Structure, Function and Selective Inhibition of ADP-ribosyltransferases

Project Leader
Docent Lari Lehtiö, Ph.D.
Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu

Background and Significance

Diphtheria toxin-like human ADP-ribosyltransferases (ARTDs), also known as poly (ADP-ribose) polymerases (PARPs), are enzymes that catalyze a covalent modification of target proteins – ADP-ribosylation. In the reaction NAD+ cofactor is cleaved to nicotinamide and ADP-ribose and an ADP-ribosyl group is attached to the target protein or to the growing polymer chain. The modification changes properties of the target protein and the resulting ADP-ribose polymer also interacts with various proteins. The polymer is metabolized by glucohydrolases that limit the time of the signalling event.

The human ARTD enzyme family consists of 18 multidomain proteins all sharing a conserved catalytic domain. The human enzyme family can be divided on the basis of activity to polymerases (ARTD1–6), mono ADP-ribosyltransferases (mARTDs; ARTD8–18) and the probably inactive ARTD9/13.

The widely studied human enzyme ARTD1/PARP1 is involved in many processes, but it is mainly known for its function in DNA repair mechanisms. On the other hand, ARTD1 is also linked to death of the cell, as over-activation of ARTD1, due to extensive DNA damage, leads to cell death. The involvement of ARTD1 in DNA repair implies that inhibitors could be used to enhance the effects of anticancer drugs, whereas the involvement in cell death suggests that inhibitors could be used in cases of injury and inflammation. Currently many ARTD1 inhibitors or “PARP inhibitors” are in clinical trials and the first PARP inhibitors have recently been accepted for clinical use. Notably, other members of the ARTD family also have functions that could be potentially utilized in therapeutics, especially for cancer, as many ARTD enzymes are involved in DNA repair as well as in cell survival and proliferation.

Recent Progress

Development of chemical probes and drug leads
One of the focus areas of our group is the development of chemical probes to be used as tools to study enzymes and development of drug leads to facilitate the development of therapeutics and to validate enzymes as drug targets. Our drug discovery efforts have recently been focused on tankyrases (TNKS1/ARTD5 and TNKS2/ARTD6), which are promising drug targets due to their involvement in the Wnt signalling pathway. In recent years several new inhibitors have been described and they can be classified by their binding to the nicotinamide subsite, to the adenosine subsite or to both sites. We have studied different inhibitors anchoring either to the nicotinamide or to the adenosine binding sites, which has enabled us to develop several potent and selective tankyrase inhibitors. We use protein X-ray crystallography to study protein–ligand interactions and this molecular information is combined with various techniques ranging from enzyme inhibition measurements to calorimetry (Figure 1). Several new inhibitor scaffolds to be used as drug leads have been developed together with our collaborators.

We have analysed inhibitor selectivity with activity assays, which we have now optimized for the majority of recombinantly produced human ARTDs. In addition to structure-based design we are also screening random compound libraries and this way we have been able to find new chemical probes, which are currently under characterization and further development. During 2016 we reported on a first-in-class inhibitor of a mARTD enzymes (Venkannagari et al. 2016). This discovered small molecule has specificity towards ARTD10. The compound is not toxic, rescues the cells from ARTD10 induced cell death and sensitizes cells to DNA damage likely through a role of ARTD10 in S phase DNA repair. Based on structural analysis the first mARTD inhibitor will allow us to develop further chemical probes targeting the mARTD subclass and later study their functions and perhaps validate some of them as new drug targets.


Figure 1. Ligand-binding studies using different techniques. A) Dose–response measurement of ARTD10 inhibitor with a fluorescence-based activity assay. B) Thermal shift assay of different small molecules showing various degrees of protein stabilization. C) Isothermal titration calorimetry of a ligand to a macro domain. D) Crystal structure of a tankyrase-inhibitor complex.

Structural and functional studies of multidomain ARTDs
ARTD enzymes contain a homologous transferase domain usually located at the C-terminus, and in addition to that many other domains which differ between enzymes. These domains contribute to the function and localization of the enzymes. The interplay between domains in interaction and activation processes is largely unknown and we use both biophysical and structural methods in order to understand how the enzymes function. We use X-ray crystallography to study the structures of full-length enzymes, individual domains and macromolecular complexes and combine this with low-resolution structural studies (Figure 3). Activity assays, which we use also for screening of compound libraries, allow us to study the effects of interaction partners on catalytic activity. We use calorimetry and fluorescence polarization to study affinities and stoichiometry between macromolecules and we create various truncated constructs to study the roles of individual domains. We recently reported characterization of human ARTD2 (Obaji et al. 2016), which is one of the ARTD enzymes responsible for recognition of a DNA damage and initiation of the repair process. Using multiple biophysical methods we identified that the enzyme binds all DNA oligonucleotides with high affinity, but is only robustly activated by 5’ phosphorylated oligonucleotides. The disordered N-terminal part, unique for ARTD2, appears to be a high affinity DNA binding module, while the following WGR domain is responsible for detection of 5’ phosphorylated DNA and activation of the C-terminal catalytic domain. Small angle X-ray scattering helped us to study the organization of the protein at the site of the damage. In case of a nick DNA the protein binds the site preferentially as a monomer, while at the double strand break it binds as an asymmetric dimer. This may allow ARTD2 to have distinct mechanisms of activating different DNA repair cascades.


