A discovery platform for the identification of caloric restriction mimetics with broad health-improving effects

ABSTRACT

The age-related decline in organismal fitness results in vulnerability to pathologies and eventual lethal decay. One way to counteract cellular aging and to delay and/or prevent the onset of age-related maladies is the reduction of calorie intake or the institution of fasting regimens. Caloric restriction mimetics (CRMs) have the ability to imitate the health-promoting and lifespan-extending effects of caloric restriction without the need for dietary restriction. CRMs induce an increase in autophagic flux in response to the deacetylation of cellular proteins in the absence of cytotoxicity. Here we report the development of a high-throughput discovery platform for novel CRMs that uses systems biology approaches, in vitro validation and functional tests employing in vivo disease models. This workflow led to the identification of 3,4-dimethoxychalcone (3,4-DC) as a novel CRM that stimulated TFEB (transcription factor EB)- and TFE3 (transcription factor E3)-dependent macroautophagy/autophagy. 3,4-DC showed cardioprotective effects and stimulated anticancer immunosurveillance in the context of immunogenic chemotherapy.

The prevention (or delay) of age-related disorders can be achieved by the permanent or intermittent reduction of calorie intake. The health- and lifespan-extending effects of caloric restriction (CR) have been characterized across distinct species including yeast (Saccharomyces cerevisiae), nematodes (Caenorhabditis elegans), flies (Drosophila melanogaster), rodents (Mus musculus) and non-human primates (Macaca mulatta). Such beneficial outcomes of caloric restriction are caused by complex metabolic adaptations that are accompanied by anti-inflammatory effects and result from the systemic induction of protective autophagy. Nevertheless, the maintenance of long-term CR regimens is impeded by psychosocial difficulties which are (at least in part) imposed by modern society through the excessive supply of nutriment. The concept of CR mimicry has been developed as an alternative to fasting or CR regimens. This approach employs CR mimetic (CRM) drugs that have the ability to phenocopy the biological effects of CR.

Nutrient scarcity induces a cascade of events that involves the depletion of acetyl coenzyme A (AcCoA), prior to the subsequent deacetylation of hundreds of cytosolic proteins and the activation of the autophagic machinery. CRMs stimulate analogous biochemical events culminating in protein deacetylation and autophagy induction. The nutritional uptake of the CRM spermidine, which inhibits the autophagy-suppressive acetyltransferase EP300, increases the life expectancy of yeast, nematodes, flies and mice. Other known CRMs include resveratrol, which induces deacetylation through the activation of SIRT1 (sirtuin 1), and hydroxycitrate, an inhibitor of the AcCoA-generating enzyme ACLY (ATP citrate lyase).

In mice, CRMs elicit broad cardioprotective effects and improve the efficacy of anticancer chemotherapies by stimulating adaptive immune responses against tumor-associated antigens. Consistently, epidemiological studies reveal that high spermidine uptake is associated with reduced cardiovascular pathologies and cancer-related mortality in humans. Recently, using a yeast chronological aging assay, we identified yet another CRM, the flavonoid 4,4′-dimethoxychalcone (4,4ʹ-DC), which possesses a phylogenetically conserved capacity to extend the lifespan of yeast, worms and flies and demonstrated health-promoting cardio- and hepatoprotective effects in mice. 4,4ʹ-DC significantly reduces the infarction area caused by myocardial ischemia and dampens ethanol-induced liver damage. Autophagy activation by 4,4ʹ-DC depends on the regulation of specific GATA transcription factors, but does not rely on TORC1 signaling.

Functionally, CRMs can be defined as nontoxic pharmacological agents or natural compounds that reduce cellular protein acetylation, thereby increasing autophagic flux and improving cellular and organismal health. To discover new CRMs, we assembled a multistep discovery pipeline for the biosensor-based identification of novel CRMs. This discovery pipeline includes an automated high-throughput high-content screening (HTHCS) approach employing cellular biosensor models that measure different CRM-related effects such as the lipidation of green fluorescent protein (GFP)-tagged MAP1LC3B/LC3 (microtubule associated proteins 1 light chain 3 beta) into the forming autophagosome membrane as an indicator for the onset of autophagy, the deacetylation of cytosolic proteins, as well as the absence of morphological signs of toxicity. This allows for a phenotypic hit identification, still with the general limitation of false positives. Lysosomal inhibitors largely phenocopy the effects of autophagy inducers. Thus, at a second step, hits identified in the initial HTHCS undergo in vitro validation by autophagic flux measurements in several cellular models from distinct species including cells engineered to express a tandem GFP-RFP-LC3 fusion protein as well as assays that monitor the turnover of autophagic cargo such as mutated HTT (huntingtin) protein with abnormally long polyglutamine expansion.

Confirmed hits then undergo a final in vivo validation that usually evaluates several disease models. First, lead compounds are tested in transgenic mice that express the GFP-LC3 biosensor. Tissue samples including heart and liver are analyzed for the formation of LC3 puncta in response to the CRM candidates. Disease models can include the evaluation of cardio-protective effects in instances of ischemic injury, hepato-protective properties in conditions of ethanol-mediated liver damage and the reinstatement of anticancer immunosurveillance in immunocompetent tumor-bearing mice. (Figure 1A). CRM candidates can be further evaluated in chronological aging and longevity studies in yeast and flies, respectively. Using this discovery pipeline on a custom-arrayed library composed of polyphenols and polyamines, we validated the effect of 4,4ʹ-DC and identified yet another CRM, namely, 3,4-dimethoxychalcone (3,4-DC) as being a potent yet nontoxic inducer of GFP-LC3 puncta formation and cytoplasmic protein deacetylation [1]. When added to multiple different human cell lines, 3,4-DC induces global protein deacetylation and stimulated autophagic flux. In vivo, 3,4-DC induces autophagy in yeast and in mouse organs, mediates autophagy-dependent cardioprotective effects and improves the efficacy of anthracycline-based anticancer chemotherapy. The novel CRM 3,4-DC was further subjected to an in-depth mechanistic deconvolution. We found that 3,4-DC differs in its pro-autophagic mode of action from other known CRMs. As compared to the closely-related 4,4ʹ-DC, which depends on GATA2 and GATA3, 3,4-DC requires TFEB- and TFE3-dependent gene transcription to induce autophagy (Figure 1B). Thus, the discovery platform is able to identify novel CRMs that activate protective autophagy with a previously unrecognized mode of action.

Figure 1.

Discovery platform for the identification of novel caloric restriction mimetics. (A) The workflow for the identification and validation of novel caloric restriction mimetics (CRMs) includes a primary HTHCS campaign that measures the induction of autophagy, the deacetylation of cytoplasmic proteins and morphological parameters of toxicity. This initial approach is followed by secondary hit validations involving several cellular model systems and autophagic flux measurements. Lead compounds are further tested in GFP-LC3 transgenic animals for their effects in vivo. Health-promoting effects are evaluated in several disease models including cardio- and hepato-protection as well as the reinstatement of anticancer immunosurveillance. CRM candidates can be further tested in aging models in yeast and flies (B) The novel CRM 3,4-DC differs in its autophagy-inducing mode of action from the closely-related 4,4ʹ-DC. 3,4-DC requires activation of pro-autophagic TFEB and TFE3, whereas 4,4ʹ-DC depends on the inhibition of specific autophagy-inhibitory GATA transcription factors.

Disclosure statement

O.K., D.C-G., F.M. and G.K. are the scientific co-founders of Samsara Therapeutics.