1–5 μM range, as can be seen in 2+ detection using different nanoplatforms (probes), see The emission spectra of PAA-NaYbμM range , indicatμM range . The flug et al. . These r0.5Nd0.5F4@NaYb0.5Gd0.49Tm0.01F4@NaGdF4 UCNPs under 980 nm excitation toward Cu2+ ions was further investigated by testing the PAA-UCNPs response toward a wide range of metal ions, particularly, equimolar Al3+, Ba2+, Ca2+, Co2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, and Zn2+. 2+ ions response in 2+, indicating that the current PAA-UCNPs based sensing platform is most sensitive toward the Cu2+ ions. Furthermore, to confirm the competitive selectivity of the Cu2+ ion by PAA-UCNPs, the upconversion emission spectra of PAA-UCNPs in presence of mixed metal ions i.e., Al3+, Ba2+, Ca2+, Co2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, and Zn2+ at different concentrations  were measured and presented in 2+ do not show substantial difference in the emission intensity, however, as the Cu2+ ions were added to the solution containing other metal ions, the emission intensity was significantly decreased monotonically with increase in the Cu2+ concentration, see 2+ and PAA-UCNPs that leads to the efficient fluorescence quenching of PAA-UCNPs. These results demonstrate that the PAA-UCNPs based platform can serve as an upconversion detector for the detection of the Cu2+ with high sensitivity and selectivity. The preferred selectivity of the Cu2+ ions can be explained by considering the binding constants of the multi-valent ions to the PAA. Many researchers have measured the binding constants of the different multivalent metal ions and reported in the literature  and after (red line) the Cu2+ addition with obvious emission intensity difference, are shown in 2+ detection is reversible. It is also noted that the PAA-UCNPs based platform responds very quickly within a minute for both the Cu2+ ions to quench the emission and the EDTA solution to recover the quenched emission. We believe that the strong affinity of the Cu2+ toward EDTA is responsible for the dissociation of the copper carboxylate complex thereby recovering the emission intensity of the PAA-UCNPs.We also checked the reversibility of the Cu2+ to PAA-UCNPs. Generally, upon 980 nm excitation, the Yb3+ ions are excited from their 2F7/2 ground state to the 2F5/2 excited state, subsequently, the cooperative ET from Yb3+-trimer to the different level of the Tm3+ produces the visible emission (Jean-Claude and Claude, 2+ ions, the cooperative ET from Yb3+-trimer to Tm3+ is hampered by the quenching processes caused by Cu2+, which leads to the upconversion emission quenching of the PAA-UCNPs (Saleh et al., 2+ addition, is caused by the disturbance of the Yb3+ → Tm3+ ET by the heavy metal ion. Upon adding EDTA, the Cu2+ ions leave the copper carboxylate complex to bind with the EDTA because of their higher affinity toward EDTA than the PAA and thus the upconversion emission gets recovered.2+ ions. With increase in the Cu2+ concentration, the upconversion emission intensity of the PAA-UCNPs is quenched monotonically within the 0.125–3.125 μM range due to the ET between PAA-UCNPs and Cu2+. The LOD for the Cu2+ is calculated to be 0.1 μM. It has been found that the PAA-UCNPs based nanoplatform can detect Cu2+ with higher sensitivity and selectivity even from the mixture of the various metal ions under 980 nm laser excitation. We believe that the PAA-UCNPs based nanoplatform under NIR excitation can be used in a wide range of practical bio-applications due to their superior characteristics such as no autofluorescence from bio-samples and interference from scattered excitation source, and high penetration depth. This makes PAA-UCNPs based nanoplatform a promising candidate for the biological applications in copper ion-related diseases i.e., in detection and diagnosis.We have developed a simple water-dispersible PAA-UCNPs based nanoplatform for the detection of CuThe original contributions presented in the study are included in the article/HW conceived the study. SS, HW, and XC designed the experiments. HW and DAH wrote the manuscript. SS, ZM, and GT carried out the material synthesis, characterization, and measurements. SS, HW, XC, and DAH analyzed the data. All authors contributed to the article and approved the submitted version.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.


The N-Myc downstream-regulated gene (NDRG) family belongs to the α/β-hydrolase fold and is known to exert various physiologic functions in cell proliferation, differentiation, and hypoxia-induced cancer metabolism. In particular, NDRG3 is closely related to proliferation and migration of prostate cancer cells, and recent studies reported its implication in lactate-triggered hypoxia responses or tumorigenesis. However, the underlying mechanism for the functions of NDRG3 remains unclear. Here, we report the crystal structure of human NDRG3 at 2.2 Å resolution, with six molecules in an asymmetric unit. While NDRG3 adopts the α/β-hydrolase fold, complete substitution of the canonical catalytic triad residues to non-reactive residues and steric hindrance around the pseudo-active site seem to disable the α/β-hydrolase activity. While NDRG3 shares a high similarity to NDRG2 in terms of amino acid sequence and structure, NDRG3 exhibited remarkable structural differences in a flexible loop corresponding to helix α6 of NDRG2 that is responsible for tumor suppression. Thus, this flexible loop region seems to play a distinct role in oncogenic progression induced by NDRG3. Collectively, our studies could provide structural and biophysical insights into the molecular characteristics of NDRG3. The α/β-hydrolase fold superfamily is one of the largest groups of structurally related proteins since its discovery in 1992 . In spitThe N-Myc downstream-regulated gene (NDRG) family is a member of the α/β-hydrolase superfamily and is repressed by Myc expression ,6. GivenCompared with the tumor suppressive NDRG members, NDRG3 has been reported to be oncogenic. NDRG3 is upregulated in epithelial prostate cancer cells and prostatic stromal cells at both mRNA and protein levels, and overexpression of NDRG3 induces cell proliferation and migration . InteresIn lieu of the lactate-mediated NDRG3 signaling, NDRG3 provokes anti-apoptotic processes during the hypoxia postcondition by regulating adenosine A2a receptors , activat® DNA polymerase  and subcloned in the expression vector, pET-28a(+)  to produce recombinant protein containing an N-terminal hexahistidine tag (MGSSHHHHHHSSGLVPRGSH). The recombinant plasmid was transformed into BLR(DE3), an Escherichia coli strain , and the cells were cultured in Luria–Bertani medium  supplemented with 30 μg/mL kanamycin. When the transformed cells were in the mid-log growth phase, 0.5 mM of isopropyl β-D-thiogalactopyranoside (IPTG) was added for induction of NDRG3 ΔNC overexpression. The cells were additionally incubated for 16 h at 293 K, then pelleted by centrifugation at 6000× g for 10 min at 277 K and disrupted by sonication in a buffer containing 20 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) titrated with hydrochloric acid to pH 7.5, 500 mM sodium chloride, and 35 mM imidazole supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The crude lysate was centrifuged at 36,000× g for 50 min at 277 K and the resultant supernatant was loaded onto a nickel-charged HiTrap™ Chelating HP 5 ml column . After washing unbound proteins, the column-bound proteins were eluted by the addition of a buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM sodium chloride, and an imidazole gradient increasing from 35 to 1000 mM. During an imidazole gradient elution, NDRG3 ΔNC protein was divided into monomer and dimer fractions  and further purified using a HiTrap™ Q HP 5 ml column  with a linear gradient from 50 to 500 mM of sodium chloride. The eluted proteins were loaded onto a HiLoad™ 16/600 Superdex 200 prep grade column  equilibrated with 10 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 1% glycerol, and 0.1 mM TCEP. The purified monomer protein was concentrated to 40 mg/mL using an Amicon Ultra-15 centrifugal filter unit  for further studies. NDRG3 ΔNC dimer fractions were purified using the same protocol as for the NDRG3 ΔNC monomer . The truncated NDRG3 gene  was amplified by PCR using PrimeSTARractions . NDRG3 Δ monomer .