A group of scientists from China has successfully created an effective senolytic prodrug that targets the enzyme β-galactosidase . This prodrug, called SSK1, has demonstrated impressive results across various cell types and advantages over some existing senolytics.
Cellular senescence is a phenomenon in which cells stop dividing and supporting the tissues of which they are part. It is a major hallmark of aging, and it is associated with chronic inflammation, fibrosis , and other detrimental effects. Senescent cells accumulate in the body, secreting damaging chemicals and inducing senescence in nearby cells as well. Chemicals released by senescent cells, collectively known as the SASP (senescence-associated secretory phenotype), are thought to significantly contribute to aging and cancer. Senescence itself may be caused by various factors, such as telomere erosion, irradiation, and oxidative stress. The removal of senescent cells has been shown to attenuate various age-related conditions and to lengthen healthspan in mice .
Researchers have developed a class of drugs called senolytics in order to remove senescent cells. Some of them have shown potential and are currently undergoing human trials. Senolytics are supposed to cause a long-overdue death by apoptosis in senescent cells. However, according to the authors of this study, creating a senolytic that excels in both efficacy and selectivity may be challenging due to the diversity of senescence mechanisms across various cell types.
One of the characteristics shared by all senescent cells is the greatly increased production of the enzyme β-galactosidase (β-gal). β-gal is one of about 60 various enzymes found in lysosomes – cellular organelles charged with breaking down obsolete or unused biomolecules from both inside and outside the cell. It is not entirely understood why senescence causes a spike in β-gal levels, but this phenomenon presents a convenient target for intervention.
The researchers of this new study set out to create a prodrug – a compound that is therapeutically neutral until it encounters a trigger chemical in the body. In this case, the trigger is β-gal, and the prodrug is expected to be cleaved by the enzyme, releasing an apoptosis-inducing compound. After screening hundreds of FDA-approved drugs in search of such a compound, the researchers settled on gemcitabine, a known chemotherapy medication. Gemcitabine had been previously shown to effectively induce apoptosis in both human and murine cells. It also has little systemic toxicity due to its short circulation time in the body.
The resulting prodrug, SSK1, was tested first on mouse embryonic fibroblasts (MEFs), both on healthy cells and on their counterparts rendered senescent by replication. The authors report that SSK1 was found to effectively and selectively kill β-gal-positive senescent cells within a wide therapeutic window (i.e. across a wide spectrum of dosages).
To establish that the prodrug indeed targets β-gal, the researchers administered another gemcitabine-based chemical that lacked the β-gal-sensitive component. This second chemical had indiscriminately killed both senescent and non-senescent cells, as expected of an apoptosis-inducing drug. The scientists then used genetically modified cells in which GLB1 gene, the one responsible for β-gal encoding, was significantly downregulated. Senescent cells with the gene knocked down demonstrated regular levels of other senescence markers and were unharmed by SSK1. This was yet another way to demonstrate that SSK1 works by specifically targeting β-gal.
To examine the specificity of SSK1 for both murine and human cells, the authors performed a battery of additional tests. First, they used the prodrug on MEFs in which senescence had been induced by three different factors: ionizing radiation, oncogene overexpression and genotoxic stress. In all three cases, SSK1 successfully eliminated senescent cells while causing little to no damage to the healthy ones. Similar results were later recreated in mice that had artificially induced lung fibrosis. SSK1 succeeded in removing senescent cells from the affected lungs and in lowering various senescence and inflammation markers.
Other in vitro tests involved human embryonic fibroblasts (HEFs), human umbilical vein endothelial cells (HUVECs) and human preadipocytes (the precursors of mature fat cells). Yet again, SSK1 demonstrated the ability to selectively kill senescent cells and mostly spare healthy ones across various cell types and senescence stimuli.
Since some non-senescent cells also tend to express higher levels of β-gal, the researchers tested SSK1 on tissues from the two organs with the highest concentrations of such cells: the kidney and the submandibular gland. Following treatment with SSK1, only a negligible number of healthy cells underwent apoptosis, leading the researchers to conclude that the prodrug, overall, is extremely safe.
The authors also compared SSK1 to three other senolytic candidate drugs (ABT263, dasatinib plus quercetin, and fisetin) on HEFs, HUVECs, and human preadipocytes. They report that although all drugs showed senolytic abilities, SSK1 proved superior in the combined categories of potency, specificity and non-toxicity.
There was another interesting facet to this research. Macrophages are normally an important element of our immune system. However, activated macrophages that accumulate in aging tissues induce chronic inflammation  and exhibit elevated levels of β-gal, not unlike senescent cells. Building on this latter quality, the researchers tested SSK1 on macrophages and discovered that the prodrug successfully lowered the number of activated macrophages in the livers of aged mice.
Finally, upon discovering that SSK1 significantly reduces chronic inflammation locally and systemically, the researchers decided to run functional tests on aged mice treated with the novel prodrug. The tests showed improvements in motor function, balance ability, exercise endurance, skeletal muscle capacity, and spontaneous exploration compared to the untreated mice. Here, SSK1 yet again proved to be superior overall to other senolytics, even though they also demonstrated beneficial effects.
Cellular senescence is a major hallmark of aging, contributing to age-related chronic inflammation and other detrimental phenomena. Fortunately, senolytic drugs may be our best hope of dealing a serious blow to aging in the near future, and this prodrug strategy has yielded highly promising results in terms of efficacy, specificity, and non-toxicity.
 Cai, Y., Zhou, H., Zhu, Y. et al. Elimination of senescent cells by β-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice. Cell Res (2020).
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