Inhibitor

Aging is a gradual process of natural change that occurs after reaching sexual maturity. It is also a known risk factor for many chronic diseases. Recent research has shown that senolytics can extend the lifespans and health spans of model organisms


Introduction
Aging is characterized by the gradual progression of organ tissue or organ dysfunction, an increased population of senescent cells, imbalances in homeostasis, decreased repair ability, pathological changes, and an elevated risk of disease and mortality (Alichniewicz et al., 2012;Marchand et al., 2011).It is considered one of the risk factors for multiple chronic diseases (López-Otín et al., 2013), which primarily affect the elderly.These diseases include atherosclerosis, cardiovascular disease, stroke, cancers, and neurodegenerative diseases among others.Furthermore, aging also makes individuals more susceptible to agerelated syndromes that compromise immunity and physical resilience.
Cellular senescence is a significant characteristic of aging.It refers to the stable stagnation of the cell cycle accompanied by predictable changes, such as a decrease in cell regeneration capacity and loss of function (Campisi and d'Adda di Fagagna, 2007).Various factors, such as oxidative stress, DNA damage, tumor genes, or external factors, can induce cellular senescence (Calcinotto et al., 2019).When cells encounter toxic stress, the increased expression of cell-cycle factors can halt cell division (Di Micco et al., 2021).In aging organisms, this regulatory system may become less efficient and deplete the regenerative potential of progenitor cells, resulting in the accumulation of senescent cells (Campisi, 2005).These senescent cells release specific patterns of cytokines called senescence-associated secretory phenotypes (SASPs), which include proinflammatory factors, metalloproteinases, and chemokines (Borghesan et al., 2019;Coppé et al., 2008;Gorgoulis et al., 2019).SASPs can influence the growth of neighboring healthy cells, promote chronic inflammation, and contribute to dysfunction and the development of age-related diseases (Coppé et al., 2010;Coppé et al., 2008;Kennedy et al., 2014;Schafer et al., 2020;Zhu et al., 2014).
The elimination of p16 INK4a -positive senescent cells through senolytics has been shown to improve age-related diseases and extend the lifespan of Ercc1− /− mice, which serve as a mouse model for human progeria syndrome (Fuhrmann-Stroissnigg et al., 2017).These drugs are classified as senotherapeutics and can be further divided into two categories: senolytics which selectively kill senescent cells, and senomorphics, which attenuate the adverse effects of pathologic SASPs.In 2015, the first senolytics (in combination with Dasatinib (D) and Quercetin (Q)) were found to effectively induce the apoptosis of senescent cells (Zhu et al., 2015).Furthermore, D + Q was also found to alleviate a variety of age-related diseases, including weakness, osteoporosis, insulin resistance, vasomotor dysfunction, pulmonary fibrosis and skeletal muscle dysfunction, and could prolong the life span of elderly mice (Wissler Gerdes et al., 2020).
The PI3K/AKT and mTOR signal transduction pathways are classical pathways that respond to insulin signals and regulate cell growth, survival, and proliferation (Xu et al., 2020).The PI3K/AKT pathway plays a role in regulating age-related diseases such as cancer, cardiovascular disease, diabetes and Alzheimer's disease (AD).It is also a crucial regulator of cell survival during cellular stress (Datta et al., 1999).Given the diverse nature of aging and aging-related diseases, there is an ongoing need to develop additional senotherapeutics to broaden the range of available treatments that target senescent cells.

Drug preparation
All small-molecule inhibitors in the molecular library were dissolved in DMSO with a concentration of 10 μM.In addition, ABT-737 and PF-04691502 were dissolved in DMSO, the stock solution was at a concentration of 50 μM, stored at − 80 • C, diluted to 10 μM, and the smallmolecule inhibitors in the molecular library were held at − 20 • C. The final working concentration was obtained by diluting in the culture medium, and the same DMSO concentration was used as the negative control.

Assay to identify senotherapeutics
NIH-3T3 cells were inoculated into 96-well plates at 3 × 10 3 cells per well and incubated in 5 % CO 2 at 37 • C in constant-temperature incubators for 24 h.Then, the medium was treated with doxorubicin (1 μM) for 3 days to induce the senescent cell model.Similarly, NIH-3T3 cells were inoculated into 96-well plates at 3 × 10 3 cells per well and treated with H 2 O 2 (400 μM) for 3 days to induce the senescent cell model.After the senescent cell model was established, all compounds used in this study were dissolved in DMSO (Table S1) (10 μM), or DMSO (the same volume of DMSO was used as blank control), were added to the fresh culture medium for 24 h-48 h.The proven senolytic ABT-737 (Selleck, CAS: 852808-04-9) was used as a positive control.To determine the number of cells remaining after drug treatment, we used a DAPI staining reagent (Thermo Fisher Scientific) to calibrate the number of remaining cells.The positive and negative controls were compared to calculate the cell death rate of the treated group.Each drug treatment was replicated in 3-5 wells and the mean and standard deviation were calculated.Additionally, we counted the number of cells remaining in the control cell population in the 96-well plate (Fig. 1F).Cell images acquired by Olympus IX73 were used to measure cell diameters by Image J.

