Structure-based design, synthesis and biological evaluation of a NAD+ analogue targeting Pseudomonas aeruginosa NAD kinase.

Multidrug resistance is a major public health problem that requires the urgent development of new antibiotics and therefore the identification of novel bacterial targets. The activity of nicotinamide adenine dinucleotide kinase, NADK, is essential in all bacteria tested so far, including many human pathogens that display antibiotic resistance leading to the failure of current treatments. Inhibiting NADK is therefore a promising and innovative antibacterial strategy since there is currently no drug on the market targeting this enzyme. Through a fragment-based drug design approach, we have recently developed a NAD+ -competitive inhibitor of NADKs, which displayed in vivo activity against Staphylococcus aureus. Here, we show that this compound, a di-adenosine derivative, is inactive against the NADK enzyme from the Gram-negative bacteria Pseudomonas aeruginosa (PaNADK). This lack of activity can be explained by the crystal structure of PaNADK, which was determined in complex with NADP+ in this study. Structural analysis led us to design and synthesize a benzamide adenine dinucleoside analogue, active against PaNADK. This novel compound efficiently inhibited PaNADK enzymatic activity in vitro with a Ki of 4.6 µm. Moreover, this compound reduced P. aeruginosa infection in vivo in a zebrafish model.

Structural Basis for Inhibition of ROS-Producing Respiratory Complex I by NADH-OH.

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0 Šresolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.

Supplementing media with NAD+ precursors enhances the in vitro maturation of porcine oocytes.

In vitro maturation (IVM) is an important reproductive technology used to produce embryos in vitro. However, the developmental potential of oocytes sourced for IVM is markedly lower than those matured in vivo. Previously, NAD+-elevating treatments have improved oocyte quality and embryo development in cattle and mice, suggesting that NAD+ is important during oocyte maturation. The aim of this study was to examine the effects of nicotinic acid (NA), nicotinamide (NAM) and nicotinamide mononucleotide (NMN) on oocyte maturation and subsequent embryo development. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation medium supplemented with NA, NAM and resveratrol or NMN. Mature oocytes were artificially activated and presumptive zygotes cultured for 7 days. Additionally, oocytes were matured without treatment then cultured for 7 days with NMN. Supplementing the IVM medium with NA improved maturation and blastocyst formation while NAM supplementation improved cleavage rates compared with untreated controls. Supplementing the IVM or embryo culture media with NMN had no effect on maturation or embryo development. The results show that supplementing the maturation medium with NA and NAM improved maturation and developmental potential of porcine oocytes.

Improved Detection Sensitivity of an Antigen Test for SARS-CoV-2 Nucleocapsid Proteins with Thio-NAD Cycling.

Antigen tests for infectious diseases are inexpensive and easy-to-use, but the limit of detection (LOD) is generally higher than that of PCR tests, which are considered the gold standard. In the present study, we combined a sandwich enzyme-linked immunosorbent assay (ELISA) with thionicotinamide-adenine dinucleotide (thio-NAD) cycling to improve the LOD of antigen tests for coronavirus disease 2019 (COVID-19). For recombinant nucleocapsid proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the LOD of our ELISA with thio-NAD cycling was 2.95 × 10-17 moles/assay. When UV-irradiated inactive SARS-CoV-2 was used, the minimum detectable virions corresponding to 2.6 × 104 RNA copies/assay were obtained using our ELISA with thio-NAD cycling. The assay volume for each test was 100 µL. The minimum detectable value was smaller than that of the latest antigen test using a fluorescent immunoassay for SARS-CoV-2, indicating the validity of our detection system for COVID-19 diagnosis.

Synthesis of [4S-2 H]NADH, [4R-2 H]NADH, [4-2 H2 ]NADH and [4-2 H]NAD+ cofactors through heterogeneous biocatalysis in heavy water.

This practitioner protocol describes the synthesis of a family of deuterated nicotinamide cofactors: [4S-2 H]NADH, [4R-2 H]NADH, [4-2 H2 ]NADH and [4-2 H]NAD+ . The application of a recently developed H2 -driven heterogeneous biocatalyst enables the cofactors to be prepared with high (>90%) 2 H-incorporation with 2 H2 O as the only isotope source.

