Ribose Units Research Articles

RNA-Templated Peptide Bond Formation Promotes L-Homochirality.

The world in which we live is homochiral. The ribose units that form the backbone of DNA and RNA are all D-configured and the encoded amino acids that comprise the proteins of all living species feature an all-L-configuration at the α-carbon atoms. The homochirality of α-amino acids is essential for folding of the peptides into well-defined and functional 3D structures and the homochirality of D-ribose is crucial for helix formation and base-pairing. The question of why nature uses only encoded L-α-amino acids is not understood. Herein, we show that an RNA-peptide world, in which peptides grow on RNAs constructed from D-ribose, leads to the self-selection of homo-L-peptides, which provides a possible explanation for the homo-D-ribose and homo-L-amino acid combination seen in nature.

RNA‐Vermittelte Peptidbindungen Fördern die L‐Homochiralität

AbstractDie Welt, in der wir leben, ist homochiral. Die Riboseeinheiten, die das Rückgrat von DNA und RNA bilden, sind alle D‐konfiguriert, und die kodierten Aminosäuren, aus denen die Proteine aller lebenden Arten bestehen, weisen an den α‐Kohlenstoffatomen eine L‐Konfiguration auf. Die Homochiralität der α‐Aminosäuren ist unerlässlich für die Faltung der Peptide in wohldefinierte und funktionelle 3D‐Strukturen und die Homochiralität der D‐Ribose ist entscheidend für die Ausbildung einer Helix und die Basenpaarung. Die Frage, warum die Natur nur kodierte L‐α‐Aminosäuren verwendet, ist nicht geklärt. Wir zeigen hier, dass eine RNA‐Peptid‐Welt, in der Peptide an aus D‐Ribose aufgebauten RNAs wachsen, zur Selbstselektion von homo‐L‐Peptiden führt, was eine mögliche Erklärung für die in der Natur beobachtete Kombination von homo‐D‐Ribose und homo‐L‐Aminosäuren liefert.

Abstract 4698: Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery

Abstract Inhibitors of poly (ADP-ribose) polymerases (PARPs) have revolutionized cancer therapy by highlighting the potential of synthetic lethal drugs to target the DNA damage response (DDR) network. More recently, the development of PARG inhibitors was spurred by the discovery that inhibiting PARG (poly (ADP-ribose) glycohydrolase) results in the accumulation of poly ADP-ribose on DDR proteins and induces cell death. Adding PARG or PARP inhibitors to a cell of interest and quantifying the resulting levels of PARylation can provide valuable insight on compound membrane permeability and target engagement in a cellular context. Nevertheless, assessing PARylation (poly-ADP-ribosylation in which multiple ribose units are added in linear or branched forms) in cells is difficult compared to biochemical assays, impeding the progress of candidate drug development. A sandwich ELISA-based assay was developed to analyze the total protein PARylation present in cellular extracts. Experimental protocols were optimized to discern differences in cellular PARylation levels resulting from activating the DDR and from exposure to PARP inhibitors and/or PARG inhibitors. It is worth noting that our assay specifically detects PARylation, measuring the effects associated with PARP family members 1 to 5, as other family members primarily catalyze MARylation (mono-ADP ribosylation in which a single ADP-ribose unit is added). Titration of known PARP inhibitors and PARG inhibitors was performed to validate the assay. 1) PARG inhibitor PDD00017273, used in combination with DNA damaging agent H2O2, increased the level of cellular PARylation, exhibiting a similar IC50 value across the four cell lines tested. This is consistent with the notion that PARG is the major de-PARylating enzyme in most cells. The potency of other known PARG inhibitors PDD00017272 and PDD00017238 ranked the compounds as expected from published results (PDD00017272< PDD00017273< PDD00017238). 2) Titration of the three PARP1/2 inhibitors Talazoparib, Olaparib and AZD5305, in combination with a DNA damage agent and PDD00017273 inhibitor, shows a dose-dependent reduction in cellular PARylation in HEK293 cells, as expected since PARP1 is activated in response to DNA damage and this activation is blocked by the inhibitors (IC50 values, Olaparib = 0.013 µM, AZD5305 = 0.0024 µM, Talazoparib = 0.0016 µM). These IC50 are consistent with published values and underscore the usefulness of the assay in measuring dose response effects, allowing accurate determination of compound IC50 in living cells. In summary, our optimized experimental cell treatment protocols and ELISA-based assay allow quantification of total cellular PARylation and accurately rank candidate drugs targeting PAR erasers or PAR writers in a high-throughput format, using living cells. Citation Format: Caila Pilo, Mohammad Awad, Veronique T. Baron, Fernando Martins, Jonathan Mikolosko, Steven Plouffe, Zhihua Tao, Katarzyna Zientara-Rytter, Henry Zhu, Pavel Shashkin. Measuring cellular PARylation to gain insight in PARP/PARG-targeted drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4698.