β-Galactosidase staining
The cells were washed 3 times with PBS for 3 min each and fixed with 4 % paraformaldehyde for 15 min.After washing 3 times with PBS, we added dye working solution (prepared according to the instructions of the Solarbio β-Galactosidase Staining Kit) and incubated overnight at 37 • C.After washing with PBS 3 times to terminate the enzymatic reaction, cells were stained with DAPI reagent and observed and counted under a fluorescence microscope.Quantitative analysis was performed using ImageJ software.The relative number of SA-β-Gal cells was calculated by dividing the SA-β-Gal area by the total number of cells and calculating the number of SA-β-Gal cells based on the total number of cells calculated by DAPI.In an experiment, the percentage of positive aging cells was determined by counting over 500 cells in 10 random fields.

Cell viability assay
The CCK-8 (Bimake) assay was performed using a CCK8 kit following the manufacturer's protocol.Briefly, after 24 h of adding potential senolytics to senescent or non-senescent cells, replace the 100 μL culture medium of NIH-3T3 cells with a fresh culture medium.Ten microliters of CCK-8 solution was added into each well, continued to culture in a 37 • C incubator for 1-4 h, and then measured the optical density at 450 nm with a microplate reader and the cell proliferation activity.Each drug (including ABT-737) treatment was repeated in 3-5 wells, and their mean values and standard deviations were calculated accordingly.

RNA extraction and qRT-PCR
The total RNA of cells was extracted by TRIZOL reagent, and complementary DNA (cDNA) was synthesized by reverse transcription kit with gDNA and RT mix.qRT-PCR was performed in a real-time PCR system using the SYBR Green master mix.The relative expression of genes was measured by fluorescence quantitative comparison CT value method and normalized to internal control gene 18S.The primers used are shown in the attached table (Table S2).

Quantification and statistical analysis
All statistical analyses were carried out using GraphPad Prism version 8.00.Statistical analysis of technical repetition and biological repetition was carried out.At least three biological replicates were carried out for each group; error bars represent standard deviations.Student's t-test was used to calculate significance.Statistical significance was considered reached when p < 0.05.

Flow cytometry
For flow cytometry, the NIH-3T3 cells were seeded in a 6-well culture plate with a convergence rate of 70-80 % and incubated with H 2 O 2 for 24 h.For non-senescent cells, 5 × 10 5 cells were seeded per well for use.After adding the drug, incubated at 5 % CO 2 for 24 h.Then, cells were collected and stained using the Cell Apoptosis Detection Kit (KeyGEN Bio TECH).Specifically, the sample was digested with trypsin (-EDTA) and collected into a centrifuge tube.After centrifugation, it was resuspended in a 500uL Bind buffer.Added 5 μL Annexin V and gently mixed, then added 5 μL of Propidium Iodide (PI) and mixed well; incubated at room temperature and avoided light for 10 min, performed machine detection and analysis (Beckman, USA), and analyzed flow cytometry data using the software CytExpert.

