Defining regorafenib as a senomorphic drug: therapeutic potential in the age-related lung disease emphysema

Senescence, a hallmark of aging, is a factor in age-related diseases (ARDs). Therefore, targeting senescence is widely regarded as a practicable method for modulating the effects of aging and ARDs. Here, we report the identification of regorafenib, an inhibitor of multiple receptor tyrosine kinases, as a senescence-attenuating drug. We identified regorafenib by screening an FDA-approved drug library. Treatment with regorafenib at a sublethal dose resulted in effective attenuation of the phenotypes of βPIX knockdown- and doxorubicin-induced senescence and replicative senescence in IMR-90 cells; cell cycle arrest, and increased SA-β-Gal staining and senescence-associated secretory phenotypes, particularly increasing the secretion of interleukin 6 (IL-6) and IL-8. Consistent with this result, slower progression of βPIX depletion-induced senescence was observed in the lungs of mice after treatment with regorafenib. Mechanistically, the results of proteomics analysis in diverse types of senescence indicated that growth differentiation factor 15 and plasminogen activator inhibitor-1 are shared targets of regorafenib. Analysis of arrays for phospho-receptors and kinases identified several receptor tyrosine kinases, including platelet-derived growth factor receptor α and discoidin domain receptor 2, as additional targets of regorafenib and revealed AKT/mTOR, ERK/RSK, and JAK/STAT3 signaling as the major effector pathways. Finally, treatment with regorafenib resulted in attenuation of senescence and amelioration of porcine pancreatic elastase-induced emphysema in mice. Based on these results, regorafenib can be defined as a novel senomorphic drug, suggesting its therapeutic potential in pulmonary emphysema.


Screening of an FDA-approved drug library
For screening of senescence-modulating drugs, transfection of IMR-90 cells was performed using siPIX RNAs for 24 h followed by incubation with each drug (0.5 μM) from the library for 24 h 1 .
Staining of cells was performed using SA-β-Gal solution, followed by observation under a microscope (Olympus, Japan). Drugs that reduced the staining intensity by less than 50% of siPIXtreated cells were selected in the first round of screening. The second round of screening was conducted, and Western blotting was performed for the cell cycle inhibitors, p16 INKa and p21 WAF1 for confirmation of these candidate drugs.
Spectrophotometry was performed at 570 nm for quantification of formazan.

TUNEL assay
Regorafenib (5 or 15 mg/kg) was administrated orally once a day for two weeks in mice (eight weeks old). To perform TUNEL assay, the lungs were prepared to paraffin-embedded sections. TUNEL staining was performed on paraffin-embedded lung sections of mice using TUNEL Assay Kit-BrdU-Red (Abcam) according to the manufacturer's protocol. Paraffin-embedded mouse lung sections were deparaffinized and rehydrated. After washing with PBS, the slides were incubated with TUNEL reaction mixture at 37°C for 1 h, washed with PBS, and incubated with BrdU-Red mixture for 30 min in a humidified chamber. After washing with PBS, the slides were mounted with mounting medium containing DAPI (Invitrogen). Fluorescence images were analyzed with ImageJ software.

TMT-labeling and peptide fractionation
Labeling of trypsin-digested peptides was performed using 10-plex TMT reagent (Thermo Fisher Scientific Inc., MA). Peptide samples (50 μg each) were labeled TMT reagents, respectively. Then, all TMT-labeled peptides were combined and dried using a Speed-Vac for High-pH fractionation. Fractionation of TMT-labeled peptides was performed by increasing acetonitrile stepgradient elution using a high pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific Inc.). First, acetonitrile and 0.1% trifluoroacetic acid (TFA) were applied for equilibration of the column. Second, the mixed labeled peptide samples and pure water were loaded and then desalted by low-speed centrifugation. Finally, the column was combined with increasing concentrations of high-pH acetonitrile solution. The peptides were subjected to gradient elution of nine fractionations, and vacuum drying was performed on each eluted peptide sample.

LC-MS/MS analysis and quantification
For conduct of proteome analysis, LC-MS/MS analysis was performed using an EASY-nLC 1200 UPLC (Thermo Fisher Scientific Inc.) coupled to a Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific Inc.). Trapping of peptides was performed on a 75 μm × 2 cm C18 precolumn (nanoViper, Acclaim PepMap100, Thermo Fisher Scientific Inc.) prior to separation on an analytical C18 column (75 μm × 50 cm PepMap RSLC, Thermo Fisher Scientific Inc.) at a flow rate 7 of 250 nL/min. The mobile phases A and B were composed of 0 and 80% acetonitrile containing 0.1% formic acid, respectively. The LC gradient began with 5% B and was maintained at 5% B for 5 min, ramped to 38% B for 90 min, to 95% B for 5 min, and remained at 95% B over 9 min. Finally, it was ramped to 5% B for 1 min. Re-equilibration of the column was performed by application of 5% B for 10 min prior to the next run. Voltage of 2000 V was applied for production of an electrospray. During the chromatographic separation, data-dependent mode was used in operation of the Orbitrap Fusion Previously fragmented ions were excluded for 30 sec.
Analysis of MS/MS spectra was performed according to the following software analysis protocol using the Uniprot human database (released on 06-02-2020). The reversed sequences of all proteins were appended into the database for calculation of the false discovery rate (FDR). The peptides were identified using ProLuCID 2 in Integrated Proteomics Pipeline software; IP2 (www.integratedproteomics.com), with a precursor mass tolerance of 10 ppm, and a fragment ion mass tolerance of 100 ppm. Filtering and sorting of the output data files was performed in order to compile a list of proteins with two and more peptide assignments for identification of protein at a false positive rate less than 0.01. TMT reporter ion analysis was performed using Census 3 in Integrated Proteomics Pipeline software within a mass tolerance of 20 ppm. Perseus 4 software (version 1.6.14) was used in performance of statistical analysis. Comparison of protein expression between samples was performed using a multiple sample test with p value set at < 0.05 and hierarchical clustering was constructed for DEPs after calculation of the z-score. A volcano plot was generated after performance of a 2-sample T-test. A list of proteins with log2 fold change of > 0.4 or < -0.4 is shown (Table 1). All