Senolytic Drugs: Reducing Senescent Cell Viability to Extend Health Span

Abstract

Senescence is the consequence of a signaling mechanism activated in stressed cells to prevent proliferation of cells with damage. Senescent cells (Sncs) often develop a senescence-associated secretory phenotype to prompt immune clearance, which drives chronic sterile inflammation and plays a causal role in aging and age-related diseases. Sncs accumulate with age and at anatomical sites of disease. Thus, they are regarded as a logical therapeutic target. Senotherapeutics are a new class of drugs that selectively kill Sncs (senolytics) or suppress their disease-causing phenotypes (senomorphics/senostatics). Since 2015, several senolytics went from identification to clinical trial. Preclinical data indicate that senolytics alleviate disease in numerous organs, improve physical function and resilience, and suppress all causes of mortality, even if administered to the aged. Here, we review the evidence that Sncs drive aging and disease, the approaches to identify and optimize senotherapeutics, and the current status of preclinical and clinical testing of senolytics.

Keywords: aging; senescence; senescence-associated secretory phenotype; senolytics; senomorphics.

Figure 1

Events that drive cellular senescence and events driven by senescence. Senescence can be driven by different types of cellular stress, including genotoxicity, telomere shortening, epigenetic dysregulation, oncogene activation, mitochondrial dysfunction, and metabolic and oxidative stress. This leads to signaling events that result in senescence. Senescent cells have a robust SASP, which can reinforce and spread senescence, locally and systemically, inhibiting stem cell function and disrupting tissue homeostasis while increasing sterile inflammation, termed inflammaging. Figure adapted, with permission, from the original figure by Dr. Rajesh Vyas. Abbreviation: SASP, senescence-associated secretory phenotype.

Figure 2

Characteristics of a Snc that can be exploited as biomarkers to detect and quantify senescence. Cells undergoing senescence due to damage and stress (see Figure 1) have activated signaling pathways, including the DNA damage response/ATM, GATA-4, IKK/NF-κB, JAK/STAT, and mTOR signaling pathways. There is also upregulation of the cell cycle inhibitors p53, p16^INK4a, and p21^CIP1. In addition, Sncs can show evidence of telomere shortening or damage, called TAFs; damage elsewhere in the genome, called SADFs; and epigenetic changes, called SAHFs. Sncs also have decreased levels of Lamin B1 and increased SA-βgal activity. Many Sncs have a SASP composed of chemokines, inflammatory factors and interleukins, growth factors and regulators, extracellular matrix components, soluble receptors, proteases and regulators, reactive metabolites, bioactive lipids, microRNAs, and extracellular vesicles, and early in senescence they secrete HMBG1, a key DAMP (an endogenous molecule that activates the innate immune system), which amplifies the SASP. There is no Snc-specific marker and not all Sncs express the same markers, especially in terms of the SASP. Figure adapted, with permission, from the original figure by Matthew Moore. Abbreviations: cgDNA, cytoplasmic genomic DNA; DAMP, danger-associated molecular pattern; ROS, reactive oxygen species; SA-βgal, senescence-associated β-galactosidase; SADFs, senescence-associated DNA damage foci; SAHFs, senescence-associated heterochromatin foci; SASP, senescence-associated secretory phenotype; Snc, senescent cell; TAFs, telomere-associated foci.

Figure 3

Senescent MEF-based screening assay. MEFs deficient in the DNA repair endonuclease ERCC1-XPF are used to increase physiologically relevant, oxidative stress–induced genotoxic stress and thereby senescence. Greater than 50% of these primary MEFs become SA-βgal positive by passage 5–6 at 20% O₂. These cells can then be aliquoted onto a 96- or 384-well plate for drug screening. After drug application, the total number of cells (DAPI-positive nuclei), the number of cells positive for SA-βgal (C₁₂FDG fluorescence), and cell morphology are measured in a high-content fluorescent plate reader. The use of this approach facilitates the identification of senolytic drugs, by which there is a reduction in the number of Sncs preferentially, and senomorphic drugs, by which there is no reduction in the number of cells but a reduction in SA-βgal-positive cells. It is also possible to identify drugs that increase the percent of Sncs on the plate without affecting cell number (prosenescent drugs) and drugs that increase the total number of cells on the plate (pro-proliferative drugs). This approach can address the specificity of the drugs for Sncs over non-Sncs. Subsequent assays are needed to validate the senolytic activity of the hits identified in this screen because some compounds may affect the detection of SA-βgal activity. Figure adapted, with permission, from the original figure by Dr. Rajesh Vyas. Abbreviations: C₁₂FDG, 5-dodecanoylaminofluorescein-di-β-d-galactopyranoside; DAPI, 4^′,6-diamidino-2-phenylindole; MEF, mouse embryonic fibroblast; SA-βgal, senescence-associated β-galactosidase; Snc, senescent cell.

Figure 4

Development of novel approaches to selectively kill Sncs. (a) Selectively targeting specific SCAP factors can be enhanced by PROTAC. Here, a drug that binds a SCAP is linked directly to an E3 ligase–targeting moiety to direct rapid and efficient Ub-dependent proteasomal degradation of the SCAP, rendering the Snc vulnerable to apoptosis. (b) To target a cytotoxic drug specifically to Sncs, one approach involves linking a cytotoxic agent to galactoside, which can be cleaved by lysosomal β-galactosidase, the enzyme selectively increased in Sncs (SA-βgal) to release an active toxin. (c) Optimizing senolytic activity by SAR. To optimize the activity of a senolytic, a series of analogs can be generated for testing in different Snc assays. Additional rounds of SAR can be performed to optimize senolytic activity and to improve the drug-like properties of the senolytic. Figure adapted, with permission, from the original figure by Dr. Lei Zhang and Dr. Carolina Soto Palma. Abbreviations: PROTAC, proteolysis-targeting chimera; SA-βgal, senescence-associated β-galactosidase; SAR, structure-activity relationship; SCAP, senescence-associated antiapoptotic pathway; Snc, senescent cell; Ub, ubiquitin.