The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1−/Δ mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1−/Δ mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

BackgroundSenescence is a tumor suppressor mechanism activated in stressed cells to prevent replication of damaged DNA. Senescent cells have been demonstrated to play a causal role in driving aging and age-related diseases using genetic and pharmacologic approaches. We previously demonstrated that the combination of dasatinib and the flavonoid quercetin is a potent senolytic improving numerous age-related conditions including frailty, osteoporosis and cardiovascular disease. The goal of this study was to identify flavonoids with more potent senolytic activity.MethodsA panel of flavonoid polyphenols was screened for senolytic activity using senescent murine and human fibroblasts, driven by oxidative and genotoxic stress, respectively. The top senotherapeutic flavonoid was tested in mice modeling a progeroid syndrome carrying a p16INK4a-luciferase reporter and aged wild-type mice to determine the effects of fisetin on senescence markers, age-related histopathology, disease markers, health span and lifespan. Human adipose tissue explants were used to determine if results translated.FindingsOf the 10 flavonoids tested, fisetin was the most potent senolytic. Acute or intermittent treatment of progeroid and old mice with fisetin reduced senescence markers in multiple tissues, consistent with a hit-and-run senolytic mechanism. Fisetin reduced senescence in a subset of cells in murine and human adipose tissue, demonstrating cell-type specificity. Administration of fisetin to wild-type mice late in life restored tissue homeostasis, reduced age-related pathology, and extended median and maximum lifespan.InterpretationThe natural product fisetin has senotherapeutic activity in mice and in human tissues. Late life intervention was sufficient to yield a potent health benefit. These characteristics suggest the feasibility to translation to human clinical studies.FundNIH grants P01 AG043376 (PDR, LJN), U19 AG056278 (PDR, LJN, WLL), R24 AG047115 (WLL), R37 AG013925 (JLK), R21 AG047984 (JLK), P30 DK050456 (Adipocyte Subcore, JLK), a Glenn Foundation/ (AFAR) BIG Award (JLK), Glenn/AFAR (LJN, CEB), the Ted Nash Long Life and Noaber Foundations (JLK), the Connor Group (JLK), Robert J. and Theresa W. Ryan (JLK), and a Minnesota Partnership Grant (AMAY-UMN#99)-P004610401–1 (JLK, EAA).

Aging is the main risk factor for many chronic degenerative diseases and cancer. Increased senescent cell burden in various tissues is a major contributor to aging and age-related diseases. Recently, a new class of drugs termed senolytics were demonstrated to extending healthspan, reducing frailty and improving stem cell function in multiple murine models of aging. To identify novel and more optimal senotherapeutic drugs and combinations, we established a senescence associated β-galactosidase assay as a screening platform to rapidly identify drugs that specifically affect senescent cells. We used primary Ercc1 −/− murine embryonic fibroblasts with reduced DNA repair capacity, which senesce rapidly if grown at atmospheric oxygen. This platform was used to screen a small library of compounds that regulate autophagy, identifying two inhibitors of the HSP90 chaperone family as having significant senolytic activity in mouse and human cells. Treatment of Ercc1 −/∆ mice, a mouse model of a human progeroid syndrome, with the HSP90 inhibitor 17-DMAG extended healthspan, delayed the onset of several age-related symptoms and reduced p16INK4a expression. These results demonstrate the utility of our screening platform to identify senotherapeutic agents as well as identified HSP90 inhibitors as a promising new class of senolytic drugs.

Detailed knowledge of lithospheric structure is essential for understanding the long-term evolution and dynamics of continents. We present an image of lithospheric structure across the central and western North China Craton (NCC), derived using S and P receiver functions from a dense seismic array. A negative velocity discontinuity is identifi ed at ~80-100 km depth within the thick lithosphere (~160-200 km), similar to that observed in many other cratonic regions and roughly at the same depth as the base of the lithosphere in the eastern NCC. The intralithospheric discontinuity may indicate an ancient, mechanically weak layer within the overall strong cratonic lithosphere, and probably also existed beneath the eastern NCC before the Mesozoic. The presence of such a weak layer could have facilitated simultaneous lithospheric modifi cation at the base and the middle of the lithosphere in the eastern NCC, especially under the strong infl uence of the Mesozoic Pacifi c subduction, leading to the severe lithospheric thinning and destruction recorded in this region. The weak layer probably did not strongly affect the stability and evolution of the central and western NCC and other cratonic regions where effects from plate boundary processes were weak. Our seismic images, integrated with geological data, provide new insights into structural heterogeneities in the subcontinental lithospheric mantle and their roles in the dynamic evolution of continents.

Given the significant body of data supporting an essential role for c-jun-N-terminal kinase (JNK) in neurodegenerative disorders, we set out to develop highly selective JNK inhibitors, with good cell potency, and good brain penetration properties. The structure activity relationships (SAR) around a series of aminopyrimidines was evaluated utilizing biochemical and cell- based assays to measure JNK inhibition, and brain penetration in mice. Microsomal stability in three species, P450 inhibition, inhibition of generation of reactive oxygen species (ROS), and pharmacokinetics in rats were also measured. Compounds 9g, 9i, 9j, and 9l had greater than 135-fold selectivity over p38, and cell-based IC50 values < 100 nM. Moreover, compound 9l showed an IC50= 0.8 nM for inhibition of ROS and had good pharmacokinetic properties in rat, along with a brain-to-plasma ratio of 0.75. These results suggest that biaryl substituted aminopyrimidines represented by compound 9l may serve as the first small molecule inhibitors to test efficacy of JNK inhibitors in neurodegenerative disorders.