Fluorescent Vitamin B12–Platinum(II) Derivatives as Potential Metallotheranostic Agents for the Treatment and Imaging of Tumors

Vitamin B12 (cyanocobalamin) is an essential nutrient with very low bioavailability. Compared with normal cells, tumor cells show an increased demand for vitamin B12 to support their abnormal proliferation, which is a feature that can be exploited for the tumor-specific delivery of therapeutic and/or diagnostic agents by functionalizing vitamin B12 with suitable metallodrugs and/or luminescent probes. In this context, we report on the design of fluorescent vitamin B12–metal conjugates of the type [FLUO–B12–{M}] in which cyanocobalamin is functionalized at the 5′-site of the ribose unit with a fluorophore (FLUO: rhodamine 6G), whereas the Co(III)–cyano moiety is N-coordinated to a metal-based anticancer scaffold ({M}: Pt(II) substrate bearing enantiopure phenylamino-oxime ligands derived from R- or S-limonene). Two novel fluorescent cyanocobalamin–platinum(II) derivatives and their corresponding non-fluorescent counterparts were successfully generated and fully characterized, including the evaluation of their lipophilicity and luminescent properties. Although they exhibit low antiproliferative activity (IC50 = 40–70 μM), both fluorescent vitamin B12–platinum(II) conjugates showed an enhanced capability to inhibit cell viability compared with the inactive metal precursors and the non-fluorescent vitamin B12–platinum(II) analogues, confirming the beneficial effect of functionalization with the rhodamine 6G scaffold not only for imaging purposes but also with the aim of improving their biological activity.

Biogenic Polymer-Based Fluorescent Assemblies: Versatile Platforms for Ultrasensitive ATP Detection and Enzyme Assay.

Here, we investigated the optical properties of biocompatible supramolecular assemblies formed through electrostatic interactions between anionic fluorescent dyes and biogenic polymers. The dynamic equilibrium between the monomeric form (fluorescent) and aggregates (nonfluorescent) of dye molecules is responsible for the stimuli-responsive behavior of these polymer composites, which can respond to changes in pH, temperature, and ionic strength. Furthermore, we employed supramolecular assemblies for the purpose of turn-on fluorescence sensing of adenosine triphosphate (ATP) at physiological pH. Notably, no interference was observed even in the presence of well-known competing analytes such as pyrophosphate. In addition to its outstanding selectivity, the present system can detect ATP at concentrations as low as 4.8 nM. The superior detection capabilities are achieved through multiple interactions with biogenic polymers, involving the adenine ring, ribose unit (through hydrogen bonding), and phosphate groups (via charge pairing) of ATP. Given the remarkable sensitivity to ATP, we have applied the present system for the detection of a dephosphorylating enzyme, alkaline phosphatase.

Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High-Performance Liquid Chromatography, and Capillary Electrophoresis.