Cell senescence model screen showed that PF-04691502 has potential as a senolytic agent
DNA damage is a major cause of senescence in mouse and human cells.Doxorubicin hydrochloride (ADR) is a widely used anticancer drug in clinical practice.Numerous studies have demonstrated that doxorubicin can cause damage to most normal cells, resulting in the production of high levels of reactive oxygen species (ROS), which in turn leads to DNA damage and promotes cell senescence (Cappetta et al., 2017).In this study, we took advantage of this property, we used a concentration of 1 μM doxorubicin to induce senescence in NIH-3T3 cells, and on day 5, >90 % of these cells were positive for β-Gal, as detected by the SAβ-Gal kit.The doxorubicin-treated group showed a significant increase in senescent cells compared to the control group (Fig. 1A and B).Furthermore, cell cycle arrest was observed in senescent cells, as evidenced by the up-regulation of CDK inhibitors p16 INK4a and p21 Cip1 expression (Fig. 1C and D).Additionally, we observed an upregulation in the mRNA levels of Senescence-Associated Secretory Phenotype (SASP) cytokines such as il6, cxcl1, igf2r, mmp3, tnf-α and gdf-15 (Fig. 1E).The increased expression of these cell-senescence markers indicates the successful establishment of a doxorubicin-induced cell senescence model in this study.
The model described above was used as a senolytic drug screening system for the follow-on study (Fig. 1F).To screen for senolytics that can kill aging cells without causing damage to proliferating cells, NIH-3T3 cells were induced to age through ADR, and proliferating cells were used as a control The PI3K/AKT/mTOR pathway, known to be overactivated in the process of senescence, was targeted for inhibition as it has been shown to have an excellent anti-aging effect.For instance, rapamycin inhibits mTOR protein and exhibits anti-aging activity (Papadopoli et al., 2019).Therefore, a library of 156 drugs (Table S1) targeting the PI3K/AKT/mTOR pathway by 14 different targets (Fig. 2A) was screened using this system.ABT-737, a BCL-2 targeted inhibitor, was used as a positive control due to its significant senolytic activity.All small molecules were screened at a concentration of 10 μM for primary screening.Among them, 55 drugs showed a clearing effect on ADRinduced aging model cells within 48 h (Table S1).After conducting two rounds of ADR-induced senescence model cell clearance experiments and cell viability detection for a total of 55 drugs, it was found that only 9 compounds exhibited a significant reduction in the number of senescent cells, with a clearance rate ranging from 50 % to 90 %.These 9 compounds were identified as having senolytic potential (Fig. 2B).An ideal senolytic should selectively remove senescent cells with minimal toxicity or side effects on normal cells.Therefore, further verification was necessary to determine if these nine compounds are indeed senolytic drugs.
To evaluate the clearance effect of candidate compounds on senescent cells, we conducted an experiment with a group of non-senescent cell control and drug-treated groups.Our findings indicate that PF-04691502 effectively clears senescent cells (Fig. 2B) while showing no significant effect on non-senescent cells (Fig. 2C, Table 1).Therefore, we focused our further mechanism exploration on PF-04691502.Detailed concentration gradient experiments revealed that PF-04691502 at 5 μM and 10 μM did not significantly affect normal cells, but specifically cleared senescent cells in both the 5 μM and 10 μM PF-04691502 groups (Fig. 2D, and E).Cell viability assays further confirmed that treatment with 5 μM and 10 μM of PF-04691502 significantly cleared ADR-induced senescent cells, while having no significant effect on non-senescent cells (Fig. 2F and G).These results suggest that PF-04691502 specifically targets the elimination of senescent cells in a doxorubicin-induced NIH-3T3 model of cellular senescence.

PF-04691502 showed senolytic activity in the hydrogen peroxideinduced senescent model
To evaluate the senolytic activity of PF-04691502 in a different model, we used the H 2 O 2 -induced stress senescent model (Chen and Ames, 1994;Wang et al., 2013b) and excluded the influence of the doxorubicin model (Fig. S1).Our results showed that PF-04691502 significantly reduced the ratio of SA-β-Gal-positive cells at concentrations of 5 μM and 10 μM, indicating effective clearance of senescent cells while normal cells were unaffected (Fig. 3A-D).
Furthermore, when, we added 5 μM of PF-04691502 to cells H 2 O 2-treated cells, we observed significant toxic effects on senescent cells, as evidenced by changes in their size (Fig. 3E and F).Increasing the concentration of PF-04691502 further enhanced its toxicity to senescent cells (Fig. 3G).Importantly, we found that PF-04691502 had no significant impact on the viability of normal cells at 5 and 10 μM (Fig. 3H).
The concentration used corresponds to the lowest concentration of the drug that exhibits senolytic effects without affecting normal cell survival, we used 5 μM as the subsequent experimental concentration.It was further confirmed that PF-04691502 could be used as a new senolytic drug in both the doxorubicin senescent model and the H 2 O 2 senescent model.

PF-04691502 reduced molecular markers associated with senescent
To validate the results of SA-β-Gal staining, we examined other senescence-related indicators including the expression of cyclindependent kinase inhibitors p16 INK4a and p21 Cip1 , as well as the expression of SASP factors such as il6, il1α, igf2r and tnf-α.In the hydrogen peroxide-induced model of cellular senescence, treatment with PF-04691502 at a concentration of 5 μM, for 24 h significantly decreased the mRNA levels of p16 INK4a and IL6 proteins (Fig. 4A), along with a considerable down-regulation of p21 Cip1 mRNA and protein levels (Fig. 4B, C).Interestingly, even in non-senescent cells, treatment with PF-04691502 at a concentration of 5 μM also led to a decrease in the protein levels of p16 INK4a and p21 Cip1 (Fig. S2).Furthermore, PF- 04691502 exhibited a significant down-regulation of mRNA expression levels of SASP cytokines (Fig. 4D).These findings suggest that PF-04691502 has the potential to reduce the expression of age-related molecular markers and may serve as a senolytic drug in NIH-3T3.

