Structural Basis for Inhibitor Specificity in Human Poly(ADP-ribose) Polymerase-3 † Lari Lehtio ¨, ‡,§ Ann-Sofie Jemth, | Ruairi Collins, ‡ Olga Loseva, | Andreas Johansson, ‡ Natalia Markova, ‡,⊥ Martin Hammarstro ¨m, ‡ Alex Flores, ‡ Lovisa Holmberg-Schiavone, ‡,§ Johan Weigelt, ‡ Thomas Helleday,* ,|,# Herwig Schu ¨ler,* ,‡ and Tobias Karlberg ‡ Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden. Department of Genetics, Microbiology, and Toxicology, Stockholm UniVersity, SE-10691 Stockholm, Sweden. iNoVacia AB, SE-11251 Stockholm, Sweden. Gray Institute for Radiation Oncology & Biology, UniVersity of Oxford, Oxford OX3 7DQ, United Kingdom ReceiVed January 15, 2009 Poly(ADP-ribose) polymerases (PARPs) activate DNA repair mechanisms upon stress- and cytotoxin-induced DNA damage, and inhibition of PARP activity is a lead in cancer drug therapy. We present a structural and functional analysis of the PARP domain of human PARP-3 in complex with several inhibitors. Of these, KU0058948 is the strongest inhibitor of PARP-3 activity. The presented crystal structures highlight key features for potent inhibitor binding and suggest routes for creating isoenzyme-specific PARP inhibitors. Introduction PolyADP ribosylation is a ubiquitous protein modification involved in the regulation of transcription, cell proliferation, differentiation, and apoptosis. 1,2 Of the 17 human poly(ADP ribose) polymerase (PARP) a enzymes, at least PARP-1, PARP-2 and tankyrase-1 (PARP-5a) are required for the maintenance of genome stability. 3 PARP-1 has important roles in DNA single-strand break and base excision repair. 4 Inhibition of PARP-1 activity may have beneficial effects in a variety of diseases, including stroke, myocardial infarction, heart failure, and diabetes mellitus, where extensive DNA damage may lead to fatigue and tissue necrosis. 5 PARP inhibitors also efficiently and selectively kill BRCA2 defective tumors in monotherapy. 6,7 While PARP inhibitors are in numerous clinical trials, 8 it is unclear whether they act on PARP-1 alone or also on other PARP family members. Given the high degree of conservation of the active sites among these proteins, off-target effects may be expected. Therefore, to better understand the effects of PARP inhibition in disease treatment, information on the specificity and selectivity of PARP inhibitors is urgently needed. Results and Discussion PARP-3 is a poorly characterized family member with extensive homology to PARP-1 and -2. We determined the crystal structure of the PARP domain (residues 178-532) of human PARP-3. Details of the structure determination and refinement are sum- marized in Table S1 in the Supporting Information. The PARP-3 structure consists of an N-terminal R-helical domain and a C-terminal R/-domain (Figure 1). As expected based on sequence similarity (∼35% identity between the PARP domains), this structure is similar to the previously reported structures of the PARP-1 and PARP-2 PARP domains. 9-11 The C-terminal domain contains the PARP signature motif (-R-loop--R) on its inner surface, forming the NAD + donor binding crevice located between the two domains. The C-terminal domain is also similar to that of PARP-5a/tankyrase-1. 12 The residues lining the active site are largely conserved between PARP-1 and -3, apart from a few notable exceptions. The most important differences between the PARP-3 and PARP-1 PARP domains lie in the loops surrounding the active site. The donor site loop (D-loop) in PARP-3 (residues † PDB IDs for the PARP-3 inhibitor complexes: 3FHB, 3C4H, 3C49, and 3CE0. * To whom correspondence should be addressed. For T.H.: phone, +468162914; fax, +468164315; E-mail, helleday@gmt.su.se. For H.S.: phone, +46852486843; fax, +46852486868; E-mail, herwig.schuler@ mbb.ki.se. ‡ Structural Genomics Consortium, Karolinska Institutet. § Present address: For L.L.: Department of Biochemistry and Pharmacy, Åbo Akademi, FI-20520 Turku, Finland. For L.H.S.: Structural Chemistry Laboratory, AstraZeneca R&D, SE-43150 Mo ¨lndal, Sweden. | Department of Genetics, Microbiology and Toxicology, Stockholm University. ⊥ iNovacia AB. # Gray Institute for Radiation Oncology & Biology, University of Oxford. a Abbreviations: ITC, isothermal titration calorimetry; NAD + , nicotina- mide adenine dinucleotide; PARP, poly(ADP ribose) polymerase. Figure 1. Comparison of PARP-3 and PARP-1 structures. Overall comparison of the catalytic fragments of PARP-3 (PDB entry 3C4H) and PARP-1 (PDB entry 2RD6). Blue to red, PARP-3; gray, PARP-1. Ligand 2 from structure 3C4H is shown as a ball-and-stick model to indicate the position of the active site between the domains. Loops surrounding the acceptor site that differ the most between PARP-1 and PARP-3 are labeled, as well as the D-loop lining the NAD + binding site. The D-loop in PARP-3 (residues 398-411) adopts a different conformation, which affects packing of the long helix (residues 233-241) in the N-terminal domain. In addition the D-loop is four residues shorter in PARP-3. These differences in the D-loops may affect the binding affinities of the known PARP inhibitors. PARP-3 structure 3C4H superposes with an rmsd of 1.4 Å to human PARP-1 (2RD6; 319 CR atoms). J. Med. Chem. 2009, 52, 3108–3111 3108 10.1021/jm900052j CCC: $40.75  2009 American Chemical Society Published on Web 04/08/2009