Researchers publishing in the Journal of Clinical Investigation have developed a new method of screening for compounds, and they found one that appears to directly attack senescent cells involved in lung fibrosis.
A disease of senescence
Senescent cells are a major part of an age-related lung disease known as idiopathic pulmonary fibrosis (IPF) [1]. Specifically, cells positive for the senescent marker p16INK4a have been implicated, and a previous paper had suggested that targeting them could be effective [2].
As these researchers note, developing a senolytic that works in living animals is not as easy as developing one that works in cellular cultures, as a living organism has a far greater range of environments. Even in cultures, cell types and what drives them into senescence can have significant effects on what they are and how they react [3].
Therefore, these researchers previously developed INKBRITE, a genetic reporter that directly correlates fluorescence with p16INK4a in a mouse model. With this, they showed that the levels of this compound correspond to various senescent cell types in the lungs [4]. For this newest experiment, they wanted to screen for a senolytic compound that targets senescent cells that are directly taken from diseased tissues, then test that compound in living animals.
Making the target glow
Two weels after using bleomycin to induce senescence-causing injury in INKBRITE mice, the researchers extracted cells from the animals and found six subtypes that had been previously documented in fibrotic mouse lungs [5]. The researchers also found specific pathological subtypes that they did not find in their own previous study because that study had used napthalene rather than bleomycin to injure the lungs.
The researchers also conducted an experiment in which they injured the lungs of animals that did not express p16INK4a. Compared to animals that did, these model mice experienced less lung fibrosis after injury. However, before injury, the lungs of the animals were largely the same.
Therefore, they then began their next experiment: testing a library of nearly two thousand small molecules to determine which is the most effective at killing p16INK4a-positive cells while leaving cells without it alive. The fluorescent reporter of the INKBRITE mice was instrumental in determining this. Previous senolytics, most notably dasatinib, quercetin, and fisetin, did not meet the strong threshold of three standard deviations that the researchers used to screen for the best compounds.
Further experimentation with the doses of these compounds revealed three strong candidates that continued to work even at low concentrations. Testing these three against precision-cut lung slices revealed that one of them was likely to be ineffective in living organisms. In total, XSP888, an inhibitor of heat shock protein 90, was found to be the strongest candidate.
Living animals and human cells
The researchers tested XSP888 along with four other promising candidates in the lungs of bleomycin-treated INKBRITE mice. XSP888 was the only one that was found to reduce the percentage of p16INK4a cells in these animals. Fibrosis in total was also reduced, even when the senolytic combination of dasatinib and quercetin did not have any effect in this regard.
Importantly, these findings were replicated in human cells taken from people with IPF. The same senescence markers are found in people as in mice. The researchers found that XSP888 preferentially targets p16INK4a-positive human cells, reducing this marker of pathology.
However, there is no data as to whether or not XSP888 is safe and effective for treating lung fibrosis in people. Further testing, along with a clinical trial, would need to be conducted to determine if this is a drug that might one day make it to the clinic. Hopefully, this and other drugs discovered through these high-throughput screening techniques will eradicate each of the many harmful subsets of senescent cells that drive age-related diseases.
Literature
[1] Barnes, P. J., Baker, J., & Donnelly, L. E. (2019). Cellular senescence as a mechanism and target in chronic lung diseases. American journal of respiratory and critical care medicine, 200(5), 556-564.
[2] Schafer, M. J., White, T. A., Iijima, K., Haak, A. J., Ligresti, G., Atkinson, E. J., … & LeBrasseur, N. K. (2017). Cellular senescence mediates fibrotic pulmonary disease. Nature communications, 8(1), 14532.
[3] Hernandez-Segura, A., de Jong, T. V., Melov, S., Guryev, V., Campisi, J., & Demaria, M. (2017). Unmasking transcriptional heterogeneity in senescent cells. Current Biology, 27(17), 2652-2660.
[4] Reyes, N. S., Krasilnikov, M., Allen, N. C., Lee, J. Y., Hyams, B., Zhou, M., … & Peng, T. (2022). Sentinel p16 INK4a+ cells in the basement membrane form a reparative niche in the lung. Science, 378(6616), 192-201.
[5] Tsukui, T., Sun, K. H., Wetter, J. B., Wilson-Kanamori, J. R., Hazelwood, L. A., Henderson, N. C., … & Sheppard, D. (2020). Collagen-producing lung cell atlas identifies multiple subsets with distinct localization and relevance to fibrosis. Nature communications, 11(1), 1920.