Fluorescent detection of PARP activity in unfixed tissue.

Poly-ADP-ribose-polymerase (PARP) relates to a family of enzymes that can detect DNA breaks and initiate DNA repair. While this activity is generally seen as promoting cell survival, PARP enzymes are also known to be involved in cell death in numerous pathologies, including in inherited retinal degeneration. This ambiguous role of PARP makes it attractive to have a simple and fast enzyme activity assay, that allows resolving its enzymatic activity in situ, in individual cells, within complex tissues. A previously published two-step PARP activity assay uses biotinylated NAD+ and streptavidin labelling for this purpose. Here, we used the fluorescent NAD+ analogues ε-NAD+ and 6-Fluo-10-NAD+ to assess PARP activity directly on unfixed tissue sections obtained from wild-type and retinal degeneration-1 (rd1) mutant retina. In standard UV microscopy ε-NAD+ incubation did not reveal PARP specific signal. In contrast, 6-Fluo-10-NAD+ resulted in reliable detection of in situ PARP activity in rd1 retina, especially in the degenerating photoreceptor cells. When the 6-Fluo-10-NAD+ based PARP activity assay was performed in the presence of the PARP specific inhibitor olaparib, the activity signal was completely abolished, attesting to the specificity of the assay. The incubation of live organotypic retinal explant cultures with 6-Fluo-10-NAD+, did not produce PARP specific signal, indicating that the fluorescent marker may not be sufficiently membrane-permeable to label living cells. In summary, we present a new, rapid, and simple to use fluorescence assay for the cellular resolution of PARP activity on unfixed tissue, for instance in complex neuronal tissues such as the retina.

Dihydronicotinamide riboside promotes cell-specific cytotoxicity by tipping the balance between metabolic regulation and oxidative stress.

Nicotinamide adenine dinucleotide (NAD+), the essential cofactor derived from vitamin B3, is both a coenzyme in redox enzymatic processes and substrate in non-redox events; processes that are intimately implicated in all essential bioenergetics. A decrease in intracellular NAD+ levels is known to cause multiple metabolic complications and age-related disorders. One NAD+ precursor is dihydronicotinamide riboside (NRH), which increases NAD+ levels more potently in both cultured cells and mice than current supplementation strategies with nicotinamide riboside (NR), nicotinamide mononucleotide (NMN) or vitamin B3 (nicotinamide and niacin). However, the consequences of extreme boosts in NAD+ levels are not fully understood. Here, we demonstrate the cell-specific effects of acute NRH exposure in mammalian cells. Hepatocellular carcinoma (HepG3) cells show dose-dependent cytotoxicity when supplemented with 100-1000 µM NRH. Cytotoxicity was not observed in human embryonic kidney (HEK293T) cells over the same dose range of NRH. PUMA and BAX mediate the cell-specific cytotoxicity of NRH in HepG3. When supplementing HepG3 with 100 µM NRH, a significant increase in ROS was observed concurrent with changes in the NAD(P)H and GSH/GSSG pools. NRH altered mitochondrial membrane potential, increased mitochondrial superoxide formation, and induced mitochondrial DNA damage in those cells. NRH also caused metabolic dysregulation, altering mitochondrial respiration. Altogether, we demonstrated the detrimental consequences of an extreme boost of the total NAD (NAD+ + NADH) pool through NRH supplementation in HepG3. The cell-specific effects are likely mediated through the different metabolic fate of NRH in these cells, which warrants further study in other systemic models.

Real-time monitoring of PARP1-dependent PARylation by ATR-FTIR spectroscopy.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

Ultrafast Fluorescence Signals from ß-Dihydronicotinamide Adenine Dinucleotide: Resonant Energy Transfer in the Folded and Unfolded Forms.

