A molecular-logic gate for COX-2 and NAT based on conformational and structural changes: visualizing the progression of liver disease† †Electronic supplementary information (ESI) available. See DOI: 10.1039/d0sc00574f

Lighting up the relevant lesion boundaries during operations is vital for guiding the effective resection of hepatopathic tissue.


Procedures section
A.R. grade of solvents and reagents were used in this work. Column chromatographic was used to purify compounds and silica gel (200-300 mesh) was used as fillers. Cyclooxygenase-2 (COX-2, Enzyme Commission Number 3.6.1.3) and N-acetyltransferase (NAT, Enzyme Commission Number 2.3.1.5) were obtained from Sigma Chemical Co. (USA). Doubly purified water was used in all experiments, which was prepared using by a Milli-Q system. IAN derivatives as stock were used in spectrographic determination and cell experiments. HPLC (Agilent 1290 Infinity)-HRMS (Bruker microToF II, Bruker Co., Switzerland) with an auto sampler operated inline with a quantum triple quadrupole instrument was carried out in mass spectral studies on ESI positive or negative ion mode. NMR spectra were obtained from Avance 400 and 600 MHz spectrometer (Bruker Co., Switzerland).

Spectrographic determination in vitro.
A Lambd 950 spectrophotometer from PerkinElmer (USA) and a LS-55 spectrophotometer from PerkinElmer (USA) were used to measure absorption spectra and fluorescence spectra, respectively. In all spectral experiments, the final solutions contained < 5 ‰ DMSO. Each experiment was carried out in five replicates (n = 5). The relative fluorescence quantum yields were determined using Rhodamine B (Φ F = 0.97 in methanol) by the following equation: (1) where Φ represents quantum yield; F is the integrated area under the corrected emission spectrum; A is absorbance at the excitation wavelength; λ ex is the excitation wavelength; n is the refractive index of the solution (because of the low concentrations of the solutions, 10 -7 -10 -8 mol/L, the refractive indices of the solutions were replaced with those of the solvents); and the subscripts x and s refer to the unknown and the standard, respectively.
The detection limit was calculated by three times the standard deviation divided by the slope of the blank. The data were obtained from replicate experiments (n = 5) To avoid fluorescence quenching caused by excessive concentration, the concentration (3.0 μM) of probes was used into the all fluorescence spectrum test. To improve the accuracy of the experimental values (absorbance value), that is, the absorbance value is at the range of 0.3-0.7, the concentration (30 μM) of probes was used into the all absorption spectra test.

Quantum Calculations.
The geometric structure of IAN derivatives was calculated using Gaussian 16 program suite. S1 The functional ωB97X-D and TZVP basis set was used in the Density functional theoretical (DFT) methods. S2 The electronic transition energies and corresponding oscillator strengths were calculated by time-dependent density functional theory (TD-DFT) at ωB97X-D/TZVP level. Water is used as solvent in the CPCM calculations to evaluate the solvent effect. S3

Photostability in solution.
IAN derivatives were dissolved in  all at concentrations of 3.0 μM. The solutions were irradiated by a 500W iodine-tungsten lamp situated 250 mm away for 7 h. An aqueous solution of sodium nitrite (50 g/L) was placed between the samples and the lamp as a light filter (to cut off the light shorter than 400 nm) and as a heat filter. The photostabilities were expressed in terms of remaining absorption (%) calculated from the changes of absorbance at the absorption maximum before and after irradiation by iodine-tungsten lamp. The absorbance was determined. The data were obtained from replicate experiments (n = 5).

Photostability in cells.
The fluorescence intensity was determined, after cell staining with 3.0 μM IAN derivatives for 30 min at 37 °C under 5% CO 2 , by spectral confocal multiphoton microscopy (Olympus, FV1200) with a high-performance mode-locked titanium-sapphire laser source (MaiTai, Spectra-Physics, USA). The change of fluorescence intensity with scan time was determined by spectral confocal multiphoton microscopy (Olympus, FV1200) in the consecutive t-scan mode for 60 min. The intensity of IAN derivatives were recorded with the relevant emission.
Excitation wavelength: 800 nm with constant intensity. The data were obtained from replicate experiments (n=5).

Cell and human hepatopathic tissue samples.
HepG 2 cell lines were obtained from the Chinese Academy of Medical Sciences. The human hepatopathic tissue samples from individuals were obtained from Department of Hepatobiliary and pancreatic surgery, Henan Provincial People's Hospital (Zhengzhou, China) and Xinxiang Medical University (Henan, China, ethics statement Reference No. 2015016) was followed for all experiments performed during this study. (1) The first type of experiment: The hepatopathic tissue samples were sliced up to obtain 10 μm deep tissue slices by freezing microtome, and the hepatopathic tissue samples were incubated with 3IAN4 (10 μM of 3IAN4 for 15 min) before imaging. Olympus spectral confocal multiphoton microscope (FV1200) with MaiTai femtosecond laser source (Spectra-Physics) was used for the imaging experiment. (2) The second type of experiment: hematoxylin-eosin staining was used to detect the histopathology of all tissues. (3) The third type of experiment: Tissues were spayed with 3IAN4 (10 μM of 3IAN4 for 15 min). The samples were then imaged under the hand held lamp. All samples were incinerated after the experiment according to the ethics guidelines (Reference No. 2015016).

Fluorescence Imaging in Cells in hepatopathic tissue samples.
Olympus spectral confocal multiphoton microscope (FV1200) with MaiTai femtosecond laser source (Spectra-Physics) was used in image experiment. Inverted microscope was used in cell and in hepatopathic tissues imaging. The imaging parameters are as follow. Internal PMTs = 16 bit, pixels = 1024 × 1024. Lasers: 405 nm and 488 nm. The scan ranges were ascertained according to fluorescence of dye.