Poly(ADP-ribose) (PAR) is a homopolymer made of two or more adenosine diphosphate ribose (ADP-ribose) units. The polymer is usually conjugated to protein as a posttranslational modification playing key roles in cellular processes, such as DNA repair, RNA metabolism, and biomolecular condensate formation. Emergent data revealed that PAR length is highly regulated and determines the selection of and affinity towards protein binders. Here, we describe several fluorescence-based methods that quantify PAR length distributions. Briefly, we use the bioconjugation technique ELTA (enzymatic labeling of terminal ADP-ribose) to fluorescently label PAR, which can be isolated from in vitro and cellular samples. We describe a novel capillary electrophoresis method to separate and quantify PAR length and compare the profile to gel electrophoresis- and high-performance liquid chromatography-based methods. The capillary electrophoresis method is rapid and automatable, enabling accurate determination of the length profiles from subfemtomole quantities of PAR.

Intermolecular interactions in microhydrated ribonucleoside and deoxyribonucleoside: A computational study

The nature of interactions between the ribonucleoside and deoxyribonucleoside with water molecule was examined employing the dispersion corrected density functional theory and wave functional analysis. In the nucleosides, the water binds with the 5′–OH hydrogen and N3 nitrogen or N7-H group. In the deoxynucleosides, the water binds through 3′–OH and N3 nitrogen or carbonyl oxygen. The interaction energy, binding energy, hydration entropy and enthalpy do not show any appreciable change for the nucleoside-water complexes, emulating the experimental findings. Electrostatic potential analysis shows that the most positive region is observed near the 5′–OH hydrogen atom of the ribose unit in nucleosides. In deoxynucleosides, the 3′–OH hydrogen has Vs,max values equivalent to 5′–OH hydrogen atom. Hence, the water binds with the 3′–OH hydrogen and the nucleobase. QTAIM analysis shows the presence of medium-strength hydrogen bonds between water and nucleoside. In deoxynucleoside-water complexes, in addition to the 3′–OH hydrogen bonding with water, the nearby 2′CH in ribose unit bonds with oxygen in the water molecule. QTAIM and EDA analysis show the intermolecular bonds are noncovalent and electrostatic dominant. The 2D RDG plot shows, additional spikes due to the interaction of 2′C hydrogen with water oxygen. The 3D spatial diagram shows the existence of several green patches in the deoxynucleoside-water complexes, associated with the weak van der Waal’s interactions which make them more stable.

The PARP Way to Epigenetic Changes.

ADP-ribosylation, is a reversible post-translational modification implicated in major biological functions. Poly ADP-ribose polymerases (PARP) are specialized enzymes that catalyze the addition of ADP ribose units from “nicotinamide adenine dinucleotide-donor molecules” to their target substrates. This reaction known as PARylation modulates essential cellular processes including DNA damage response, chromatin remodeling, DNA methylation and gene expression. Herein, we discuss emerging roles of PARP1 in chromatin remodeling and epigenetic regulation, focusing on its therapeutic implications for cancer treatment and beyond.

Magnetic bead-based electrochemical and colorimetric methods for the detection of poly(ADP-ribose) polymerase-1 with boronic acid derivatives as the signal probes

Poly(ADP-ribose) polymerase-1 (PARP-1), as a highly conserved nuclear enzyme, can catalyze the transfer of multiple ADP-ribose units from β-nicotinamide adenine dinucleotide (NAD+) to substrate proteins, including PARP-1 itself. In this work, two boronic acid derivatives, ferroceneboronic acid (FcBA) and 4-mercaptophenylboronic acid (MPBA) were used as the signal probes for electrochemical and colorimetric detection of PARP-1, respectively. The detection platform was constructed by modification of double-stranded DNA (dsDNA) on the surface of magnetic beads (MBs). Once PARP-1 was captured by the MB-dsDNA, auto-PARylation was initiated and the PAR products consisting of abundant ribose units were covalently bound to FcBA or MPBA through the formation of boronate ester covalent bonds. Sequestration of FcBA or MPBA by the PAR-covered MBs led to the decrease in the electrochemical signal of FcBA or preventing the MPBA-triggered aggregation of gold nanoparticle indicators. The change of oxidation current or adsorption intensity (color) was dependent upon the concentration and activity of PARP-1. A wide linear range of 0.1 ∼ 50 U was obtained for the electrochemical or colorimetric assays. The methods have been used to evaluate the inhibition efficiency and monitor the PARP-1 activity in living and apoptotic cancer cells.