ß-Dihydronicotinamide adenine dinucleotide (NADH) plays a critical role in biological redox processes. Inside the cell, NADH can be in a folded or an unfolded conformation, depending on the chemical environment that surrounds it. It is known that selective excitation of adenine in NADH can induce energy transfer events from this nucleotide to the reduced nicotinamide chromophore. From the anticipated time scales, this process must compete with adenine's internal conversion channel, which is known to occur in the sub-picosecond time scale. In this work, we studied the dynamics of the excited states of both chromophores in NADH through the time resolution of the spontaneous emission from both nucleotides. Through these experiments, we extend the knowledge about the competition between the different photophysical channels both in the folded and unfolded states. The study involved the folded and unfolded states of NADH by experiments in water and methanol solutions. Our femtosecond fluorescence results were complemented by the first nuclear magnetic resonance through space magnetization transfer measurements on NADH, which establish the solvent-dependent folded versus unfolded states. We determined the dynamics of the excited states by direct excitation of dihydronicotinamide at 380 nm and adenine at 266 nm. From this, we were able to measure for the folded state, a time constant of 90 fs for energy transfer. Additionally, we determined that even in what is referred to as an unfolded state in methanol, non-negligible excitation transfer events do take place. Spontaneous emission anisotropy measurements were used in order to confirm the presence of a minor energy transfer channel in the methanol solutions where the unfolded state dominates.

NAD Analogs in Aid of Chemical Biology and Medicinal Chemistry.

Nicotinamide adenine dinucleotide (NAD) serves as an essential redox co-factor and mediator of multiple biological processes. Besides its well-established role in electron transfer reactions, NAD serves as a substrate for other biotransformations, which, at the molecular level, can be classified as protein post-translational modifications (protein deacylation, mono-, and polyADP-ribosylation) and formation of signaling molecules (e.g., cyclic ADP ribose). These biochemical reactions control many crucial biological processes, such as cellular signaling and recognition, DNA repair and epigenetic modifications, stress response, immune response, aging and senescence, and many others. However, the links between the biological effects and underlying molecular processes are often poorly understood. Moreover, NAD has recently been found to tag the 5'-ends of some cellular RNAs, but the function of these NAD-capped RNAs remains largely unrevealed. Synthetic NAD analogs are invaluable molecular tools to detect, monitor, structurally investigate, and modulate activity of NAD-related enzymes and biological processes in order to aid their deeper understanding. Here, we review the recent advances in the design and development of NAD analogs as probes for various cellular NAD-related enzymes, enzymatic inhibitors with anticancer or antimicrobial therapeutic potential, and other NAD-related chemical biology tools. We focus on research papers published within the last 10 years.

Metabolism and biochemical properties of nicotinamide adenine dinucleotide (NAD) analogs, nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD).

Nicotinamide adenine dinucleotide (NAD) is an important coenzyme that regulates various metabolic pathways, including glycolysis, ß-oxidation, and oxidative phosphorylation. Additionally, NAD serves as a substrate for poly(ADP-ribose) polymerase (PARP), sirtuin, and NAD glycohydrolase, and it regulates DNA repair, gene expression, energy metabolism, and stress responses. Many studies have demonstrated that NAD metabolism is deeply involved in aging and aging-related diseases. Previously, we demonstrated that nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD), which are analogs of NAD, are significantly increased in Nmnat3-overexpressing mice. However, there is insufficient knowledge about NGD and NHD in vivo. In the present study, we aimed to investigate the metabolism and biochemical properties of these NAD analogs. We demonstrated that endogenous NGD and NHD were found in various murine tissues, and their synthesis and degradation partially rely on Nmnat3 and CD38. We have also shown that NGD and NHD serve as coenzymes for alcohol dehydrogenase (ADH) in vitro, although their affinity is much lower than that of NAD. On the other hand, NGD and NHD cannot be used as substrates for SIRT1, SIRT3, and PARP1. These results reveal the basic metabolism of NGD and NHD and also highlight their biological function as coenzymes.

Escherichia coli Strain Designed for Characterizing in Vivo Functions of Nicotinamide Adenine Dinucleotide Analogues.

