One-liner: ETTA is an early-stage biotechnology company developing AI/ML-designed novel molecules for simultaneously targeting senescent cells, mTOR, and insulin signaling to extend lifespan and treat serious diseases, such as chronic kidney disease.
Project PI: Kyle Brewer
Longevity Dealflow WG team
- Scientific evaluation: to be done
- Business evaluation: to be done
- Shepherd: Paolo Binetti
- Squad members: Tuan Dinh, Laurence Ion, Vivian Qu, Derek Underwood
- Sourced by: Lutz Kummer
ETTA Biotechnology is using its proprietary artificial intelligence (AI) and machine learning (ML) platform to generate novel molecules to extend healthy mammalian lifespan and demonstrate treatment of chronic kidney disease in clinical trials. ETTA has a lead novel compound in hand verified to target senescent kidney cells, without toxicity to normal cells. ETTA has also discovered a FDA approved drug that can clear senescent kidney cells, which will be improved with the AI/ML platform due to some apparent toxicity to normal cells.
37 million people in the US with chronic kidney disease will eventually die as a result of disease progression, caused by the build up of senescent cells in their kidneys during aging. Without a transplant, chronic kidney disease leads to severe illness and death.
During aging, the kidney builds up senescent cells, leading to chronic inflammation, causing the kidneys to begin to perform poorly. As excess damage occurs from inflammation and senescent cells, fibrosis occurs in the kidney, which further exacerbates the decline of renal function. The anti-cancer drug ABT-263 (Navitoclax) was shown to clear senescent cells for a chronic kidney disease model in aged mice, leading to improved kidney function, reduced kidney fibrosis, and even regeneration of the kidney through cellular proliferation . In addition, dasatinib plus quercetin has been shown to limit renal fibrosis . However, many senolytics such as ABT-263 and dasatinib cannot be used safely in older, frail populations due to toxic side effects. Also, patients with chronic kidney disease do not deal with toxin challenges well, making the use of ABT-263, dasatinib, and similar drugs even more of a problem.
ETTA’s novel small molecule drugs act by a mechanism targeting inflammatory (mTOR) and insulin signaling (PI3K/AKT) pathways, allowing senescent cells, normally resistant to apoptosis, to undergo apoptosis. Clearing senescent cells in the kidney enables resumption of proper renal function and even kidney regeneration in aged mice . In recent years, natural products, such as fisetin, have been discovered that can simultaneously clear senescent cells, reduce inflammation, inhibit insulin signaling, and extend mammalian lifespan. Furthermore, many of these compounds are present in low concentrations in common fruits and vegetables, offering great promise for safe treatments targeting kidney disease3 during aging that are also safe enough for millions of people to use regularly. However, these natural molecules have poor solubility, bioavailability, and half-life, which is the main hurdle to implementing these discoveries as therapeutics.
For preclinical development, ETTA has created a proprietary AI/ML platform, which combines currently available data from natural molecule-based senolytics to recognize and predict the most effective senotherapeutics in silico. In addition, 15 additional criteria related to safety, bioavailability, and metabolism are applied to choose the best possible drugs to maximize clinical success probability from the outset. ETTA has generated nearly 2000 proprietary small molecules that are predicted to outperform natural molecule senolytics.
After synthesizing our first candidates from the platform, we induced senescence in normal kidney cells in culture. We then incubated the cells with each compound for 48 hours. Of our first lead candidate molecules, we found ETTA1 can clear senescent cells (Fig. 1), while the total number of cells was unaffected. We found fisetin can also clear senescent cells, as published previously by our advisor Matt Yousefzedah , albeit not to the same extent as previously published, likely due to kidney cells being used here, while mouse embryonic fibroblasts and IMR90 fibroblasts were used in Dr. Yousefzedah’s publication. In addition, fisetin appears to not have the same selectivity or efficacy as ETTA1, which demonstrates the power of our platform.
ETTA2 and ETTA3 only trended toward lower senescence, likely because our machine learning training dataset currently contains only 108 molecules, so our results expectedly show prediction of senescent cell targeting-small molecules is imperfect, but practical. We expect to have even more accurate predictions as we increase the number of molecules we discover to deepen the training dataset. Noteworthy is that ETTA2 and ETTA3 do not affect total cell number, likely owing to the fact that safety is much easier to predict with AI/ML approaches due to thousands of small molecules in existence that have some safety and/or toxicity data.
