Senolytic compounds

Abstract

The present invention refers to specific compounds for use as senolytic agents to selectively induce death of senescent cells, in a method for the treatment and / or prevention of senescence-associated diseases or disorders.

Description

[0001] SENOLYTIC COMPOUNDS

[0002] FIELD OF THE INVENTION

[0003] The present invention refers to the medical field. Particularly, the present invention refers to specific compounds for use as senolytic agents to selectively induce death of senescent cells, in a method for the treatment and / or prevention of senescence-associated diseases or disorders.

[0004] STATE OF THE ART

[0005] Senescence is a cellular programme that imposes a stable arrest on damaged or old cells to avoid their replication. As well as growth arrest, senescent cells undergo profound phenotypic changes that include chromatin reorganisation, increase of 3 -galactosidase activity (referred to as senescence-associated P-galactosidase or SA-P-Gal) and secretion of multiple factors, mainly pro-inflammatory, that are collectively referred to as the senescence-associated secretory phenotype (SASP).

[0006] Replicative senescence is activated upon serial passage of cells in culture (or as cells become older in an organism). Senescence can also be induced by a range of different insults that include oncogene activation, irradiation and exposure to chemotherapeutic drugs.

[0007] Senescent cells accumulate during age and are associated with many diseases, including cancer, fibrosis and many age-related pathologies. Recent evidence suggests that senescent cells are detrimental in multiple pathologies and their elimination confers many advantages, ameliorating multiple pathologies and increasing healthspan and lifespan.

[0008] Senescent cells are present in many pre-neoplastic lesions, fibrotic tissues (e.g., in the liver, kidney, heart, pancreas) and old tissues. Senescent cells are also associated with a long list of other pathologies, including neurological (e g. brain aneurysm, Alzheimer's and Parkinson), pulmonary (e.g. idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and cystic fibrosis), ophthalmological (e.g. cataracts, glaucoma, macular degeneration), musculoskeletal (e g. sarcopenia, disc degeneration, osteoarthritis), cardiovascular (e g. atherosclerosis, cardiac fibrosis, aorta aneurysm), renal (e.g. kidney disease, transplant complications) and others such as diabetes, mucositis, hypertension and osteomyelofibrosis (OMF).

[0009] While senescent cells have a protective role against cancer and limit most types of fibrosis, the accumulation of senescent cells during aging and many other diseases is thought to be detrimental. To date, there is no unequivocal explanation of why senescent cells are detrimental.

[0010] Evidence for the many detrimental effects of senescent cells (and the benefits caused by their selective elimination) has been provided by many authors, and proof-of-concept studies have led to the identification of compounds that can selectively eliminate senescent cells (so-called “senolytics”), most of them having significant side-effects.

[0011] Indeed, senescent cells can be detrimental in the context of cancer. While cellular senescence is initially a beneficial process as it acts as a tumor-suppressive mechanism to prevent the proliferation of damaged or potentially cancerous cells, senescent cells can have negative consequences under certain conditions:

[0012] • Senescence-Associated Secretory Phenotype (SASP): Senescent cells often adopt a SASP, which involves the secretion of pro-inflammatory cytokines, growth factors, proteases, and other molecules. This secretory profile can alter the tissue microenvironment and promote chronic inflammation. In cancer, SASP can have mixed effects: Tumor Promotion: SASP factors can create a pro-tumorigenic environment by enhancing the growth and invasiveness of nearby cancer cells, supporting angiogenesis, and suppressing anti -turn or immune responses. Immune Evasion: SASP can sometimes contribute to the immune system’s inability to clear senescent or cancerous cells effectively, allowing tumor cells to persist or progress.

[0013] • Impact on Surrounding Tissues: Senescent cells can affect their neighbouring cells through paracrine signalling. In the context of cancer, SASP factors can lead to changes in the extracellular matrix and support the epithelial-to-mesenchymal transition (EMT) in nearby cells, promoting metastasis and cancer progression.

[0014] • Immunosuppression: While senescent cells can initially attract immune cells to clear them, persistent senescent cells might contribute to an immunosuppressive environment. This can inhibit the immune system’s ability to detect and eliminate emerging cancer cells, aiding tumor progression.

[0015] • DNA Damage and Genomic Instability: Senescent cells often carry DNA damage and genomic instability. Their presence can lead to a higher likelihood of mutations in neighbouring cells due to the inflammatory milieu induced by SASP. This can increase the risk of oncogenic transformations. • Resistance to Apoptosis: Senescent cells typically resist apoptosis, which makes them difficult to remove from the tissue. This resistance allows them to persist for a long time, continuing to influence their environment detrimentally and contributing to cancer development and progression.

[0016] In summary, while senescence initially acts as a barrier to cancer, its long-term presence, especially through SASP, can contribute to a tumor-supportive environment and overall cancer progression.

[0017] Regarding fibrosis, senescent cells play a detrimental role in the development and progression of fibrosis. Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) components, leading to tissue scarring and impaired organ function. The involvement of senescent cells in fibrosis can be explained through the following mechanisms:

[0018] • Senescence-Associated Secretory Phenotype (SASP): Senescent cells release a variety of SASP factors, including pro-inflammatory cytokines (e.g., IL-6, IL-8), growth factors, and proteases. These secreted molecules can (i) promote fibroblast activation (SASP can stimulate the proliferation and activation of fibroblasts, which are responsible for ECM production. Activated fibroblasts (myofibroblasts) deposit collagen and other ECM components, contributing to fibrotic tissue formation) or (ii) induce chronic inflammation (SASP factors create a pro-inflammatory microenvironment that can maintain and exacerbate chronic inflammation, a key driver of fibrosis).