Quantification of adenosine Mono-, Di- and triphosphate from royal jelly using liquid chromatography - Tandem mass spectrometry.

Nucleotides are composed of nitrogen bases, ribose units and phosphate groups. Adenine (Ade), adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) all play important roles in physiological metabolism. Royal jelly, a secretion produced by worker bees, contains a variety of natural ingredients and several studies have shown that royal jelly can serve as a source of nutrition for humans. In this study, a rapid and effective LC/ MS method coupled with pre-processing methods was developed and validated for the accurate quantification of Ade, AMP, ADP and ATP in royal jelly. To achieve the best extraction efficiency, two pretreatment methods, namely, solid-phase extraction (SPE) and dispersive solid-phase extraction (dSPE), were developed and investigated. Silica-based cyanopropyl (CN) liquid chromatography was employed using pH programming with a quaternary mobile phase system for the analyses. The total LC/MS run time was less than 12 min with a constant flow rate of 0.25 mL/min. The linear range were 2.5–1000 ng/mL with a correlation coefficient r = 0.9995. The limit of detection (LOD) of Ade, AMP, ADP and ATP was 1, 1, 2.5 and 5 ng/mL; the limit of quantitation (LOQ) was 2.5, 2.5, 5 and 10 ng/mL, respectively. Precision (RSD% <10.5%) and accuracy (recovery 81.3–118.4%) were satisfactory for both two pre-processing methods. Nucleotides were successfully quantified from 2-day and 3-day royal jelly samples, with concentrations within 6.2–2126.0 mg/kg.

Abstract 1038: A potent and selective PARP14 inhibitor decreases pro-tumor macrophage function and elicits inflammatory responses in tumor explants

Abstract PARPs (poly-ADP-ribose polymerases) are a family of enzymes that regulate a wide variety of important cellular processes including cellular stress signaling pathways implicated in inflammation and cancer. PARP14 is a member of the monoPARP sub-family of PARPs, which catalyze transfer of a single ADP ribose unit (mono-ADP-ribosylation or MARylation) to their substrates, unlike their polyPARP counterparts which construct chains of ADP ribose units (poly-ADP-ribosylation or PARylation). PARP14 is an interferon-stimulated gene (ISG) that is overexpressed in tumors compared to normal tissues and has been implicated by genetic knockout studies to promote pro-tumor macrophage polarization and suppress antitumor inflammatory response due to its role in modulating IL-4 and IFN-γ signaling pathways. Efforts to further explore and validate the role of PARP14 catalytic activity in these pathways have been hampered by a lack of potent, highly selective PARP14 inhibitors. Here we describe the discovery of the first such chemical probe, RBN012759, which inhibits PARP14 with an IC50 of 0.003 µM and exhibits &amp;gt;300-fold selectivity over all mono- and polyPARP family members. Medicinal chemistry efforts that began with an unselective micromolar screening hit were enabled by a detailed understanding of the PARP14 and broader PARP family binding pockets. X-ray co-crystal structures of RBN012759 and key early analogs bound to PARP14 clearly illustrate the origins of their potency and selectivity. RBN012759 is a cell permeable, soluble probe that demonstrates robust, dose-dependent stabilization of endogenous PARP14 and inhibition of MARylation in primary human macrophages. RBN012759 reverses IL-4 driven (pro-tumor) gene expression in macrophages, confirming published data generated with PARP14 KO and supporting an immune suppressive role of PARP14 in tumors. Moreover, we demonstrate that PARP14 inhibition in primary human tumor explants can induce an inflammatory mRNA signature similar to immune checkpoint therapy. Summary: Structure-based design of the first potent and highly selective inhibitor of PARP14, RBN012759, is described. The discovery of this chemical probe enabled exploration of the role of PARP14 in macrophage polarization and inflammatory pathways. RBN012759 reverses IL-4 driven gene expression in macrophages and induces an inflammatory mRNA signature in human tumor explants, data which support an immune suppressive role of PARP14 in tumors. Citation Format: Laurie Schenkel, Jennifer Molina, Kerren Swinger, Ryan Abo, Danielle Blackwell, Anne Cheung, William Church, Kristy Kuplast-Barr, Alvin Lu, Elena Minissale, Mario Niepel, Melissa Vasbinder, Tim Wigle, Victoria Richon, Heike Keilhack, Kevin Kuntz. A potent and selective PARP14 inhibitor decreases pro-tumor macrophage function and elicits inflammatory responses in tumor explants [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1038.