® HS DNA polymerase; Takara, Kusatsu, Japan). NDRG3 S255A/N281A mutant plasmid was transformed into BLR(DE3) E. coli strain . Each cell containing NDRG3 ΔNC wild type (WT) plasmid and S255A/N281A plasmid was cultured and induced by 0.5 mM of IPTG, and additionally incubated for 16 h at 293 K. The cells were pelleted by centrifugation at 6000× g for 10 min at 277 K and disrupted by sonication in a buffer containing 20 mM Tris-HCl pH 7.5, 500 mM sodium chloride, and 35 mM imidazole supplemented with 1 mM PMSF. The crude lysates were centrifuged at 36,000× g for 50 min at 277 K and the resultant supernatant was loaded onto a nickel-charged HiTrap™ Chelating HP 5 ml column . After washing unbound proteins, the column-bound proteins were eluted by addition of a buffer containing 20 mM Tris-HCl pH 7.5, 500 mM sodium chloride, and 300 mM imidazole. Then, 0.5 mg of eluted NDRG3 ΔNC WT and S255A/N281A proteins were loaded onto Superdex™ 200 Increase 10/300 GL  pre-equilibrated with 10 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 1% glycerol, and 0.5 mM TCEP at flow rate of 0.75 mL/min using the ÄKTA Pure FPLC system . NDRG3 C30S, I171M/S176H, and C30S/I171M/S176H mutated plasmids were transformed into SoluBL21™, E. coli strain . The overexpression and purification steps were the same as for the purification of NDRG3 ΔNC WT. Each purified monomeric mutant was concentrated to 20 mg/mL using an Amicon ultra-15 centrifugal filter unit  for further studies.The NDRG3 ΔNC mutants  were produced by PCR-based site-directed mutagenesis (PrimeSTARThe human NDRG3 ΔNC (residues 29–320) was diluted to 20 mg/mL, and initial crystallization experiments were carried out with commercially available crystal screening kits using the sitting-drop vapor diffusion method at 295 K. First crystals were obtained by mixing 0.5 μL of 20 mg/mL NDRG3 ΔNC and 0.5 μL of a solution containing 200 mM sodium citrate tribasic dihydrate and 20% polyethylene glycol (PEG)3,350 . Since the crystals did not well diffract, we optimized the crystals with a matrix screening containing 100–300 mM sodium citrate tribasic dihydrate and 10–30% PEG3,350 using the hanging-drop vapor diffusion method. The best diffracting crystal was grown in a drop mixed with 1 μL of 10 mg/mL protein solution, 0.8 μL of a crystallization solution containing 200 mM sodium citrate tribasic dihydrate and 20% PEG3,350, and 0.2 μL of the crystallization solution containing microseeds of the initial crystals. Initial crystals of NDRG3 C30S were obtained with the same crystallization solution as for NDRG3 ΔNC crystals. The best diffracting crystal of C30S mutant was obtained by mixing 15 mg/mL protein supplemented with 0.01 mM TCEP. First crystals of NDRG3 I171M/S176H were obtained by mixing 0.5 μL of 13 mg/mL protein and an equal volume of crystallization solution containing 200 mM ammonium citrate tribasic (pH 7.0) and 20% PEG3,350 buffer condition . The crystals of I171M/S176H mutant were optimized with the crystallization solution supplemented with 3% dextran sulfate sodium salt (Mr 5000). All crystals were cryoprotected with paratone oil, then flash-frozen in a liquid nitrogen gas flow at 100 K prior to data collection.C-centered monoclinic symmetry with six monomers in an asymmetric unit (ASU) to describe the disulfide bonds between each Cys30 and dimer. The structure was solved using molecular replacement method with the structure of human NDRG2b protein (PDB Id: 2XMQ) [free value calculated from 5.0% of the independent reflections. Because of merohedral twinning of NDRG3 ΔNC crystal, the intensity-based twin law option in REFMAC5 was applied for all refinement processes. The space group of NDRG3 C30S crystal was P3121 and it contains four molecules in an ASU. The crystal structure of NDRG3 C30S was determined using PHASER-MR in the PHENIX software and refined using REFMAC5. We adjusted the TLS refinement option of which parameters were calculated by PDB-REDO [221 and the structure was determined using PHASER-MR in the PHENIX software. Since the diffraction data were predicted to contain merohedral twinning, twin law was adjusted to mutant refinement at the processing in REFMAC5 as for the NDRG3 ΔNC WT crystal. The stereochemical qualities of the NDRG3 ΔNC and mutants models were checked using MolProbity [X-ray diffraction data of NDRG3 ΔNC and C30S mutant crystals were collected at synchrotron beam line BL-7A at the Pohang Light Source , using a Quantum Q270 CCD detector . While the HKL2000 program  predicted: 2XMQ)  as a sead: 2XMQ) . The modd: 2XMQ)  in the Cd: 2XMQ)  and Wincd: 2XMQ) . All refPDB-REDO . X-ray dlProbity . The datlProbity . Coordin™ 10/300 Superdex 200 GL  column was pre-equilibrated with a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM sodium chloride, and 0.5 mM TCEP, and was normalized using ovalbumin. 100 μL of monomer and dimer NDRG3 ΔNC at 2.0 mg/mL were injected into the machine at flow rate of 0.4 mL/min, respectively. Data were analyzed using the Zimm model for fitting static light-scattering data and graphed using EASI graph with a UV peak in the ASTRA V software .SEC-MALS was implemented with an FPLC machine  connected to a Wyatt MiniDAWN TREOS MALS instrument and a Wyatt Optilab rEX differential refractometer . A HiLoad™-plus CD Spectrometer  at 298 K with a wavelength range from 260 nm to 180 nm. NDRG3 ΔNC, NDRG3 mutants, and NDRG2 proteins were diluted to 0.4 mg/mL with a buffer containing 20 mM potassium phosphate dibasic pH 7.5 and 50 mM sodium fluoride. The maximum absorbances in CD wavelength were adjusted to have ranges from 0.80 to 0.85. The bandwidth was 1.5 nm and the time per point value was 0.5. The temperature was set to 298 K.CD spectroscopy was implemented with the Chiranscanhttp://ffas.burnham.org/XtalPred-cgi/xtal.pl) [https://www.phosphosite.org) [α atoms of each subunit compared with chain A as a reference, overall subunits were structurally similar and the helix α6 region is disordered in all the subunits. Interestingly, helix α8 of chain D, E, and F were structurally different from that of chain A. In the crystal packing of NDRG3 ΔNC, helix α8 of chain D, E, and F were influenced by adjacent molecules while that of chain A, B, and C were away from adjacent molecules. Therefore, the crystal structure of chain A, B, and C of NDRG3 ΔNC seems to represent the structure of NDRG3 in solution than that of chain D, E, and F (The human NDRG3 protein (375 amino acids) was predicted to contain flexible N- and C-terminal regions by the Xtalpred server  . Additioite.org)  indicateite.org) . The NDRE, and F .2) as that of the interface between chain A and chain D (999.3 Å2). However, the predicted solvation free energy of the dimeric interface between chain B and chain F was −11.5 kcal/mol, which is more stable than that of chain A and chain D . To clarify the dimeric interface of NDRG3 ΔNC, we mutated Ser255 and Asn281 which are key residues involved in hydrogen bond interactions between chain B/F . SEC-MALS results indicated that the molecular weight of monomer and dimer fractions of NDRG3 ΔNC was calculated as 31.4 kDa and 64.1 kDa, respectively A. While hain B/F E. After hain B/F F. Taken Paraburkholderia xenovorans  and malate complexed esterase (EST) from Thermogutta terrifontis , the overall structure of NDRG3 ΔNC was superposed to the α/β-hydrolases with r.m.s.d. of equipositional Cα atoms at 2.8 Å and 2.9 Å, respectively  experiments to compare secondary structure elements of NDRG3 ΔNC WT, NDRG3 mutants, and NDRG2 D. While Since the first characterization of NDRG1 in 1999, the NDRG family has been reported to play promiscuous roles in tumorigenesis or tumor suppression. Then a pro-tumorigenic activity was reported for a member of the NDRG family, NDRG3, when it was known to be associated with prostate cancer . Later, While NDRG3 ΔNC mainly exists as a monomer, we observed a stabilized dimer fraction during purification. SEC-MALS under reducing conditions indicates that the disulfide bond does not seem to contribute to its dimerization in solution. To characterize the dimeric association of NDRG3, it was worth noting that the dimeric interface between chain B/F and chain A/D faced different directions in our crystal structure. As shown in https://www.phosphosite.org) [NDRG3 has been known to be degraded by PHD2/VHL mediated ubiquitination under normoxia condition, and Pro294 hydroxylation of NDRG3 by PHD2 is a critical step of NDRG3 ubiquitination. To get a clue of ubiquitination of NDRG3, we analyzed ubiquitination sites on NDRG3 using data from the PhosphoSitePlus web server (ite.org) . Our truite.org) , while Kite.org) . Surprisite.org) . FurtherSequence comparisons and structural studies revealed that proteins of the NDRG family contain an α/β-hydrolase fold domain. Although NDRG3 has a high structural similarity to the α/β-hydrolase family, the substitution of the residues of the enzymatic catalytic triad and narrow folded cavity due to the presence of helix α7 and helix α10 indicate that NDRG3 is a nonenzymatic α/β-hydrolase fold protein. A previous report on the crystal structure of NDRG2 revealed the structural features of helix α6 compared with α/β-hydrolase family proteins and showed that helix α6 plays a key role in regulating TCF/β-catenin signaling as a nonenzymatic α/β-hydrolase fold protein. . To our We reported the crystal structure of human NDRG3 and demonstrated the dimeric conformation of NDRG3 from crystal packing in an ASU. Compared with NDRG2, the helix α6 region of NDRG3 is a flexible loop. Considering that helix α6 of NDRG2 regulates TCF/β-catenin signaling, the unfolded helix α6 of NDRG3 would play a distinctive role upon interacting with physiologically relevant binding partners. Our structural studies on NDRG3 will shed light on the characterization of NDRG family proteins, providing a fundamental source for understanding the molecular mechanism of NDRG3 in hypoxia responses.


Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been reported to use aminopeptidase N (APN) as a cellular receptor. Recently, the role of APN as a receptor for PEDV has been questioned. In our study, the role of APN in PEDV and TGEV infections was studied in primary porcine enterocytes. After seven days of cultivation, 89% of enterocytes presented microvilli and showed a two- to five-fold higher susceptibility to PEDV and TGEV. A significant increase of PEDV and TGEV infection was correlated with a higher expression of APN, which was indicative that APN plays an important role in porcine coronavirus infections. However, PEDV and TGEV infected both APN positive and negative enterocytes. PEDV and TGEV Miller showed a higher infectivity in APN positive cells than in APN negative cells. In contrast, TGEV Purdue replicated better in APN negative cells. These results show that an additional receptor exists, different from APN for porcine coronaviruses. Subsequently, treatment of enterocytes with neuraminidase (NA) had no effect on infection efficiency of TGEV, implying that terminal cellular sialic acids (SAs) are no receptor determinants for TGEV. Treatment of TGEV with NA significantly enhanced the infection which shows that TGEV is masked by SAs. Coronaviruses are known as human and animal pathogens that mainly infect the epithelium of the respiratory or intestinal tract. Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and its variant porcine respiratory coronavirus (PRCV) are classified as Alphacoronavirus. They are enveloped viruses containing a single-stranded, positive-sense RNA genome of approximately 28.5 kb. The positive ssRNA serves as mRNA for the generation of viral replicative proteins by translation of open reading frame (ORF) 1a and ORF1b. The genome contains a 5′ untranslated region (UTR), a 3′ UTR, and at least seven ORFs. ORF1a and ORF1b make up two-thirds of the viral genome and encode the non-structural replicase polyproteins (replicases 1a and 1b), which further guide the viral replication and translation, regulate cellular processes, and potentially fulfill other unknown functions. The remaining proximal third of the genome encodes four structural proteins (spike (S), envelope (E), membrane (M), and nucleocapsid (N) by ORF2, ORF4, ORF5, and ORF6, respectively). The S protein is a type I glycoprotein that projects from the virions surface forming the corona-like appearance of viral particles. It contains an N-terminal ectodomain (S1) and a short C-terminal cytoplasmic tail (S2). It is known that the peripheral S1 portion is a globular cellular receptor binding subunit while the transmembrane S2 portion is required to mediate fusion of viral and cellular membranes [The replication of viruses in their host cells is firstly mediated by the virus binding to the cellular receptors. Some Alphacoronaviruses, including human coronavirus 229E (HCoV-229E), feline infectious peritonitis virus (FIPV), canine coronavirus (CCoV), TGEV, and PEDV, may use aminopeptidase N (APN) as a cellular receptor ,7,8,9,10N-glycans, O-glycans, and glycosphingolipids. TGEV shows a hemagglutinating activity with α-2,3-linked sialic acids on the erythrocyte surface as target. Among different types of sialic acids, N-glycolylneuraminic acid was recognized by TGEV more efficiently than N-acetylneuraminic acid [In addition to APN, a sialic acid binding capacity has been reported for TGEV and PEDV ,16. Sialnic acid . Sialic nic acid . Howevernic acid . This sinic acid . Furthernic acid .In the present study, the co-culture system of primary porcine enterocytes that was established in our laboratory was used to study the role of APN in PEDV and TGEV infection in their target cells . Furtherv/v fungizone . Hydrocortisone, spermidine, and Wnt agonist were purchased from Sigma-Aldrich . Porcine insulin was purchased from Protein specialists . The small intestinal contents (IC) were collected from the duodenum of a three-day-old suckling piglet. After euthanasia, a 20 cm long segment of duodenum was closed by two surgical clamps and was removed from a piglet. Then, one clamp was removed and the intestinal contents were released from the lumen into a 15 mL centrifugation tube. In order to collect all the intestinal contents, the lumen was washed once by filling it with 5 mL Dulbecco’s modified Eagle’s medium (DMEM) containing 1000 U/mL penicillin, 1 mg/mL streptomycin, 1 mg/mL gentamicin, and 0.01% v/v fungizone. Then, the DMEM was released from the lumen into the 15 mL tube that already contained the intestinal contents. After centrifugation , the supernatant of the intestinal contents was collected and stored at −20 °C until use.Primary porcine enterocytes were isolated from the ileum of three-day-old piglets and co-cultured with porcine myofibroblasts . EuthaniThree- and seven-day-old primary porcine enterocytes were fixed in HEPES-buffered glutaric aldehyde  for scanning electron microscopy as described previously . After 2Twenty-four h post co-cultivation, monolayers of enterocytes were cultured with medium containing hydrocortisone (1 and 10 µg/mL), spermidine (50 and 500 µM), insulin (1 and 10µg/mL), Wnt agonist (0.1 µM), or intestinal contents (1%) for 24 h. The cells were fixed with 4% paraformaldehyde for 10 min and immunofluorescence staining was conducted for APN expression analysis.Cells were incubated with mouse monoclonal anti-porcine APN antibodies  containing 10% normal goat serum for 1 h at 37 °C, followed by goat anti-mouse-IgG FITC labeled antibodies for 1 h at 37 °C. Nuclei were stained with Hoechst for 10 min at RT. The percentage of APN positive cells were analyzed by fluorescence microscopy (Leica Microsystems GmbH).5.6 TCID50/mL or 107 viral RNA copies/mL of fecal suspension with 10 µg/mL trypsin. After 60 min of incubation at 37 °C, unbound viral particles were removed by three washing steps with DMEM. Cells were further incubated in medium containing 1 µg/mL hydrocortisone, 50 µM spermidine, 1 µg/mL insulin, 0.1 µM Wnt agonist, or 1% intestinal contents for 24 h  and fixed with 4% paraformaldehyde for immunofluorescence staining.At twenty-four h of co-cultivation, monolayers of enterocytes were treated with 1 µg/mL hydrocortisone, 50 µM spermidine, 1 µg/mL insulin, 0.1 µM Wnt agonist, or 1% intestinal contents for another 24 h. Then, the susceptibility of treated enterocytes to PEDV and TGEV was tested. Cells were inoculated with 200 µL of TGEV Miller at a multiplicity of infection (m.o.i.) of 1 and 200 µL of the PEDV CV777 Vero adapted strain at 10To determine the co-localization of viral antigens and APN, co-cultured enterocytes were infected with TGEV Miller and Purdue and PEDV Vero adapted and non-adapted strains. After 24 h incubation, cells were fixed with 4% paraformaldehyde for 10 min and immunofluorescence staining was performed. Cells were incubated with mouse monoclonal anti-porcine APN antibodies containing 10% normal goat serum for 1 h at 37 °C, followed by goat anti-mouse-IgG1 FITC labeled antibodies for 1 h at 37 °C. Then, cells were permeabilized with 0.1% Triton X-100 for 5 min at RT and incubated with mouse monoclonal anti-porcine PEDV antibodies  or swine polyclonal anti-TGEV antibodies  containi2). Then, cells were fixed with 4% paraformaldehyde for 10 min at RT. Immunofluorescence was performed to measure the percentage of infected cells. The cells were permeabilized with 0.1% Triton X-100 for 5 min at RT. Then, cells were incubated with swine polyclonal anti-TGEV antibodies containing 10% normal goat serum for 1 h at 37 °C, followed by goat anti-swine-IgG FITC labelled antibodies for 1 h at 37 °C. Nuclei were stained with Hoechst for 10 min at RT. The percentages of infected cells were determined by fluorescence microscopy.To remove SAs from enterocytes, monolayers of co-cultured enterocytes were washed three times with warm DMEM. Then, cells were incubated with 50 mU/mL NA from Vibrio Cholerae  at 37 °C for 1 h. Cells that were mock-treated were incubated with DMEM and underwent the same manipulations as NA-treated cells. To remove SAs from the virus, virus suspensions were incubated with 50 mU/mL NA from Vibrio Cholerae at 37 °C for 1 h. the mock-treated virus was incubated in DMEM and underwent the same manipulations as the NA-treated virus. Afterwards, cells were inoculated with either NA-treated or mock-treated virus (m.o.i of 1 for TGEV Purdue and Miller). After 60 min incubation at 37 °C, cells were washed three times with medium and further incubated in medium for 24 h  for 1 h at 37 °C. The lectin was subsequently stained with Streptavidin-FITC (Invitrogen) for 1 h at 37 °C. Then, cells were permeabilized with 0.1% Triton X-100 for 5 min at RT and incubated with swine polyclonal anti-TGEV antibodies containing 10% normal goat serum for 1 h at 37 °C, followed by goat anti-swine-IgG Texas Red labeled antibodies. Nuclei were stained with Hoechst for 10 min at RT and results were analyzed by a Leica TCS SPE laser scanning spectral confocal system linked to a DM B fluorescence microscope.To characterize the attachment of TGEV to primary enterocytes, direct virus-binding studies were carried out with TGEV particles. Cells were chilled on ice for 5 min and washed three times with DMEM. Then, cells were inoculated with TGEV Miller and Purdue particles at a m.o.i. of 10 for 1 h at 4 °C. Unbound virus particles were removed by three washings with DMEM. Cells were fixed with 4% paraformaldehyde for 10 min and a double immunofluorescence staining was performed. Cells were incubated with biotinylated Maackia amurensis lectin II or mouse monoclonal anti-porcine APN antibodies for 1 h at 37 °C, followed by Streptavidin-FITC or goat anti-mouse-IgG1 FITC labeled antibodies for 1 h at 37 °C. Then, cells were permeabilized with 0.1% Triton X-100 for 5 min at RT and incubated with swine polyclonal anti TGEV antibodies containing 10% goat serum for 1 h at 37 °C, followed by goat anti-swine-IgG Texas Red labeled antibodies. Nuclei were stained with Hoechst for 10 min at RT and the results were analyzed by a Leica TCS SPE laser scanning spectral confocal system linked to a DM B fluorescence microscope.t-test). The data are represented as means with standard deviation (SD) of three independent experiments. Results with p-values of <0.05 were considered significant.Data were statistically processed by SPSS  was observed at seven days cultivation compared to three days cultivation (66%). The expression of APN at seven days cultivation (28.7 ± 2.3%) was significantly higher than at three days cultivation  and 22 ± 9.9% (p = 0.3), respectively . When cells were pretreated with Wnt agonist, a significant increase of infection (87 ± 23 infected cells per well) was observed (p = 0.018). An increased trend of infection (but not significantly different) was observed after pretreatment with hydrocortisone , spermidine , and porcine insulin . For CV777 fecal suspension, the highest infection was observed when cells were pretreated with the Wnt agonist (12 ± 7 infected cells per well), showing a four-fold higher infection compared to mock-treated cells (3 ± 4 infected cells per well). When cells were pretreated with intestinal contents, 9 ± 6 cells were infected. For TGEV Miller, incubation with intestinal contents, porcine insulin, and Wnt agonist significantly increased the virus infection from 1.0 ± 0.3% to 3.6 ± 0.9%, 3.2 ± 0.9% and 3.0 ± 0.5%, respectively. Hydrocortisone and spermidine increased the virus infection to 2.7 ± 0.5% and 2.5 ± 0.6%  was observed in cells that were pretreated with intestinal contents. Because of the variation between the three replicates, the treatment with intestinal contents was not significantly different from the mock treatment  than in APN positive cells  and SA negative cells (1.7 ± 0.2%). The TGEV Purdue replicated slightly better in SAs negative cells (4.5 ± 0.3%) compared to SAs positive cells . The percentage of APN positive cells with bound TGEV Purdue (1.5 ± 0.7%) was lower than the percentage of APN negative cells with bound TGEV Purdue (3.9 ± 0.7%), but was not significantly different (p = 0.07). The percentage of Miller particles that colocalized with APN (65 ± 11%) was significantly higher than non-colocalized particles (35 ± 11%). The percentage of Purdue particles that colocalized with APN (33 ± 2%) was significantly lower than non-colocalized particles  at 4 °C. The binding of TGEV to APN positive/negative and SAs positive/negative cells was examined by double immunofluorescence staining. No significant differences were observed between the percentage of APN positive cells with bound TGEV Miller (2.3 ± 0.7%) and the percentage of APN negative cells with bound TGEV Miller  and with bound TGEV Purdue (5.0 ± 0.9%) was significantly higher than the percentage of SA positive cells with bound TGEV Miller (0.9 ± 0.4%) and with bound TGEV Purdue (2.1 ± 0.3%). The percentage of TGEV Miller particles (29 ± 13%) and TGEV Purdue (32 ± 12%) that colocalized with SA was significantly lower than the particles that did not colocalize, with Miller at 71 ± 13% and Purdue at 68 ± 12% .Porcine epithelial cells of the small intestines are the target cells for PEDV and TGEV in vivo. These cells show a high surface expression of APN, and APN has been reported to be the cellular receptor for PEDV and TGEV ,9. HowevThe epithelium of the small intestines is continuously and rapidly renewed in a process involving cell generation and migration from the multi-potent stem cells in the crypts to the differentiated cells at the tips of the villi within 2–3 days. In our study, after three days cultivation, the primary enterocytes were positive for sucrase and iso-maltase, which are considered as differentiation markers for epithelial cells . HoweverTo date, coronaviruses use four different proteins as cellular receptors. Mouse hepatitis virus (MHV) initiates the infection by binding to the carcinoembryonic cell adhesion molecule 1 on hepatocyte membranes and intestinal brush border membranes . Next, AIn addition to PEDV, APN has been identified as a major cellular receptor for TGEV. In the present study, it was shown that a higher expression of APN significantly increased the replication of TGEV in enterocytes, indicating that APN plays an important role as a cellular receptor for TGEV. Whitworth and colleagues demonstrated that APN knockout pigs were resistant to TGEV infection, indicating that APN is the sole functional receptor for TGEV . HoweverBased on our previously established primary enterocyte co-culture system, which is very relevant for the in vivo situation, it was shown that a higher expression of APN on the enterocytes resulted in a higher infectivity of TGEV and PEDV. However, TGEV and PEDV could also infect APN negative enterocytes, indicating that an additional receptor exists in enterocytes besides APN. TGEV did not show binding activity on SAs on the surface of enterocytes. These new insights stimulate the search for unknown receptors for PEDV and TGEV, which can assist further research on antiviral intervention.