[0019] • Impaired Tissue Regeneration: Senescent cells accumulate in tissues over time and impair the ability of organs to regenerate. In fibrotic conditions, the persistent presence of senescent cells can inhibit normal wound healing and tissue repair processes, shifting the balance toward fibrosis rather than regeneration.

[0020] • ECM Remodelling: Senescent cells can secrete matrix metalloproteinases (MMPs) and other enzymes that modify the ECM. This remodelling can disrupt the normal tissue architecture and create an environment conducive to fibrotic changes.

[0021] • Crosstalk with Immune Cells: The SASP profile of senescent cells can recruit and influence immune cells, such as macrophages and T-cells, leading to a cycle of persistent immune activation. This immune response can either clear senescent cells or, if dysregulated, contribute to chronic inflammation and fibrotic tissue deposition. Resistance to Apoptosis: Senescent cells have an increased resistance to cell death, making them difficult to remove naturally. Their continued presence in tissues can perpetuate fibrotic signalling pathways and ECM production.

[0022] In summary, while senescent cells play a critical role in wound healing and tissue maintenance, their prolonged presence and SASP activity can lead to chronic inflammation and excessive ECM deposition, driving the progression of fibrosis in various organs.

[0023] So, there is an unmet medical need of identifying further compounds with senolytic properties.

[0024] DESCRIPTION OF THE INVENTION

[0025] Brief description of the invention

[0026] The present invention refers to specific compounds for use as senolytic agents or as senotherapeutic agents designed to selectively remove senescent cells and / or targeting the SASP.

[0027] More specifically, the senotherapeutic agent is a molecule able to either i) reduce the accumulation of senescent cells for example by inducing their death via apoptosis or inhibition of the SASP component and ii) trigger the re-differentiation of cells once they have been dedifferentiated. Both reduced levels of senescence and enhancement of re-differentiation restore the capacity of the tissues to restore regeneration. The purpose of the “senotherapeutic agents” is to delay, prevent, alleviate, or reverse age-related diseases which are caused by the persistence and accumulation of senescent cells as senescence via SASP also induces inflammation, reprogramming and dedifferentiation of neighbouring cells, being therefore one of the key factors involved in tissue degeneration and progression of aging-associated diseases.

[0028] So, the compound of the invention selectively induces death of senescent cells and can be used in the treatment and / or prevention of senescence-associated diseases or disorders.

[0029] Particularly, the compound 96558 of Formula I was assayed in the present invention. Formula I

[0030] Such as it can be seen in Example 2, compound 96558 was validated as a senolytic agent in A549 bleomycin-induced senescent cells (Example 2.2) and under ionizing radiation-induced senescence (Example 2.3). Moreover, compound 96558 was explored in a different human tumor context, such as the U87MG glioma cell line (Example 2.4) and in a cellular aging model (Example 2.5), confirming that the senolytic effect of compound 96558 is mediated by the activation of the apoptotic signalling pathway (Example 2.6).

[0031] So, the proof of concept shown in Example 2 makes plausible the use the compound 96558 in the treatment and / or prevention of any senescence-associated disease or disorder [Chaib, S., Tchkonia, T., & Kirkland, J. L. (2022). Cellular senescence and senolytics: the path to the clinic. Nature medicine, 28(8), 1556- 1568. https: / / doi.org / 10.1038 / s41591-022-01923-y].

[0032] So, the first embodiment of the present invention refers to the compound of Formula I, or salts derived thereof, for use as a medicament.

[0033] In a preferred embodiment, the present invention refers to the compound of Formula I, or salts derived thereof, for use as a senolytic agent to selectively induce death of senescent cells.

[0034] The person skilled in the art, using common general knowledge, could recognize or assess whether the death of senescent cells has taken place. Examples of techniques that could be used are:

[0035] • Biomarkers of Senescence: These include testing for senescence-associated - galactosidase (SA- -gal) activity, which is a common marker of cellular senescence. Cells that have died may no longer display this marker.

[0036] • Cell Viability Assays: Assays such as trypan blue exclusion, MTT, or resazurin-based tests can indicate whether cells are alive or dead. Loss of cell viability in a senescent cell population can signal cell death.

[0037] • Flow Cytometry with Apoptosis Markers: Flow cytometry can be used with dyes and antibodies that mark apoptosis (e g., annexin V / propidium iodide staining). This helps identify whether the senescent cells are undergoing programmed cell death.

[0038] • Caspase Activity Assays: Measuring caspase activity, particularly caspase-3 / 7, can indicate apoptosis in senescent cells, as these enzymes are involved in the execution phase of cell death. • TUNEL Assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling): This method labels DNA breaks that occur during apoptosis, allowing the detection of dead or dying cells, including senescent cells.

[0039] • Microscopy Techniques: Staining methods such as DAPI or Hoechst 33342 for nuclear integrity combined with SA-P-gal staining can show the presence of cell death among senescent cells under a microscope.

[0040] • Western Blotting for Apoptosis Proteins: The expression of pro-apoptotic and anti- apoptotic proteins such as Bax, Bcl-2, and cleaved PARP can be assessed to identify if senescent cells are undergoing apoptosis.

[0041] • LDH Release Assay: The lactate dehydrogenase (LDH) release assay measures cell membrane integrity. An increase in LDH in the culture medium indicates cell membrane rupture, suggesting necrosis or cell death.

[0042] These techniques can help distinguish between living, apoptotic, or necrotic states of senescent cells and can be tailored based on the cell type and research context.

[0043] In a preferred embodiment, the present invention refers to the compound of Formula / , or salts derived thereof, for use in the treatment and / or prevention of senescence-associated diseases or disorders.

