Experiments

1.Chemical compound

WGX-50 was offered by Prof. Dr. Dongqing Wei's laboratory at Shanghai Jiao Tong University (SJTU, Shanghai, China). In C. elegans experiments, WGX-50 was dissolved in sterilized and purified water containing 0.1% of ethanol (Sangon Biotech, China). Other chemicals with source records were clearly described elsewhere.

2.Animals

Although experiment with a mammalian model is compelling, it is time-consuming and limited by the presence of ethical concerns. C. elegans has been proved as a reasonable model organism for biological research on aging because of its advantageous features.[1] Although its anatomical structure is simple, the tissues and organs, such as muscles, nervous system, gastrointestinal tract, and gonads of C. elegans, are similar to that of higher animals.[2] In addition, its complete genome sequence is available, and about 50% of human protein-coding sequences have identifiable homologous genes in nematodes. Similar to humans and other higher mammals, its behavior changes and descending physiological indexes are accompanied by aging. Moreover, there are highly evolutionary conserved mechanisms controlling physiological phenomena, such as development, aging, and disease. Homologous or functionally similar forms of the main enzymes, genes, and transcription factors involved in metabolism have been found in higher animals and C. elegans.[3] No ethics required for the nematodes. The w.t Bristol N2 C. elegans and transgenic CL2070 strain (dvIs70 [hsp-16.2p::GFP + rol-6(su1006)], referred as hsp-16.2::GFP) were obtained from the Caenorhabditis Genetic Center (CGC, https://cgc.umn.edu/). Escherichia coli strain OP50 (CGC, USA) was aerobically cultured overnight at 37°C in 200 mL of Luria-Bertani (LB) media. Aliquots of concentrated OP50 (20 mg/mL, 250 L) for each 6 mm nematode growth media (NGM) petri plate were seeded, air dried, and used as food source. Unstarved at least three generations of populations were ramp up, synchronized (the sodium hypochlorite method), maintained on NGM plates containing 10 µM of antifungal nystatin (Sangon Biotech, China) with standard techniques at 15 or 20 °C until they developed to the fourth larval stage (L4). Afterwards, they were transferred to desired conditions for assays.

3.Survival analysis

In longevity experiments, w.t N2 worms were allowed to grow to adulthood before exposure to any stimuli (i.e., drugs or RNAi) to eliminate the contribution of any developmental effects on lifespan. Lifespan assays were performed essentially as described previously.[4] Embryos were synchronized and allowed to develop on NGM plates for about 3 days to L4 stage at 20 °C. Adult animals were then transferred to new plates that had been coated with WGX-50 or vehicle with overnight culture of fresh droplets of E. coli OP50 or RNAi feeding bacteria. Growing adult worms at 20°C were scored and well maintained every other day. Animals were judged as dead when they ceased pharyngeal pumping and did not respond to prodding with a platinum wire. Worms that crawled off the plate, exploded, or died from internal hatching were excluded from analysis. Statistics for survival curves were made as described in statistical analysis part.

4.RNA-seq

5.RNA interference in C. elegans

6.RT-qPCR (Real-time polymerase chain reaction)

Nematodes were collected from plates by washing with M9 buffer three times. Total RNA from C. elegans or collected using RNAiso plus reagent kit (Takara Bio, Japan). Reverse transcription (RT) was performed by PrimeScript™ RT reagent kit with gDNA eraser (Takara Bio, Japan) using 1 μg of RNA. For quantitative PCR (qPCR), 2 μL of synthetic cDNA was added into 2× Hieff® qPCR SYBR® green master mix (Yeasen Biotech, China) with forward and reverse gene-specific primers. 3 ~4-fold diluted cDNA was amplified in 40 ~ 60-cycle runs according to the manufacturer's instructions. Data were acquired by a CFX Connect system (Bio-Rad, Singapore) controlled by CFX manager software v3.1. Percentage relative mRNA levels were measured by 2^-∆∆Ct method using C. elegans gpd-1 (GAPDHase-l) as housekeeping gene. Results are representative of two or three independent experiments, showing in % normalized relative mRNA levels for three or 6 biological replicates per condition.

7.Fluorescence microscopy

All worm strains were maintained at 25°C unless clearly stated elsewhere. Prepare nematodes after being treated with 50 μM of WGX-50 or control (0.1 % ethanol) as described elsewhere. Afterwards, they were transferred on 1 % agarose pads (1 mm) over glass slides with 25 mM of levamisole (Aladdin, China) to anesthetize. Images of worms were captured at 20/40× magnification using an inverted fluorescence microscope (RVL-100-G, ECHO, USA) under constant exposure time. ImageJ (Fiji, NIH, USA) was used for the quantification of fluorescent images. Results were presented as the integrated density of gray values ± SEM. Assays were repeated three times with at least 10 animals per treatment in each independent trial. We visualized hsp-16.2::GFP with the microscope through GFP filter sets on day 5, and scored its levels for fluorescence intensity of the two groups of nematodes.

