Gene/Protein Disease Symptom Drug Enzyme Compound
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The poly(ADP-ribose) (PAR) post-translational modification is essential for diverse cellular functions, including regulation of transcription, response to DNA damage, and mitosis. Cellular PAR is predominantly synthesized by the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a critical node in the DNA damage response pathway, and multiple potent PARP-1 inhibitors have been described, some of which show considerable promise in the clinic for the treatment of certain cancers. Cellular PAR is efficiently degraded by poly(ADP-ribose) glycohydrolase (PARG), an enzyme for which no potent, readily accessible, and specific inhibitors exist. Herein we report the discovery of small molecules that effectively inhibit PARG in vitro and in cellular lysates. These potent PARG inhibitors can be produced in two chemical steps from commercial starting materials and have complete specificity for PARG over the other known PAR glycohydrolase (ADP-ribosylhydrolase 3, ARH3) and over PARP-1 and thus will be useful tools for studying the biochemistry of PAR signaling.
ACS Chem Biol 2012 Mar 16
PMID:Selective small molecule inhibition of poly(ADP-ribose) glycohydrolase (PARG). 2222 Sep 26

Werner syndrome is a premature aging disorder that is caused by defects in the Werner protein (WRN). WRN is a member of the RecQ helicase family and possesses helicase and exonuclease activities. It is involved in various aspects of DNA metabolism such as DNA repair, telomere maintenance, and replication. Poly(ADP-ribose) polymerase 1 (PARP1) is also involved in these processes by catalyzing the formation of the nucleic-acid-like biopolymer poly(ADP-ribose) (PAR). It was previously shown that WRN interacts with PARP1 and that WRN activity is inhibited by PARP1. Using several bioanalytical approaches, here we demonstrate that the enzymatic product of PARP1, i.e., PAR, directly interacts with WRN physically and functionally. First, WRN binds HPLC-size-fractionated short and long PAR in a noncovalent manner. Second, we identified and characterized a PAR-binding motif (PBM) within the WRN sequence and showed that several basic and hydrophobic amino acids are of critical importance for mediating the PAR binding. Third, PAR-binding inhibits the DNA-binding, the helicase and the exonuclease activities of WRN in a concentration-dependent manner. On the basis of our results we propose that the transient nature of PAR produced by living cells would provide a versatile and swiftly reacting control system for WRN's function. More generally, our work underscores the important role of noncovalent PAR-protein interactions as a regulatory mechanism of protein function.
ACS Chem Biol 2013 Jan 18
PMID:Site-specific noncovalent interaction of the biopolymer poly(ADP-ribose) with the Werner syndrome protein regulates protein functions. 2308 94

Poly(ADP-ribosyl)ation is an essential post-translational modification with the biopolymer poly(ADP-ribose) (PAR). The reaction is catalyzed by poly(ADP-ribose) polymerases (PARPs) and plays key roles in cellular physiology and stress response. PARP inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality. We have developed an accurate and sensitive bioanalytical platform based on isotope dilution mass spectrometry in order to quantify steady-state and stress-induced PAR levels in cells and tissues and to characterize pharmacological properties of PARP inhibitors. In contrast to existing PAR-detection techniques, the LC-MS/MS method uses authentic isotope-labeled standards, which provide unequivocal chemical specificity to quantify cellular PAR in absolute terms with femtomol sensitivity. Using this platform we analyzed steady-state levels as well as stress-induced dynamics of poly(ADP-ribosyl)ation in a series of biological systems including cancer cell lines, mouse tissues, and primary human lymphocytes. Our results demonstrate a rapid and transient stress-induced increase in PAR levels by >100-fold in a dose- and time-dependent manner with significant differences between cell types and individual human lymphocyte donors. Furthermore, ex vivo pharmacodynamic studies in human lymphocytes provide new insight into pharmacological properties of clinically relevant PARP inhibitors. Finally, we adapted the LC-MS/MS method to quantify poly(ADP-ribosyl)ation in solid tissues and identified tissue-dependent associations between PARP1 expression and PAR levels in a series of different mouse organs. In conclusion, this study demonstrates that mass spectrometric quantification of cellular poly(ADP-ribosyl)ation has a wide range of applications in basic research as well as in drug development.
ACS Chem Biol 2013 Jul 19
PMID:Quantification of cellular poly(ADP-ribosyl)ation by stable isotope dilution mass spectrometry reveals tissue- and drug-dependent stress response dynamics. 2363 32