[0044] In a preferred embodiment, the present invention refers to the compound of Formula I, or salts derived thereof, for use in the treatment and / or prevention of senescence-associated diseases or disorders selected from: cardiovascular disorder, metabolic disease, an inflammatory disease or disorder, a pulmonary disease or disorder, a neurological disease or disorder, a proliferative disorder, a renal disorder or disease, an eye disease or disorder, and / or a dermatological disorder or disease or cancer.

[0045] In a preferred embodiment, the present invention refers to the compound of Formula I, or salts derived thereof, for use in the treatment and / or prevention of senescence-associated diseases or disorders selected from: (a) an inflammatory or autoimmune disease or disorder selected from osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, kyphosis or herniated intervertebral disc; (b) a neurological disease or disorder selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment, macular degeneration or motor neuron dysfunction; (c) a metabolic disease selected from diabetes, diabetic ulcer, metabolic syndrome or obesity; (d) a pulmonary disease selected from pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis or age-related loss of pulmonary function; (e) an eye disease or disorder selected from macular degeneration, glaucoma, cataracts, presbyopia or vision loss; (f) an age-related disorder selected from renal disease, renal failure, frailty, hearing loss, muscle fatigue, skin conditions, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucosa fibrosis or sarcopenia; (g) a dermatological disease or disorder selected from eczema, psoriasis, hyperpigmentation, nevi, rashes, atopic dermatitis, urticaria, diseases and disorders related to photosensitivity or photoaging, rhytides, pruritis, dysesthesia, eczematous eruptions, eosinophilic dermatosis, reactive neutrophilic dermatosis, pemphigus, pemphigoid, immunobullous dermatosis, fibrohistocytic proliferations of skin, cutaneous lymphomas or cutaneous lupus; or (h) a cardiovascular disease selected from atherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, brain aneurysm or stroke.

[0046] The second embodiment of the present invention refers to a pharmaceutical composition comprising a compound of Formula I, or salts derived thereof, and, optionally, pharmaceutically acceptable excipients and / or carriers.

[0047] The third embodiment of the present invention refers to a method for treating and / preventing any senescence-associated disease or disorder which comprises administering a therapeutically effective dose or amount of the compound or Formula I of the invention or a pharmaceutical composition comprising thereof along with pharmaceutically acceptable excipient or carrier.

[0048] In the context of the present invention the following terms are defined:

[0049] • The term "comprising" means including, but it is not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0050] • By "consisting of’ means including, and it is limited to, whatever follows the phrase “consisting of’. Thus, the phrase "consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. • “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0051] • By “therapeutically effective dose or amount” of a composition comprising the compounds of the invention is intended an amount that, when administered as described herein, brings about a positive therapeutic response in a subject having a senescence- associated disease or disorder. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0052] Description of the figures

[0053] Figure 1. Schematic flowchart illustrating the screening strategy for identifying senolytic compounds. Beginning with a library of 101,024 compounds, we conducted a primary screening to identify those with cytotoxic activity, resulting in 1,038 compounds. These were subsequently subjected to a selectivity screening focused on assessing cytotoxic activity specifically in senescent cells. The selectivity screening identified 64 candidate compounds, which were further subjected to IC50 validation to determine dose dependent senolytic effects.

[0054] Figure 2. Dot plot displaying the z scores of the 64 selected compounds in the senolytic screening. The red line denotes the threshold (z score < 3) for compounds considered to exhibit strong activity. Compounds within the orange boxes are those with known identities and activities. Compounds enclosed in yellow circles were excluded for failing the IC50 test. Compounds within the green circle represent the four most potent and validated senolytic compounds.

[0055] Figure 3. IC50 validation of Compound 96558 in bleomycin induced senescent A549 cells. Cell viability graphs (upper part) and IC50 curves (bottom part) of A549 cells treated with decreasing concentrations of 96558. Senescence was induced using bleomycin, and the cell viability assay was conducted at different time points (24, 48 and 72 hours). The figure provides a visual representation of the compound's differential effects on senescent and proliferative cells, highlighting its potential as a senolytic agent, n = 5 independent experiments for the 24- hour time point and n = 3 for the 48- and 72-hour time points. Data are presented as mean ± s.d. Statistical significance: ***p < 0.001; **p < 0.01; *p < 0.05 (multiple unpaired t-test comparisons for each concentration).

[0056] Figure 4. Selectivity of senolytic compound for senescent over quiescent A549 cells. Cell viability analysis of A549 cells treated with Compound 96558 for 24 hours, comparing its effect on senescent and quiescent cells. Despite the concentrations used, quiescent cells maintained viability, whereas senescent cells showed significant cytotoxicity, demonstrating the compound's preferential targeting of senescent cells. n = 3 independent experiments. Data are presented as mean ± s.d. Statistical significance: ***p < 0.001; **p < 0.01; *p < 0.05 (multiple unpaired t-test comparisons for each concentration).

[0057] Figure 5. Flow cytometry analysis of co-cultures treated with Compound 96558. Flow cytometry analysis of a co-culture of proliferating RFP+ and senescent GFP+ A549 cells treated for 24 hours with DMSO, navitoclax or Compound 96558. Green fluorescence was measured in the B525-A channel, and red fluorescence in the Y585-A channel. In the upper left quadrant, proliferating RFP+ cells are shown, and in the lower right quadrant, senescent GFP+ cells. Both navitoclax and Compound 96558 led to a reduction in green fluorescence intensity (senescent cells) and an increase in red fluorescence intensity (proliferating cells), indicating selective elimination of senescent cells. n = 3 independent experiments. All data represent the average ± s.d. Statistical significance was determined by one-way ANOVA with post-hoc correction: ***p < 0.001; **p < 0.01.