An Escherichia coli strain was constructed for the efficient import of nicotinamide adenine dinucleotide (NAD) analogues into cells by limiting extracellular degradation while expressing an efficient NAD importer. In vivo functions of three NAD analogues were characterized. Nicotinamide hypoxanthine dinucleotide was identified as an inhibitor of NAD synthesis. Nicotinamide cytosine dinucleotide had excellent biocompatibility and was used for characterizing a growth-dependent degradation of in vivo nicotinamide cofactors.

Eosinophil peroxidase oxidizes isoniazid to form the active metabolite against M. tuberculosis, isoniazid-NAD.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD+ adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD+ using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λmax = 326 nm) in the presence of NAD+. This adduct was confirmed to be INH-NAD+ using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD+, was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD+ adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment.

Titin truncations lead to impaired cardiomyocyte autophagy and mitochondrial function in vivo.

Titin-truncating variants (TTNtv) are the most common genetic cause of dilated cardiomyopathy. TTNtv occur in ~1% of the general population and causes subclinical cardiac remodeling in asymptomatic carriers. In rat models with either proximal or distal TTNtv, we previously showed altered cardiac metabolism at baseline and impaired cardiac function in response to stress. However, the molecular mechanism(s) underlying these effects remains unknown. In the current study, we used rat models of TTNtv to investigate the effect of TTNtv on autophagy and mitochondrial function, which are essential for maintaining cellular metabolic homeostasis and cardiac function. In both the proximal and distal TTNtv rat models, we found increased levels of LC3B-II and p62 proteins, indicative of diminished autophagic degradation. The accumulation of autophagosomes and p62 protein in cardiomyocytes was also demonstrated by electron microscopy and immunochemistry, respectively. Impaired autophagy in the TTNtv heart was associated with increased phosphorylation of mTOR and decreased protein levels of the lysosomal protease, cathepsin B. In addition, TTNtv hearts showed mitochondrial dysfunction, as evidenced by decreased oxygen consumption rate in cardiomyocytes, increased levels of reactive oxygen species and mitochondrial protein ubiquitination. We also observed increased acetylation of mitochondrial proteins associated with decreased NAD+/NADH ratio in the TTNtv hearts. mTORC1 inhibitor, rapamycin, was able to rescue the impaired autophagy in TTNtv hearts. In summary, TTNtv leads to impaired autophagy and mitochondrial function in the heart. These changes not only provide molecular mechanisms that underlie TTNtv-associated ventricular remodeling but also offer potential targets for its intervention.

3-Phosphoglycerate Transhydrogenation Instead of Dehydrogenation Alleviates the Redox State Dependency of Yeast de Novo l-Serine Synthesis.

The enzymatic mechanism of 3-phosphoglycerate to 3-phosphohydroxypyruvate oxidation, which forms the first step of the main conserved de novo serine synthesis pathway, has been revisited recently in certain microorganisms. While this step is classically considered to be catalyzed by an NAD-dependent dehydrogenase (e.g., PHGDH in mammals), evidence has shown that in Pseudomonas, Escherichia coli, and Saccharomyces cerevisiae, the PHGDH homologues act as transhydrogenases. As such, they use α-ketoglutarate, rather than NAD+, as the final electron acceptor, thereby producing D-2-hydroxyglutarate in addition to 3-phosphohydroxypyruvate during 3-phosphoglycerate oxidation. Here, we provide a detailed biochemical and sequence-structure relationship characterization of the yeast PHGDH homologues, encoded by the paralogous SER3 and SER33 genes, in comparison to the human and other PHGDH enzymes. Using in vitro assays with purified recombinant enzymes as well as in vivo growth phenotyping and metabolome analyses of yeast strains engineered to depend on either Ser3, Ser33, or human PHGDH for serine synthesis, we confirmed that both yeast enzymes act as transhydrogenases, while the human enzyme is a dehydrogenase. In addition, we show that the yeast paralogs differ from the human enzyme in their sensitivity to inhibition by serine as well as hydrated NADH derivatives. Importantly, our in vivo data support the idea that a 3PGA transhydrogenase instead of dehydrogenase activity confers a growth advantage under conditions where the NAD+:NADH ratio is low. The results will help to elucidate why different species evolved different reaction mechanisms to carry out a widely conserved metabolic step in central carbon metabolism.

