Human EXOG Possesses Strong AP Hydrolysis Activity: Implication on Mitochondrial DNA Base Excision Repair.

Most oxidative damage on mitochondrial DNA is corrected by the base excision repair (BER) pathway. However, the enzyme that catalyzes the rate-limiting reaction─deoxyribose phosphate (dRP) removal─in the multienzymatic reaction pathway has not been completely determined in mitochondria. Also unclear is how a logical order of enzymatic reactions is ensured. Here, we present structural and enzymatic studies showing that human mitochondrial EXOG (hEXOG) exhibits strong 5'-dRP removal ability. We show that, unlike the canonical dRP lyases that act on a single substrate, hEXOG functions on a variety of abasic sites, including 5'-dRP, its oxidized product deoxyribonolactone (dL), and the stable synthetic analogue tetrahydrofuran (THF). We determined crystal structures of hEXOG complexed with a THF-containing DNA and with a partial gapped DNA to 2.9 and 2.1 Šresolutions, respectively. The structures illustrate that hEXOG uses a controlled 5'-exonuclease activity to cleave the third phosphodiester bond away from the 5'-abasic site. This study provides a structural basis for hEXOG's broad spectrum of substrates. Further, we show that hEXOG can set the order of BER reactions by generating an ideal substrate for the subsequent reaction in BER and inhibit off-pathway reactions.

Retraction: Convenient synthesis of pyrimidine 2'-deoxyribonucleoside monophosphates with important epigenetic marks at the 5-position.

Retraction of 'Convenient synthesis of pyrimidine 2'-deoxyribonucleoside monophosphates with important epigenetic marks at the 5-position' by Song Zheng et al., Org. Biomol. Chem., 2020, 18, 5164-5173, DOI: 10.1039/D0OB00884B.

Profiling sirtuin activity using Copper-free click chemistry.

The mammalian sirtuins are a group of posttranslational modification enzymes that remove acyl modifications from lysine residues in an NAD+-dependent manner. Although initially proposed as histone deacetylases (HDACs), they are now known to target other cellular enzymes and proteins as well. Sirtuin-catalyzed simple amide hydrolysis has profound biological consequences including suppression of gene expression, promotion of DNA damage repair, and regulation of glucose and lipid metabolism. Human sirtuins have been intensively pursued by both academia and industry as potential therapeutic targets for the treatment of diseases such as cancer and neurodegeneration. To gain a better understanding of their roles in various cellular events, innovative chemical probes are highly sought after. This current study focuses on the development of activity-based chemical probes (ABPs) for the profiling of sirtuin activity in biological samples. Cyclooctyne-containing and azido-containing probes were synthesized to enable the subsequent copper-free "click" conjugation to either a fluorophore or biotin. The two groups of structurally related ABPs demonstrated different labeling efficiency and selectivity: the cyclooctyne-containing probes failed to label recombinant sirtuins to any appreciable level, while the azido-containing ABPs showed good isoform selectivity. The azido-containing ABPs were further analyzed for their ability to label an individual sirtuin isoform in protein mixtures and cell lysates. These biocompatible ABPs allow the study of dynamic cellular protein activity change to become possible.

Convenient synthesis of pyrimidine 2'-deoxyribonucleoside monophosphates with important epigenetic marks at the 5-position.

Methyl groups of thymine and 5-methylcytosine (5mC) bases in DNA undergo endogenous oxidation damage. Additionally, 5mC residues can be enzymatically deaminated or oxidized through either genetic alterations or the newly identified epigenetic reprogramming pathway. Several methods have been developed to measure the formation of modified DNA nucleobases including 32P-postlabeling. However, the postlabeling method is often limited by the absence of authentic chemical standards. The synthesis of monophosphate standards of nucleotide oxidation products is complicated by the presence of additional functional groups on the modified bases that require complex protection and deprotection strategies. Due to the emerging interest in the pyrimidine oxidation products, the corresponding protected 3'-phosphoramidites needed for solid-phase oligonucleotide synthesis have been reported, and several are commercially available. We report here an efficient synthesis of 3'-monophosphates from 3'-phosphoramidites and the subsequent enzymatic conversion of 3'-monophosphates to the corresponding 5'-monophosphates using commercially available enzymes.

Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity.

In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor. Nicotinamide is a precursor of NAD(+), an important cofactor linking cellular redox states with energy metabolism. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine-spermine N(1)-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD(+) levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD(+)-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.

Development of Second Generation Activity-Based Chemical Probes for Sirtuins.

NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to low calorie conditions. They are thought to mediate the beneficial effects of calorie restriction to extend longevity and improve health profiles. Novel chemical probes are highly desired for a better understanding of sirtuin's roles in various biological processes. We developed a group of remarkably simple activity-based chemical probes for the investigation of active sirtuin content in complex native proteomes. These probes harbor a thioacyllysine warhead, a diazirine photoaffinity tag, as well as a terminal alkyne bioorthogonal functional group. Compared to their benzophenone-containing counterparts, these new probes demonstrated improved labeling efficiency and sensitivity, shortened irradiation time, and reduced background signal. They were applied to the labeling of individual recombinant proteins, protein mixtures, and whole cell lysate. These cell permeable small molecule probes also enabled the cellular imaging of sirtuin activity change. Taken together, our study provides new chemical biology tools and future drug discovery strategies for perturbing the activity of different sirtuin isoforms.

Polarity of Hydrated Phosphatidylcholine Headgroups.

The headgroup (H) stratum (sometimes called the polar region) of membrane bilayers is a relevant yet poorly understood solvation phase for small molecules and macromolecules interacting with the membranes. Solvation of compounds in bilayer strata is characterized experimentally by wide- and small-angle X-ray scattering, neutron diffraction, and various NMR techniques. The quantification is tedious and only available for a limited set of small molecules. Our recently published model of liposome partitioning of small molecules shows that solvation of compounds in the H-stratum of fluid phosphatidylcholine (PC) bilayers correlates well with their solvation in hydrated diacetyl phosphatidylcholine (DAcPC), and solvation in the core (C) depends in a similar way on that in n-hexadecane. These two correlations became a basis for a model describing the location of compounds in the H- and C-strata and at the connecting interface as a nonlinear function of the fragment solvation characteristics of the compounds. In this study, refractivity of hydrated DAcPC phases with varying water contents was measured and polarity was determined using the steady-state fluorescence of indole and Nile Red. The results were compared with the published data obtained by other techniques for PC bilayers in liposomes or on solid supports. The demonstrated qualitative agreement, as well as the polarity and refractivity dependencies on the DAcPC concentration, supports the suitability of hydrated DAcPC as the H-stratum surrogate. Interestingly, depending on hydrations typical for the H-strata of fluid PC bilayers, the dielectric constant could decrease significantly from 31.0 to 7.3 for 16 and 8 water molecules per headgroup, respectively. Although additional experiments are needed for confirmation, this observation could help set proper dielectric constant magnitudes in continuum-based computational models of accumulation and crossing of the PC bilayers with varying hydration levels thanks to the temperature or the structure of fatty acid chains.

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan.

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1(H/H)) live shorter and show signs of accelerated aging. As wild-type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age-related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1-7) are a family of NAD(+)-dependent deacetylases that can delay age-related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD(+) and the ability of SIRT2 to maintain lysine-668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD(+) precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1(H/H) animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD(+) to delay diseases of aging in mammals is warranted.

Trichostatin A inhibits deacetylation of histone H3 and p53 by SIRT6.