[0058] Figure 6. IC50 validation of Compound 96558 in radiation-induced senescent A549 cells. The upper section displays cell viability graphs, while the lower section exhibits IC50 curves for A549 cells subjected to decreasing concentrations of compound 96558. Senescence was induced through ionizing radiation, and the cell viability assay was conducted at multiple time intervals (24, 48, and 72 hours). This figure visually depicts the Compound's distinct effects on senescent and proliferative cells, underscoring its potential as a senolytic agent in the context of ionizing radiation-induced senescence, n = 3 independent experiments. Data are presented as mean ± s.d. Statistical significance: ***p < 0.001; **p < 0.01; *p < 0.05 (multiple unpaired t-test comparisons for each concentration).

[0059] Figure 7. Assessment of the senolytic activity of Compound 96558 in U-87 MG and SK- MEL-103 cells with palbociclib -induced senescence. Cell viability graph and IC50 curve are represented. n = 3 independent experiments. Data are presented as mean ± s.d. Statistical significance: ***p < 0.001; **p < 0.01; *p < 0.05 (multiple unpaired t-test comparisons for each concentration).

[0060] Figure 8. Assessment of the senolytic activity of Compound 96558 in HT1080 cells with Trf2 deletion. Cell viability graph in HT1080 cells induced into senescence through Trf2 deletion (upper part) and IC50 curve for this cell line (bottom part). n = 3 independent experiments. Data are presented as mean± s.d. Statistical significance: ***p < 0.001; **p < 0.01; *p < 0.05 (multiple unpaired t-test comparisons for each concentration).

[0061] Figure 9. Mechanisms of cell death induced by Compound 96558 in senescent A549 cells. A) Flow cytometry analysis of Annexin-V and 7-AAD staining in proliferative A549 cells treated with DMSO, Navitoclax (ABT), or Compound 96558. Most cells remained viable (FITC-Annexin V-negative and 7-AAD-negative, lower left quadrant), with minimal differences in cell death across treatments. Quantification of the percentage of cell death (Annexin-V or 7-AAD positive) shows no significant changes among the three conditions. B) In senescent A549 cells, treatment with Compound 96558 resulted in a marked increase in cell death, primarily characterized by double-positive cells (FITC-Annexin V-positive and 7-AAD- positive, late apoptosis). Co-treatment with Z-VAD-FMK or Necrostatin-1 partially reduced cell death, though the effect was incomplete. The combination of both inhibitors yielded similar partial reductions. In contrast, Ferrostatin-1 completely rescued cell viability, restoring levels comparable to the DMSO control. C) Navitoclax, a senolytic compound that induces apoptosis, served as a control. Treatment increased the proportion of Annexin V-positive cells (early apoptosis), which was nearly completely reversed by Z-VAD-FMK or the combination of Z- VAD-FMK and Necrostatin-1, but not by Necrostatin-1 or Ferrostatin-1 alone. These findings confirm apoptosis as the primary mechanism of Navitoclax-induced cell death. / / - 4 independent experiments. Statistical significance was determined by one-way ANOVA with post-hoc correction: **p < 0.01; *p < 0.05.

[0062] Figure 10. Validation of ferroptosis as the main cell death mechanism induced by Compound 96558. Flow cytometry of senescent A549 cells stained with BODIPY-C11 to measure lipid peroxidation. Treatment with Compound 96558 or RSL3 (ferroptosis inducer) increased oxidized BODIPY-C11 (green fluorescence), a shift reversed by Ferrostatin-1 (Fer- 1). Quantification of mean fluorescence intensity shows significant increases in lipid peroxidation with Compound 96558 and RSL3 compared to DMSO, which were fully reversed by Fer-1. No differences were observed between Fer-l-treated samples and DMSO controls. n = 3 independent experiments. Data represent mean± s.d. Statistical significance: ***p < 0.001; **p < 0.01; *p < 0.05 (one-way ANOVA with post-hoc correction).

[0063] Figure 11. Characterization of the senescent state. This figure shows Beta-gal staining in senescent cells highlighted in blue, increased FDG fluorescence intensity in senescent cells and enlargement in cell size in senescent cells. The conditions include A) A549 proliferating cells or treated with bleomycin; B) proliferative or irradiated A549 cells; C) U-87 MG cells treated with palbociclib to induce senescence and proliferating ones; D) SK-MEL-103 after treatment with palbociclib or proliferative ones; and E) HT1080 cells with TRF2 deletion, both untreated and treated with doxycycline.

[0064] Figure 12. Senolytic Effect of Compound 96558 at 10 pM. Comparison of cell viability between proliferative and bleomycin-induced senescent A549 cells. The left image displays crystal violet staining highlighting viability differences after treatment with 96558. On the right, relative absorbance at 570 nm is shown for the four conditions: proliferative cells with / without treatment (96558) and senescent cells with / without treatment (96558). Viability percentages after 96558 treatment are also provided. n = 3 independent experiments. Data represent mean ± s.d. Statistical significance was performed by the two-tailed Student’s t-test: ***p < 0.001.

[0065] Figure 13. Characterization of quiescence. A) Cell cycle analysis by flow cytometry using propidium iodide (PI) staining (excitation: 488 nm, emission: 690 nm). Quiescent cells are arrested in G1 phase. B) P-galactosidase staining shows positivity in senescent cells but not in quiescent cells, confirming cell cycle arrest without senescence.

[0066] Figure 14. Cell death mechanisms induced by Compound 96558 in senescent A549 cells. A) Brightfield microscopy images of proliferative A549 cells treated with DMSO (vehicle) or Compound 96558, showing no significant cytotoxicity. B) Brightfield microscopy images of senescent A549 cells treated with DMSO, Compound 96558, or Compound 96558 combined with Z-VAD-FMK (pan-caspase inhibitor), Necrostatin-1 (necroptosis inhibitor), Z-VAD- FMK + Necrostatin-1, or Ferrostatin-1 (ferroptosis inhibitor). Treatment with Ferrostatin-1 fully rescued cell viability, confirming ferroptosis as the primary mechanism of cell death induced by Compound 96558.