Toddalia asiatica of Rutaceae, a Taiwan folk medicine, is used as an analgesic and anti-inflammatory herb. Cyclohexylamine (CHA) is an active compound from T. asiatica. Previous reports indicate CHA contracts rat vas deferens. However, the contractile mechanism of CHA on rat vas deferens was not yet reported. The purpose of this study was to investigate the contractile mechanism of CHA on rat epididymal portion of vas deferens.Male S.D. rats weighting between 200 g to 250 g were used. The epididymal portion of vas deferens was isolated and was added with various concentrations of serotonin, serotonin antagonists and CHA.−7M~1 × 10−4M) contractions on rat epididymal vas deferens, which were inhibited by pretreatment with ketanserin (1 × 10−8 M ~ 1 × 10−6 M), methysergide (1 × 10−5 M) and propranolol (1 × 10−4 M), respectively. CHA elicited dose-dependent (1 × 10−8M~1 × 10−4M) contractions on rat epididymal vas deferens. The contractions of CHA (1 × 10−4M) on epididymal vas deferens were enhanced by serotonin in a dose-dependent manner. Methysergide (1 × 10−7 ~1 × 10−5 M) did not affect the contractions evoked by CHA. However, the CHA elicited contraction was almost completely inhibited by ketanserin (1 × 10−5 M) and was enhanced by propranolol. The effect of propranolol at the concentration of 1 × 10−4 M on CHA was likely as CHA at high concentration alone.Serotonin elicited dose-dependent (1 × 102A subtype.From the above results, the contraction evoked by CHA on the isolated rat epididymal vas deferens might be mediated by serotonergic receptors through 5-HT Toddalia asiatica L. (Rutaceae) (T. asiatica L. (250 mg/kg) does have anti-inflammatory and analgesic effects. The potency is equivalent to 100 mg/kg naproxen (unpublished data).utaceae)  is used T. asiatica L. are cyclohexylamine, toddanone, brailin, dl-syringaresinol and isopimpinellin[1-adrenoceptor antagonist) and was not changed by ganglion blockade, bilateral adrenalectomy, or nephrectomy and persisted in decerebrate and spinal cats [The active compounds from mpinellin. The phanal cats ,15.Serotonin (5-HT) receptors presented on rat vas deferens ,17. In o3 24.9; D-Glucose 11; KH2PO4 1.2; KCl 4.6; MgSO4 · 7H2O 1.2; CaCl2 · 2H2O 1.5.Cyclohexylamine was purchased from Wako, Japan. Ketanserin tartrate and methysergide male-ate were purchased from Tocris, USA. Serotonin and propranolol were bought from Sigma-Aldrich, USA. All drugs were dissolved in millipore water. The composition of Kreb's solution is expressed as follows (mM/L): NaCl 119; NaHCO® and sacrificed by cervical dislocation. The vas deferens were removed and cleaned of the surrounding connective tissue and blood vessels. Only the epididymal portions (0.6 – 1 cm) of vas deferens were used. The tissues were mounted into 5 ml Magnus-organ baths containing Kreb's solution at 37° C and bubbled with 95% O2/5% CO2. Preparations were loaded with 1.5 g resting tension, and then Kreb's solution was refreshed every 10 min. Preparations were equilibrated for 1 hr.Male Sprague Dawley (SD) rats weighing between 200 and 250 g were purchased from National Laboratory Animal Center (NLAC), Taipei. Animals were fed with standard chow and housed in standard cages at a constant room temperature of 22 ± 1°C. Relative humidity 55 ± 5% with 12 h inverted light-dark cycle for at least one week prior to the experiment. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC), China Medical University (permit number: 2019-332). Male SD rats were deeply anesthetized by an intraperitoneal injection of 50 mg/kg of zoletil−7 M~1 × 10−4M) was administered ten minutes before CHA (1 × 10−4M) administration, observed and recorded the effect of 5-HT on the CHA-induced contraction of the epididymal vas deferens of rats.Different concentrations of 5-HT (1 × 10−7 M~1 × 10−5M) or ketanserin (1 × 10−8M~ 1 × 10−6M) was administered ten minutes before CHA (1 × 10−4M) administration, observed and recorded the effect of methysergide or ketanserin on the CHA-induced contraction of isolated epididymal vas deferens of rats.Different concentrations of methysergide (1 × 10−6M~1 × 10−4M) were given in advance, and the effect of propranolol on the CHA-induced contraction of the isolated epididymal vas deferens of rats was observed and recorded.Different concentrations of propranolol (1 × 10−7M~1 × 10−4M) were administered and the contraction of vas deferens was observed and recorded. Twenty minutes later, the drug was washed out and replaced with fresh Krebs solution in every ten minutes until the contractile response returned to baseline. The next concentration experiment was then performed.Different concentrations of 5-HT (1 × 10−5M) administration, different concentrations of ketanserin (1 × 10−8M~1 × 10−6M) or methysergide (1 × 10−7M~1 × 10−5M) was added to the organ bath. The effects of ketanserin or methysergide on the 5-HT-induced contraction of the isolated epididymal vas deferens of rats were recorded.Ten minutes before 5-HT (1 × 10−5M) administration, different concentrations of propranolol (1 × 10−6M~1 × 10−3M) was added to the organ bath. The effects of propranolol on the 5-HT- induced contraction of the isolated epididymal vas deferens of rats were recorded.Ten minutes before 5-HT  or the Student −7M ~1 × 10−4M) showed a dose-dependent potentiation on CHA (1 × 10−4M)-induced contraction of the isolated epididymal vas deferens of rats (P < 0.05~P < 0.001). Data were shown in −7M ~1 × 10−5 M) did not have any statistically significant effect on the CHA (1 × 10−4M)- induced isolated epididymal vas deferens of rats (as shown in −8M~1 × 10−5M) had a concentration-dependent inhibitory effect on the CHA (1 × 10−4M)-induced isolated epididymal vas deferens of rats (as shown in −5 M.Different concentrations of methysergide (1 × 10−6M~1 × 10−4M) showed a concentration- dependent enhancement of CHA (1 × 10−4M)- induced contraction of the rat epididymal vas deferens at the first two minutes. With the increase of the time, propranolol (1 × 10−5M and 1 × 10−6M) enhanced the CHA-induced contraction of the vas deferens. However, propranolol at the concentration of 1 × 10−4M showed a suppression effect on CHA-induced contraction (P < 0.01) and the frequency of contraction increased significantly (P < 0.01), the results were shown in Results were shown in −7M~1 × 10−4M) showed a dose-dependent increase in the contraction amplitude  showed a dose-dependent inhibition of 5-HT (1 × 10−5M) induced contraction in amplitude (−7M~1 × 10−5M) showed a dose-dependent inhibition of 5-HT (1 × 10−5M) induced contraction in amplitude, force   will be weakened, as shown in −3 M), the contractile response of 5-HT was completely suppressed. In addition, the statistical analysis showed that different concentrations of propranolol (1 × 10−6M to 1 × 10−3M) can inhibit the frequency of vas deferens produced by 5-HT, as shown in At a concentration of 1 × 10T. asiatica L. [Cyclohexylamine (CHA) is one of the active ingredients of atica L. . Howeveratica L. , will beatica L. –21. Ingeatica L. –25. CHA atica L. . The effatica L. .1A and α1B receptors. Besides, CHA also directly acts on presynaptic adrenal receptors, thus releasing endogenous catecholamine. The effects of CHA on the contraction of the epididymal vas deferens are all calcium-dependent responses (unpublished data). Besides the role of the sympathetic nerve, whether there are other action mechanisms also involved in this contraction of CHA is the primary goal of this study.There are few reports on the effects of CHA on vas deferens, and the action mechanism of CHA is not yet precise. According to our data, the contraction caused by CHA involved in the control of the sympathetic nervous system. CHA can directly act on post-synaptic adrenergic αMany kinds of literature indicate that there are serotonergic receptors on the vas deferens of rats –30. Ther1-adrenoceptor. As for the indirect contraction response of serotonin, the release of NE is involved, and this effect is the most crucial reason for the contraction of the vas deferens [Comparing the response of noradrenaline (NA) and 5-HT on rat vas deferens, the contractile response caused by NA is fast, and tonic; and 5-HT first produces rapid contraction, accompanied by a phasic and rhythmic contraction response (phasic and rhythmic), followed by a tonic contraction. Moreover, the vasoconstriction response of rats induced by 5-HT is 15 times weaker than that of NA . Serotondeferens .2 neurokinin receptors (5-HT2 receptors). While the part of the tonic contraction is not only mediated by post-synaptic serotonin receptors (5-HT2 receptors) but also related to the action of NE released from neuronal stores [2 neurokinin receptor mediates the contraction of rat vas deferens caused by serotonin [2 neurokinn blocking effect, ketanserin also has the activity of antagonizing α-adrenal receptors at a high concentration [2/5-HT1c neurokinin antagonist [1c neurokinin by methysergide.The rapid contraction produced by 5-HT is mainly mediated by the post-synaptic 5-HTl stores . Howevererotonin ,35. In antration . Similartagonist , it is p−8 M to 10−4 M. When the concentration is 10−5 M, serotonin can achieve the maximum contraction-enhancing effect. While the concentration of serotonin is higher than 10 −5 M, it shows a contraction-inhibitory effect. Therefore, serotonin initially enhance the vas deferens response of mice induced by electrical stimulation [2 neurokinin receptors on post-synaptic sites [1 neurokinin receptors , but it is not excluded that Results of involvement of postnatal 5- HT neurokinin receptors [Studying the effect of serotonin on the twitch- response contraction of the mice vas deferens produced by electrical stimulation , it was mulation .is becauic sites ,38,39, receptors .1 neurokinin antagonist, at low concentrations enhances the serotonin-induced contraction of epididymal vas deferens of the rat. The inhibition of presynaptic 5- HT1 neurokinin receptors may enhance the