Figure 3. We use small angle X-ray scattering combined with crystal structures and biophysical methods in order to understand how multidomain enzymes work. Rigid-body modelling of crystal structures of individual protein domains and DNA is shown, as fitted to the low-resolution SAXS envelope.

Future Goals

An important goal of the group is to develop effective and robust methods to screen inhibitors and especially to efficiently test the inhibitors against all members of the human ARTD family. Selective inhibitors will make it possible to study and verify the biological functions of the enzymes. This would potentially also create new drug leads, as many of the enzymes appear to have functions and interactions that are potentially of pharmaceutical interest. We have already identified selective inhibitors of some human ARTD enzymes and they are currently being evaluated using structural studies and cell-based assays.

We have expanded the set of target proteins involved in ADP-ribosylation and are currently carrying out structural and functional studies as well as screening assay development and inhibitor discovery efforts on several enzymes, which perform ADP-ribosylation, bind and recognize the modification or hydrolyse it. There are many unanswered questions regarding the functions of the enzymes involved in ADP-ribosylation and we aim to understand the molecular details using activity assays, biophysical methods and structural studies.

Selected Publications

Nathubhai A, Haikarainen T, Koivunen J, Murthy S, Koumanov F, Lloyd MD, Holman GD, Pihlajaniemi T, Tosh D, Lehtiö L, Threadgill MD. Highly potent and isoform-selective dual-site-binding tankyrase/Wnt signaling inhibitors that increase cellular glucose uptake and have anti-proliferative activity. J Med Chem 60:814-820, 2017.

Venkannagari H, Verheugd P, Koivunen J, Haikarainen T, Obaji E, Narwal M, Pihlajaniemi T, Lüscher B, Lehtiö L. Chemical probe rescues cells from ARTD10/PARP10 induced apoptosis and sensitizes cancer cells to DNA damage. Cell Chem Biol 23:1251-1260, 2016.

Schlesinger M, Vilchez Larrea SC, Haikarainen T, Narwal M, Venkannagari H, Flawiá MM, Lehtiö L, Fernández Villamil SH. Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei.  Parasit Vectors 9:173, 2016.

Obaji E, Haikarainen T, Lehtiö L. Characterization of the DNA dependent activation of human ARTD2/PARP2. Sci Rep 6:34487, 2016.

Nathubhai A, Wood PJ, Haikarainen T, Hayward PC, Muñoz-Descalzo S, Thompson AS, Lloyd MD, Lehtiö L, Threadgill MD. Structure-activity relationships of 2-arylquinazolin-4-ones as highly selective and potent inhibitors of the tankyrases. Eur J Med Chem 118:316-327, 2016.

Haikarainen T, Lehtiö L. Proximal ADP-ribose Hydrolysis in Trypanosomatids is Catalyzed by a Macrodomain. Sci Rep 6:24213, 2016.

Haikarainen T, Waaler J, Ignatev A, Nkizinkiko Y, Venkannagari H, Obaji E, Krauss S, Lehtiö L. Development and structural analysis of adenosine site binding tankyrase inhibitors. Bioorg Med Chem Lett 26:(2):328-33, 2016

Kumpan K, Nathubhai A, Zhang C, Wood PJ, Lloyd MD, Thompson AS, Haikarainen T, Lehtiö L, Threadgill MD. Structure-based design, synthesis and evaluation in vitro of arylnaphthyridinones, arylpyridopyrimidinones and their tetrahydro derivatives as inhibitors of the tankyrases. Bioorg Med Chem 23:3013-32, 2015

Nkizinkiko Y, Suneel Kumar BV, Jeankumar VU, Haikarainen T, Koivunen J, Madhuri C, Yogeeswari P, Venkannagari H, Obaji E, Pihlajaniemi T, Sriram D, Lehtiö L. Discovery of potent and selective nonplanar tankyrase inhibiting nicotinamide mimics. Bioorg Med Chem 23:4139-4149, 2015

Paine HA, Nathubhai A, Woon EC, Sunderland PT, Wood PJ, Mahon MF, Lloyd MD, Thompson AS, Haikarainen T, Narwal M, Lehtiö L, Threadgill MD. Exploration of the nicotinamide-binding site of the tankyrases, identifying 3-arylisoquinolin-1-ones as potent and selective inhibitors in vitro. Bioorg Med Chem 23:5891-908, 2015