[0067] Detailed description of the invention The present invention is illustrated by means of the Examples set below without the intention of limiting the scope of protection.

[0068] Example 1. Material and methods

[0069] Example 1.1. Cells and Reagents

[0070] A549, SK-MEL-103 and U-87 MG cell lines were acquired from ATCC. HT1080 cell line carrying a DN-TRF2 gene under the tetracycline-controlled promoter was a kind gift from Dr Oscar Fernandez-Capetillo (CNIO, Madrid, Spain). To generate stable A549 cells expressing GFP or RFP for co-culture experiments, we used the plasmids pLJMl-EGFP and pLJM3-RFP, kindly provided by Alejo Efeyan (CNIO, Madrid, Spain). Cells were cultured in a controlled environment at 37°C with a 5% CO2 humidified atmosphere and were routinely tested for mycoplasma contamination. The culture medium consisted of DMEM high glucose (4500 mg / L) (Sigma) supplemented with 10% FBS (Corning), 1% glutamine (Sigma) and 1% penicillin / streptomycin solution (Sigma). For serum deprivation, we reduced FBS concentration to 0.05% for a minimum of 3 days.

[0071] Navitoclax (ABT-263, Abbvie) was used as a positive control for senolysis. For chemotherapy- induced senescence we used Bleomycin (Mylan Pharmaceuticals) at 20 pM for 5 days. Alternatively, we also used 5 pM Palbociclib (MedChemExpress) for 7 days. Ionizing radiation at a dose of 10 Gy was applied to induce senescence. Doxycycline (Sigma), a stable tetracycline analogue, was used as an inducer for inducible expression systems at 1 pg / mL.

[0072] Example 1.2. Crystal Violet Assay

[0073] Cell viability was tested by crystal violet staining as reported previously (Feoktistova et al. 2016). Both proliferative and senescent cells from different cell lines were plated at a concentration of 1,2 x 104 cells per well in a 96-well plate. After a 24 h incubation period, they were treated with Compound 96558 at decreasing concentrations (with DMSO as the control) and at various time points. Subsequently, cells were washed with phosphate-buffered saline (PBS, Merck) and fixed with paraformaldehyde (Electron Microscopy Science) prepared at 2% in PBS for 15 minutes, followed by a 5 min staining process using a 0.1% crystal violet solution in Milli-Q water. After staining, the excess dye was removed, washed 3 times with water, and air dried. Captured crystal violet was solubilized in 10% acetic acid and incubated on an orbital shaker at room temperature for 10 min. The absorbance was measured using a BioTek microplate reader equipped with Gen5 software. Example 1.3. IC50 Calculation

[0074] The half-maximal concentration of the Compound that shows effectiveness was calculated using cell cultures induced to senescence by different stimuli and subsequent treatment with the senolytic Compound starting at a high concentration and using serial dilutions. Cell viability was evaluated following the previously mentioned protocol of crystal violet staining. The IC50 calculations were performed using GraphPad Prism® software.

[0075] Example 1.4. Senescence-associated P-Galactosidase staining

[0076] Cells were fixed at room temperature using a solution containing 2% formaldehyde and 0.2% glutaraldehyde. After washing, they were incubated overnight at 37°C with a fresh SABG staining solution composed of 1 mg / mL of 5-bromo-4-chl oro-3 -indolyl beta-D-galactoside (X- Gal) from Fisher Scientific or 20 mM fluorescein di beta D-Galactopyranoside (FDG), along with 40 mM citric acid / sodium phosphate at pH 6.0, 5 mM K3Fe[CN]6, 5 mM K4Fe[CN]6, 150 mM NaCl, and 2 mM MgC12. Subsequent quantification using light microscopy or fluorimetry. Cell size was determined using LUNA-IITM automated cell counter (Logos Biosystems, Anyang, South Korea).

[0077] Example 1.5. Apoptosis Assay

[0078] A549 cells were resuspended into a single-cell suspension and plated at a density of 3 x 105 cells per well in six-well plates. After culturing for 24 h, cells were treated with DMSO (control), the compound, Navitoclax, or a combination of the compound or Navitoclax with the pan-caspase inhibitor Z-VAD-FMK (Santa Cruz Biotech ), Necrostatin-1 (Sigma), or Ferrostatin-1 (Sigma).. Following a 24 h incubation with the drugs, the cells were harvested by centrifugation at 3,000 rpm for 5 minutes and washed twice with PBS. Subsequently, the cells were resuspended in 500 pl of binding buffer. Then, 5 pl of Annexin V-FITC and 5 pl of 7- AAD (Elabscience) were used to detect the effect of Compound 96558 on cell apoptosis. The resulting samples were immediately analyzed by flow cytometry (FACScan, BD Biosciences), using the FITC channel (excitation: 488 nm and emission: 525 nm) and the 7-AAD channel (excitation: 488 nm and emission: 670 nm).

[0079] Example 1.6. Lipid ROS detection

[0080] Lipid ROS levels were measured using the BODIPY 581 / 591 Cl 1 kit (Fisher Scientific). Cells were seeded at a density of 3 * 105cells per well in six-well plates. After 24 hours of culture, cells were treated for an additional 24 hours with DMSO, Compound 96558, 1S,3R-RSL3 (Sigma), or a combination of these compounds with Ferrostatin-1. Then, cells were then stained with 2 pM Cl l-BODIPY (581 / 591) probe following the manufacturer’s protocol. Oxidized BODIPY was detected using excitation / emission wavelengths of 488 / 525 nm (FITC channel).

