New Dual mTORC1-mTORC2 Inhibitor Extends Lifespan & Healthspan in Mice: KU0063794

#1

I know next to nothing about this because I don’t have a copy of the paper: I’m just going off of the abstract and what has previously been reported about the drug. I have some confidence in the briefly-described result because it comes from a new paper from Vadim Gladyshev with ITP leader Richard Miller, who would probably not coauthor a paper with a BS claim of healthy and total life extension:

Here, we conducted multi-tissue RNA-seq analyses across 41 mammalian species, identifying longevity signatures and examining their relationship with transcriptomic biomarkers of aging and established lifespan-extending interventions. An integrative analysis uncovered shared longevity mechanisms within and across species, including downregulated Igf1 and upregulated mitochondrial translation genes, and unique features, such as distinct regulation of the innate immune response and cellular respiration. … lifespan-extending interventions counteracted aging patterns and affected younger, mutable genes enriched for energy metabolism. The identified biomarkers revealed longevity interventions, including KU0063794, which extended mouse lifespan and healthspan.

KU0063794 is a previously-reported investigational dual mTORC1-mTORC2 inhibitor:

In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of ∼10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation.

We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest.
https://portlandpress.com/biochemj/article/421/1/29/79701

The approved mTOR inhibitors produce clinically meaningful responses, however, the responses are short-lived and almost never curative [for renal cell carcinoma] [8]–[11]. Both temsirolimus and everolimus are rapamycin analogs that target mTORC1 but not mTORC2. Therefore, it has been argued that strategies to target mTORC1 and mTORC2 may produce better clinical responses [7]. Furthermore, it has been proposed that drug resistance develops due to compensatory activation of mTORC2 signaling during treatment with temsirolimus or everolimus [12]. This argument is supported by the observation that selective inhibition of mTORC1 can increase Akt activity by removing negative feedback loops provided by mTORC1, S6K1, and IRS1 [4].

Several synthetic small molecules have been described that inhibit both mTORC1 and mTORC2 and some are already in early phase clinical trials [7], [13]–[15]. Ku0063794 is a highly specific small-molecule inhibitor of mTOR kinase that inhibits both mTORC1 and mTORC2 [16]. Ku0063794 inhibits the phosphorylation of S6K1 and 4E-BP1, which are downstream substrates of mTORC1, and it inhibits Akt phosphorylation on Ser473, which is the target of mTORC2.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054918

[mTOR] is up‐regulated after moderate to severe traumatic brain injury (TBI). Currently, the significance of this increased signalling event for the recovery of brain function is unclear; therefore, we used two different selective inhibitors of mTOR activity to discover the functional role of mTOR inhibition in a mouse model of TBI performed by a controlled cortical impact injury (CCI).

Treatment with KU0063794, a dual mTORC1 and mTORC2 inhibitor, and with rapamycin as well‐known inhibitor of mTOR, was performed 1 and 4 hours subsequent to TBI. Results proved that mTOR inhibitors, especially KU0063794, significantly improved cognitive and motor recovery after TBI, reducing lesion volumes. Also, treatment with mTOR inhibitors ameliorated the neuroinflammation associated with TBI, showing a diminished neuronal death and astrogliosis after trauma. Our findings propose that the involvement of selective mTORC1/2 inhibitor may represent a therapeutic strategy to improve recovery after brain trauma.
https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16702

4 Likes
#2

I’ll look later on Twitter… authors of the papers usually can share a special like that gives full access to the paper…

More info

and… friends in the chemistry business can buy it :slight_smile:

See purchasing options here:

https://www.selleckchem.com/products/KU-0063794.html

#3

Someone created a thread on sci-hub forum about this paper half an hour ago.

Once someone provides the article, I’ll also create a thread to get it, I still have some points left.

3 Likes
#4

Someone from the lab or who worked on the paper almost always shares the full access paper on twitter… here it is:

3 Likes
#5

This still comes out paywalled for me; do you get the full text from that link?

#6

This twitter post has the full paper link (not sure why it doesn’t work if I copy the article link from the twitter post):

https://twitter.com/JPCastro_Aging/status/1664887049122349057

1-s2.0-S0092867423004762-mainsm.pdf (2.2 MB)

2 Likes
#7

They started treatment only at 25 months, with average lifespan around 30 months, and still got 11% median life extension and 33% remaining median life extension. Also max lifespan in the treatment group is ~150 days longer than in control. Pretty impressive! How does it compare to rapamycin?

2 Likes
#8

Below are some good snippets. The paper is currently in press, so the figures are low quality at this point. I’m sure they will be better once it’s published.

Has anyone looked into ascorbyl-palmitate? Seems to be available as a supplement.