As our platform is based on natural molecules, which have had success in safety in the clinic , while other senolytics have had safety problems [6, 7], we expect our platform to outperform competitors for patient safety. In addition, drugs are filtered out of the body by the kidney, so we can expect to use lower concentrations of our molecules compared to competitors going after other organ targets. The kidney acting as a drug filter also allows us to more specifically target the kidney compared to competitors focusing on other organ targets. We expect other tissues or organs to be exposed to the drug, as with any small molecule. This fact can also be beneficial because reducing senescent cells in other organs will decrease the inflammatory cytokines circulating in the blood that may also impair kidney function. As our approach and platform are designed to excel in safety, these off-target senolytic effects are expected to help, not hinder our efforts.
To further expand our pipeline, we looked for candidates that inhibited both mTOR and PI3K/AKT pathways like fisetin does, which is the hypothesized mechanism of action to clear senescent cells. We discovered an FDA approved small molecule drug that inhibits these pathways can indeed clear senescent cells (Fig. 1). This discovery is important as it shows FDA approval is a strong possibility. However, total cell number may be affected (p=0.058), so we will use our AI/ML platform to make novel improvements for this drug.
Fig. 1 | Evaluation of lead candidates to clear senescent cells
Senescence was induced in normal kidney cells, then cells were incubated with each novel compound or fisetin at 10 µM concentration, or with DMSO vehicle only. Cells were stained with C12FDG to identify SA-β-gal+ cells, then Hoechst, as previously described . Results were compared for n=4-6 independent wells, evaluated using unpaired Student’s t-test relative to the DMSO control, and plotted as the mean±SEM. *p<0.05, **p<0.01
Chronic kidney disease is a $95 billion dollar market and affects 1 in 7 people. The risk of death from chronic kidney disease exponentially increases with increased age, as with other major age-related diseases. Despite this opportunity, the only treatments available are for controlling blood pressure and diabetes to manage kidney decline. In absence of a transplant, dialysis is the only way to filter blood due to the failure of the kidney to function normally. Decrease in inflammation and scar formation, both of which are important to treat chronic kidney disease, appeared to be the main physiological outcomes in our previous work with fisetin nanoparticles, which is why we prioritize this disease. ETTA’s treatments would be the first drugs that target the underlying cause of chronic kidney disease and would likely be taken by all patients.
Other oral senolytics have severe safety problems, such as navitoclax (ABT-263) having testicular toxicity, lymphopenia, and thrombocytopenia6, and dasatanib having side effects of pleural effusion (“water on the lungs”) and pulmonary arterial hypertension . In addition, companies such as Unity Biotechnology chose to do local injections due to the toxicity of their compounds. Fisetin, on the other hand, has shown no severe or serious side-effects in 60 subjects during ongoing clinical trials . Our compounds are AI/ML-designed derivatives of natural products such as fisetin, and at least one of our first lead candidates appears to have better senescent cell targeting than fisetin (Fig. 1). Other competitors in this area include Oisin Biotechnologies, Rubedo Life Sciences, and Cleara Biotech, but we cannot evaluate the safety of these approaches as they do not yet have relevant data from clinical studies in humans.
Relevance to longevity
ML/AI designed molecules from ETTA’s platform likely target 3 key features of aging simultaneously:
- Senescence by clearing senescent cells.
- Insulin signaling by inhibiting AKT in the insulin signaling pathway.
- Inflammation by inhibiting mTOR (the target of rapamycin).
Clearing senescent cells can extend lifespan by 17-35% in mice when treated throughout life . Fisetin, used in our ML training data set along with closely related natural molecules, was previously shown to clear senescent cells and extend lifespan by ~10% when given to aged 22-24-month-old mice . Likewise, insulin signaling inhibition with caloric restriction and mTOR inhibition with rapamycin can extend lifespan by 10-35%  and 23-26% , respectively, when mice are treated throughout life. Fisetin has also been shown to be a dual inhibitor of the insulin signaling (PI3K/AKT) and mTOR signaling pathways .