[0081] Example 2. Results

[0082] Example 2.1. Discovery of Novel Senolytic Compounds via High-Throughput Screening

[0083] We conducted an extensive search for potential senolytic compounds utilizing a library comprising 101,024 small molecules (EU-OPENSCREEN library). In this screening process, we employed the A549 human lung adenocarcinoma cell line, which had been subjected to a 5-day treatment with Bleomycin to induce a well-characterized senescent state. These senescent cells were plated in 384-well plates, and individual compounds from the library were added at a concentration of 10 pM, followed by a 24 h incubation period. Cell viability was assessed by quantifying cell numbers using Hoechst DNA staining on an Operetta system. A schematic representation of the screening strategy is presented in Figure 1.

[0084] During the primary screening, we identified 1,038 compounds displaying cytotoxic activity in senescent cells. Subsequently, for selectivity screening, we plated senescent and proliferative A549 cells in parallel and treated them with these 1,038 compounds at 10 pM in triplicate for 24 hours. This secondary screening enabled us to select 64 compounds exhibiting specific cytotoxicity in senescent cells, indicative of their potential as senolytics.

[0085] We further assessed the senolytic activity of these 64 compounds by calculating their IC50 values. Only 15 compounds demonstrated a robust and dose-dependent senolytic effect. Notably, two of these compounds (ABT-737 and A-l 155463) are known inhibitors of the BCL2 family of antiapoptotic proteins, a recognized senolytic target, thus validating the efficacy and specificity of our screening platform.

[0086] Out of these 15 compounds displaying dose-dependent senolytic activity, four exhibited significant activity. Among these four, one compound (designated as 96558 in Figure 2) had previously unknown senolytic activity and demonstrated a robust response in various cellular contexts.

[0087] Example 2.2. Validation of Compound 96558 as a Senolytic Agent in A549 Bleomycin- Induced Senescent Cells First, we sought to validate the senolytic potential of the Candidate Compound within the same cell type and under the same conditions of senescence induction as employed during the high- throughput screening. To achieve this, we used A549 cells in which senescence was induced using bleomycin (20 pM; 5 days). Subsequently, following the confirmation of the senescent status of these cells in comparison to the proliferative control, a cell viability assay was performed, employing crystal violet staining, as detailed in the Materials and Methods section.

[0088] After evaluating the senolytic activity of the Candidate Compound at the same concentration used in the screening, we conducted further tests using a range of concentrations, from 100 pM to 1.5625 pM, in A549 tumor cells. These tests were performed both in their non-treated (proliferating) state and after the induction of senescence with Bleomycin. In addition, we also assessed the drug's ability to affect cell viability at different time points (24, 48, and 72 hours) by renewing the Compound every 24 hours.

[0089] Comparing viability, we observed how Navitoclax, our positive control, acts as a senolytic at the tested concentration, since untreated cells (proliferative ones) are barely affected (with viability close to 100%). Bleomycin-induced senescent cells, on the contrary, are significantly affected.

[0090] This same effect is found with Compound 96558 at all three measured time points and specific drug concentrations. At 24, 48, and 72 hours, using low concentrations of the Compound we could observe an efficient elimination of the senescent cells while our control, proliferative ones showed intact viability.

[0091] At 24 hours, around 12.5 pM of the drug demonstrates effective cytotoxicity in senescent cells with minimal impact on proliferative cells. As time progresses, the concentration required for this favorable senolytic effect decreases. At 72 hours, a concentration of 6.25 pM proves to be the most efficient one. Nonetheless, in all cases, the senolytic effect of the Compound is confirmed.

[0092] We determined the IC50 values at different time points, revealing lower values under senescent conditions. Specifically, at the 24-hour mark, a concentration of 4.626 pM is required to induce lethality in a minimum of 50% of the senescent cells. It is noteworthy that this concentration diminishes with extended drug incubation times, resulting in an IC50 value of 2.314 pM after 72 hours. It is important to emphasize that, across all instances, the IC50 values observed for proliferative cells are consistently higher. This underscores the Compound's greater specificity in targeting senescent cells as opposed to proliferating ones. This effect is also reflected on the fact that the senolytic index, the ratio between the IC50 values on proliferating cells over senescent ones, increases with time (Figure 3, Figure 11 and Figure 12).

[0093] Example 2.3. Selectivity of Compound 96558 for senescent over quiescent cells

[0094] To determine whether Compound 96558 specifically targets senescent cells or exhibits general cytotoxicity towards non-dividing cells withdrawn from the cell cycle, we evaluated its effect on quiescent A549 cells. Quiescence was induced by serum deprivation, leading to cell cycle arrest in Gl. In this condition, we compared the cytotoxic effects of Compound 96558 on quiescent versus senescent cells using a proliferation assay like the previous one.

[0095] The results demonstrated a clear cytotoxic effect of Compound 96558 on senescent cells, with significantly less sensitivity observed in quiescent cells. Even at high concentrations of the compound (up to 40 pM), quiescent cells maintained viability despite being arrested in the cell cycle. This highlights the specificity of Compound 96558 for senescent cells over non-dividing, quiescent cells (Figure 4, Figure 13).

[0096] Example 2.4. Senolytic activity of Compound 96558 in a co-culture of proliferating and senescent cells

[0097] The next step in our evaluation was to assess the functionality of Compound 96558 in a coculture containing both proliferating and senescent cells. For this, we genetically modified A549 cells to express GFP (green fluorescence protein) or RFP (red fluorescence protein). Senescence was induced in the GFP-labelled cells by treatment with bleomycin. A co-culture was then established with a ratio of three GFP-positive senescent cells to one RFP-positive proliferating cell, as proliferating cells continue to divide during this period.