Lehtiö L, Jemth A-S, Collins R, Loseva O, Johansson A, Markova N, Hammarström M, Flores A, Holmberg-Schiavone L, Weigelt J, Helleday T, Schüler H, Karlberg T. (2009) Structural basis for inhibitor specificity in human poly(ADP-ribose) polymerase-3. J Med Chem 52:3108-3111

Lehtiö L, Collins R, van den Berg S, Johansson A, Dahlgren LG, Hammarström M, Helleday T, Holmberg-Schiavone L, Kalrberg T, Weigelt J. (2008) Zinc binding catalytic domain of human tankyrase 1. J. Mol. Biol. 379:136-145

Narwal, M., Fallarero, A., Vuorela, P. & Lehtiö, L. (2012a) Homogenous Screening Assay for Human Tankyrase. J. Biomol. Screen. 17:593-604

Narwal, M., Venkannagari, H. & Lehtiö, L. (2012b) Structural Basis of Selective Inhibition of Human Tankyrases. J. Med. Chem. 55:1360-1367

Vilchez Larrea SC, Haikarainen T, Narwal M, Schlesinger M, Venkannagari H, Flawia MM, Fernandez Villamil SH, Lehtiö L. (2012) Inhibition of poly(ADP-ribose) polymerase interferes with Trypanosoma cruzi infection and proliferation of the parasite. PLoS One 7:e46063

Narwal M, Koivunen J, Haikarainen T, Obaji E, Legala O, Venkannagari H, Joensuu P, Pihlajaniemi T, Lehtiö L. Discovery of tankyrase inhibiting flavones with increased potency and isoenzyme selectivity. J Med Chem 56:7880-7889, 2013

Haikarainen T, Koivunen J, Narwal M, Venkannagari H, Obaji E, Joensuu P, Pihlajaniemi T, Lehtiö L. para-substituted 2-phenyl-3,4-dihydroquinazolin-4-ones as potent and selective tankyrase inhibitors. ChemMedChem 8:1978-1985, 2013

Haikarainen T, Venkannagari H, Narwal M, Obaji E, Lee HW, Nkizinkiko Y, Lehtiö L. Structural basis and selectivity of tankyrase inhibition by a Wnt signaling inhibitor WIKI4. PloS One 8:e65404, 2013

Lehtiö L, Chi NW, Krauss S. Tankyrases as drug targets. FEBS J 280:3576-3593, 2013

Narwal M, Haikarainen T, Fallarero A, Vuorela PM, Lehtiö L. Screening and structural analysis of flavones inhibiting tankyrases. J Med Chem 56:3507-3517, 2013

Venkannagari H, Fallarero A, Feijs KL, Lüscher B, Lehtiö L. Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors. Eur J Pharm Sci 49:148-156, 2013.

Haikarainen T, Narwal M, Joensuu P, Lehtiö L. Evaluation and Structural Basis for the inhibition of Tankyrases by PARP Inhibitors. ACS Med Chem Lett 20:18-22, 2014

Haikarainen T, Krauss, S, Lehtiö L. Tankyrases: Structure, Function and Therapeutic Implications in Cancer. Curr Pharm Des 20:6472-6488, 2014

Doctoral Theses 2016

Harikanth Venkannagari: Discovery of selective small molecule inhibitors towards human Diphtheria toxin-like mono-ADP-ribosyltransferases. FBMM, 2016

Research Group Members


Project Leader:
Lari Lehtiö, Ph.D., Docent (Academy of Finland)

Senior and Post-doctoral Investigators:
Teemu Haikarainen, Ph.D. (Academy of Finland, Biocenter Oulu)
Alexander Ignatev, Ph.D. (Jane and Aatos Erkko Foundation)
Mirko Maksimainen, Ph.D. (Academy of Finland)

Ph.D. Students:
Harikanth Venkannagari, Ph.D. (Sigrid Jusélius Foundation, Academy of Finland)
Ezeogo Obaji, M.Sc. (Sigrid Jusélius Foundation, Jane and Aatos Erkko Foundation)
Yves Nkizinkiko, M.Sc. (Biocenter Oulu)
Sudarshan Murthy, M.Sc. (Sigrid Jusélius Foundation)
Yashwanth Ashok, M.Sc. (Biocenter Oulu)

Undergraduate Students:
Elena Rolina, B.Sc.
Reduanul Bari, B.Sc.

Foreign Scientists, 8

National and International Activities

Visiting Researchers in 2016 (over two weeks)
Dr Amos Fatokun, University of Bradford, UK

Last updated: 7.4.2017