SIRT6 is an epigenetic modification enzyme that regulates gene transcription through its deacetylase activity. In addition to histone protein, SIRT6 also modify other proteins and enzymes, some of which are central players in metabolic reprogramming and aging process. Therefore, SIRT6 has emerged as a therapeutic target for the treatment of metabolic disorder and age-related diseases. Here, we report that SIRT6 deacetylates lysine 382 of p53 in short synthetic peptide sequence and in full length p53. Further studies showed that the deacetylation of H3K9Ac and p53K382Ac are insensitive to nicotinamide inhibition, but are sensitive to trichostatin A (TSA) inhibition. Detailed kinetic analysis revealed that TSA competes with the peptide substrate for inhibition, and this inhibition is unique to SIRT6 in the sirtuin family. Taken together, this study not only suggests potential roles of SIRT6 in regulating apoptosis and stress resistance via direct deacetylation of p53, but also provides lead compound for the development of potent and selective SIRT6 inhibitors.

Chemo-enzymatic synthesis of isotopically labeled nicotinamide riboside.

As a cofactor for numerous reactions, NAD+ is found widely dispersed across many maps of cellular metabolism. This core redox role alone makes the biosynthesis of NAD+ of great interest. Recent studies have revealed new biological roles for NAD+ as a substrate for diverse enzymes that regulate a broad spectrum of key cellular tasks. These NAD+-consuming enzymes further highlight the importance of understanding NAD+ biosynthetic pathways. In this study, we developed a chemo-enzymatic synthesis of isotopically labeled NAD+ precursor, nicotinamide riboside (NR). The synthesis of NR isotopomers allowed us to unambiguously determine that NR is efficiently converted to NAD+ in the cellular environment independent of degradation to nicotinamide, and it is incorporated into NAD+ in its intact form. The versatile synthetic method along with the isotopically labeled NRs will provide powerful tools to further decipher the important yet complicated NAD+ metabolism.

Post-Translational Modifications and Diabetes.

Diabetes and its associated complications have increasingly become major challenges for global healthcare. The current therapeutic strategies involve insulin replacement therapy for type 1 diabetes (T1D) and small-molecule drugs for type 2 diabetes (T2D). Despite these advances, the complex nature of diabetes necessitates innovative clinical interventions for effective treatment and complication prevention. Accumulative evidence suggests that protein post-translational modifications (PTMs), including glycosylation, phosphorylation, acetylation, and SUMOylation, play important roles in diabetes and its pathological consequences. Therefore, the investigation of these PTMs not only sheds important light on the mechanistic regulation of diabetes but also opens new avenues for targeted therapies. Here, we offer a comprehensive overview of the role of several PTMs in diabetes, focusing on the most recent advances in understanding their functions and regulatory mechanisms. Additionally, we summarize the pharmacological interventions targeting PTMs that have advanced into clinical trials for the treatment of diabetes. Current challenges and future perspectives are also provided.

Multifunctional activity-based chemical probes for sirtuins.

The sirtuin family of NAD+-dependent protein deacylases has gained significant attention during the last two decades, owing to their unique enzymatic activities as well as their critical roles in a broad array of cellular events. Innovative chemical probes are heavily pursued for the functional annotation and pharmacological perturbation of this group of "eraser" enzymes. We have developed several series of activity-based chemical probes (ABPs) to interrogate the functional state of active sirtuins in complex biological samples. They feature a simple Ala-Ala-Lys tripeptide backbone with a thioacyl "warhead", a photoaffinity group (benzophenone or diazirine), and a bioorthogonal group (terminal alkyne or azido) for conjugation to reporters. When applied in a comparative fashion, these probes reveal the changes of active sirtuin contents under different physiological conditions. Additionally, they can also be utilized in a competitive manner for inhibitor discovery. The Nobel-winning "click" conjugation to a fluorophore allows the visualization of the active enzymes, while the covalent adduct to a biotin leads to the affinity capture of the protein of interest. Furthermore, the "clickable" tag enables the easy access to proteolysis targeting chimeras (PROTACs) that effectively degrade human SIRT2 in HEK293 cells, albeit at micromolar concentrations. These small molecule probes offer unprecedented opportunities to investigate the biological functions and physiological relevance of the sirtuin family.

Activation of SIRT6 Deacetylation by DNA Strand Breaks.