[0098] The co-culture was treated for 24 hours with DMSO, Navitoclax as a positive senolytic control, and Compound 96558 at a concentration of 10 pM. Flow cytometry analysis was used to assess the effects on both populations. In the co-culture with the vehicle, both the GFP-positive senescent cells and RFP-positive proliferating cells were observed. Upon treatment with Navitoclax, a significant reduction in the green senescent population was noted, with a corresponding enrichment of the red proliferating population. Similarly, treatment with Compound 96558 led to a marked reduction in the senescent GFP-positive cells, confirming its senolytic activity in this co-culture model (Figure 5). Example 2.5. Evaluation of Compound 96558 as a Senolytic Agent under Ionizing

[0099] Radiation-Induced Senescence

[0100] Subsequently, we aimed to assess the senolytic capability of our Compound using a senescence induction method different from the previous chemotherapy-induced approach. In this regard, we exposed A549 cells to ionizing radiation, confirmed the senescent status and conducted a similar assay as described previously. Notably, the outcomes obtained in this instance were analogous to those observed earlier, verifying the senolytic capacity in a different cellular context.

[0101] Our examination of cell viability reveals that our Compound has senolytic activity, showing a greater cytotoxic effect on senescent cells compared to their proliferating counterparts, across the 24, 48, and 72-hour treatment intervals, particularly at specific concentrations. Notably, the drug shows enhanced efficacy at approximately 12.5 pM, 6.25 pM, and 3.125 pM across the three time points. Furthermore, the IC50 curves illustrate that lower Compound concentrations are required for the eradication of senescent cells in comparison to their proliferative counterparts, a consistent observation across all examined conditions (Figure 6).

[0102] Example 2.6. Exploring Senolytic Potential of Compound 96558 in a Different Human Tumor Context

[0103] To thoroughly explore the senolytic potential of Compound 96558, we employed two different human tumor cell lines, specifically the U-87 MG glioma and SK-MEL-103 melanoma cell lines. Senescence was induced through treatment with palbociclib. This not only allowed us to test the candidate compound in different tumor cell lines but also under a different chemotherapeutic induction, increasing the range of its applicability.

[0104] Once again, we conducted a viability assay to confirm the senolytic activity in these different tumor environments. Regarding U-87 MG cells, we observed senolytic effect at certain concentrations, such as 5 pM of the compound. In the case of SK-MEL-103, a significant difference is observed between the behavior of proliferating cells and those induced into senescence with palbociclib after exposure to Compound 96558. Senescent cells are greatly affected, with an IC50 of 1.324 pM pM, while a dose of 33.06 pM is required to eliminate at least 50% of their proliferative counterparts. This results in a senolytic index of 24.97, reflecting the compound's high senolytic activity in this cellular environment. Likewise, this IC50 analysis of the glioma and the melanoma cell lines further underscores the compound’s previously proven senolytic properties (Figure 7).

[0105] Example 2.7. Assessing Compound 96558 senolytic efficacy in a cellular aging model

[0106] To assess the senolytic efficacy of the Compound in a distinct cellular context, we turned to a genetically modified cell line of fibrosarcoma, HT1080. This particular cell line was modified for the conditional deletion of the Trf2 gene, responsible for encoding the telomeric repeat binding factor 2 (Trf2). In normal circumstances, Trf2 plays a crucial role in maintaining chromosomal stability by forming specialized structures called t-loops, which shield chromosome ends from being recognized as double-strand DNA breaks by the DNA damage repair machinery (d'Adda di Fagagna et al. 2003).

[0107] The experimental knockdown of Trf2 in HT1080 cells leads to the disintegration of the t-loop structure, an event referred to as telomere uncapping. This, in turn, triggers a rapid doublestrand DNA break response, marked by p53-mediated upregulation of p21 and the induction of cellular senescence (Stansel et al. 2001). This process strikingly resembles the natural aging- related telomere shortening, which can result in critically short telomeres and subsequent uncapping (Daniali et al. 2013). Therefore, this cellular model provides valuable insights into the potential senolytic effects of the compound in a context reminiscent of cellular aging.

[0108] Similarly, we conducted an analysis of cell viability and substantiated the senolytic properties of the compound across the tested cancer cell lines, irrespective of the method employed for senescence induction or their tissue of origin. In this context, the IC50 analysis of the fibrosarcoma cell line HT1080, which was induced into senescence through the deletion of TRF2 following treatment with doxycycline, demonstrates the compound's preferential cytotoxicity towards senescent cells in comparison to proliferating ones. When these cells were exposed to Compound 96558, the senescent cells displayed a significantly higher susceptibility to the treatment, while their proliferative counterparts exhibited a comparatively reduced response (Figure 8).

[0109] Example 2.8. Cell death mechanisms induced by Compound 96558 in senescent A549 cells

[0110] To elucidate the cell death mechanism by which Compound 96558 selectively targets senescent cells, we conducted a flow cytometry assay analysing Annexin-V and 7-AAD staining. Navitoclax, a well-characterized Bcl-2 inhibitor inducing apoptosis in senescent cells as the basis for its senolytic activity, was included as a control to compare the mechanisms of action. We first confirmed that cell death was not induced in proliferative A549 cells treated with DMSO as the vehicle, Compound 96558 or Navitoclax, as most cells remained viable (FITC- Annexin V-negative and 7-AAD negative, lower left quadrant). This result highlights the selective cytotoxicity of both compounds towards senescent cells.