contractile effect of post-synaptic 5-HT2 neurokinin receptors. The reason why high doses of propranolol completely inhibit the contractile response produced by serotonin is not known, and further investigation is needed. Perhaps, there may be other mechanisms (such as β-sympathetic receptors) that are also involved in serotonin's effect on isolated rat contractile response of the epididymal vas deferens.Propranolol, a non-selective 5-HT2 antagonist - LY 53857 nor the non-selective 5-HT antagonist -1- NP could block the vas deferens caused by serotonin [However, neither the selective 5-HTerotonin . Thus, terotonin .2/5-HT1c neurokinin antagonist-ketanserin produced the dose-dependent inhibition of CHA-induced contractile responses of the rat epididymal vas deferens. It revealed the effect of cyclohexylamine is related to the regulation of neurokinin receptors. Ketanserin is a selective 5-HT2A neurokinin antagonist [2A neurokinin receptor. However, the involvement of neurokinin receptors in the CHA-induced contraction may also be an indirect effect. Because different concentrations of methysergide, 5-HT2/5-HT1c neurokinin antagonist, do not affect the response of CHA. Propranolol can enhance the contraction of CHA at concentrations of 1 × 10−6 M and 1 × 10−5 M, but at the concentration of 1 × 10−4 M appears to suppress the contraction amplitude of CHA. However, the effect of increased contraction frequency can only be deduced that perhaps the effect of cyclohexylamine on the contraction of the epididymal vas deferens may also be related to the 5-HT1c neurokinin receptors and β-sympathetic receptors.Although the actual contraction mechanism of serotonin induced vas deferens is still controversial, its mechanism is mainly related to the mediator of the post-synaptic neurokinin receptor combined with NE that released from nerve terminal. In our experiments, different concentrations of 5-HTtagonist , and neu2A subtype. The proposed action mechanism of cyclohexylamine (CHA)-induced contraction on epididymal vas deferens of rat shows in From the above results, CHA induced contraction on the isolated rat epididymal vas deferens might be mediated by serotonergic receptors especially through 5-HT


All oxindole–indole conjugates, except 6i, 9b, and 9c efficiently affected the growth of the human breast cancer MCF-7 (IC50: 0.39 ± 0.05–21.40 ± 1.58 μM) and/or MDA-MB-231 (IC50: 1.03 ± 0.04–22.54 ± 1.67 μM) cell lines, whereas bioisosteric replacement of the oxindole nucleus with indane or tetralin rings  diminished the anti-proliferative activity. In addition, hybrids 6e and 6f displayed effective cell cycle disturbance and proapoptotic capabilities in MCF-7 cells. Furthermore, the efficient anti-proliferative agents towards MCF-7 and/or MDA-MB-231 cell lines  were investigated for their potential inhibitory action toward CDK4. Hybrids 6a and 6e displayed good CDK4 inhibitory activity with IC50s equal 1.82 and 1.26 µM, respectively. The molecular docking study revealed that oxindole moiety is implicated in two H-bonding interactions via both (NH) and (C=O) groups with the key amino acids Glu94 and Val96, respectively, whereas the indole framework is stably accommodated in a hydrophobic sub-pocket establishing hydrophobic interactions with the amino acid residues of Ile12, Val20, and Gln98 lining this sub-pocket. Collectively, these results highlighted hybrids 6a and 6e as good leads for further optimization as promising antitumor drugs toward breast malignancy and CDK inhibitors. On account of their overexpression in a wide range of human malignancies, cyclin-dependent kinases (CDKs) are among the most validated cancer targets, and their inhibition has been featured as a valuable strategy for anticancer drug discovery. In this study, a hybrid pharmacophore approach was adopted to develop two series of oxindole–indole conjugates ( Cancer is a serious health problem that is considered a leading cause of death worldwide. Cancer is characterized by uncontrolled cell proliferation and differentiation mechanisms . AlthougCyclin-dependent kinases (CDKs) are categorized as protein serine/threonine kinases that are responsible for regulating the cell cycle through the complexation with regulatory proteins known as “cyclins”. These complexes are responsible for the progression of the cell cycle through its different phases ,6. This CDK4 is a key CDK cell cycle regulator that is responsible for the progression from the G1 to S phase through complexation with cyclin-D at the G1-phase ,12. Seve®, β (GSK-3β) [As a privileged scaffold, isatin  represents a leading and promising heterocyclic nucleus that is endowed with a range of interesting biological properties, chiefly antitumor activities . Upon FD(GSK-3β) , aurora (GSK-3β) , p90 rib(GSK-3β) , and mic(GSK-3β) , to nameI–III [In the current medical era, the molecular hybridization strategy is being featured as a promising drug design approach, particularly in anticancer drug discovery ,30. It iI–III ,33,34, FI–III ,36, inhiI–III ,38, in aI–III ,40.6a–i, 9a–f, and 11a,b) aiming to develop efficient antitumor agents with potential inhibitory action toward CDK4. First, the privileged indole moiety was selected to be conjugated with the isatin scaffold to furnish the first series (6a–i); thereafter, N-1 of the oxindole moiety was alkylated and benzylated to produce the second set of target hybrids (9a–f)  with a view to investigating the significance of the oxindole moiety in achieving the desired biological activities  . Finallytivities . 6a–i, 9a–f, and 11a,b) were examined for their potential cytotoxic activity towards MCF-7 and MDA-MB-231 cancer cell lines via SRB assay. Thereafter, hybrids 6e and 6f were further explored for their plausible influence on cell cycle progression and apoptosis induction possibility in breast cancer MCF-7 cells. In addition, the most efficient anti-proliferative agents towards MCF-7 and/or MDA-MB-231 cells  were examined for their inhibitory action toward CDK4, and then docked in the CDK4 binding site to justify their inhibitory action as well as to explore their binding pattern.All the reported conjugates  in boiling ethanol to produce the key intermediate 1H-indole-2-carbohydrazide 4. Final target hybrids 6a–i were obtained via condensation of 1H-indole-2-carbohydrazide 4 with the appropriate unsubstituted (5a), 5-substituted (5b–h), and 5,7-disubstituted (5i) isatin derivative in refluxing glacial acetic acid for four hours  were examined for the potential cytotoxic activity towards breast cancer MCF-7 and MDA-MB-231 cell lines, utilizing the protocol of the sulforhodamine B colorimetric (SRB) assay [6, 9, and 11 were designed with diverse substituents on C-5 and C-7 of the oxindole moiety; R and R1 in series 6a–i, and diverse substitutions on the N-1 of the oxindole moiety; and R2 in series 9a–f, while the oxindole moiety was replaced by an indane and tetralin rings in compounds 11a,b in order to explore the privilege of the oxindole moiety over other pharmacophoric groups. The IC50 values for the tested conjugates were compared as an insight of their anti-proliferative potency taking the clinically approved staurosporine as the positive control anticancer drug (All the synthesized conjugates (B) assay . With thcer drug .50 values for the tested hybrids toward MCF-7 breast cancer cells revealed that most hybrids belonging to series 6 had excellent activity against the aforementioned cell line with IC50 values spanning between 0.39 ± 0.05 μM and 6.00 ± 0.32 μM, with the exception of compounds 6h and 6i  bearing a strong electron withdrawing 5-NO2 group and 5,7-dimethyl substitution on the oxindole moiety, respectively  revealed that it had double the activity of staurosporine (IC50 = 6.81 ± 0.22 μM), which highlighted that this scaffold is a promising one. The influence of grafting different groups on the oxindole moiety reflected that substitution with a halide was advantageous for the anti-proliferative activity in case of 5-chloro substituent in 6c, as it displayed slight potency enhancement compared to its unsubstituted analogue 6a , while the 5-bromo derivative 6d (IC50 = 3.31 ± 0.11 μM) did not prove to be more effective than the unsubstituted derivative 6a (IC50 = 3.12 ± 0.14 μM), and even the 5-fluoro derivative 6b (IC50 = 6.00 ± 0.32 μM) showed about half the activity in comparison to 6a (IC50 = 3.12 ± 0.14 μM).The IC6e (IC50 = 0.39 ± 0.05 μM) markedly boosted the activity 8-fold compared to the unsubstituted prototype 6a (IC50 = 3.12 ± 0.14 μM), emphasizing that an electron donating group is advantageous for activity. In accordance with this finding, compound 6f, bearing an electron-donating 5-OCH3 group, also exhibited excellent anti-proliferative activity (IC50 = 1.85 ± 0.08 μM) with 1.7-fold efficacy enhancement as compared to 6a (IC50 = 3.12 ± 0.14 μM). Unfortunately, compound 6g, which possesses a trifluoromethoxy group, did not exhibit superior anti-proliferative activity compared to 6a, yet it still proved to be more efficient than the reference compound, staurosporine, with an IC50 value of 5.07 ± 0.21 μM.Furthermore, C-5 monosubstitution of the oxindole motif with a methyl group in 9 did not show better cytotoxic action against the MCF-7 cell line than their N-unsubstituted analogues 6a–i;  and , indicating that both N-alkylation and N-benzylation for the oxindole nucleus is not advantageous for the growth inhibitory action toward MCF-7 breast cancer cell line. N-1 substitution of the oxindole moiety with a methyl group produced compound 9a, which is slightly more potent toward the MCF-7 cell line compared to the reference staurosporine . Alternatively, compound 9b, bearing an n-propyl group, and 9c, possessing an ester group, showed very poor anti-proliferative action against MCF-7 cancer cells with IC50s equal to 40.89 ± 3.17 μM and 67.51 ± 3.02 μM, respectively. While compound 9d, bearing a benzyl ring, showed moderate anti-proliferative activity with an