SIRT6 is an emerging regulator of longevity. Overexpression of SIRT6 extends the lifespan of mice. Conversely, SIRT6 knockout mice demonstrate severe metabolic defects and a shortened lifespan. The discrepancy between SIRT6's weak in vitro activity and robust in vivo activity has led to the hypothesis that this enzyme can be activated in response to DNA damage in cells. Here, we demonstrate that the deacetylase activity of SIRT6 can be stimulated by DNA strand breaks for synthetic peptide and histone substrates. The mechanism of activation is further explored by using an integrative chemical biology approach. SIRT6 can be preferentially activated by DNA lesions harboring a 5'-phosphate. The N- and C-termini of SIRT6 are strictly required for DNA break-induced activation. Additionally, the defatty-acylase activity of SIRT6 is also sensitive to DNA breaks, although the physiological significance needs further investigation. Collectively, our study sheds important light on the cellular regulation of diverse SIRT6 activities and suggests possible strategies for effective SIRT6 activation.

Nicotinamide riboside activates SIRT5 deacetylation.

Human sirtuins play important roles in various cellular events including DNA repair, gene silencing, mitochondrial biogenesis, insulin secretion and apoptosis. They regulate a wide array of protein and enzyme targets through their NAD+ -dependent deacetylase activities. Sirtuins are also thought to mediate the beneficial effects of low-calorie intake to extend longevity in diverse organisms from yeast to mammals. Small molecules mimicking calorie restriction to stimulate sirtuin activity are attractive therapeutics against age-related disorders such as cardiovascular diseases, diabetes and neurodegeneration. Little is known about one of the mitochondrial sirtuins, SIRT5. SIRT5 has emerged as a critical player in maintaining cardiac health and neuronal viability upon stress and functions as a tumour suppressor in a context-specific manner. Much has been debated about whether SIRT5 has evolved away from being a deacetylase because of its weak catalytic activity, especially in the in vitro testing. We have, for the first time, identified a SIRT5-selective allosteric activator, nicotinamide riboside (NR). It can increase SIRT5 catalytic efficiency with different synthetic peptide substrates. The mechanism of action was further explored using a combination of molecular biology and biochemical strategies. Based on the existing structural biology information, the NR binding site was also mapped out. These activators are powerful chemical probes for the elucidation of cellular regulations and biological functions of SIRT5. The knowledge gained in this study can be used to guide the design and synthesis of more potent, isotype-selective SIRT5 activators and to develop them into therapeutics for metabolic disorders and age-related diseases.

Merging cultures and disciplines to create a drug discovery ecosystem at Virginia commonwealth university: Medicinal chemistry, structural biology, molecular and behavioral pharmacology and computational chemistry.

The Department of Medicinal Chemistry, together with the Institute for Structural Biology, Drug Discovery and Development, at Virginia Commonwealth University (VCU) has evolved, organically with quite a bit of bootstrapping, into a unique drug discovery ecosystem in response to the environment and culture of the university and the wider research enterprise. Each faculty member that joined the department and/or institute added a layer of expertise, technology and most importantly, innovation, that fertilized numerous collaborations within the University and with outside partners. Despite moderate institutional support with respect to a typical drug discovery enterprise, the VCU drug discovery ecosystem has built and maintained an impressive array of facilities and instrumentation for drug synthesis, drug characterization, biomolecular structural analysis and biophysical analysis, and pharmacological studies. Altogether, this ecosystem has had major impacts on numerous therapeutic areas, such as neurology, psychiatry, drugs of abuse, cancer, sickle cell disease, coagulopathy, inflammation, aging disorders and others. Novel tools and strategies for drug discovery, design and development have been developed at VCU in the last five decades; e.g., fundamental rational structure-activity relationship (SAR)-based drug design, structure-based drug design, orthosteric and allosteric drug design, design of multi-functional agents towards polypharmacy outcomes, principles on designing glycosaminoglycans as drugs, and computational tools and algorithms for quantitative SAR (QSAR) and understanding the roles of water and the hydrophobic effect.