4-(2-Pyridylazo)-resorcinol (PAR) functionalized thermosensitive ionic microgels (PAR-MG) were synthesized by a one-pot quaternization method. The PAR-MG microgels were spherical in shape with radius of ca. 166.0 nm and narrow size distribution and exhibited thermo-sensitivity in aqueous solution. The PAR-MG microgels could optically detect trace heavy metal ions, such as Cu(2+), Mn(2+), Pb(2+), Zn(2+), and Ni(2+), in aqueous solutions with high selectivity and sensitivity. The PAR-MG microgel suspensions exhibited characteristic color with the presence of various trace heavy metal ions, which could be visually distinguished by naked eyes. The limit of colorimetric detection (DL) was determined to be 38 nM for Cu(2+) at pH 3, 12 nM for Cu(2+) at pH 7, and 14, 79, 20, and 21 nM for Mn(2+), Pb(2+), Zn(2+), and Ni(2+), respectively, at pH 11, which was lower than (or close to) the United States Environmental Protection Agency standard for the safety limit of these heavy metal ions in drinking water. The mechanism of detection was attributed to the chelation between the nitrogen atoms and o-hydroxyl groups of PAR within the microgels and heavy metal ions.
ACS Appl Mater Interfaces 2015 Oct 07
PMID:4-(2-Pyridylazo)-resorcinol Functionalized Thermosensitive Ionic Microgels for Optical Detection of Heavy Metal Ions at Nanomolar Level. 2637 Feb 74

A stable and reusable electrochemical biosensor for the label-free detection of poly(ADP-ribose) polymerase (PARP) is designed in this work. C-kit-1, a thiol-modified G-quadruplex oligonucleotide, is first self-assembled on a gold electrode surface. The G-quadruplex structure of c-kit-1 can specifically tether and activate PARP, resulting in the generation of negatively charged poly(ADP-ribose) polymer (PAR). On the basis of electrostatic attraction, PAR facilitates the surface accumulation of positively charged electrochemical signal molecules. Through the characterization of electrochemical signal molecules, the label-free quantification of PARP is simply implemented. On the basis of the proposed method, selective quantification of PARP can be achieved over the linear range from 0.01 to 1 U with a calculated detection limit of 0.003U. Further studies also demonstrate the applicability of the proposed method to biosamples revealing the broad potential in practical applications. Furthermore, inhibitor of PARP has also been detected with this biosensor. Meanwhile, benefited from self-assembly on solid surface, this biosensor possesses two important features, i.e., reusability and stability, which are desirable in related biosensors.
ACS Appl Mater Interfaces 2016 Jul 27
PMID:Stable and Reusable Electrochemical Biosensor for Poly(ADP-ribose) Polymerase and Its Inhibitor Based on Enzyme-Initiated Auto-PARylation. 2736 74

