New Horizons: Novel Approaches to Enhance Healthspan Through Targeting Cellular Senescence and Related Aging Mechanisms

The elderly population is increasing faster than other segments of the population throughout the world. Age is the leading predictor for most chronic diseases and disorders, multimorbidity, geriatric syndromes, and impaired ability to recover from accidents or illnesses. Enhancing the duration of health and independence, termed healthspan, would be more desirable than extending lifespan merely by prolonging the period of morbidity toward the end of life. The geroscience hypothesis posits that healthspan can be extended by targeting fundamental aging mechanisms, rather than attempting to address each age-related disease one at a time, only so the afflicted individual survives disabled and dies shortly afterward of another age-related disease. These fundamental aging mechanisms include, among others, chronic inflammation, fibrosis, stem cell/ progenitor dysfunction, DNA damage, epigenetic changes, metabolic shifts, destructive metabolite generation, mitochondrial dysfunction, misfolded or aggregated protein accumulation, and cellular senescence. These processes appear to be tightly interlinked, as targeting any one appears to affect many of the rest, underlying our Unitary Theory of Fundamental Aging Mechanisms. Interventions targeting many fundamental aging processes are being developed, including dietary manipulations, metformin, mTOR (mechanistic target of rapamycin) inhibitors, and senolytics, which are in early human trials. These interventions could lead to greater healthspan benefits than treating age-related diseases one at a time. To illustrate these points, we focus on cellular senescence and therapies in development to target senescent cells. Combining interventions targeting aging mechanisms with disease-specific drugs could result in more than additive benefits for currently difficult-to-treat or intractable diseases. More research attention needs to be devoted to targeting fundamental aging processes.

Drug Metabolites: General Features and Most Applicable Analytical Methods of Studies

Drug discovery and development is a multi and interdisciplinary process that includes chemistry and biology understanding. Though, drug discovery is an expensive and time-consuming process, promoting the lack of new drugs approval in recent years. In general, most of the drugs are established based on a macromolecular target, which is vital to disease progression. However, in many cases, the mechanism of drug-macromolecular target interaction is complex and not understood. All this missed information is strongly dependent on the physicalal-chemical stability and behavior of the molecule inside the cell, which is correlated to its metabolism resistance and entrance into the cell through the cellular membrane, respectively. Thus, bioanalysis often provides enough data about these molecular characteristics and helps to figure out new information about these subjects. In this context, the study of the metabolism of parental compound plays a key role in bioanalysis and, consequently in drug discovery. For a long time, the study of metabolism represented a huge challenge in medicinal chemistry, but with the technological advancements, many powerful techniques were developed and, currently, the metabolomics fields are essential steps in the process of discovering a new drug. Herein, we briefly discuss the biological aspects of the drug metabolism, focusing on the most used analytical tools to better understand the metabolite generation, and consequently, their chemical and biological characteristics.

In Vitro Exploration of Healthy and Stressed Gut Microbiota Metabolism (P20-002-19)

Objectives From a military perspective, the gut microbiome serves as an ideal tool to enhance Soldier gut and immune health and to improve survivability and performance. Our work employs in vitro tools as a means to elucidate systematic processes of colonic bacterial metabolism of dietary inputs under native and stressed conditions. This talk will focus on the use of in vitro fermentation to investigate both the prebiotic potential of cranberry proanthocyanidins (PAC) within a healthy microbiome and stress-induced alterations in microbial inter-species competition for fermentable fiber. Methods Fermentations were performed in triplicate, utilizing fecal inocula derived from at least three individuals, in a nutrient-rich anaerobic medium with sampling at 0 and 24 hrs. Within PAC supplementation studies, samples were analyzed for bacterial identification (16 s rRNA sequencing) and metabolite content (GC-FID and GC/MS). Stressed metabolism studies, which utilized fecal samples before and after a 21-day change in diet challenge (habitual vs. Meal Ready-to-Eat), employed media supplemented with resistant starch under ascending colon domain-specific conditions; samples taken were analyzed for bacterial identification (16 s rRNA) and enumeration of select organisms (qPCR). Results Bacterial population dynamics within PAC supplementation studies indicated a dose-dependent increase in several beneficial taxa, including Ruminococcus spp (P < 0.05). Phenolic metabolite generation as a function of PAC dosage identified several compounds associated with anti-inflammatory activity, including 3-(4-OH-phenyl) propionic acid (P < 0.001). Within stressed metabolism studies, Lactobacillus spp. growth was attenuated as a function of sudden change in diet (P ≤ 0.001), whereas growth of R. bromii was enhanced (P ≤ 0.05), indicating potential for an acute stressor to impact gut bacteria functional metabolism. Conclusions In vitro fermentation elucidated both a potential prebiotic effect of cranberry PAC on gut microbiota and the impact of sudden change in diet on inter-species competition for nutritional substrates. Understanding of gut microbiota metabolism dynamics could direct future dietary supplementation strategies to build resiliency against military-relevant stressors and offset negative impacts of dysbiosis. Funding Sources Defense Health Program.

Metatox - Web application for generation of metabolic pathways and toxicity estimation

Xenobiotics biotransformation in humans is a process of the chemical modifications, which may lead to the formation of toxic metabolites. The prediction of such metabolites is very important for drug development and ecotoxicology studies. We created the web-application MetaTox ( http://way2drug.com/mg ) for the generation of xenobiotics metabolic pathways in the human organism. For each generated metabolite, the estimations of the acute toxicity (based on GUSAR software prediction), organ-specific carcinogenicity and adverse effects (based on PASS software prediction) are performed. Generation of metabolites by MetaTox is based on the fragments datasets, which describe transformations of substrates structures to a metabolites structure. We added three new classes of biotransformation reactions: Dehydrogenation, Glutathionation, and Hydrolysis, and now metabolite generation for 15 most frequent classes of xenobiotic’s biotransformation reactions are available. MetaTox calculates the probability of formation of generated metabolite — it is the integrated assessment of the biotransformation reactions probabilities and their sites using the algorithm of PASS ( http://way2drug.com/passonline ). The prediction accuracy estimated by the leave-one-out cross-validation (LOO-CV) procedure calculated separately for the probabilities of biotransformation reactions and their sites is about 0.9 on the average for all reactions.

Interaction between Iron Oxide Nanoparticles and HepaRG Cells: A PreliminaryIn VitroEvaluation

Nowadays, the use of iron oxide nanoparticles is widespread to label cells for magnetic resonance imaging tracking. More recently, magnetic labeling provides promising new opportunities for tissue engineering by controlling and manipulating cells through the action of an external magnetic field. The present work describes nonspecific labeling of metabolically competent HepaRG hepatocytes with anionic iron oxide nanoparticles. An interaction was observed between nanoparticles and studied cells, which were easily attracted when exposed to a magnet. No cytotoxicity was detected in the hepatocytes after 24 hours of incubation with iron oxide nanoparticles. Impact on HepaRG metabolization activity was assessed. Although a slight decrease in the metabolite generation was observed after exposure to nanoparticles (2 mM in iron), the enzymatic capacity was maintained. These results pave the way for 3D cultures of magnetic labeled HepaRG cells by using a magnetic field.