An in vivo screening platform identifies senolytic compounds that target p16INK4a+ fibroblasts in lung fibrosis.

The appearance of senescent cells in age-related diseases has spurred the search for compounds that can target senescent cells in tissues, termed senolytics. However, a major caveat with current senolytic screens is the use of cell lines as targets where senescence is induced in vitro, which does not necessarily reflect the identity and function of pathogenic senescent cells in vivo. Here, we developed a new pipeline leveraging a fluorescent murine reporter that allows for isolation and quantification of p16Ink4a+ cells in diseased tissues. By high-throughput screening in vitro, precision-cut lung slice (PCLS) screening ex vivo, and phenotypic screening in vivo, we identified a HSP90 inhibitor, XL888, as a potent senolytic in tissue fibrosis. XL888 treatment eliminated pathogenic p16Ink4a+ fibroblasts in a murine model of lung fibrosis and reduced fibrotic burden. Finally, XL888 preferentially targeted p16INK4a-hi human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF), and reduced p16INK4a+ fibroblasts from IPF PCLS ex vivo. This study provides proof of concept for a platform where p16INK4a+ cells are directly isolated from diseased tissues to identify compounds with in vivo and ex vivo efficacy in mice and humans, respectively, and provides a senolytic screening platform for other age-related diseases.

Author Correction: Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptional profiling and therapeutic targeting of oxidative stress in neuroinflammation.

Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells identified a core oxidative stress gene signature coupled to coagulation and glutathione-pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen and oxidative stress gene network analysis identified the glutathione-regulating compound acivicin, with potent therapeutic effects that decrease oxidative stress and axonal damage in chronic and relapsing multiple sclerosis models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable discovery of selective neuroprotective strategies.

Stress Resistance Screen in a Human Primary Cell Line Identifies Small Molecules That Affect Aging Pathways and Extend Caenorhabditis elegans' Lifespan.

Increased resistance to environmental stress at the cellular level is correlated with the longevity of long-lived mutants and wild-animal species. Moreover, in experimental organisms, screens for increased stress resistance have yielded mutants that are long-lived. To find entry points for small molecules that might extend healthy longevity in humans, we screened ∼100,000 small molecules in a human primary-fibroblast cell line and identified a set that increased oxidative-stress resistance. Some of the hits fell into structurally related chemical groups, suggesting that they may act on common targets. Two small molecules increased C. elegans' stress resistance, and at least 9 extended their lifespan by ∼10-50%. We further evaluated a chalcone that produced relatively large effects on lifespan and were able to implicate the activity of two, stress-response regulators, NRF2/skn-1 and SESN/sesn-1, in its mechanism of action. Our findings suggest that screening for increased stress resistance in human cells can enrich for compounds with promising pro-longevity effects. Further characterization of these compounds may reveal new ways to extend healthy human lifespan.

Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch.

The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6ß or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination.

Natural product libraries to accelerate the high-throughput discovery of therapeutic leads.

A high-throughput (HT) paradigm generating LC-MS-UV-ELSD-based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology, an extract of the Indo-Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including latrunculins (1-4, 10), fijianolides (5, 8, 9), mycothiazole (11), aignopsanes (6, 7), and sacrotride A (13). Compounds 1-5 and 8-11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including aignopsanoic acid B (13), apo-latrunculin T (14), 20-methoxy-fijianolide A (15), and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly, and 15 demonstrated modest microtubule-stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only one or two major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and/or new molecular structures using LC-MS-UV-ELSD-based libraries.

Identification and optimization of inhibitors of Trypanosomal cysteine proteases: cruzain, rhodesain, and TbCatB.

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas' disease and African sleeping sickness, respectively. Both parasites rely on essential cysteine proteases for survival: cruzain for T. cruzi and TbCatB/rhodesain for T. brucei. A recent quantitative high-throughput screen of cruzain identified triazine nitriles, which are known inhibitors of other cysteine proteases, as reversible inhibitors of the enzyme. Structural modifications detailed herein, including core scaffold modification from triazine to purine, improved the in vitro potency against both cruzain and rhodesain by 350-fold, while also gaining activity against T. brucei parasites. Selected compounds were screened against a panel of human cysteine and serine proteases to determine selectivity, and a cocrystal was obtained of our most potent analogue bound to cruzain.

The aignopsanes, a new class of sesquiterpenes from selected chemotypes of the sponge Cacospongia mycofijiensis.

A survey of individual specimens of northern Papua New Guinea derived Cacospongia mycofijiensis has yielded novel sesquiterpenes, aignopsanoic acid A (1), methyl aignopsanoate A (2), and isoaignopsanoic acid A (3). The structures and absolute configurations of 1-3 were established using NMR data, X-ray crystallography results, and an analysis of CD properties. Two of these metabolites, 1 and 2, were moderately active against Trypanosoma brucei, the parasite responsible for sleeping sickness.