Proposing a Better Approach to the Discovery of Calorie Restriction Mimetics

Calorie restriction slows aging and extends life span in near all species tested to date. The short term effects in humans are beneficial, and there is good evidence for the practice of calorie restriction to reduce the risk of age-related disease. The size of the effect on life span is much smaller in long-lived mammals than is the case in short-lived mammals, unfortunately, as is the case for all approaches based on increased activity of stress response mechanisms. Nonetheless, there is considerable interest in the discovery and development of calorie restriction mimetics, compounds that provoke some of the same beneficial alterations in metabolism as occur in calorie restricted individuals. So far this has been a painfully slow and expensive process, and thus it is entirely understandable that some groups are working on ways to improve the efficiency of this part of the field of aging research. Even so, when the benchmark is resveratrol, a noted failure, it seems hard to imagine this line of work producing meaningful results when it comes to human longevity. Stress response upregulation is a poor approach to age-related degeneration when compared to targeted repair of the biochemical damage that causes aging.

Caloric restriction (CR) is defined as a reduction of caloric intake by 30-40% of ad libitum consumption, without causing malnutrition. CR can cause lifespan extension by triggering a shift from a physiological state of proliferation and growth, to repair and maintenance. Studies have shown that CR reduces oxidative damage, retards age-related functional decline such as deteriorations in DNA repair capacity, and causes a 30% increase in maximal lifespan of mammals. Nevertheless, the amount and duration of CR necessary to extend lifespan is not practical in humans. A feasible solution lies in developing a CR mimetic that can directly target biochemical pathways affected by CR and similarly achieve lifespan extension.

Natural products represent a good starting point for drug discovery, and there is great interest in synthesizing analogs of these compounds in order to explore the mechanism of action, and enhance bioactivity and bioavailability. Polyketides are functionally and structurally diverse secondary metabolites produced in bacteria, fungi, and plants. Many of these bioactive natural products have significant medical applications. Because of the chemical and structural complexity of polyketides and their derivatives, chemical synthesis is difficult. Current research in the engineering and structural characterization of polyketide synthases (PKSs) has facilitated their use as biocatalysts to generate novel polyketides, which can serve as potential drug leads.

The conventional way of anti-ageing drug screening is via lifespan assays. However, lifespan assays are time-consuming and impractical for screening a large library of bioactive compounds. This study aims to develop a medium throughput screening methodology by conducting mitochondrial function assays on C. elegans exposed to various compounds using an Extracellular Flux Analyzer. By periodically introducing pharmacological agents such as electron transport chain inhibitors to manipulate mitochondrial activity and respiratory function, the mitochondrial biology of C. elegans can be examined to establish a correlation between oxygen consumption rates, CR mimetics, and lifespan extension.

Here, we show that by establishing a combinatorial biosynthetic route in Escherichia coli and exploring the substrate promiscuity of a mutant PKS from alfalfa, 413 potential anti-ageing polyketides were biosynthesized. In this approach, novel acyl-coenzyme A precursors were utilized by PKS to generate polyketides which were then fed to Caenorhabditis elegans to study their potential efficacy in lifespan extension. It was found that CR mimetics like resveratrol can counter the age-associated decline in mitochondrial function and increase the lifespan of C. elegans. Using the mitochondrial respiration profile of C. elegans supplemented for 8 days with 50 μM resveratrol as a blueprint, we can screen our novel polyketides for potential CR mimetics with improved potency. This study highlights the utility of synthetic enzymology in the development of novel anti-ageing therapeutics.



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