8.Target protein modelling

To predict potential targets of WGX-50, we used online machine learning (ML) platform SwissTargetPrediction (http://swisstargetprediction.ch/) both for human and mice molecules. Then we checked ZINC/ChEMBL hits ZINC1225873/CHEMBL252000 (WGX-50) via SEA in ChEMBL 20 library setting the affinity threshold at 5 nM. More deeply, both extended connectivity fingerprints (ECFP, by rdkit) and maximum tanimoto coefficient (Tc) parameters were used for target prediction that involved searching database ChEMBL (https://www.ebi.ac.uk/chembl/) for proteins of which the ligands share similar structural features as WGX-50. ECFP was utilized to represent chemical structure of the query. Tc was employed as similarity metric for comparing fingerprints. For identified targets, a lower p-value suggests more significant association between the compound and target.

9.Molecular dynamics simulations

A combination of molecular docking, MD simulations, binding free energy calculations were applied to investigate the binding mode of WGX-50 with HSP90 (PDB#2XJX) protein. in our previous work. Briefly, the lowest-energy conformations of ligand were obtained using CREST of the XTB program.[9] For conformation search of the ligand, the root mean square deviation (RMSD) threshold was set at 0.5 Å, and no constraint was used. It was found that all low-energy conformers at the DFT level are within 3 kcal/mol relative to the lowest energy conformers at the GFN2-xTB method. Molecular docking was performed using AutoDock Vina33. Docking poses of substrates to the enzyme were generated with the molecular docking method in the “Receptor-ligand Interaction” module. To obtain the force field parameters for the substrates, calculations of electrostatic potential surfaces were performed at the level of HF/6-31G(d), and then a two-step restrained electrostatic potential charge fitting method[10] was applied to generate the bonds, angles, dihedral angles and van der Waals radii parameters for the substrates by using the Antechamber package. MD simulations were performed using Amber 22 and AmberTools 22 packages.[7] Protonation states of the titratable residues such as His, Glu, and Asp were determined from the PDB2PQR server using PROPKA.[11] Through tleap, the systems were solvated in an octahedral box of TIP3P water with the thickness of the external water layer exceeding 10 Å, while sodium ions were added to achieve charge neutralization for the calculated system. To remove atomic collisions, the solvated systems were subjected to energy minimization of the water molecules and then of the overall system. The system was gradually heated up from 0 to 300 K in 50 ps using the Langevin thermostat and switched to constant pressure and temperature (NPT) using Langevin temperature regulation and the Berendsen barostat for constant pressure controlling and equilibrated for 50 ps with a collision frequency of 2 ps^-1 to adjust to the correct density. Each system was first performed with 10 parallel experiments which include the above minimization, heating, equilibration and 1 ns production run. Three of the ten with stable hydrogen bond networks were chosen to run extension. The Particle Mesh Ewald (PME) method was employed to account for long-range electrostatic interactions. Residues with binding contributions over 1.0 kcal/mol then were selected for further hydrophobic interaction and hydrogen bonding analysis with the CPPTRAJ toolset.[12]

10.Noncovalent interaction analysis

The noncovalent interaction (NCI) analysis including IGMH (independent gradient model based on Hirshfeld partition of molecular density) analysis and AIM (atoms in molecules) was performed.[13][14] Briefly speaking, IGMH replaces all ρ^free involved in the IGM (independent gradient model) equations with atomic densities derived by the popular Hirshfeld partition of the actual electron density. Another difference of IGMH compared to IGM is that the sign(λ₂) r calculated based on the actual electron density is employed instead of the sign(λ₂) r under the promolecular approximation. In Bader’s famous AIM theory[14], a critical point (CP) refers to the position where the magnitude of gradient of r is zero. The so-called (3, -1) type of CP commonly appears between two attracting atoms and hence is referred to as bond critical point (BCP). The (3, +1) type of CP often implies that electron is locally depleted at this position it generally occurs at the center of a ring. The standard for distinguishing (3, - 1) and (3,+1) types of CPs is the sign of λ₂. In AIM theory, BCP is often recognized as the most representative position of a notable interaction, and values of various functions at a BCP are very useful in characterizing the interaction nature and strength. A bond path is generated by tracing the steepest ascent path from a BCP, and it may be viewed as the most representative path revealing an interatomic interaction. Maps were produced using VMD (v1.9.3) and Multiwfn (v3.7) software packages.[15]

11.Statistical analysis

Reference