[0111] In senescent A549 cells, Compound 96558 induced significant cell death, with a notable shift towards late apoptosis (FITC-Annexin V-positive and 7-AAD positive, upper right quadrant). In contrast, Navitoclax treatment primarily shifted the cell population towards early apoptosis (FITC-Annexin V-positive and 7-AAD negative, lower right quadrant), though an increase in late apoptotic cells was also observed. These findings suggest that while both compounds are senolytic, their cell death mechanisms differ.

[0112] To investigate the pathways involved in Compound 96558-induced cell death, we co-treated senescent cells with specific cell death inhibitors. The pan-caspase inhibitor Z-VAD-FMK partially reversed the cytotoxic effect of Compound 96558, reducing the proportion of late apoptotic cells and increasing cell viability. However, this rescue was not complete, indicating that apoptosis alone does not fully account for the senolytic effect of Compound 96558. Similarly, treatment with Necrostatin-1, an inhibitor of necroptosis, led to a partial reduction in cell death, suggesting that necroptosis also contributes to cell death. Combining Z-VAD- FMK and Necrostatin-1 resulted in a similar reduction of cell death. However, the reversal remained incomplete, implying the involvement of additional pathways.

[0113] In contrast, treatment with Ferrostatin-1, a ferroptosis inhibitor, completely rescued the viability of senescent cells treated with Compound 96558, restoring the population to levels comparable to the DMSO control. This indicates that ferroptosis is the primary cell death pathway activated by Compound 96558 in senescent cells. Notably, Ferrostatin-1 had no protective effect against Navi tocl x-induced cell death, consistent with the established pro- apoptotic mechanism of Navitoclax. Furthermore, Z-VAD-FMK nearly completely reversed Navitoclax-induced cell death; similar results were observed with the combination of Z-VAD- FMK and Necrostatin-1. Neither Necrostatin-1 nor Ferrostatin-1 alone reversed Navitoclax- induced senolysis, further supporting apoptosis as the dominant pathway for this compound.

[0114] These results demonstrate that Compound 96558 induces cell death in senescent cells primarily through ferroptosis, with minor contributions from apoptotic and necroptotic pathways (Figure 9, Figure 14).

[0115] Example 2.9. Validation of ferroptosis as the main cell death mechanism Ferroptosis is a distinctive form of regulated cell death driven by the lethal accumulation of lipid peroxides in plasma membranes. To confirm ferroptosis as the primary mechanism underlying the senolytic effect of Compound 96558, we performed flow cytometry using the fluorescent probe B0DIPY-C11. This assay detects the oxidized form of the probe, which is indicative of lipid peroxide accumulation.

[0116] We treated senescent cells with DMSO (vehicle), Compound 96558, or 1S,3R-RSL3 (RSL3, a known ferroptosis inducer used as a positive control). Both Compound 96558 and RSL3 induced a significant increase in the oxidized form of B0DIPY-C11, confirming enhanced lipid peroxidation. This result strongly implicates ferroptosis as a key mechanism of cell death for Compound 96558.

[0117] To further validate the role of ferroptosis, we co-treated cells with Ferrostatin-1 in combination with either Compound 96558 or RSL3. Ferrostatin-1 completely reversed the increase in oxidized BODIPY-C11, restoring lipid peroxidation levels to those observed in vehicle-treated controls. This reversal corroborates that the accumulation of lipid peroxides is necessary to the cell death induced by Compound 96558 and that Ferrostatin-1 effectively prevents ferroptosis lipid damage (Figure 10).

Claims

CLAIMS1. Compound of Formula 7, or salts derived thereof,for use as a medicament.

2. Compound of Formula I, or salts derived thereof, for use, according to claim 1, as senolytic agent to selectively induce death of senescent cells.

3. Compound of Formula I, or salts derived thereof, for use, according to any of the claims 1 or 2, in a method for the treatment and / or prevention of senescence-associated diseases or disorders.

4. Compound of Formula I, or salts derived thereof, for use, according to any of the claims 1 to 3, in a method for the treatment and / or prevention of senescence-associated diseases or disorders selected from: cardiovascular disorder, metabolic disease, an inflammatory disease or disorder, a pulmonary disease or disorder, a neurological disease or disorder, a proliferative disorder, a renal disorder or disease, an eye disease or disorder, and / or a dermatological disorder or disease or cancer.

5. Compound of Formula I, or salts derived thereof, for use, according to any of the claims 1 to 4, in a method for the treatment and / or prevention of senescence-associated diseases or disorders selected from: (a) an inflammatory or autoimmune disease or disorder selected from osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, kyphosis or herniated intervertebral disc; (b) a neurological disease or disorder selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment, macular degeneration or motor neuron dysfunction; (c) a metabolic disease selected from diabetes, diabetic ulcer, metabolic syndrome or obesity; (d) a pulmonary disease selected from pulmonary fibrosis, chronic obstructivepulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis or age-related loss of pulmonary function; (e) an eye disease or disorder selected from macular degeneration, glaucoma, cataracts, presbyopia or vision loss; (f) an age-related disorder selected from renal disease, renal failure, frailty, hearing loss, muscle fatigue, skin conditions, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucosa fibrosis or sarcopenia; (g) a dermatological disease or disorder selected from eczema, psoriasis, hyperpigmentation, nevi, rashes, atopic dermatitis, urticaria, diseases and disorders related to photosensitivity or photoaging, rhytides, pruritis, dysesthesia, eczematous eruptions, eosinophilic dermatosis, reactive neutrophilic dermatosis, pemphigus, pemphigoid, immunobullous dermatosis, fibrohistocytic proliferations of skin, cutaneous lymphomas or cutaneous lupus; or (h) a cardiovascular disease selected from atherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, brain aneurysm or stroke.

6. Pharmaceutical composition comprising a compound of Formula I, or salts derived thereof,Formula I and, optionally, pharmaceutically acceptable excipients and / or carriers.

Images