Two patents filed
- Patent 1: Oral Fisetin Nanoparticles
- Patent 2: Topical Fisetin Nanoparticles
- AI/ML designed novel molecules to clear senescent cells and treat kidney disease, inflammation, fibrosis, and aging.
Tissue Culture Screening and Lead Candidate Determination
ETTA will synthesize additional top novel small molecule candidates from our AI/ML platform based on predicted efficacy, safety, bioavailability, metabolism, and chemical diversity. We will test these novel compounds applied to senescent kidney cells and normal kidney cells over the course of 3 days. Molecules that clear senescent cells with a p-value <0.05 (senescent cells compared to normal cells) will be considered appropriate for further testing to narrow down to a lead candidate. These molecules will then be tested at different concentrations to determine the half maximal effective concentration (EC50) for both clearing senescent cells and normal cells. Novel molecules with the lowest EC50 for senescent cell clearance and the highest ratio of EC50 for normal cells to EC50 for senescent cells will be tested in vivo.
Lifespan Study in C. elegans
We will apply our novel lead small molecules to C. elegans to determine any effect on lifespan. As molecules in our machine learning dataset also feature mTOR inhibition and insulin signaling inhibition beyond just clearing senescent cells, C. elegans screening may determine effects of our novel compounds on these pathways, which are conserved from worms to humans. Any hits on lifespan extension will be repeated in a daf-2 background to elucidate if the lifespan extension is due to insulin signaling inhibition or another effect.
Kidney Disease Model in Aged Mice
Based on ETTA’s previous work with fisetin nanoparticles, we have chosen kidney disease as a preferred indication to examine in a preclinical model. We will use the unilateral renal ischemia-reperfusion model of kidney injury in 22-month-old mice as described previously for ABT-263 senolytic treatment1. The mice will be treated by oral gavage with water (negative control) or our top lead novel molecule from our AI/ML platform. Kidney function will be determined by blood urea nitrogen and cystatin C levels in serum. Serum cytokines will also be examined to determine inflammatory markers. Senescent cell burden will be determined by p21 staining. Fibrosis will be assessed by picrosirius red staining in kidney tissues slices, and renal cell proliferation will be determined by Ki67 staining. Clinical observations will also be made for the mice (body weight, appearance, and health evaluation scoring) to determine any overt physical effects of the treatment, which may also correlate with effects on physical aging.
- Medicinal chemistry: $25,000
- In vitro screening: $5,000
- In vivo lifespan in C. elegans: $5,000
- In vivo chronic kidney disease model in aged mice: $35,000
- Patent filing: $20,000
- Overhead (10%): $10,000
- Total: $100,000
Financing and VitaDAO funding terms
ETTA has raised $100K pre-seed funding and is now raising a $1.5M seed round using the recently obtained data with the first compounds we have discovered. ETTA also has soft commitments of $100-200K from other investors, as well as interest from additional investors.
VitaDAO would fund $100K under Molecule’s sponsored development agreement terms for the line items in the budget. The seed round funds will be used for further determination of the optimal drug formulation, drug dose, and drug safety in mice, as well as further development of the platform and additional lead molecules, such as the FDA approved drug candidate we have discovered to clear senescent kidney cells.
Kyle founded ETTA Biotechnology to create the most effective treatments targeting aging currently possible. The first therapies he designed have the potential to extend healthy human lifespan by 10-30 years if preclinical mouse studies apply similarly to humans. Kyle has raised $100,000 for ETTA Biotechnology thus far.
Kyle is a scientist and entrepreneur with >15 years of expertise in aging and biotechnology, with a special focus on delivery of small molecules, DNA, mRNA, and proteins to extend lifespan. Kyle was the second scientist at Rejuvenation Technologies, a Stanford startup out of Helen Blau’s lab dedicated to using mRNA nanoparticles to extend telomeres, where he learned how to run and manage an early biopharma startup.
Kyle researched synaptic vesicle exocytosis in collaboration with Nobel Prize winner Thomas Südhof during his PhD in biophysics at the University of Texas Southwestern. He also investigated how circulating proteins in the bloodstream can influence the aging brain during his postdoc at Stanford in the lab of Tony Wyss-Coray, collaborating with Nobel Prize winner Carolyn Bertozzi.