PF-04691502 inhibited PI3/AKT and mTOR pathways and promoted apoptosis of senescent cells
The PI3K/AKT/mTOR pathway is known to regulate autophagy, apoptosis, and cellular senescence (Fuhrmann-Stroissnigg et al., 2017;Papadopoli et al., 2019).In this study, we investigated the inhibitory effect of PF-04691502 on the PI3K/AKT/mTOR pathway in an H 2 O 2 -induced senescent cell model.Western blot analysis revealed a significant down-regulation of phosphorylated AKT and S6K proteins (p-AKT and the p-S6K) in senescent cells treated with PF-04691502 (Fig. 5A  and B).To assess the involvement of the apoptotic pathway, we measured the mRNA expression levels of apoptosis-related genes including caspase family members caspase 3, caspase6, caspase9 and bax (Fig. 5C).The results showed a significant up-regulation of these genes, indicating that PF-04691502 can induce apoptosis in senescent cells.The decreased expression of the DNA repair enzyme gene parp1 further supported the activation of apoptotic signals (Fig. 5D).Additionally, the mRNA and protein levels of p53, a key regulator of the endogenous apoptosis pathway, were found to be up-regulated, further confirming the occurrence of apoptosis (Fig. 5E and F).These findings suggest that PF-04691502 can function as a senolytic agent by inhibiting the PI3K/ AKT/mTOR pathway and inducing apoptosis in senescent cells.To determine whether PF-04691502 preferentially triggers apoptosis in senescent cells, Annexin V/PI staining was performed on cultures of senescent and non-senescent cells, and analysis was performed using flow cytometry (Fig. 5G).The use of Annexin V (phosphatidylserine binding compounds for early apoptosis) and Propidium Iodide (membrane impermeable dyes typically excluded from living cells, PI) can detect cells at different stages of cell death.Compared with untreated cells, treatment with PF-04691502 increased in Annexin V/PI doublepositive cells.On the contrary, the apoptosis of non-senescent cells did not significantly increase, supporting the conclusion that PF-04691502 selectively kills senescent cells (Fig. 5H).

Discussion
As one of the hallmarks of aging, cellular senescence has recently become a popular target for improving age-related diseases and prolonging life expectancy (López-Otín et al., 2023).Studies have confirmed that the targeted elimination of senescent cells can extend lifespan and alleviate age-related diseases in model organisms such as fruit flies and mice (Xu et al., 2018;Xu et al., 2022;Yousefzadeh et al., 2018).Therefore, we developed a screening platform to identify and test senescent therapeutics, compounds that specifically kill senescent cells (senolytic) using primary mouse embryonic fibroblasts (Fig. 1).By using the cellular senescence model, a total of 156 drugs with different functions were tested, including PI3K/AKT/mTOR, IκB/AKK and GSK-3 inhibitors, fatty acid synthase and autophagy inducers (Table S1).In particular, we found that PF-04691502 can play a senolytic role by inhibiting PI3K/AKT and mTOR pathways, and can reduce the expression level of cellular senescence markers.We also found that PF-04691502 can inhibit the phosphorylation of S6K and AKT and induce apoptosis in senescent cells.Our results imply that PF-04691502 has the potential as a new senolytic anti-senescence drug.This enriches the senolytic library and provides a new direction for the future preclinical research of PF-04691502.
Senescent cells exhibit resistance to apoptosis and upregulate anti-  apoptotic pathways to protect themselves from their own inflammatory SASP, enabling their survival (Kirkland and Tchkonia, 2020).Studies have shown that targeting anti-apoptotic proteins with ABT-263 and ABT-737 has senolytic effects (Yosef et al., 2016;Zhu et al., 2016).PF-04691502 acts as a dual inhibitor, reducing the phosphorylation levels of AKT and S6K, which are key factors in the anti-apoptotic pathways PI3K/AKT and mTOR (Fig. 5A and B) (Pungsrinont et al., 2021) (Fuhrmann-Stroissnigg et al., 2017).Additionally, we observed upregulation of the pro-apoptotic protein bax and caspases, which are directly involved in regulating apoptosis, as indicated by the mRNA levels of apoptosis-related proteins (Fig. 5C).In terms of the intrinsic pathways' role in apoptosis, a subset of p53 family genes plays a crucial role in regulating both pro-survival and pro-apoptosis members of the Bcl-2 family, including bax.The cellular senescence program induces stable cell cycle arrest through complex interactions between Rb and p53 tumor suppression pathways in response to various types of cellular stress (Beauséjour et al., 2003;Narita et al., 2006).In a previous study, Ines Sturmtechnier et al. demonstrated that the transcription levels of MDM2 and RNASE4 were also elevated in senescent cells to inhibit p53-mediated cell apoptosis and promote survival.Once cells become fully senescent, it is necessary to control p53 activity to inhibit its proapoptotic function and ensure the survival of aging cells (Sturmlechner et al., 2022).Therefore, we hypothesize that activating p53 activity in an aging state can promote apoptosis of aging cells.P53 interacts with antiapoptotic Bcl-XL and pro-apoptotic Bcl-2 family proteins, resulting in the release of proapoptotic effector bax/bak (Mijit et al., 2020).The up-regulation of p53 further confirmed that PF-04691502 plays a senolytic role by promoting the apoptosis of senescent cells (Fig. 5E and F).This is consistent with the previous report that PF-04691502 can trigger the apoptosis of hepatocellular carcinoma cells and induce cell death (Wang et al., 2013a).Furthermore, our conclusion demonstrates that PF-04691502 preferentially triggers apoptosis in senescent cells and exerts senolytic activity (Fig. 5G and H).Recent in vivo studies in model animals have demonstrated that targeting anti-apoptotic pathways to eliminate senescent cells can restore tissue regeneration ability (Ferreira-Gonzalez et al., 2022;Ritschka et al., 2020).Future in vivo experimental studies on PF-04691502 may provide further relevant evidence.To facilitate the widespread application of PF-04691502, it is crucial to verify its ability to extend healthy lifespan and exert senolytic activity in model organisms.To address this issue, our planned work includes lifespan experiments in drosophila and mice, as well as injection experiments.