IC50 value of 9.56 ± 0.61 μM, compound 9e, with a fluorobenzyl group, proved to be three-fold more potent compared to its counterpart 9d with an unsubstituted benzyl group with an IC50 of 3.70 ± 0.19 μM, On the other hand, the tested hybrids of series 9f, bearing an n-propyl group, displayed about two-fold enhanced activity (IC50 = 19.26 ± 0.65 μM) toward the MCF-7 cell line than its C-5 unsubstituted counterpart 9b (IC50 = 40.89 ± 3.17 μM), highlighting that C-5 substitution may be beneficial for the growth inhibitory activity of the N-substituted oxindole-based hybrids against MCF-7 cells. Moreover, it indicates that a chloro substitution at the oxindole C-5 is beneficial for activity, as also seen in compound 6c.Moreover, the 5-chlorooxindole-based hybrid 50 profile for the tested hybrids against the MDA-MB-231 cell line unravels very interesting results. All the compounds showed a narrower range of IC50 values against MDA-MB-231 as compared to their corresponding values toward the MCF-7 cell line. The IC50s were ranging from 1.03 ± 0.04 μM to 22.54 ± 1.67 μM, which proves that the examined conjugates exhibited more potent growth inhibitory action against TNBC, MDA-MB-231, with the exception of conjugates 6i, 9c, 11a, and 11b, which showed no activity toward the tested cell line. Regarding the IC50 values of series 6a–i, the unsubstituted derivative, 6a, did not show superior activity to staurosporine , whereas C-5 substitution of the oxindole nucleus with a halide was suggested to be advantageous for the anti-proliferative action regarding the fluoro substitution in 6b (IC50 = 6.36 ± 0.29 μM), which is more potent than the reference compound (IC50 = 10.29 ± 0.72 μM). Moreover, grafting a chloro group afforded 6c with comparable potency to staurosporine , while substitution with a bromo group afforded 6d (IC50 = 18.33 ± 0.71 μM), which is much less potent than staurosporine. This proves that increasing the size of the halide group is detrimental for the cytotoxic activity against MDA-MB-231. Unexpectedly, grafting a methyl group was not advantageous for the growth inhibitory action toward breast cancer MDA-MB-231 cells in contrast to activity recorded against MCF-7, as the IC50 of 6e against MDA-MB-231 was 22.54 ± 1.67 μM, which is almost half the potency of staurosporine. Fortunately, grafting a stronger electron donating group, a methoxy group, boosted the activity of 6f (IC50 = 2.85 ± 0.9 μM) 3.6-fold as compared to staurosporine (IC50 = 10.29 ± 0.72 μM). In addition, substitution with a trifluoromethoxy group imparted 6g, which is more potent than the reference drug . Surprisingly, grafting a strong electron withdrawing group, a nitro group, afforded 6h, which is 5.56-fold more potent compared to staurosporine . Increasing the alkyl group to a propyl group afforded 9b which has a greater IC50 value (18.25 ± 0.85 μM), suggesting that increasing the bulkiness of the alkyl group is unfavorable for the anti-proliferative activity. Conversely, substitution with a benzyl group afforded 9d, which exhibited good anti-proliferative activity in comparison to staurosporine . Replacement of the benzyl ring with a fluorobenzyl group even boosted the activity ten-fold as compared to staurosporine .Regarding the antiproliferative action of series 11a,b), which did not exert any significant anti-proliferative action toward MCF-7 and MDA-MB-231 cancer cell lines  and the anti-apoptotic marker Bcl-2 was investigated to prove that our tested compounds exert their antiproliferative activity by driving the cell to apoptosis. Auspiciously, both conjugates evidently enhanced the level of the proapoptotic Bax protein by 8.5- and 7-fold, respectively. Homogeneously, both conjugates were able to decrease the level of the anti-apoptotic Bcl-2 protein by 2.12-, and 1.7-fold, respectively  were initially evaluated for their percent inhibition of CDK4 at a dose of 20 μM (6a (92%) and 6e (93%) showed stunning inhibitory activities, the rest of the examined hybrids showed low to moderate activity with percent inhibition spanning in the range of 12% to 46%   . The presented results in 6a and 6d were the best developed CDK2 inhibitors here with percent inhibition of 78% and 85%, respectively. In addition, compounds 6c, 6e, 6f, and 6h displayed percent inhibition equal to or higher than 50% toward this isoform at a concentration of 20 μM.On the other hand, inhibition of CDK2 ranged from good to weak, with percent inhibition range of 45%–85% . In partIn conclusion, the results of inhibition of the three tested CDK isoforms  disclosed the inability of the target compounds to inhibit CDK9 significantly, with concomitant good inhibitory activities against CDK2 and CDK4. Accordingly, further optimization for the here reported oxindoles considering their plausible dual CDK2 and CDK4 inhibitory actions are currently in development and expected to be conveyed in the future.A protein data bank (PDB) search showed that there are not any CDK4 protein structures co-crystalized with an inhibitor available (holoprotein). The available structures are apoprotein structures for CDK4 in complex with cyclin D such as PDB ID 2W96, 2W9Z, 2W9F, and 2W99 . On the First, the molecular docking protocol was validated by performing self-docking for the added oxindole ligand in the vicinity of the CDK4 binding site. The self-docking validation replicated the binding pattern for the added ligand, efficiently demonstrating the aptness of the used setup for the planned docking study, and this was confirmed by the low RMSD of 0.879 Å between the docked pose and the added ligand (energy score (S) = −9.72 kcal/mol) .6a–i showing promising cytotoxic as well as CDK4 inhibitory activity were docked in the CDK4 binding site in order to justify their inhibitory action and investigate their binding patterns. In general, hybrids 6a–i achieved similar binding patterns, where the oxindole framework was fitted in the ATP adenine binding pocket like that of the added ligand in the hinge region. As observed in 9 with N-substituted derivatives. Moreover, the oxindole ring interacts by its phenyl part through π–π stacking interaction with the side chain phenyl of Phe93 and through hydrophobic interaction with the side chains of Phe93 and Ala157. A hydrophobic substituent with the appropriate size on the oxindole ring 5-position could improve the binding affinity, which rationalizes the promising activity of 6e with a 5-methyl substitution. Thus, derivatives in this series carrying substituents with hydrophilic characters, 6b, 6f, and 6g, or bulky size, 6c, 6d, 6h, and 6i, showed lower CDK4 inhibitory activity as reflected in their CDK4 inhibitory actions and docking scores . All coupling constant (J) values were expressed in hertz. The used abbreviations are as following: s, singlet; d, doublet; m, multiplet. Compound 3 [8a–f [4 [6a–d [Infrared (IR) spectra were recorded as KBr disks by the use of a Schimadzu FT-IR 8400S spectrophotometer. NMR spectra were recorded on JEOL ECA-500 II (500/125 MHz) or Bruker (400/100 MHz) NMR spectrophotometers using deuterated dimethylsulfoxide  in ethyl alcohol (25 mL). The resulting reaction mixture was heated under reflux for two hours. Thereafter, it was cooled to r.t. and then poured over crushed ice. The formed solid was filtered-off, washed with water (3 × 5 mL), and recrystallized from isopropyl alcohol to produce the key intermediate 1H-indole-2-carbohydrazide 4. An excess of 99% hydrazine monohydrate  was added to a hot solution of ethyl indole-2-carboxylate 4  in glacial acetic acid (15 mL) was treated with the appropriate isatin derivative 5a–i and 8a–f (1.2 mmol), or ketones 10a and 10b (1.2 mmol). The resulting reaction mixture was refluxed for four hours then cooled to r.t. The obtained precipitate was collected by filtration and dried to get a powder that was recrystallized from glacial acetic acid to furnish the titled conjugates 6a–i, 9a–f, and 11a,b.A solution of indole-2-carbohydrazide 4) and the target hybrids  are provided in the Details for the full characterization of the key intermediate  are provided in the Details for experimental protocols used in the diverse biological assays for the targeted hybrids  that obtained from American Type Culture Collection. Cytotoxicity was assessed following the SRB colorimetric assay protocol [Target compounds protocol , as repoprotocol ,51.6e and 6f at their IC50 , by BD FACS Calibur flow cytometer, as described previously [Flow cytometric analysis (FACS) was performed to assess the cell cycle distributions in MCF-7 breast cancer cells after treatment by hybrids eviously .6e and 6f towards MCF-7 cells was evaluated through an FITC Annexin V apoptosis detection kit by flow cytometry, in accordance with the manufacturer’s protocol and referring to the reported procedures [Assessment of the levels for the proapoptotic markers  as well as the antiapoptotic marker (Bcl-2) was carried out through the use of ELISA colorimetric kits in accordance with the manufacturer’s instructions, as mentioned earlier . In addiocedures .http://www.reactionbiology.com) Kinase HotSpotSM service.The in vitro CDK kinase inhibition assay was performed by Reaction Biology Corp.  (MatchMaker command available in UCSF Chimera package 1.12 [The CDK2 protein structure (PDB ID: 4FX3) co-crystalized with an oxindole inhibitor and the CDK4 protein structure (PDB ID: 2W96)  were dowage 1.12 . Then, tFor carrying out the molecular docking for the target hybrids in the CDK4 binding site, the following procedures were adopted. Pose mode [(A) Ligand preparation: The synthesized hybrids as well as the added oxindole ligand were built as 3D structures using Discovery Studio Visualizer 2017R2 . The OMEose mode ,56.