Chemical Synthesis and Biological Application of Modified Oligonucleotides.

RNA plays a myriad of roles in the body including the coding, decoding, regulation, and expression of genes. RNA oligonucleotides have garnered significant interest as therapeutics via antisense oligonucleotides or small interfering RNA strategies for the treatment of diseases ranging from hyperlipidemia, HCV, and others. Additionally, the recently developed CRISPR-Cas9 mediated gene editing strategy also relies on Cas9-associated RNA strands. However, RNA presents numerous challenges as both a synthetic target and a potential therapeutic. RNA is inherently unstable, difficult to deliver into cells, and potentially immunogenic by itself or upon modification. Despite these challenges, with the help of chemically modified oligonucleotides, multiple RNA-based drugs have been approved by the FDA. The progress is made possible due to the nature of chemically modified oligonucleotides bearing advantages of nuclease stability, stronger binding affinity, and some other unique properties. This review will focus on the chemical synthesis of RNA and its modified versions. How chemical modifications of the ribose units and of the phosphatediester backbone address the inherent issues with using native RNA for biological applications will be discussed along the way.

Role of APD-Ribosylation in Bone Health and Disease.

The transfer of adenosine diphosphate (ADP)-ribose unit(s) from nicotinamide adenine dinucleotide (NAD+) to acceptor proteins is known as ADP-ribosylation. This post-translational modification (PTM) unavoidably alters protein functions and signaling networks, thereby impacting cell behaviors and tissue outcomes. As a ubiquitous mechanism, ADP-ribosylation affects multiple tissues, including bones, as abnormal ADP-ribosylation compromises bone development and remodeling. In this review, we describe the effects of ADP-ribosylation in bone development and maintenance, and highlight the underlying mechanisms.

One-Carbon Metabolism Supports S-Adenosylmethionine and Histone Methylation to Drive Inflammatory Macrophages

Activated macrophages adapt their metabolic pathways to drive the pro-inflammatory phenotype, but little is known about the biochemical underpinnings of this process. Here, we find that lipopolysaccharide (LPS) activates the pentose phosphate pathway, the serine synthesis pathway, and one-carbon metabolism, the synergism of which drives epigenetic reprogramming for interleukin-1β (IL-1β) expression. Glucose-derived ribose and one-carbon units fed by both glucose and serine metabolism are synergistically integrated into the methionine cycle through de novo ATP synthesis and fuel the generation of S-adenosylmethionine (SAM) during LPS-induced inflammation. Impairment of these metabolic pathways that feed SAM generation lead to anti-inflammatory outcomes, implicating SAM as an essential metabolite for inflammatory macrophages. Mechanistically, SAM generation maintains a relatively high SAM:S-adenosylhomocysteine ratio to support histone H3 lysine 36 trimethylation for IL-1β production. We therefore identify a synergistic effect of glucose and amino acid metabolism on orchestrating SAM availability that is intimately linked to the chromatin state for inflammation.