Cytotoxicity.
HepG 2 cell lines were prepared for cell viability studies in 96-well plates (1 × 10 5 cells per well that were incubated in 100 μL). The cells were incubated for an additional 24 h with 3IAN4 in different concentrations.

Molecular docking.
All works of molecular docking were conducted in Yinfo Cloud Platform (http://cloud.yinfotek.com/). The UCSF DOCK 6.9 software was used for the molecular docking. The chemical structure of the small molecule 3IAN4 was drawn by JSME and converted to 3D structure with energy minimization in MMFF94 force field. The crystal structure of COX-2 was taken from the RCSB Protein Data Bank. PDB encoding is 3NT1. The DMS tool was employed to build molecular surface of the receptor using a probe atom with a 1.4 Å radius. The binding pocket was defined by the crystal ligand and spheres were generated filling the site by employing the Sphgen module in UCSF Chimera. A box enclosing the spheres was set with center of (25.455, 20.111, 23.683) and sizes of (-55.760, -62.573, -33.680), within which grids necessary for rapid score evaluation were created by the Grid module. Finally, DOCK 6.9 program was utilized to conduct semi-flexible docking where 10000 different orientations were produced. Clustering analysis were performed (RMSD threshold was set 2.0 Å) for candidate poses and the best scored ones were output.

Molecular dynamics simulation.
MD was performed using the sander module implemented in the Amber 18 suite, with the ff14SB force field used for the protein system, and the GAFF force field used for the ligand. ANTECHAMBER module was used to calculate ligands 3IAN4 in the presence and absence of COX-2 atom charges. Hydrogen atoms and sodium ions (to neutralize the negative charges) were added to protein with the leap utility. The simulation system was immersed in a truncated octahedral box of TIP3P explicit water, extended 10 Å outside the protein on all sides. To start the MD simulation, initial structure of the complex was relaxed to minimize energy during 10,000 minimization steps (5,000 steepest descent steps, SD, and 5,000 conjugate-gradient steps, CG). After energy minimization, the system was gradually heated in the NVT ensemble from 0 to 300 K over 50 ps using the Berendsen coupling algorithm. This procedure was followed by 50 ps of NPT simulation at 300 K and 1 atm pressure using the Langevin dynamics algorithm. After equilibration, 50ns production MD simulation was performed. In all MD simulations, bonds involving hydrogen atoms were constrained using the SHAKE algorithm.
Long range interactions were treated using the particle-mesh Ewald (PME) method and a non-bonded interaction cutoff of 10 Å was used. A time step of 2.0 fs was used and coordinates of the system were saved every 50 ps.
Binding free energy calculation of the ligand was performed using the MMGBSA module in amber.

1.10
The incubation time of celecoxib and nimesulide.
In order to build appropriate models for the different activity of NAT and COX-2 in HepG 2 cells, we evaluated the inhibitory effect of celecoxib and nimesulide. Through experimental measurement, the inhibitory concentration of celecoxib to COX-2 (IC50 35 μM) is lower than that of the probe 3IAN4 (IC50 75 μM), indicating that the binding of celecoxib and COX-2 is stronger than that of the probe 3IAN4 and COX-2. The incubation time of celecoxib (10 mM and 100 μM) and nimesulide (10 mM) are 2-6 h. And the activities of NAT and COX-2 in every sample were detected using an ELISA assay to make sure that cell model is the model we want. And then, the appropriate cell models were washed three times by PBS.

ELISA assay.
(1) The sample preparation: After the normal incubation, the HepG 2 cells were crushed to obtain the supernatant by centrifugation (2000-3000rpm (2) Enzyme activity of COX-2: 100 μL of the sample was added to the corresponding plate well and incubated at 37 o C for 90 min. Then, the liquid in the plate was discarded, 100 μL of biotinylated antibody working liquid was immediately added and incubated at 37 o C for 60 min. All the liquid was discarded and the sample washed 3 times.
Then 100 μL of HRP enzyme binding solution was added to each well, and incubated at 37 o C for 30 min, the liquid was discarded and the sample washed 5 times. Then 90 μL of substrate solution was added to each well and incubated at 37 o C for about 15 min. Finally, 50 μL of termination fluid was added into each well. Then the well was immediately read at 450 nm by a microplate reader and the data processed.
(3) Enzyme activity of NAT: 40 μL of the sample was added to the corresponding plate well and the mixture gently shook. Then, 100 μL of the enzyme-labeled reagent was added to each well, except for blank wells. The plate was sealed by plate film, and incubated at 37 o C for 60 min. The liquid was discarded, ad each well was filled with washing liquid, and left to stand for 30 seconds, before discard the liquid, this process was repeated 5 times.

Synthetic procedures for IAN derivatives and intermediates. The synthesis of A-1
Synthesis of A-1 was synthesized by the previously reported method. S4

The synthesis of 3IAN2
To a solution of 3IA2 (1.0 mmol) in ethanol (20 mL), excessive Zn powders and crystallization of calcium chloride were added and the mixture was refluxed for 3.0 h. After then, the solvent was removed. The solid was purified by silica gel column chromatography by using CH 3 OH/CH 2 Cl 2 =1:10 as eluent, affording 3IAN2 as yellow solid.

The binding affinities and binding free energies
1BM0) were -19.60 kcal/mol and -18.76 kcal/mol, respectively. The data indicated 3IAN4 and 3IAO4 can not bind with albumin.