Aging is intimately related to health and disease. The incidence of major fatal pathological conditions, including cancer, is strongly associated with aging. The finding that aging is malleable by genetic or pharmacological perturbations has offered enormous hope for transforming our understanding and treatment of aging and age-related diseases. Yet few valid longevity drugs have been developed or translated for treating age-related diseases as target identification and a mechanistic understanding of their activity is largely missing.

Metabolism plays a pivotal role in aging. Metabolic interventions, such as dietary restriction, significantly modulate aging and aging-related symptoms. Recently, emerging evidences has shown that metabolites can directly modulate aging as well. Studies of these endogenous “elixirs” provide insights on the biological regulatory networks acted upon by the metabolites as well as potential therapeutic interventions for aging and aging-related symptoms.

Our lab aims to discover novel longevity metabolites and explore their applications in aging-related symptoms. Here we present several novel longevity metabolites, which are α-ketoglutarate (α-KG), 2-hydroxyglutarate (2-HG), and α-ketobutyrate (α-KB). By combining target identification, epistasis, metabolomics, and bioenergetics analyses, we successfully uncover the molecular mechanisms of the longevity effects of these compounds. Specifically, we, for the first time, showed that α-KG and 2-HG extend lifespan of C. elegans by inhibiting ATP synthase and TOR, while α-KB delays aging through interruption of pyruvate oxidation and activation of AMPK. Interestingly, we discover an unexpected growth inhibitory effects of α-KG and 2-HG on tumor cells through targeting ATP synthase, and find that α-KG may confer beneficial effects for tumor treatment. In brief, our results identify novel potent longevity compounds and reveal their underlying mechanisms. More importantly, our work suggests a potential translational application of longevity metabolites in aging-related symptoms and provides novel directions for metabolic therapy.

As the elderly population is now the fastest growing age group in the United States, there is an imminent need to understand the molecular mechanisms of aging and develop new approaches to counter aging and the maladies that come with it. Modulation of metabolism, either by dietary restriction or pharmacologic or genetic alteration of metabolic pathways, can extend lifespan and delay age-related diseases in evolutionarily diverse organisms. Here we demonstrate, for the first time, a role for alpha-ketoglutarate (alpha-KG) in the regulation of aging. Alpha-KG serves many functions in the cell, including being an intermediate of the tricarboxylic acid cycle, a precursor for several amino acids, and a substrate for alpha-KG-dependent oxygenases. We show that alpha-KG extends the mean lifespan of adult Caenorhabditis elegans by about 50%, and provide evidence to suggest that this is not due to the metabolism of alpha-KG into other metabolites. Other metabolites have been shown to extend lifespan as well, but without a well defined mechanism. Research on small molecules in aging has been hindered by the inability to identify drug targets. We use a label-free, unbiased drug target identification strategy termed DARTS (drug affinity responsive target stability) to uncover a new binding target of alpha-KG, the beta subunit of the ATP synthase. We reveal that alpha-KG inhibits ATP synthase in in vitro and in vivo assays using both worm and mammalian models. Consistently, genetic or pharmacological inhibition of ATP synthase yields the same phenotypes as alpha-KG: increased lifespan, decreased ATP levels, lower oxygen consumption rates, elevated levels of reactive oxygen species, and reduced target of rapamycin signaling. However, unlike genetic inhibition of ATP synthase, alpha-KG does not induce the mitochondrial unfolded protein response, cause larval arrest, or slow pharyngeal pumping rates. Remarkably, alpha-KG does not further increase the lifespan of dietary restricted animals and is elevated during starvation, a testament to it being a key regulator of dietary restriction mediated lifespan extension. Like dietary restriction, alpha-KG also induces autophagy, and we provide evidence to suggest that autophagy may play an essential role for the longevity of alpha-KG treated animals. Our findings posit alpha-KG as a key regulator of metabolic pathways in response to nutrient status, and suggest new strategies for the prevention and treatment of aging and age-related diseases.