Lurong recently joined as ETTA co-founder. Lurong is an expert in using ML and AI to generate novel molecules that outperform existing real world molecules. She founded Ainnocence, a company geared towards this goal that develops small molecules and antibodies using an AI approach. She was also the Director of Computation Science at UAB. In addition, Lurong is a Senior Investigator/Advisor to the Global Health Drug Discovery Institute, a Gates Foundation funded effort to advance pharmaceutical research and translational medicine.
- Matt Yousefzadeh. Assistant Professor, University of Minnesota, discovered the natural molecule fisetin can clear senescent cells and can extend lifespan.
- Sam Roosz. Chief Executive Officer, Crescendo Health, founder of Datavant, a unicorn company with focus on clinical data analytics. MBA from Stanford Business School.
- Colin Maraganore. Co-Founder and Principal of Abeja Ventures. Venture Fellow at Healthspan Capital. Early Stage Biotechnology Operations and Strategy Specialist. Previously the Head Of Business Development at Rejuvenation Technologies, Scientist at BioAGE, and Senior Research Associate at Vium.
- Mylonas, K. J., O’Sullivan, E. D., Humphries, D., Baird, D. P., Docherty, M.-H., Neely, S. A., Krimpenfort, P. J., Melk, A., Schmitt, R., Ferreira-Gonzalez, S., Forbes, S. J., Hughes, J., & Ferenbach, D. A. (2021). Cellular senescence inhibits renal regeneration after injury in mice, with senolytic treatment promoting repair. In Science Translational Medicine (Vol. 13, Issue 594). American Association for the Advancement of Science (AAAS). https://doi.org/10.1126/scitranslmed.abb0203 - The senolytic navitoclax (ABT-263) restored kidney function and even led to renal regeneration in normal, aged mice.
- Li, C., Shen, Y., Huang, L., Liu, C., & Wang, J. (2020). Senolytic therapy ameliorates renal fibrosis postacute kidney injury by alleviating renal senescence. In The FASEB Journal (Vol. 35, Issue 1). Wiley. https://doi.org/10.1096/fj.202001855rr - The senolytic cocktail of dasatinib plus quercetin limits kidney fibrosis (scarring).
- Ren, Q., Guo, F., Tao, S., Huang, R., Ma, L., & Fu, P. (2020). Flavonoid fisetin alleviates kidney inflammation and apoptosis via inhibiting Src-mediated NF-κB p65 and MAPK signaling pathways in septic AKI mice. In Biomedicine & Pharmacotherapy (Vol. 122, p. 109772). Elsevier BV. https://doi.org/10.1016/j.biopha.2019.109772 - Fisetin limits renal inflammation and apoptosis in a common kidney injury model.
- Yousefzadeh, M. J., Zhu, Y., McGowan, S. J., Angelini, L., Fuhrmann-Stroissnigg, H., Xu, M., Ling, Y. Y., Melos, K. I., Pirtskhalava, T., Inman, C. L., McGuckian, C., Wade, E. A., Kato, J. I., Grassi, D., Wentworth, M., Burd, C. E., Arriaga, E. A., Ladiges, W. L., Tchkonia, T., … Niedernhofer, L. J. (2018). Fisetin is a senotherapeutic that extends health and lifespan. In EBioMedicine (Vol. 36, pp. 18–28). Elsevier BV. https://doi.org/10.1016/j.ebiom.2018.09.015 - The natural molecule fisetin can clear senescent cells and extend the healthy lifespan of normal, 22-24-month-old mice.
- Verdoorn, B. P., Evans, T. K., Hanson, G. J., Zhu, Y., Langhi Prata, L. G. P., Pignolo, R. J., Atkinson, E. J., Wissler‐Gerdes, E. O., Kuchel, G. A., Mannick, J. B., Kritchevsky, S. B., Khosla, S., Rizza, S. A., Walston, J. D., Musi, N., Lipsitz, L. A., Kiel, D. P., Yung, R., LeBrasseur, N. K., … Kirkland, J. L. (2021). Fisetin for COVID‐19 in skilled nursing facilities: Senolytic trials in the COVID era. In Journal of the American Geriatrics Society (Vol. 69, Issue 11, pp. 3023–3033). Wiley. https://doi.org/10.1111/jgs.17416 - Fisetin has shown no severe or serious side-effects in 60 subjects during ongoing clinical trials.