Fig. 1 .
Fig. 1.The cell senescence model was constructed by doxorubicin.(A) SA-β-Gal staining results of NIH-3T3 cells before (left) and after (right) doxorubicin induction; blue represents positive SA-β-Gal staining results (the scale bar is 50 μm).(B) Quantitative statistics of SA-β-Gal-positive cells in Fig. (A).*** p < 0.001, n = 3. (C) Western blot determination of p16 INK4a protein expression in normal and senescent cells.** p < 0.01, n = 3. (D) Western blot determination of p21 Cip1 protein expression in normal and senescent cells.* p < 0.05, n = 3. (E) mRNA expression level of SASP senescence markers detected by qRT-PCR.* p < 0.05, ** p < 0.01, *** p < 0.001, n = 3. (F) Scheme of possible outcomes after treating senescent NIH-3T3 cells with potential senolytics.Note: The expression levels were shown with relative fold changes, and Student's t-test was used to analyze the significance of the differences.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .
Fig. 5. PF-04691502 acts as a senolytic by inducing apoptosis of senescent cells.(A) Western blot analysis of the protein expression of phosphorylated AKT after treatment with PF-04691502 at 5 μM for 24 h.** p < 0.01, n = 3. (B) Western blot was used to detect the protein expression of mTOR pathway marker phosphorylated p70 S6K following PF-04691502 treatment at 5 μM for 24 h.*** p < 0.001, n = 3. (C) mRNA expression levels of apoptosis-related genes were detected by qRT-PCR.* p < 0.05, ** p < 0.01, *** p < 0.001, n = 3. (D) mRNA expression level of DNA repair enzyme parp1 was detected by qRT-PCR.*** p < 0.001, n = 3. (E) The mRNA expression level of p53 was detected by qRT-PCR.*** p < 0.001, n = 3. (F) Western blot was used to detect the protein expression of p53 following PF-04691502 treatment at 5 μM for 24 h.** p < 0.01, n = 3. (G) Flow cytometry analysis of cell death in senescent NIH-3T3 cell cultures treated with PF-04691502 via Annexin V/PI staining.NIH-3T3 cells are either senescent (Top) or non-senescent (Bottom).Live cells show dual negative results for PI and Annexin V (bottom left quadrant), early apoptotic cells show positive results for Annexin V (bottom right quadrant), late apoptotic cells show positive results for Annexin V and PI (top right quadrant), and dead cells only show positive results for PI (top left quadrant).(H) Quantification of flow cytometry data.Apoptosis of senescent and non-senescent cells was calculated by summing up all Annexin V positive cells.* p < 0.05, n = 3. Note: The expression levels were shown with relative fold changes, and Student's ttest was used to analyze the significance of the differences.