Senolytics

A new wide spectrum senolytic SSK1 in development.  They imply that it works on all senescent cell types and works better than Dasatinib, Fisetin, and Quercetin.

https://www.nature.c...1422-020-0314-9

In this study, we designed a new prodrug based on the major senescence marker — the elevated β-gal — to selectively target senescent cells. This prodrug, SSK1, specifically killed both human and mouse senescent cells independent of senescent cell types and inducers. In a mouse lung injury model, SSK1 cleared stress-induced senescent cells in vivo and alleviated associated symptoms. Importantly, SSK1 also effectively reduced naturally occurring senescent cells in aged mice, decreased the senescence- and age-associated gene signatures, down-regulated SASP both locally and systemically, and restored physical functions. These results demonstrated the robustness and specificity of our prodrug in reducing senescent cells.

We successfully developed a new prodrug strategy that directed gemcitabine to kill senescent cells in a highly selective manner. The foundation of our prodrug strategy was to select a highly specific senescence marker and identify an appropriate potent drug to efficiently kill senescent cells. First, the elevated enzymatic activity of β-gal, a universal senescence biomarker both in vitro and in vivo,18,52 was utilized to direct the prodrug to specifically target senescent cells. Second, through the screening of hundreds of FDA-approved drugs, we found gemcitabine to be one of the most potent agents in killing non-dividing senescent cells (Fig. 1a; Supplementary information, Fig. S1b, c, and Table S1). In addition, gemcitabine is a widely used FDA-approved drug with proven safety and short plasma circulation time.25,26 Our designed prodrug SSK1 is activated to release gemcitabine selectively in senescent cells (Fig. 1c), following which senescent cells were effectively eliminated (Fig. 1d). Our further study showed that gemcitabine activates p38 to induce apoptosis in non-dividing senescent cell (Supplementary information, Fig. S2a, b). As a nucleoside analog, gemcitabine could kill senescent cells through inducing mitochondrial DNA damage (Supplementary information, Fig. S2d), similar to a reported nucleoside analog, Ganciclovir.33 These results demonstrated that prodrug SSK1 is superior to current reported senolytics in terms of design strategy and specificity.

Cellular senescence is a highly heterogeneous process due to the different cell origins and stimuli,6,53 whereas the key feature of SSK1 is the ability to efficiently clear senescent cells with a broad spectrum of cell types and senescence inducers, including replication, irradiation, oncogene and genotoxic stress (Fig. 2a, c). In this study, we compared SSK1 with other reported senolytics on HEFs, human preadipocytes and HUVECs, which were used to test senolytics. ABT263 (a classical anti-apoptosis BCL-2 inhibitor) eliminated senescent HEFs and HUVECs, but showed little effect on human preadipocytes (Supplementary information, Fig. S3b, f, j), which was also reported by other previous studies.12,13 The combination of dasatinib (a pan-tyrosine kinase inhibitor) and quercetin (a plant flavonoid) killed all three types of senescent cells in a dose-dependent manner (Supplementary information, Fig. S3c, g, k), but had a high toxic effect on non-senescent cells in consistence with others’ results.11,16,36 Another natural flavonoid fisetin, reported as a potential senotherapeutic agent,15 showed modest effect on eliminating senescent HEFs and preadipocytes even at higher concentrations (Supplementary information, Fig. S3d, h) which was comparable to other’s results.54 Most importantly, SSK1 could overcome these limitations, including cell-type dependency, high toxicity on non-senescent cells, and low efficiency on senescent cells (Supplementary information, Fig. S3a, e, i). Therefore, SSK1 possessed a better senolytic activity regarding of specificity and efficiency on a wider range of cell types, demonstrating the superiority of β-gal-based prodrug strategy to target senescence.