Aging and aging related diseases are closely related to wellness and lifespan of aged population. Modulating aging can be achieved by specific gene mutations, intrinsic signaling regulation (e.g. by dietary restriction) and pharmacological perturbations. Recent studies on the role of metabolism and endogenous metabolites in aging process provided hope for new anti-aging approaches to counter aging and aging related diseases. Here we identified a novel anti-aging metabolite, α-ketobutyrate (α-KB), that extends the lifespan of adult Caenorhabditis elegans and aged mice. Utilizing drug affinity responsive target stability assay, in-vitro enzymatic kinetics analysis, and epistasis, we demonstrated that α-KB depends on pyruvate dehydrogenase perturbation as a competitive alternative substrate to extend lifespan. Consistently, rewired pyruvate metabolism was detected in both α-KB treatment and genetic inhibition of pyruvate dehydrogenase in mammalian models. Furthermore, AMP-activated protein kinase (AMPK) pathway activation is required to induce longevity by α-KB. In an Alzheimer’s disease C. elegans model, epistasis demonstrated that α-KB requires pyruvate dehydrogenase and AMPK to delay the age dependent paralysis progression. Besides lifespan extension, α-KB also improves fitness in aged mice, including maintaining healthy hair growth. In the search of its mechanism and additional hair regenerative compounds, we discovered the essential role of autophagy in hair growth cycle and potential application of autophagy activators in hair loss therapy. As a shared effector of anti-aging signaling pathways, including TOR and AMPK pathways, autophagy is successfully induced by known aging modulators, α- ketoglutarate, α-KB, rapamycin, and metformin in mouse skin and confers their hair regeneration effect. Co-treatment of specific autophagy inhibitors blocks the stimulation of hair regeneration, which further confirmed the requirement of autophagy activation by these agents. Consistently, dynamically regulated autophagy level is detected during hair cycle: upregulation from telogen throughout anagen phase and downregulation from catagen until telogen. Our finding that α-KB combats aging and aging related disorders in worms and mice has implications for human anti-aging remedies development.

With increasing age comes increasing susceptibility to age-related diseases, including cancer and neurodegenerative diseases. A novel approach to treating such diseases is to treat aging itself. However, not much is known about the molecular targets of the aging pathway. Utilizing the Drug Affinity Responsive Target Stability (DARTS) approach developed by our laboratory, we have been working to identify the molecular targets of the aging pathway and to analyze the anti-aging mechanisms of various small molecules that have been shown to increase life expectancy. One such small molecule, resveratrol (RSV), a polyphenol found in the skin of red grapes, has been shown to possess various health benefits, including the extension of lifespan, in several different model organisms. However, the underlying molecular mechanisms of RSV’s beneficial effects remain unclear and highly controversial. My thesis work entails the identification of a novel molecular target of resveratrol, deleted in breast cancer 1 (DBC1), and analysis of its mechanism of action through the protein deacetylase sirtuin 1 (SIRT1), which has been shown to be required for some of RSV’s various health benefits. I have also identified DBC1 as a target of RSV’s metabolites, the 3- and 4’-glucuronides and sulfates, and analyzed the metabolites’ mechanisms of action through SIRT1. And finally, I have discovered that resveratrol induces the phosphorylation of DBC1 and activates a distinct ATM-dependent but DBC1-independent DNA damage response from etoposide that does not result in apoptosis. Taken together, my thesis provides a novel molecular mechanism of resveratrol and its metabolites through the novel molecular target DBC1, resolving some of the controversy surrounding resveratrol’s SIRT1-dependent mode of action, and identifies a distinct ATM-dependent and DBC1-independent DNA damage response that resveratrol activates, suggesting another novel molecular mechanism of resveratrol that could contribute to its beneficial health effects.

Heart attack is one of the major causes of death in elderly populations worldwide. Ischemia-reperfusion (I/R) injury is significantly involved in diseases such as heart attack and stroke, and it occurs as a result of the disruption and subsequent restoration of blood supply. The reperfusion process of the ischemic tissue is essential for the survival and recovery of the tissue, but at the same time initiates secondary damage. The I/R injury differs from the primary ischemic damage and is detrimental to patient survival. The I/R injury has been identified to be a result of several mitochondrial dysfunctions during reperfusion stage, including the production of reactive oxygen species (ROS), opening of the mitochondrial permeability transition pore (mPTP), loss of membrane potential, and activation of downstream cell death signaling pathways. In clinical practice, there is a lack of effective treatment for I/R injury.

Here we report that tricarboxylic acid cycle intermediate α-ketoglutarate (α-KG), when administered at the onset of reperfusion, confers robust cardioprotection against I/R injury and reduces myocardial infarct size in vivo by ~70%. Previously, α-KG has been shown to bind to the mitochondrial F1Fo-ATP synthase/ATPase using drug affinity responsive target stability (DARTS), a small-molecule target identification method. The structure of the mPTP has been studied for decades. It was suggested that ATP synthase plays a critical role in the mPTP opening. Here we provide evidence that α-KG regulates the interaction between ATP synthase to CypD. The opening of the mPTP has been suggested to be regulated by the interaction between ATP synthase to CypD. We further showed that cardioprotection by α-KG against I/R injury is abolished by CypD deficiency. Our findings link a basic metabolite to a critical mitochondrial component and provide novel approaches for cardioprotection against IR injury.