The enzyme poly(ADP-ribose) glycohydrolase (PARG) performs a critical role in the repair of DNA single strand breaks (SSBs). However, a detailed understanding of its mechanism of action has been hampered by a lack of credible, cell-active chemical probes. Herein, we demonstrate inhibition of PARG with a small molecule, leading to poly(ADP-ribose) (PAR) chain persistence in intact cells. Moreover, we describe two advanced, and chemically distinct, cell-active tool compounds with convincing on-target pharmacology and selectivity. Using one of these tool compounds, we demonstrate pharmacology consistent with PARG inhibition. Further, while the roles of PARG and poly(ADP-ribose) polymerase (PARP) are closely intertwined, we demonstrate that the pharmacology of a PARG inhibitor differs from that observed with the more thoroughly studied PARP inhibitor olaparib. We believe that these tools will facilitate a wider understanding of this important component of DNA repair and may enable the development of novel therapeutic agents exploiting the critical dependence of tumors on the DNA damage response (DDR).
ACS Chem Biol 2016 11 18
PMID:First-in-Class Chemical Probes against Poly(ADP-ribose) Glycohydrolase (PARG) Inhibit DNA Repair with Differential Pharmacology to Olaparib. 2768 88

Poly(ADP-ribosyl)ation (PARylation), i.e., the formation of the nucleic acid-like biopolymer poly(ADP-ribose) (PAR), is an essential posttranslational modification carried out by poly(ADP-ribose) polymerases (PARPs). While PAR levels are low under physiological conditions, they can transiently increase more than 100-fold upon induction of genotoxic stress. The accurate quantitation of cellular PAR with high sensitivity is of critical importance to understand the role of PARylation in cellular physiology and pathophysiology and to determine the pharmacodynamic efficiencies of clinically relevant PARP inhibitors, which represent a novel class of promising chemotherapeutics. Previously, we have developed a bioanalytical platform based on isotope dilution mass spectrometry (LC-MS/MS) to quantify cellular PAR with unequivocal chemical specificity in absolute terms with femtomol sensitivity (Martello et al. ACS Chem Biol 8(7):1567-1575, 2013). This method enables the analysis of steady-state levels, as well as stress-induced levels of PAR in various biological systems including cell lines, mouse tissues, and primary human lymphocytes. It has a wide range of potential applications in basic research, as well as in drug development (Martello et al. ACS Chem Biol 8(7):1567-1575, 2013; Mangerich et al. Toxicol Lett 244:56-71, 2016). Here, we present an improved and adjusted version of the original protocol by Martello/Mangerich et al., which uses UPLC-MS/MS instrumentation.
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PMID:Quantitation of Poly(ADP-Ribose) by Isotope Dilution Mass Spectrometry. 2869 99

Poly(ADP-ribose) polymerase-1 (PARP-1), over expression in vast majority of cancer cells, is a potential biomarker for clinical diagnosis. However, very limited detection methods have been developed so far, especially for in situ intracellular imaging. Here, we developed a spectral-resolved single-particle detection method for detection of PARP-1 in vitro and in situ intracellular imaging with dark-field microscopy (DFM). A gold nanoparticle (50 nm) modified with active DNA duplex (Au50-dsDNA) was used as a scattering probe. Under the function of active dsDNA, PARP-1 catalyzed to synthesize the hyperbranched poly (ADP-ribose) polymer (PAR) by using nicotinamideadenine dinucleotide as substrates, forming Au50-dsDNA@PAR. Then, negatively charged PAR adsorbed positively charged AuNPs (8 nm) to form Au50-dsDNA@PAR@Au8. As a result, a notable red shift occurred in localized surface plasmon resonance scattering spectra of Au50, accompanying with obvious color change. Thus, PARP-1 has been detected with a linear range from 0.2 to 10 mU based on the scattering spectra change. The detection limit was 2 orders of magnitude lower than previously reported methods. Probes showed distinct different colors in cancer cells and normal cells, realizing in situ imaging of intracellular PARP-1 at a single-particle level. Compared with previously reported fluorescence imaging methods, the proposed strategy avoided sophisticated label procedures, which has great potential to be used for clinical diagnosis and PARP-1 inhibitor research.
ACS Sens 2020 04 24
PMID:Single-Particle Assay of Poly(ADP-ribose) Polymerase-1 Activity with Dark-Field Optical Microscopy. 3220 31