c-Jun N-terminal kinase 3␣1 (JNK3␣1) is a mitogen-activated protein kinase family member expressed primarily in the brain that phosphorylates protein transcription factors, including c-Jun and activating transcription factor-2 (ATF-2) upon activation by a variety of stress-based stimuli. In this study, we set out to design JNK3-selective inhibitors that had >1000-fold selectivity over p38, another closely related mitogen-activated protein kinase family member. To do this we employed traditional medicinal chemistry principles coupled with structurebased drug design. Inhibitors from the aminopyrazole class, such as SR-3576, were found to be very potent JNK3 inhibitors (IC 50 ‫؍‬ 7 nM) with >2800-fold selectivity over p38 (p38 IC 50 > 20 M) and had cell-based potency of ϳ1 M. In contrast, indazole-based inhibitors exemplified by SR-3737 were potent inhibitors of both JNK3 (IC 50 ‫؍‬ 12 nM) and p38 (IC 50 ‫؍‬ 3 nM). These selectivity differences between the indazole class and the aminopyrazole class came despite nearly identical binding (root mean square deviation ‫؍‬ 0.33 Å ) of these two compound classes to JNK3. The structural features within the compounds giving rise to the selectivity in the aminopyrazole class include the highly planar nature of the pyrazole, N-linked phenyl structures, which better occupied the smaller active site of JNK3 compared with the larger active site of p38.Because the initial reports on the discovery of p38 (1) and c-Jun N-terminal kinase (JNK) 2 (2-6) these mitogen-activated protein kinase family members have generated great interest as drug targets. p38 especially has garnered considerable interest, particularly for the treatment of rheumatoid arthritis and Crohn disease, and numerous compounds have entered clinical trials for these indications (7-11). Because most of the p38 inhibitors are competitive versus ATP (12-17), and there are 518 kinases in the genome, it was crucial to develop compounds that are selective against a broad panel of kinases so that compounds could be advanced to clinical development. The molecular basis that gives rise to selective p38 inhibitors from numerous structural classes has been reported (18 -20) and is centered on amino acid differences at the so-called "gatekeeper" Thr-106 residue in p38 (Met in all of the JNK isoforms and Gln in extracellular regulated kinase, the other mitogenactivated protein kinase family member). Many compounds have been synthesized that take advantage of this deeper hydrophobic pocket in p38, compared with JNK3, and the structures of the compounds have included trifluoromethyl and other large moieties, which all contribute to p38 selectivity (21). In contrast to p38, there have been fewer reports for selective JNK inhibitors, and the clinical development of JNK inhibitors also lags that of p38. Despite the paucity of highly selective JNK inhibitors that have advanced to clinical development, numerous recent reports have begun to emerge that show compounds from various structural classes (benzothiazole pyrimidines, aminopyr...

There are currently no drugs to treat neurodegeneration in Parkinson’s disease (PD) and all existing medications only treat symptoms, lose efficacy over time, and produce untoward side effects. In the current work, we report the first highly selective, orally bioavailable, c-jun-N-terminal kinase (JNK) inhibitor for protection of dopaminergic neurons in vitro and in vivo. At 300 nM this compound showed statistically significant protection of primary dopaminergic neurons exposed to 1-methyl-4-phenylpyridinium (MPP+), had pharmacokinetic properties in rodents consistent with twice daily (b.i.d.) dosing, and was orally efficacious at 30 mg/kg in a mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease. Moreover, a dose-dependent target modulation of c-jun phosphorylation served as a biomarker for demonstrating on-target inhibition of JNK as the mechanism of action for this compound. Collectively these results suggest that this JNK inhibitor could be a promising therapeutic neuroprotective agent in the treatment of Parkinson’s disease.

Isolated rat hepatocytes bind and internalize the iron-binding protein lactoferrin (Lf) by a set of high-affinity, recycling, Ca2+-dependent binding sites. We have purified a 45-kDa membrane protein (p45) from rat hepatocytes that exhibits Ca2+-dependent receptor activity. In this study, we found p45 to be identical to the major subunit (RHL-1) of the rat asialoglycoprotein receptor. Two tryptic fragments of p45 showed 100% identity with RHL-1 internal sequences (Leu121 --> Lys126 and Phe198 --> Lys220), and monospecific antisera against p45 and RHL-1 cross-reacted equally well with each protein. Molar excesses of anti-p45 IgG, anti-RHL-1 IgG, asialoorosomucoid, and asialofetuin competitively blocked the binding of 125I-Lf to isolated rat hepatocytes at 4 degrees C. Similarly, either excess anti-p45 or Lf blocked the binding of 125I-asialoorosomucoid to cells at 4 degrees C. We did not detect the minor subunits of the rat asialoglycoprotein receptor (RHL-2/3) in p45 preparations from Triton X-100 extracts of hepatocytes and 125I-Lf bound to purified RHL-1 but not to RHL-2/3 immobilized on nitrocellulose. Nonetheless, anti-RHL-2/3 IgG reduced the binding of 125I-Lf to hepatocytes at 4 degrees C. Exoglycosidases were used to remove terminally-exposed N-acetylneuraminyl, alpha- and beta-galactosyl, and N-acetylhexosaminyl sugars from human and bovine Lf glycans, and lectin blotting confirmed that glycosidase-treated Lfs lacked detectable terminal galactosyl sugars. Unexpectedly, these deglycosylated Lfs exhibited no loss in their ability to compete with unmodified Lfs for binding to isolated hepatocytes. In addition, molar excess of beta-lactose but not sucrose competitively blocked the binding of 125I-Lf to cells, indicating that Lf bound at or very near the carbohydrate-recognition domain of RHL-1. We conclude that RHL-1 is the Ca2+-dependent Lf receptor on hepatocytes and that it binds Lf at its carbohydrate-recognition domain yet in a galactose-independent manner.