Since chronic rapamycin treatment also leads to glucose intolerance in mice,[131] we we performed a glucose tolerance test on a separate cohort of 24-month-old male mice subjected to KU0063794 for 2 months. KU0063794 did not affect glucose tolerance in old mice as there was no difference in glucose clearance dynamics between the control and treated groups (Figures 7H and 7I).

By comparing expression profiles of treated mice with age matched control samples, we identified compound-induced ECs in murine organs and examined their association with biomarkers of aging and longevity (Table S6B). Consistent with CMap predictions, all selected compounds generated changes positively associated with an aggregated signature of at least one longevity model (Figure 7A, lower). Moreover, pro-longevity effects of KU0063794, ascorbyl-palmitate, AZD8055, and GDC0941 were supported simultaneously by aggregated biomarkers of lifespan-extending interventions (in the kidney and the liver; p adjusted < 0.004) and long-lived species (in kidney; p adjusted < 6 3 10"4 ), along with multiple individual signatures (GH deficiency, CR, maximum and median lifespan, etc.). To test if compounds that induce longevity-associated ECs extend murine lifespan and healthspan, we subjected 25-month-old C57BL/6 male mice to a diet containing a top hit from our analysis, KU0063794 (Figure 7B). KU0063794 at 10 ppm extended the remaining median and ML of old mice by 32.6% and 10.9%, respectively (log-rank test p = 0.038) (Figure 7C), with no effect on animal body weight (Figure 7D). KU0063794 also improved mouse gait speed measured at 30 months (Figure 7E). The frailty index of mice before and 5 months after the treatment initiation showed no difference between the control and experimental groups prior to drug supplementation (Figure S7A); however, mice subjected to KU0063794 were significantly less frail following the treatment (Figure 7F). Detailed analyses also revealed a KU0063794- induced improvement of coat and eye-related features (Figure S7B).

4 Likes
#9

Do we have any chemists here?

MATERIALS AND METHODS

Materials

Protein G–Sepharose and glutathione–Sepharose were purchased from Amersham Bioscience. [γ-32P]ATP was from PerkinElmer; IGF1 (insulin-like growth factor) was from Invitrogen. Tween 20, DMSO, PMA and dimethyl pimelimidate were from Sigma, and CHAPS and rapamycin were from Calbiochem. Akti-1/2, PI-103 and PD0325901-CL were synthesized by Dr Natalia Shpiro at the MRC Protein Phosphorylation Unit, University of Dundee. Ku-0063794 was synthesized at AstraZeneca. The wild-type control mLST8+/+ and mLST8−/− knockout MEFs (mouse embryonic fibroblasts) were described previously [17] and provided by David Sabatini (Whitehead Institute for Biomedical Research, Cambridge, MA, U.S.A.). The wild-type control Rictor+/+ and Rictor−/− knockout MEFs were described previously [29] and provided by Mark Magnuson (Vanderbilt University School of Medicine, Nashville, TN, U.S.A.). The wild-type control Sin1+/+ and Sin1−/− knockout MEFs were described previously [16] and provided by Bing Su (Yale University School of Medicine, New Haven, CT, U.S.A.).

#10

I’ve been taking it for a few years now… research looks positive, no obvious downsides:

Screen Shot 2023-06-07 at 1.00.29 PM
Screen Shot 2023-06-07 at 1.00.29 PM1920×658 80.6 KB

See on Amazon:
https://www.amazon.com/gp/product/B001F0R66A/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1

Also - some interesting information on the other compounds mentioned:

AZD8055

The serine/threonine kinase mammalian target of rapamycin (mTOR) is crucial for cell growth and proliferation, and is constitutively activated in primary acute myeloid leukemia (AML) cells, therefore representing a major target for drug development in this disease. We show here that the specific mTOR kinase inhibitor AZD8055 blocked mTORC1 and mTORC2 signaling in AML. Particularly, AZD8055 fully inhibited multisite eIF4E-binding protein 1 phosphorylation, subsequently blocking protein translation, which was in contrast to the effects of rapamycin. In addition, the mTORC1-dependent PI3K/Akt feedback activation was fully abrogated in AZD8055-treated AML cells. Significantly, AZD8055 decreased AML blast cell proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependent apoptosis in leukemic cells but not in normal immature CD34+ cells. Interestingly, AZD8055 strongly induced autophagy, which may be either protective or cell death inducing, depending on concentration. Finally, AZD8055 markedly increased the survival of AML transplanted mice through a significant reduction of tumor growth, without apparent toxicity. Our current results strongly suggest that AZD8055 should be tested in AML patients in clinical trials.

More here:
https://www.nature.com/articles/leu2011339

Preclinical pharmacology
AZD8055 is a potent, selective inhibitor of mTOR kinase, targeting both mTORC1 (rapamycin-sensitive) and mTORC2 (rapamycin insensitive) complexes. AZD8055 is specific against mTOR (IC50 value of 0.8 ± 0.2 nM using an immunoprecipitate of full length mTOR from HeLa cells in an ELISA-based kinase assay). AZD8055 was inactive in a counter screen against 260 kinases.