Small Molecule Regulators Targeting NAD+ Biosynthetic Enzymes.

Nicotinamide adenine dinucleotide (NAD+) is a key player in many metabolic pathways as an activated carrier of electrons. In addition to being the cofactor for redox reactions, NAD+ also serves as the substrate for various enzymatic transformations such as adenylation and ADP-ribosylation. Maintaining cellular NAD+ homeostasis has been suggested as an effective anti-aging strategy. Given the importance of NAD+ in regulating a broad spectrum of cellular events, small molecules targeting NAD+ metabolism have been pursued as therapeutic interventions for the treatment of mitochondrial disorders and agerelated diseases. In this article, small molecule regulators of NAD+ biosynthetic enzymes will be reviewed. The focus will be given to the discovery and development of these molecules, the mechanism of action as well as their therapeutic potentials.

Plasmodium falciparum Nicotinamidase as A Novel Antimalarial Target.

Inhibition of Plasmodium falciparum nicotinamidase could represent a potential antimalarial since parasites require nicotinic acid to successfully recycle nicotinamide to NAD+, and importantly, humans lack this biosynthetic enzyme. Recently, mechanism-based inhibitors of nicotinamidase have been discovered. The most potent compound inhibits both recombinant P. falciparum nicotinamidase and parasites replication in infected human red blood cells (RBCs). These studies provide evidence for the importance of nicotinamide salvage through nicotinamidase as a central master player of NAD+ homeostasis in P. falciparum.

Emerging Role of Nicotinamide Riboside in Health and Diseases.

Among all the NAD+ precursors, nicotinamide riboside (NR) has gained the most attention as a potent NAD+-enhancement agent. This recently discovered vitamin, B3, has demonstrated excellent safety and efficacy profiles and is orally bioavailable in humans. Boosting intracellular NAD+ concentrations using NR has been shown to provide protective effects against a broad spectrum of pathological conditions, such as neurodegenerative diseases, diabetes, and hearing loss. In this review, an integrated overview of NR research will be presented. The role NR plays in the NAD+ biosynthetic pathway will be introduced, followed by a discussion on the synthesis of NR using chemical and enzymatic approaches. NR's effects on regulating normal physiology and pathophysiology will also be presented, focusing on the studies published in the last five years.

Facile synthesis of photoactivatable adenosine analogs.

Adenosine and its derivatives are important building blocks of the biological system. They serve as the universal energy currency, amplify intracellular signals for various signal transduction pathways, and can also be used as the co-substrates for enzymatic transformations. The synthesis and regulation of adenosine and its analogs rely on the adenosine binding proteins (ABPs). Dysregulated ABP activity contributes to numerous diseases such as cancer, metabolic disorders, and neurodegenerative diseases. Presently, there is intense interest in targeting ABPs for therapeutic purposes. A large fraction of the human ABP family remains poorly characterized. The need for innovative chemical probes to investigate ABP function in the native biological matrix is apparent. In this study, an adenosine analog, probe 1, with a photoaffinity group and biotin tag was synthesized using concise synthetic strategies. This probe was able to label and capture individual recombinant ABPs with good target selectivity. Probe 1 was also evaluated for its ability to label spiked ABP in complex cell lysates. This chemical probe, together with the labeling and enrichment assay, is of great value to interrogate the biological functions of ABPs and to elucidate their diversity under different physiological conditions.

Sirtuins inhibitors: the approach to affinity and selectivity.

Accumulating evidence has indicated the importance of sirtuins (class III histone deacetylases) in various biological processes. Their potential roles in metabolic and neurodegenerative diseases have encouraged scientists to seek potent and selective sirtuin inhibitors to investigate their biological functions with a view to eventual new therapeutic treatments. This article surveys current knowledge of sirtuin inhibitors including those discovered via high-throughput screening (HST) or via mechanism-based drug design from synthetic or natural sources. Their inhibitory affinity, selectivities, and possible inhibition mechanisms are discussed.