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Recently accepted papers

Sister chromatid cohesion establishment factor ESCO1 operates by substrate-assisted catalysis

In order for cohesion to be established, the cohesin subunit SMC3 needs to be acetylated by a homolog of the ESCO1/Eco1 acetyltransferases, the enzymatic mechanism of which has remained unknown. Here we report the crystal structure of the ESCO1 acetyltransferase domain in complex with acetyl-coenzyme A, and show by SAXS that ESCO1 is a dimer in solution. The structure reveals an active site that lacks a potential catalytic base side chain. However, mutation of glutamate 789, a surface residue that is close to the automodification target lysine 803, strongly reduces autoacetylation of ESCO1. Moreover, budding yeast Smc3 mutated at conserved residue D114, adjacent to the cohesion-activating acetylation site K112, K113, cannot be acetylated in vivo. This indicates that ESCO1 controls cohesion through substrate-assisted catalysis. Thus, this study discloses a key mechanism for cohesion establishment. Published in Kouznetsova et al. Structure, 2016.

Identification of inhibitors of Pseudomonas aeruginosa Exotoxin-S ADP-ribosyltransferase activity.

The purpose of this study was to formulate a real-time assay of purified recombinant ExoS activity for high-throughput application. We characterized the turnover kinetics of the fluorescent dinucleotide 1,N6-etheno-NAD+ as co-substrate for ExoS. Further, we found that the toxin relied on any of five tested isoforms of human 14-3-3 to modify vH-Ras and the Rho-family GTPases Rac1, -2, and -3 and RhoC. We then used 14-3-3β-stimulated ExoS modification of vH-Ras to screen a collection of low-molecular-weight compounds selected to target the poly-ADP ribose polymerase family and identified 3-(4-oxo-3,5,6,7-tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-2-yl)propanoic acid as an ExoS inhibitor with micromolar potency. Thus, we present an optimized method to screen for inhibitors of ExoS activity that is amenable to high-throughput format and an intermediate affinity inhibitor that can serve both as assay control and as a starting point for further development. Published in Pinto et al. Journal of Biomolecular Screening, 2016.

Sirtuins are unaffected by PARP inhibitors containing planar nicotinamide bioisosteres.

We report here that while the activities of the four human sirtuin isoforms SIRT1, SIRT2, SIRT3 and SIRT6 are blocked by sirtuin inhibitor Ex527 in vitro, they are unaffected by the seven clinical and commonly used PARP inhibitors niraparib, olaparib, rucaparib, talazoparib, veliparib, PJ34, and XAV939. These findings indicate that PARP inhibitors containing planar nicotinamide mimetics do not bind to sirtuin cofactor sites. In conclusion, a simple commercially available assay can be used to rule out interference of novel PARP inhibitors with sirtuin NAD+ binding. Published in Ekblad & Schüler. Chemical Biology and Drug Design, 2015.

Lack of ADP-Ribosyltransferase Activity in PARP13

PARP13/ARTD13, also called zinc finger antiviral protein (ZAP), has roles in viral immunity and microRNA mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used X-ray crystallography, molecular dynamics simulations and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD+ binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity. Published in Karlberg et al. Journal of Biological Chemistry, 2015.


A selective inhibitor of PARP3

The small compound ME0328 inhibits PARP3 (ARTD3) in vitro and in cells, resulting in delays in DNA repair. The fluorescent image of DAPI- and gammaH2AX-stained A549 cells (background) was captured by Mareike Hesse. Molecular graphics, layout and composition of the image were prepared and designed by Tobias Karlberg. Inhibitor of PARP3 (ARTD3) that elicits specific effects in cells at sub-micromolar concentrations. Published in Lindgren et al. ACS Chemical Biology, 2013. Further, we found that the stereochemistry is of great importance for both selectivity and potency when evaluating 55 compounds in this class, published in Lindgren et al. Journal of Medicinal Chemistry, 2013.


Recognition of Mono-ADP-Ribosylated PARP10 substrates by PARP14 Macrodomains

PARP14 (ARTD8) Macrodomains 2 and 3 are readers of mono-ADP-ribosylation both in vitro and in cells. The figure shows the solved crystal structure of PARP14 macrodomain 3 in complex with ADP-ribose modelled together with a substrate protein. Published in Forst et al. Structure, 2013.


Discovery of ligands for ADP-Ribosyltransferases via docking-based virtual screening

One isomer of the most promising hit compound A16(E) from docking-based virtual screening bound to PARP14 (ARTD8) in a complex crystal structure. Thermal shift assays show binding to PARP15 (ARTD8) and PARP14. Both isomers confirmed binding in the low micromolar range using isothermal titration calorimetry. These results form a starting point in the development of chemical tools for the study of the role and function of PARP14 and PARP15. Published in Andersson et al. Journal of Medicinal Chemistry, 2012.


Crystal structure of human PARP16

Crystal structure of human PARP16 (ARTD15) reveals a novel putative regulatory domain. The canonical PARP inhibitor 3-aminobenzamide (3-AB), a nicotinamide mimic, is bound in the active site. The helical domain is shown in blue, and the transferase domain in green and orange. Published in Karlberg et al. Journal of Biological Chemistry, 2012.


Family wide chemical profiling of PARP inhibitors

Family wide chemical profiling of PARP inhibitors. Principal component analysis of the complete screening data set showing the activity space of PARP inhibitors according to the first and second components. Positive values of component F1 indicate stabilization of PARP1-4, and positive values of component F2 indicate stabilization of TNKS1-2. Green indicates the PARP1-4 selective, yellow the unselective and red the tankyrase-selective primary hit compounds. Published in Wahlberg et al. Nature Biotechnology, 2012.

PARP target practice Science Business Exchange by Joanne Kotz
PARP family portraits Nature Chemical Biology by Joanne Kotz
News and Views Profiling PARP inhibitor Nature Biotechnology by Philip Jones