AZD8055 inhibited downstream targets of both mTORC1 (phosphorylation of S6 at serine 235/236) and mTORC2 (phosphorylation of AKT at serine 473) in several in vitro models in a dose- and time-dependent manner. Oral treatment of mice bearing U87-MG human glioma xenografts twice daily with 2.5, 5, and 10 mg/kg/day AZD8055 for 10-days resulted in a dose-dependent tumour growth inhibition of 33%, 48% and 77%, respectively. All doses were well tolerated. The growth inhibitory effect of once daily administration with 10 and 20 mg/kg of AZD8055 for 10-days was 57 and 85%, respectively. This tumour growth inhibitory effect was observed in a number of xenograft models tested in vivo. Furthermore, the antitumour activity in vivo was in good agreement with growth inhibition observed in cell lines in vitro.

More here:

https://openinnovation.astrazeneca.com/preclinical-research/preclinical-molecules/azd8055.html

GDC0941 / Pictilisib

Purpose

This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal tolerated dose (MTD), dose limiting toxicities (DLTs), pharmacokinetics, pharmacodynamics and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent and selective inhibitor of the Class I phosphatidylinositol-3-kinases (PI3K)

Results

Pictilisib was well-tolerated. The most common toxicities were grade 1-2 nausea, rash and fatigue while the DLT was grade 3 maculopapular rash (450mg, 2 of 3 patients; 330mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in platelet rich plasma at 3 hours post-dose at the MTD and in tumor at pictilisib doses associated with AUC >20uM.hr. Significant increase in plasma insulin and glucose levels, and >25% decrease in 18F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively.

Conclusion

Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100mg and signs of antitumor activity. The recommended Phase II dose was continuous dosing at 330mg once-daily.

A Phase 2 trial with another drug/combination:

Conclusion

Adding pictilisib to anastrozole significantly increases suppression of tumor cell proliferation in luminal B primary breast cancer.

Phase II Randomized Preoperative Window-of-Opportunity Study of the PI3K Inhibitor Pictilisib Plus Anastrozole Compared With Anastrozole Alone in Patients With Estrogen Receptor–Positive Breast Cancer

https://ascopubs.org/doi/10.1200/jco.2015.63.9179

Availabilty and Pricing:

3 Likes
#12

Excessive saturated free fatty acids (SFFAs; e.g. palmitate) in blood are a pathogenic factor in diabetes, obesity, cardiovascular disease and liver failure. In contrast, monounsaturated free fatty acids (e.g. oleate) prevent the toxic effect of SFFAs in various types of cells. The mechanism is poorly understood and involvement of the mTOR complex is untested. In the present study, we demonstrate that oleate preconditioning, as well as coincubation, completely prevented palmitate-induced markers of inflammatory signaling, insulin resistance and cytotoxicity in C2C12 myotubes. We then examined the effect of palmitate and/or oleate on the mammalian target of rapamycin (mTOR) signal path and whether their link is mediated by AMP-activated protein kinase (AMPK). Palmitate decreased the phosphorylation of raptor and 4E-BP1 while increasing the phosphorylation of p70S6K. Palmitate also inhibited phosphorylation of AMPK, but did not change the phosphorylated levels of mTOR or rictor. Oleate completely prevented the palmitate-induced dysregulation of mTOR components and restored pAMPK whereas alone it produced no signaling changes. To understand this more, we show activation of AMPK by metformin also prevented palmitate-induced changes in the phosphorylations of raptor and p70S6K, confirming that the mTORC1/p70S6K signaling pathway is responsive to AMPK activity. By contrast, inhibition of AMPK phosphorylation by Compound C worsened palmitate-induced changes and correspondingly blocked the protective effect of oleate. Finally, metformin modestly attenuated palmitate-induced insulin resistance and cytotoxicity, as did oleate. Our findings indicate that palmitate activates mTORC1/p70S6K signaling by AMPK inhibition and phosphorylation of raptor. Oleate reverses these effects through a metformin-like facilitation of AMPK.

From:

Palmitate activates mTOR/p70S6K through AMPK inhibition and...

Excessive saturated free fatty acids (SFFAs; e.g. palmitate) in blood are a pathogenic factor in diabetes, obesity, cardiovascular disease and liver failure. In contrast, monounsaturated free fatty acids (e.g. oleate) prevent the toxic effect of...

#13

It doesn’t seem that palmitate (palmitic acid) is the same (functionally) as Ascorbyl palmitate

Palmitates are the salts and esters of palmitic acid. The palmitate anion is the observed form of palmitic acid at physiologic pH (7.4). Palmitic acid is the most common SFA found in plants, animals, and many microorganisms.

1 Like