Moreover, we found SSK1 exerted its elimination effect on senescent cells in vivo. SSK1 eliminated stress-induced senescent cells effectively and decreased different senescence markers in bleomycin-induced lung injury model, highlighting its effectiveness in vivo (Fig. 3). SSK1 treatment also relieved lung fibrosis (Fig. 3d; Supplementary information, Fig. S4c, d) and attenuated the impaired physical function as tested by treadmill assay (Supplementary information, Fig. S4f). In this study, we also treated naturally aged mice with SSK1 and tested its effects on senescent cells, chronic inflammation and physical function. First, SSK1 could remove senescent cells in multiple tissues and decrease the senescence-associated signatures as shown by the GSEA analysis (Fig. 4b–g). Second, SSK1 could decrease the expression of SASP-associated genes in aged livers and kidneys and reduce chronic low-grade inflammation in the blood (Fig. 5a, b, e–h). Third, SSK1 ameliorated the impaired motor function, balance, exhausted exercise, muscle strength, and spontaneous exploration in aged mice (Fig. 6a–e). Most importantly, the performance of rotarod and beam balance in the SSK1-treated group was improved compared with that in the initial pretreatment condition (Supplementary information, Fig. S8e, f). Collectively, our prodrug SSK1 targeting β-gal-positive cells exerted very significant biological effects in naturally aged mice.

While SA-β-gal is widely used as a marker of cellular senescence,20,21,22 its elevated activity can be found in some other cells such as activated macrophages.48,55 These SA-β-gal-positive macrophages can be harmful and have been found to accumulate in injured and aged tissues contributing to chronic inflammation.44,45 Importantly, we have shown that SSK1 decreases the number of SA-β-gal-positive macrophages in injured lungs and aged livers (Supplementary information, Fig. S6g–j), which is consistent with our observation of reduced secretion of chronic inflammation-related cytokines. Therefore, eliminating macrophage accumulation by SSK1 might reduce chronic inflammation and benefit aged organisms. In addition, activated macrophages play crucial roles in acute inflammation and cytokine storm,56 especially those induced by virus infection. During virus-induced acute inflammation, macrophages produce pro-inflammatory factors and trigger initiation of cytokine storms.57 For instance, the depletion of macrophages could protect mice from coronavirus-induced lethal infection.58 Accordingly, activated macrophages could be potential targets for treating acute inflammation and cytokine storms via SSK1. The future potential for SSK1 in treatment of acute inflammation, injury, and age-induced chronic inflammation is promising.

An understanding of the toxicological effects of SSK1 in vivo is critical to clinical applications. Importantly, our data showed that high concentration (100 mg/kg) and high frequency SSK1 treatment had no apparent systemic toxicities (Supplementary information, Fig. S9). This provides strong evidence for the in vivo safety of SSK1. This safety profile is further supported by comparison to gemcitabine, an SSK1 effector and approved clinical drug. First, our effective in vivo dosage of SSK1 was approximately 60-fold lower than the clinical dosage of gemcitabine (30 mg/kg) and implies a greatly reduced risk of in vivo toxicity of SSK1.24 Second, gemcitabine is a nucleoside analog that potently affects rapidly dividing cells and the off-target effects of gemcitabine could be shown in different proliferating cell types.59,60 SSK1, however, targets only β-gal-positive cells. Notably, in addition to macrophages, we found that the majority of non-senescent β-gal-positive cells are rapidly dividing epithelial cells (Supplementary information, Fig. S10a, b). As a result, the potential types of proliferating cells with SSK1 sensitivity are greatly narrowed relative to gemcitabine. Even though small numbers of β-gal-positive epithelial cells are targeted by SSK1 treatment, these cells have robust self-renewal ability.61 Consistent with this self-renewal property, we found that epithelial tubular cells in the kidney with elevated β-gal activity had a high percentage of Ki67-positive cells (Supplementary information, Fig. S10c). Following SSK1 treatment, we observed only a few cells of this tubular cell population undergoing apoptosis (Supplementary information, Fig. S10d), and such low-level impairment could be easily repaired by the rapid self-renewal of tubular cells during SSK1 treatment. Moreover, the short duration of SSK1 treatment might avoid significant impairment in related tissues and further minimize its side effects. In summary, our study demonstrates the superiority and safety of this prodrug strategy by targeting β-gal to selectively remove senescent cells of multiple cell types. These findings open a new avenue for the treatment of age-associated diseases and provide a clinical opportunity for intervention into the aging process.