Regulation of poly(ADP-Ribose) polymerase 1 functions by post-translational modifications.

The poly(ADP-ribose) polymerases (PARPs) catalyze poly(ADP-ribosyl)ation, a post-translational modification of proteins. This consists of the attachment of mono- or poly-adenosine diphosphate (ADP)-ribose units from nicotinamide adenine dinucleotide (NAD+) to specific polar residues of target proteins. PARP1 is the most abundant and best-characterized member of the family of PARP enzymes. PARP1 plays key roles in DNA repair, as well as a wide variety of cellular processes, including transcriptional regulation, chromatin modulation, cellular signaling pathway, inflammation, cellular stress responses and so on. Hence, PARP1 inhibitors have become a promising therapeutic approach for human diseases including cancer. Recent studies indicate that post-translational modifications (PTMs) such as phosphorylation, acetylation, and methylation are crucial for the regulation of PARP1 activity, and dysregulation of modifications on PARP1 is observed in human cancer. In this review, we describe the importance of PTMs to regulate the activity of PARP1, and the involvement of dysregulation of PTMs in human cancer.

Tricyclanos: conformationally constrained nucleoside analogues with a new heterotricycle obtained from a d-ribofuranose unit.

A novel type of nucleoside analogue in which the sugar part is replaced by a new tricycle, 3,7,10-trioxa-11-azatricyclo[5.3.1.05,11]undecane has been prepared by substrate-controlled asymmetric synthesis. 1,5-Dialdehydes obtained from properly protected or unprotected uridine, ribothymidine, cytidine, inosine, adenosine and guanosine by metaperiodate oxidation reacted readily with tris(hydroxymethyl)aminomethane to provide the corresponding tricyclic derivatives with three new stereogenic centers. Through a double cyclisation cascade process the tricyclic compounds were obtained in good to high yields, with very high diastereoselectivity. Formation of one stereoisomer, out of the eight possible, was observed in all cases. The absolute configuration of the new stereotriad-containing tricyclic systems was aided by conventional NMR experiments followed by chemical shift calculations using an X-ray crystal structure as reference that was in good agreement with H-H distances obtained from a new ROESY NMR method. The synthesis was compatible with silyl, trityl and dimethoxytrityl protecting groups. A new reagent mixture containing ZnCl2, Et3SiH and hexafluoroisopropanol was developed for detritylation of the acid-sensitive tricyclano nucleosides.

Abstract 3218: Molecular function of PARP inhibition in glioblastoma

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor, whose major biomarkers are the DNA methylation status of the promoter for O6-methylguanine-DNA-methyltransferase (MGMT) and the loss/mutation of the tumor suppressor phosphatase and tensin homolog (PTEN). Despite treatment combining surgery, radiotherapy and chemotherapy with the alkylating agent temozolomide (TMZ), patient survival remains poor and recurrence is virtually inevitable, due to TMZ resistance. Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear protein involved in multiple facets of DNA repair and transcriptional regulation. This enzyme catalyses the transfer and polymerization of ADP ribose units from NAD+ to form branched polymers of ADP-ribose, called poly(ADP-ribose) (PAR), covalently attached to heterologous acceptor proteins or PARP-1 itself. There is a growing interest in PARP inhibitors (PARPi) for their clinical potential in cancer treatment. Because of the importance of PARP-1 activity in the maintenance of genomic integrity and DNA repair, treatment with PARPi as adjuvant to TMZ chemotherapy can be beneficial in GBM. PARPi are currently in clinical trials for patients with GBM (NCT02152982, NCT01390571, NCT01514201), and one of them is a phase II/III study of the PARPi veliparib for patients with newly-diagnosed GBM (NCT02152982). However, the molecular role of PARP-1 and its activity in GBM remains elusive. Using cellular and molecular approach, we are assessing the effectiveness of the PARPi veliparib compared to standard treatment with TMZ in PTEN-mutated GBM and TMZ-resistant GBM cell lines. By understanding the biology of PARP inhibition in GBM, we will establish a specific molecular signature that predicts the efficacy of PARPi treatment in patients diagnosed with GBM. Citation Format: Christopher Aldrighetti, Barbara Huebert, Delphine Quenet. Molecular function of PARP inhibition in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3218. doi:10.1158/1538-7445.AM2017-3218

Using Clickable NAD+ Analogs to Label Substrate Proteins of PARPs.

