Schüler group - Highlights

Structural basis for potency and promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase inhibitors

Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects. Published in Thorsell et al. Journal of Medicinal Chemistry, 2017.

Small molecule microarray based discovery of PARP14 inhibitors

PARP14 in complex with H10

Most small-molecule PARP inhibitors developed to date have been against PARP1, and suffer from poor selectivity. Herein, we describe a small molecule microarray-based strategy for high-throughput synthesis, screening of >1000 potential bidentate inhibitors of PARPs, and the successful discovery of a potent PARP14 inhibitor H10 with >20-fold selectivity over PARP1. Co-crystallization of the PARP14/H10 complex indicated H10 bound to both the nicotinamide and the adenine subsites. Further structure–activity relationship studies identified important binding elements in the adenine subsite. In tumor cells, H10 was able to chemically knockdown endogenous PARP14 activities. Published in Peng et al. Angewandte Chemie International Edition, 2017.

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.

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 ADP-ribose transferase PARP16 reveals a novel putative regulatory domain

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