AbstractAs organisms age, they often experience declining health and an increased risk of debilitating diseases, ultimately leading to mortality. With the aging population growing rapidly in the United States and globally, medical expenditures for treating age-related conditions are skyrocketing. Decades of research into aging have accumulated vast knowledge, paving the way for the development of treatments and therapeutic strategies to combat these issues. The extraordinary findings have shown that aging processes can be regulated, as evidenced by model organisms whose lifespan has been doubled through specific gene mutations, offering promising avenues for controlling aging and associated diseases. Here we show that supplementation with the endogenous metabolite α-ketobutyrate (α-KB) prolongs the healthspans and lifespans of C. elegans and mice. Using an unbiased biochemical approach, drug affinity responsive target stability (DARTS), we identified microtubule-actin cross-linking factor (MACF1) protein to be the target of α-KB, as MACF1 exhibited resistance to proteolysis in the presence of α-KB. MACF1, a cytoskeletal linker protein critical for cellular and tissue integrity, has not previously been studied for its influence on longevity pathways. Our research revealed that α-KB's longevity effects require VAB-10, the worm ortholog of mammalian MACF1. Knockdown of VAB-10 via RNA interference increased the phosphorylation of AMP-activated protein kinase (AMPK), a sensor and regulator of cellular energy balance. We found that AMPK was also activated by α-KB treatment in wildtype animals. Furthermore, AMPK downstream effectors CRTC-1 and NHR-49 were found to be essential for lifespan extension by α-KB in this study. AMPK activation has been shown to preserve mitochondrial homeostasis during aging, and we found that α-KB treated worms had maintained youthful mitochondrial network during aging. α-KB’s longevity effect is abrogated by loss-of-function mutation of mitochondrial fusion component. These findings collectively propose a model wherein α-KB promotes longevity by modulating AMPK via VAB-10/MACF1 interaction. Crucially, our study demonstrated that α-KB prolonged the lifespan of aged male mice and enhanced the healthspan of both male and female mice. Our findings provide hope for developing therapeutic strategies that utilize endogenous metabolites to target longevity pathways linked to cellular energy metabolism and mitochondrial dynamics.

Small molecule target identification is a monumental task. Knowledge of the binding interactions between small molecule compounds and cellular macromolecules such as proteins is crucial for understanding their biological properties and mechanisms of action, yet discovering these interactions remains a considerable challenge. To overcome the limitations of current target ID methods, we developed a novel technique that can identify direct binding interactions using the native, unmodified compounds. This method, called drug affinity responsive target stability (DARTS), takes advantage of the stabilization of protein structure upon drug binding that provides the target protein with resistance to protease degradation. When a small molecule is incubated with protein lysates, it binds and protects target proteins from proteolysis while degradation of non-target proteins is unaffected. Using proteomics, target proteins are then identified based on their enrichment in the compound-treated sample versus a control sample post-proteolysis. After demonstration of DARTS using a variety of known drug-protein pairs, DARTS was used to discover that the protein translation initiation machinery is a novel target of resveratrol. Then, to improve the sensitivity of DARTS, it was combined with a powerful gel-free fractionation technique and mass spectrometry analysis, which allowed for the identification of several potential new targets for the anti-cancer marine natural product didemnin B (DB). The TCP1 chaperonin complex (TRiC) was validated to be a target of DB using both biochemical and cell-based assays. Importantly, this is the first non ATP-mimetic compound reported to bind and inhibit TRiC, and could help design more specific and potent inhibitors of this complex. Finally, we demonstrated that DARTS can also be used for mapping the binding domains of small molecules within their target proteins. DARTS provided the first experimental evidence that DB binds both domains 1 and 2 of the eukaryotic elongation factor-1 alpha and verified the binding of pateamine A and hippuristanol to the N-terminal and C-terminal domains of the eukaryotic translation initiation factor 4a, respectively. Collectively, the results presented in this dissertation establish DARTS as a powerful technique for identifying and studying protein-small molecule interactions that will have a profound impact on the chemical biology field.

This thesis presents the design and implementation of a hardware language, Pyrope. It first describes Pyrope’s distinct language features with their design rationales and compares Pyrope with the mainstream language, SystemVerilog. It then shows the implementation of the Pyrope compilation flow that translates the language specification into low-level intermediate representation in a compiler framework, LiveHD. Several improvements to LiveHD as part of this work are also presented. Finally, the thesis shows the result of compiling an example design written in Pyrope.

Prostatic adenocarcinomas can recur with aggressive and lethal small cell neuroendocrine carcinoma (SCNC). Since glycolysis is a feature of malignancy and the degree generally correlates with tumor aggressiveness, glucose metabolism and the molecular mechanisms involved between the two tumor types was studied. LNCaP and PC-3 cell lines modeled adenocarcinoma and SCNC, respectively, to compare glycolytic features and metabolomics. CD44’s role was studied by RNA knockdown and overexpression. Human tissue microarrays were analyzed for glycolytic enzyme expression. Glycolytic features were found to be higher in PC-3 over LNCaP cells. PFKFB4 was overexpressed in SCNC. CD44 regulated glucose metabolism, intracellular ROS, and cell proliferation in PC-3 cells. CD44 inhibition sensitized PC-3 cells to carboplatin. The data suggests different pathways of glucose metabolism may contribute to the differing biological behaviors of the two tumor types. CD44 was found to be an important regulator of glucose metabolism in SCNC and potential therapeutic target.