(B) Protein preparation: The generated structure for CDK4 in step 3.2.5.1. with the added oxindole inhibitor was used in this step. Using Discovery Studio Visualizer 2017R2 , the proChemgauss4 scoring function [(C) Molecular docking: The HYBRID docking module of “OEDocking 3.2.0.2” program in OpenEye package was utilized to carry out the molecular docking for the generated conformers of the herein reported hybrids as well as the oxindole inhibitor in the CDK4 binding site using the function ,58,59,60First, the molecular docking protocol was validated by performing self-docking for the added oxindole ligand in the CDK4 binding site, producing a docking pose with energy score (S) and RMSD equal to −9.72 kcal/mol and 0.879Å, respectively. Then, this validated docking setup was adopted to investigate the ligand–target interactions in the CDK4 active site for the prepared hybrids to explore their binding pattern and to justify their binding affinity. PoseView 1.1.2 was used to generate the 2D figures of the ligand–target interactions ,62,63,646a–i and 9a–f) and carbocycle–indole hybrids  as efficient antitumor agents with potential inhibitory action toward CDK4. All the prepared hybrids  were examined for the potential cytotoxic activity towards MCF-7 and TNBC MDA-MB-231 breast cancer cell lines via SRB assay. The synthesized oxindole–indole conjugates, except 6i, 9b and 9c, efficiently affected the growth of the human breast cancer MCF-7 (IC50: 0.39 ± 0.05–21.40 ± 1.58) and/or MDA-MB-231 (IC50: 1.03 ± 0.04–22.54 ± 1.67) cell lines, whereas bioisosteric replacement of the oxindole motif with indane or tetralin rings  diminished the anti-proliferative activity, indicating the importance of the oxindole scaffold for the antitumor activity. Moreover, hybrids 6e and 6f triggered cell cycle arrest and apoptosis in MCF-7 cancer cells as explicated by their capabilities to considerably boost the Bax/Bcl-2 ratio, and to up-regulate the level of caspase-3 and p53. Furthermore, single dose (20 μM) inhibitory activity of the most efficient anti-proliferative agents  was initially evaluated toward CDK4. Hybrids 6a and 6e showed the most potent inhibitory activity of 92% and 93%, respectively, whereas the rest of the examined hybrids showed low to moderate activity with percent inhibition of 12%–46%. Thereafter, the IC50 values for hybrids 6a and 6e in CDK4 were further determined and were found to be 1.82 and 1.26 µM, respectively. The molecular docking study revealed that the oxindole moiety was engaged in two hydrogen bonding interactions via both (NH) and (C=O) functionalities with the key amino acids Glu94 and Val96, respectively, rationalizing the lower activity of series 9 with N-substituted derivatives. The oxindole ring interacted by its phenyl part through π–π stacking interactions with the side chain phenyl of Phe93 and through hydrophobic interaction with the hydrophobic side chains of Phe93 and Ala157. In addition, the indole framework was stably accommodated in a hydrophobic sub-pocket, establishing a hydrophobic interaction with the hydrophobic side chains of amino acids Ile12, Val20, and Gln98 lining this sub-pocket. Finally, compounds 6a–h, 9a, and 9e were screened for their potential inhibitory activities against CDK2 and CDK9 isoforms. While CDK9 was weakly inhibited with percent inhibition range of 4%–32%, inhibition of CDK2 ranged from good to weak, with percent inhibition range of 45%–85%. Collectively, these results highlighted hybrids 6a and 6e as good leads for further optimization as promising antitumor drugs toward breast malignancy and CDK inhibitors.The current study presents the development of two series of oxindole–indole hybrids (


Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. Thus, analyzing the effects of the astrocyte secretome may provide valuable insight into these neuroprotective mechanisms. Previously, we characterized a potent neuroprotective activity mediated by retinal astrocyte conditioned media (ACM) on retinal and cortical neurons in metabolic stress models. However, the molecular mechanism underlying this complex activity in neuronal cells has remained unclear. Here, a chemical genetics screen of kinase inhibitors revealed phosphoinositide 3-kinase (PI3K) as a central player transducing ACM-mediated neuroprotection. To identify additional proteins contributing to the protective cascade, endogenous PI3K was immunoprecipitated from neuronal cells exposed to ACM or control media, followed by MS/MS proteomic analyses. These data pointed toward a relatively small number of proteins that coimmunoprecipitated with PI3K, and surprisingly only five were regulated by the ACM signal. These hits included expected PI3K interactors, such as the platelet-derived growth factor receptor A (PDGFRA), as well as novel RNA-binding protein interactors ZC3H14 (zinc finger CCCH-type containing 14) and THOC1 (THO complex protein 1). In particular, ZC3H14 has recently emerged as an important RNA-binding protein with multiple roles in posttranscriptional regulation. In validation studies, we show that PI3K recruitment of ZC3H14 is necessary for PDGF-induced neuroprotection and that this interaction is present in primary retinal ganglion cells. Thus, we identified a novel non–cell autonomous neuroprotective signaling cascade mediated through PI3K that requires recruitment of ZC3H14 and may present a promising strategy to promote astrocyte-secreted prosurvival signals. This result is consistent with evidence that homeostatic ACM contains an array of neurotrophic factors that activate class I PI3K signaling , 16, 17.Here we utilize this untargeted approach to identify and validate a unique neuronal interactome that explores the shift in endogenous PI3K binding preferences following protective ACM exposure, compared with control cell-free media (CFM). These hits included expected PI3K interactors, such as the platelet-derived growth factor receptor A (PDGFRA), as well as the novel RNA-binding protein (RBP) interactors ZC3H14 (zinc finger CCCH-type containing 14) and THOC1 (THO complex protein 1). In particular, ZC3H14 has emerged as an important RBP with multiple roles in posttranscriptional regulation, including mRNA stability and transport , 24, 25,A). This protection is illustrated by the total screen results that show a clear distinction in viability between ACM- and CFM-treated samples, demonstrating that most of the kinase inhibitors had no effect on ACM-induced protection at 1 μM . It is remarkable that, of the total library, only 10 kinase inhibitors met the selection criteria showing loss of ACM-induced protection, but no apparent toxicity, as defined in the methods. Further, seven of these ten compound hits targeted the PI3K/AKT pathway, strongly implicating a role for this signaling pathway in transducing the protective ACM effect . This effect is notably more robust than the original screening result (50 = 0.7–210 nM) (50 = 20–890 nM) (D). None of the validated kinase inhibitors was toxic when tested at the specified concentrations in either model employed. Finally, we assessed whether ACM treatment is sufficient to activate the PI3K pathway by immunoblotting for downstream AKT phosphorylation following ACM addition in Ht22 cells. ACM treatment rapidly induced AKT phosphorylation, as reflected by the significantly increased phospho-AKT signal by 30 min, normalized to total AKT (E). Taken together, these data indicate that PI3K signaling was the only robustly supported hit from the kinase inhibitor screen.To validate the screening results, the effect of PI3K/AKT inhibition was verified independently using fresh compound by hand in both Ht22 cells and in primary cortical neurons. A known PI3K/AKT inhibitor, PX866 (IC.1–88 nM ), which g result  and may –210 nM) , 30 and –890 nM)  similarl–890 nM) D. None ootal AKT E. Taken A).The strong evidence for PI3K/AKT in ACM-mediated activity, although interesting, provided limited detail into the protective mechanism, as this cascade can initiate pleotropic signaling through a variety of pathways , 33. IdeFig. 2B and Fig. 2B). Subsequent Coomassie staining of blots showed a specific capture of PI3K, as reflected by generally clear eluate lanes compared with the corresponding input and unbound lanes .A panel of PI3K antibodies was first probed for their ability to effectively deplete and elute PI3K from cross-linked Ht22 cell lysates . Based oFig. 2D). Together, these results indicate that the PI3K complex was highly purified.Proteome Discoverer sequencing of the LC-MS data from PI3K (P85) immunoprecipitates identified 293 proteins with 12,117 peptide-to-spectrum matches (PSMs) at or exceeding 95% confidence. An inclusion criterion of at least two spectral counts was established to identify quantified proteins. Additional bioinformatic analyses further narrowed down the list of interactors to filter out nonspecific binders using the negative control as a reference and including only reproducible hits across the replicates. The initial protein list was sorted based on abundance, determined by PSM. Two key observations in these data highlight the selectivity of the affinity capture: (1) Both the target P85 regulatory and P110 catalytic PI3K subunits were the most abundant in the interactome, with more than 2000 PSMs for each hit , indicatFig. 