- Xiong, H., Pradhan, R. S., Nada, A., Krivoshik, A. P., Holen, K. D., Rhodes, J. W., … Awni, W. M. (2014). Studying navitoclax, a targeted anticancer drug, in healthy volunteers–ethical considerations and risk/benefit assessments and management. Anticancer Research, 34(7), 3739–3746. https://ar.iiarjournals.org/content/34/7/3739.long - The senolytic navitoclax (ABT-263) has testicular toxicity, lymphopenia, and thrombocytopenia.
- Nekoukar, Z., Moghimi, M., & Salehifar, E. (2021). A narrative review on adverse effects of dasatinib with a focus on pharmacotherapy of dasatinib-induced pulmonary toxicities. In BLOOD RESEARCH (Vol. 56, Issue 4, pp. 229–242). The Korean Society of Hematology. https://doi.org/10.5045/br.2021.2021117 - The senolytic dasatinib has pleural effusion (“water on the lungs”) and pulmonary arterial hypertension as harmful side-effects.
- Baker, D. J., Childs, B. G., Durik, M., Wijers, M. E., Sieben, C. J., Zhong, J., A. Saltness, R., Jeganathan, K. B., Verzosa, G. C., Pezeshki, A., Khazaie, K., Miller, J. D., & van Deursen, J. M. (2016). Naturally occurring p16Ink4a-positive cells shorten healthy lifespan. In Nature (Vol. 530, Issue 7589, pp. 184–189). Springer Science and Business Media LLC. Naturally occurring p16Ink4a-positive cells shorten healthy lifespan | Nature - Clearing senescent cells extends lifespan of normal mice by 17-35%.
- Acosta-Rodríguez, V., Rijo-Ferreira, F., Izumo, M., Xu, P., Wight-Carter, M., Green, C. B., & Takahashi, J. S. (2022). Circadian alignment of early onset caloric restriction promotes longevity in male C57BL/6J mice. In Science (Vol. 376, Issue 6598, pp. 1192–1202). American Association for the Advancement of Science (AAAS). https://doi.org/10.1126/science.abk0297 - Insulin signaling inhibition by caloric restriction increases lifespan in mice by 10-35%
- Miller, R. A., Harrison, D. E., Astle, C. M., Fernandez, E., Flurkey, K., Han, M., Javors, M. A., Li, X., Nadon, N. L., Nelson, J. F., Pletcher, S., Salmon, A. B., Sharp, Z. D., Van Roekel, S., Winkleman, L., & Strong, R. (2014). Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. In Aging Cell (Vol. 13, Issue 3, pp. 468–477). Wiley. https://doi.org/10.1111/acel.12194 - Rapamycin inhibition of mTOR can increase lifespan in mice by 23-26%.
- Adhami, V. M., Syed, D. N., Khan, N., & Mukhtar, H. (2012). Dietary flavonoid fisetin: A novel dual inhibitor of PI3K/Akt and mTOR for prostate cancer management. In Biochemical Pharmacology (Vol. 84, Issue 10, pp. 1277–1281). Elsevier BV. https://doi.org/10.1016/j.bcp.2012.07.012 - Fisetin is a dual insulin signaling and mTOR inhibitor.
The slide deck is at this link.
- ML/AI designed molecules may target 3 key features of aging: 1. Cell senescence, 2. Insulin signaling, 3. Inflammation.
- Many of the molecules used in the machine learning training data set have been used safely in humans already.
- The ML/AI platform predicts key features important for successful drug development including efficacy, safety, metabolism, and bioavailability.
- A novel molecule generated by the ML/AI platform has been validated yet to clear senescent kidney cells in vitro.
- Clear clinical target
- The novel molecules generated by the ML/AI platform have not been validated yet to treat an in vivo mouse model of disease or aging (this goal is part of this proposal).
- Traditional pharma approach of “one disease, one target, one drug” is different from this approach, which is why targeting senescence, insulin signaling, and mTOR simultaneously is possible.
- Competition from other longevity biotech companies also going for senolytics
- Early stage: need to build the team further.
- Revisions Requested (Detail in Comments)