ADP-ribosylation has been well known as an important posttranslational modification, which is catalyzed by a family of enzymes called poly(ADP-ribose) polymerases (PARPs). PARPs transfer of a single or multiple adenine diphosphate ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD+) to specific amino acids on substrate proteins. Through mono- or poly-ADP-ribosylation enzymatic activities, PARPs regulate various biological processes, including DNA damage repair, chromatin remodeling, transcriptional regulation, RNA processing and metabolism. Notably, PARP inhibitors are in clinical trials to treat human diseases, in particular cancer. To further investigate the biological function of PARPs, and to achieve better therapeutic effect of PARP inhibitors, it is important to identify the physiological substrates of PARPs. Here we describe a protocol to use clickable analog of nicotinamide adenine dinucleotide (NAD+) that can be applied for the detection, affinity purification and identification of substrate proteins of PARPs.

Magnesium pyrophosphates in enzyme mimics of nucleotide synthases and kinases and in their prebiotic chemistry

Derivatives of ribosyl pyrophosphate have been synthesized, and examined with magnesium salts in the coupling of the ribose unit to various nucleophiles, including pyrazole and 2-chloroimidazole. Only with the magnesium salt present did they generate the ribosyl cation by binding to the leaving group and then couple the ribose derivative with nucleophiles. The role of magnesium salts in phosphorylation of methanol by ATP was also examined. Here a remarkable effect was seen: phosphorylation by ATP was slowed with low concentrations of Mg(2+) but accelerated by higher concentrations. Related effects were also seen in the effect of Mg(2+) on phosphorylation by ADP. The likely mechanisms explain these effects.

Effective Antibiofilm Polyketides against Staphylococcus aureus from the Pyranonaphthoquinone Biosynthetic Pathways of Streptomyces Species.

Streptomyces bacteria are renowned for their ability to produce bioactive secondary metabolites. Recently, synthetic biology has enabled the production of intermediates and shunt products, which may have altered biological activities compared to the end products of the pathways. Here, we have evaluated the potential of recently isolated alnumycins and other closely related pyranonaphthoquinone (PNQ) polyketides against Staphylococcus aureus biofilms. The antimicrobial potency of the compounds against planktonic cells and biofilms was determined by redox dye-based viability staining, and the antibiofilm efficacy of the compounds was confirmed by viable counting. A novel antistaphylococcal polyketide, alnumycin D, was identified. Unexpectedly, the C-ribosylated pathway shunt product alnumycin D was more active against planktonic and biofilm cells than the pathway end product alnumycin A, where a ribose unit has been converted into a dioxane moiety. The evaluation of the antibiofilm potential of other alnumycins revealed that the presence of the ribose moiety in pyranose form is essential for high activity against preformed biofilms. Furthermore, the antibiofilm potential of other closely related PNQ polyketides was examined. Based on their previously reported activity against planktonic S. aureus cells, granaticin B, kalafungin, and medermycin were also selected for testing, and among them, granaticin B was found to be the most potent against preformed biofilms. The most active antibiofilm PNQs, alnumycin D and granaticin B, share several structural features that may be important for their antibiofilm activity. They are uncharged, glycosylated, and also contain a similar oxygenation pattern of the lateral naphthoquinone ring. These findings highlight the potential of antibiotic biosynthetic pathways as a source of effective antibiofilm compounds.