2A). The preliminary results yielded a total of 122 proteins reliably quantified from three or more MS3 spectra with reporter ion signals representing all six immunoprecipitated groups  . Previously identified interactors included two 14-3-3 adaptor proteins, which are critical to propagating PI3K pathway signaling by stabilizing the insulin receptor substrate-2 (IRS2)  , 35, 36.2 (IRS2) , 40, 41,2 (IRS2) , 43. How2 (IRS2) , 25, 26.C). Effective PI3K capture and elution was verified by probing a parallel blot . ZC3H14 is alternatively spliced to produce several protein isoforms. The largest isoform, described as ZC3H14 isoform 1, was identified in the interactome, but it may also encompass isoforms 2 and 3 . In this case a control blot verified ZC3H14 capture and elution . These data provide further biochemical evidence to support an interaction between ZC3H14 and PI3K.In the fully processed data set, ZC3H14 was the most highly regulated ACM-induced interactor, and to our knowledge, this protein has not been previously investigated in the context of PI3K signaling. ZC3H14 modulates several steps in the posttranscriptional regulation of gene expression, including regulating polyadenosine (poly(A)) tail length and nuclear export , 24, 26.Fig. 3 2 and 3 , 45. Thi 2 and 3  Fig. S4C. EffectA). Next, we investigated whether recombinant PDGFbb is sufficient to generate HT22 protection from the glutamate injury model previously used for screening. The experiment demonstrated a robust corresponding protective effect mediated by recombinant PDGFbb at 50 ng/ml . It is unclear why only the higher concentration elicited a protective effect, but the functional assay is performed at a much later time point than the phosphorylation assay, leading to potential discrepancies in phosphorylation time course and the roles of additional phosphorylation sites or complexed proteins, among other variables. Finally, we tested the addition of the receptor tyrosine kinase inhibitor Ki 8751, which targets PDGFRA (IC50 = 67 nM), along with VEGFR-2 . Of note, this observation is supported by complementary ZC3H14 depletion in the unbound fraction of PDGF-treated cells compared with the vehicle control . As a control, depletion and elution of PI3K itself was consistent for each condition .As PDGFRA and ZC3H14 were both components of the PI3K complex in the ACM interactome, we then investigated whether PDGF treatment affects PI3K–ZC3H14 binding. A PI3K coimmunoprecipitation was carried out as previously, following treatment with 20 ng/ml PDGFbb or vehicle. For each IP, equal concentrations of cell lysate, antibody, and beads were used. The resulting blots show a dramatic increase in ZC3H14 elution with treatment of PDGF C, I. Of  control C, I. As ondition C, II.D). This experiment demonstrates that ZC3H14 is critical for PDGF-induced protection. Taken together, these data suggest that PDGF treatment increases PI3K recruitment of ZC3H14 to mediate its protective signal.To test whether ZC3H14 is required for the PDGF-induced protection of cells, we used a validated siRNA  Fig. S4Fig. S4 tFig. 4A), as well as colocalization between ZC3H14 and THOC1 (A). The same subcellular localization was observed in cultured RGCs under control conditions (B). This pattern is consistent with previous reports showing primarily a nuclear localization of ZC3H14 (B and C). This result reproduces previous studies that have detected ZC3H14 in the cytoplasmic fraction of neurons (D).To assess whether these ACM-induced interactions are relevant in the context of primary neurons, we assessed PI3K and ZC3H14 colocalization in retinal ganglion cell (RGCs). The retina provides an excellent model to study this interaction given the close association between RGCs and neighboring astrocytes. Immunostaining of mouse retinal sections revealed prominent localization of PI3K and ZC3H14 to the ganglion cell layer A, as welnd THOC1 . Of notend THOC1 A. The sanditions B. This pf ZC3H14  and was f ZC3H14 , 41. Upof ZC3H14 , B and C neurons , 50. Of  neurons D.Figure E). PDGFbb treatment produced little difference in this signal. However, treatment with PDGFab resulted in a striking increase in the PLA signal, particularly in the perinuclear region . Quantification of these results confirmed them be highly significant and reproducible . Therefore, endogenous PI3K and ZC3H14 interact in RGCs, and this interaction is enhanced by PDGFab.To test for direct interactions between endogenous PI3K and ZC3H14 in primary RGCs, which are more limited in number than immortalized cells, we employed a proximity ligation assay. This technique generates an amplified fluorescent signal only when the target proteins identified by two antibody probes are in direct proximity, indicating a direct interaction with high subcellular specificity . ApplicaFig. 5in vitro and in vivo models  RNA and contributes to a range of posttranscriptional functions , 80, incdysfunction is linked to neurodegenerative disease, including postsynaptic density protein 95 (Psd95), ATP synthase lipid-binding protein (Atp5g1), and microtubule-associated protein tau (MAPT)  . Furtheru (MAPT) . Given t5 cells/well. ACM or cell-free control medium (CFM) was harvested after 24 h incubation and stored at –80 °C. Neuronal Ht22 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 10% FBS/1% penicillin/streptomycin  for nondenaturing applications, supplemented with protease/phosphatase inhibitor cocktail. Protein concentrations were quantified using bicinchoninic acid assay (Thermo), and equal amounts were loaded for SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membrane and blocked with 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 and incubated overnight at 4  °C with primary antibodies raised against PI3 Kinase p85 (CST), pan AKT (CST), Phospho-Akt (Ser473) (CST), GAPDH , ZC3H14 , and THOTo optimize PI3K capture, Ht22 cells were plated in 15-cm dishes and grown to 70% confluency. Following a 1-h incubation with either ACM or CFM, cells were fixed with 0.5% formalin in PBS for 10 min to preserve labile and transient protein–protein interactions. Formalin was neutralized with 150 mM Tris base, 150 mM glycine solution for 10 min. Cell lysates were collected in 0.2% NP-40 lysis buffer, containing 150 mM Tris, 150 mM NaCl, with addition of protease/phosphatase inhibitor cocktail. A library of PI3K antibodies (CST) targeting a variety of subunits were screened to choose the antibody with the most efficient capture . Before The preparation and analysis of immunoaffinity preparations followed published methods , 94. Sevhttp://string-db.org)  and Sequest HT search engines within Proteome Discoverer software  as well as PEAKS studio 8.5  using the mouse international protein index (IPI) database . The search was constrained to peptides with fully tryptic cleavage and up to two missed cleavages per peptide, fixed iTRAQ 8-plex modification at amino-termini and lysine residues, fixed pyridylethylation at cysteine residues, precursor mass deviation within 20 ppm, and product ion mass deviation up to 0.4 Da. Deamidation at asparagine or glutamine, oxidation at methionine, and phosphorylation at serine, threonine, or tyrosine were allowed variable modifications. The false discovery rate was fixed at 0.05 and determined by Percolator with validation based on q-value in Proteome Discoverer or with the PEAKS algorithm. iTRAQ reporter ion signals were extracted from MS3 spectra in the QUANTITATION algorithm of PEAKS and the Reporter Ions Quantifier in Proteome Discoverer. Reporter Ions Quantifier integration tolerance was 20 ppm, and integration was based on the most confident centroid. Included in protein quantification were PSMs scoring above false discovery rate cutoffs. Protein levels were normalized to one ACM replicate (ACM3), which was arbitrarily chosen as an internal reference. To determine the extent to which PI3K interactions changed as a result of ACM treatment, the data was filtered over three main stages: (1) Proteins with negative control:ACM3 ratios >0.4 were filtered out to eliminate nonspecific binders. (2) Candidate interactors were those proteins having an ACM1:ACM3 or ACM2:ACM3 ratio between 0.65 and 1.35, an indicator of high reproducibility between ACM triplicates. (3) Among highly reproducible candidates, PI3K interactor proteins with a CFM:ACM of <0.65 were considered ACM-induced interactors and those of >1.35 were considered to have been dissociated by ACM . Known i-db.org) .5 cells per well in 6-well plates in 10% FBS DMEM. Each well received 4 μl of Lipofectamine 2000 (Thermo), diluted in 150 μl of Optimem media (Thermo); siRNA was also diluted in 150 μl of Optimem media. Lipofectamine 2000-siRNA complexes were then combined with 1200 μl of serum-free DMEM and applied to cells for 6 h. The medium was then replaced by fresh 10% FBS DMEM. ZC3H14 knockdown was evaluated by immunoblotting 48 h following transfection.A previously validated siRNA sequence was used for ZC3H14 knockdown  (TGACTGAA proximity ligation assay was performed for PI3K and ZC3H14 probes according to the manufacturer's directions (Sigma). Briefly, rat RGCs were plated at 8000 cells per well on 96-well plates. Cells were fixed in 4% paraformaldehyde, washed 2× in PBS and 1× in PBST, then blocked for 1 h in donkey serum and incubated with primary antibodies overnight at 4 °C. The next morning, cells were washed and incubated with 30 μl diluted Duolink probes and incubated for 1 h at 37 °C. The prepared ligase mixture was then added to washed cells and incubated for 30 min at 37 °C, followed by polymerase mixture for 100 min at 37 °C. Finally, cells were washed, with DAPI added to the final wash. Negative controls consisted of wells missing one or both primary antibodies. PLA images were obtained using a Nikon Confocal Microscope to detect a neu-N neuronal marker antibody staining at 488 nm, and the PLA signal at 561 nm, with the same settings across groups. Nikon NIS-Elements Advanced Research software, v 4.51, was used to acquire corresponding images using the 488-nm signal to threshold for neurons and measure mean intensity of the 561-nm PLA signal per cell.t test (C and F) or one-way ANOVA with either Dunnet (D and 4B) or Tukey (D) post hoc analyses. Figure 4D was analyzed by two-way ANOVA with Tukey multiple comparisons test. Corresponding p values are reported in each figure or legend.For all experiments, n refers to the number of biological replicates or animals. Data were assessed for normality and statistical analyses were performed by t test , C and Fr Dunnet D and 4B or Tukey D post hoAll data are included in the article or are available upon request. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (96) with the dataset identifier PXD022200.The authors declare that they have no conflicts of interest with the contents of this article.