NAD(P)HX dehydratase (NAXD) deficiency: a novel neurodegenerative disorder exacerbated by febrile illnesses.

Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX dehydratase (NAXD) is essential for intracellular repair of NAD(P)HX. Here we present a series of infants and children who suffered episodes of febrile illness-induced neurodegeneration or cardiac failure and early death. Whole-exome or whole-genome sequencing identified recessive NAXD variants in each case. Variants were predicted to be potentially deleterious through in silico analysis. Reverse-transcription PCR confirmed altered splicing in one case. Subject fibroblasts showed highly elevated concentrations of the damaged cofactors S-NADHX, R-NADHX and cyclic NADHX. NADHX accumulation was abrogated by lentiviral transduction of subject cells with wild-type NAXD. Subject fibroblasts and muscle biopsies showed impaired mitochondrial function, higher sensitivity to metabolic stress in media containing galactose and azide, but not glucose, and decreased mitochondrial reactive oxygen species production. Recombinant NAXD protein harbouring two missense variants leading to the amino acid changes p.(Gly63Ser) and p.(Arg608Cys) were thermolabile and showed a decrease in Vmax and increase in KM for the ATP-dependent NADHX dehydratase activity. This is the first study to identify pathogenic variants in NAXD and to link deficient NADHX repair with mitochondrial dysfunction. The results show that NAXD deficiency can be classified as a metabolite repair disorder in which accumulation of damaged metabolites likely triggers devastating effects in tissues such as the brain and the heart, eventually leading to early childhood death.

Combining Chemical Genetics with Proximity-Dependent Labeling Reveals Cellular Targets of Poly(ADP-ribose) Polymerase 14 (PARP14).

Poly(ADP-ribose) polymerase 14 (PARP14) is a member of the PARP family of enzymes that transfer ADP-ribose from NAD+ to nucleophilic amino acids on target proteins, a process known as mono-ADP-ribosylation (MARylation). PARP14 is involved in normal immune function through the IL-4 signaling pathway and is a prosurvival factor in multiple myeloma and hepatocellular carcinoma. A mechanistic understanding of the physiological and pathophysiological roles of PARP14 has been limited by the dearth of PARP14-specific MARylation targets. Herein we engineered a PARP14 variant that uses an NAD+ analog that is orthogonal to wild-type PARPs for identifying PARP14-specific MARylation targets. Combining this chemical genetics approach with a BioID approach for proximity-dependent labeling of PARP14 interactors, we identified 114 PARP14-specific protein substrates, several of which are RNA regulatory proteins. One of these targets is PARP13, a protein known to play a role in regulating RNA stability. PARP14 MARylates PARP13 on several acidic amino acids. This study not only reveals crosstalk among PARP family members but also highlights the advantage of using disparate approaches for identifying the direct targets of individual PARP family members.

Anodic reactions of NADH model compound by utilizing both light irradiation and riboflavin as a redox mediator.

Both light and a redox mediator riboflavin (RF) were utilized to promote the electro-oxidation of an NADH model compound (1-benzyl-1,4-dihydronicotinamide, BNAH), which is a key process for enzymatic biofuel cells to obtain a high performance. At the cathode, H+ ions were simultaneously reduced to produce H2 gas. To elucidate the cell reactions of this photogalvanic cell, which is significant information about the fabrication of enzymatic biofuel cells with a high performance, the effect of the BNAH and RF concentrations on the cell current, the light wavelength dependence on the current, and reduction of the RF concentration were evaluated. The obtained results strongly suggest that the anodic reactions were composed of the following reactions: 1) the photo-excitation of RF, 2) the attack of the excited RF on the BNAH and the generation of the radical species of BNAH and RF, and 3) the chain reactions between the radical species.