Targeted Screens for Small Molecules that Slow Aging

The identification of small molecules that impact human aging is of obvious value. A true antiaging compound would be expected to not only increase life span but delay the onset of most age-associated diseases. With the development of high-throughput life-span assays in model systems (see High-Throughput Approaches to Measuring Life Span in Simple Eukaryotes), the ability to carry out small molecule screens for compounds that slow aging has become reality. This type of unbiased screening is likely to result in a plethora of small molecules that increase the life span of simple eukaryotic models such as yeast, worms, and flies, which can then be tested in mammalian systems.

Traditional gene-specific approaches are also being taken to identify small molecules that impact aging or age-associated disease, through the development of high-throughput screening methods to identify specific inhibitors or activators of putative aging genes.

For example, resveratrol, a polyphenolic molecule found in red wine, has received much attention as a possible antiaging drug and CR mimetic based on this type of approach. The link between resveratrol and aging came out of a screen for small molecules that activate SirT1, a human protein orthologous to yeast Sir2 (Howitz et al., 2003). Sir2-family proteins (Sirtuins) have been reported to increase life span in yeast (Kaeberlein et al., 1999), worms (Tissenbaum and Guarente, 2001), and flies (Rogina and Helfand, 2004) when overexpressed, suggesting that Sirtuin activators might also slow aging.

A high-throughput screening method (Fluor-de-Lys) was developed to identify small molecule activators of Sirtuins (Howitz et al., 2003). Sirtuins are NAD-dependent protein deacetylases (Imai et al., 2000; Landry et al., 2000; Tanner et al., 2000), and the Fluor-de-Lys method monitors deacetylation of a peptide substrate that has been covalently linked to a fluorescent moiety. Resveratrol and several related compounds were isolated from this screen as activators of SirT1. Resveratrol treatment was reported to increase life span in yeast, worms, and flies (Howitz et al., 2003; Wood et al., 2004), apparently confirming its antiaging properties, at least in simple eukaryotes. More recently, however, two reports have independently determined that activation of Sirtuins by resveratrol is an in vitro artifact caused by the Fluor-de-Lys substrate (Borra et al., 2005; Kaeberlein et al., 2005a). The fluorophore inhibits interaction of enzyme with substrate, an effect "suppressed" by resveratrol, and resveratrol has no effect on in vitro deacetylation of a native peptide lacking the fluorescent moiety. Further, one study finds no detectable in vivo activation of Sir2 (Kaeberlein et al., 2005a). This example points out a potential flaw in high-throughput methods that alter the fundamental properties of the standard low-throughput assay system. Novel "hits" from a high-throughput screen must always be validated using well-characterized methods.

In addition to Sirtuins, several other genes have been identified that play a role in determining aging rate in multiple eukaryotic models. For example, stress and nutrient responsive kinases, including TOR and Akt homologs increase life span in several different organisms when function is decreased (Fabrizio et al., 2001; Kaeberlein et al., 2005b; Kapahi et al., 2004; Vellai et al., 2003), and are likely to represent viable small molecule targets. Indeed, inhibitors of TOR, such as rapamycin and rapamycin analogues, are currently in clinical trials as anticancer agents. In general, genes that function to promote aging (increase longevity when function is reduced) are likely to make better candidates for high-throughput small molecule screening, as it is much easier to identify protein inhibitors rather than activators.

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