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 likely inactive ARTD13.

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 - tankyrases
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. Several new inhibitor scaffolds to be used as drug leads have been developed together with our collaborators.

In order to develop potent and selective tankyrase inhibitors we have recently used a hybridization strategy to combine features from existing and newly discovered inhibitors to form improved compounds. In order to create a sub-nM inhibitor we used a nicotinamide binding small molecule and extended the compound towards a adenosine binding site. This formed a so called dual-site binder, which spans the whole NAD+ binding cleft in the active site (Figure 1A). The compound was selective for tankyrases over the other ARTD enzymes, around 30 nM in cell-based Wnt reporter assay, enhanced insulin uptake, and inhibited growth of colorectal cancer cells (Nathubhai et al. 2017). Hybridization of features from two adenosine site binding inhibitors using was a key in a discovery of a 6 nM inhibitor with extremely good target selectivity within the ARTD enzyme family and 20 nM IC50 in Wnt inhibition assay (Figure 1B). We also demonstrated that the resulting compound had a good ADME profile with no inhibition of kinases as well as good oral bioavailability in mouse, rat and dog (Anumala et al. 2017). The compound was also efficient in xenograft cancer models.

Figure 1. Highly potent and selective tankyrase inhibitors reported in 2017. A) A potent dual site binder crystal structure with tankyrase 2 (Nathubhai et al. 2017). B) A crystal structure of a tankyrase inhibitor with good ADME profile (Anumala et al. 2017).

Structural and functional studies DNA repair 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. 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. With help of the crystal structures of the ARTD WGR domain with various oligonucleotides and mutagenesis we were able to assign roles for the residues recognizing the DNA damage and transferring the activation signal to the catalytic domain (Figure 2). The structures also show how ARTD2 is able to join and protect loose DNA ends in the case of the double strand break (Obaji et al. submitted).

Figure 2. Crystal structure of ARTD2 WGR domain with DNA break consisting of two double stranded blunt end DNA molecules with a 5´-phosphate.

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. In addition to tankyrase inhibitors which are in hit-to-lead development phase, we have also identified selective inhibitors of other 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.

Publications 2017-

Anumala UR, Waaler J, Nkizinkiko Y, Ignatev A, Lazarow K, Lindemann P, Olsen PA, Murthy S, Obaji E, Majouga AG, Leonov S, von Kries JP, Lehtiö L, Krauss S, Nazaré M. Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach. J. Med. Chem. 60:10013-10025, 2017.

Haikarainen T, Schlesinger M, Obaji E, Fernández Villamil SH, Lehtiö L. Structural and Biochemical Characterization of Poly-ADP-ribose Polymerase from Trypanosoma brucei. Sci. Rep. 7:3642, 2017.

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 Antiproliferative Activity. J. Med. Chem. 60:814-820, 2017.

Haikarainen T, Maksimainen MM, Obaji E, Lehtiö L. Development of an Inhibitor  Screening Assay for Mono-ADP-Ribosyl Hydrolyzing Macrodomains Using AlphaScreen Technology. SLAS Discov. in press, 2018.

Nkizinkiko Y, Desantis J, Koivunen J, Haikarainen T, Murthy S, Sancineto L, Massari S, Ianni F, Obaji E, Loza MI, Pihlajaniemi T, Brea J, Tabarrini O, Lehtiö L. 2-Phenylquinazolinones as dual-activity tankyrase-kinase inhibitors. Sci. Rep. in press, 2018.

Research Group Members 2017


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

Senior and Post-doctoral Investigators:
Mirko Maksimainen, Ph.D. (Academy of Finland)
Alexander Ignatev, Ph.D. (Jane and Aatos Erkko Foundation)
Yashwanth Ashok, Ph.D. (Academy of Finland, Jane and Aatos Erkko Foundation)
Albert Galera Prat, Ph.D. (Jane and Aatos Erkko Foundation)
Teemu Haikarainen, Ph.D. (University of Oulu)

Ph.D. Students:
Ezeogo Obaji, M.Sc. (Academy of Finland, Orion Research Foundation)
Yves Nkizinkiko, M.Sc. (Magnus Ehrnrooth Foundation)
Sudarshan Murthy, M.Sc. (Sigrid Jusélius Foundation)
Sven Sowa, M. Sc. (Jane and Aatos Erkko Foundation)

Undergraduate Students:
Elena Rolina, B. Sc.
Reduanul Bari, B. Sc.
Sarah Wazir, M. Sc.
Danilo Kimio Hirabae De Oliveira, B. Sc.

Foreign Scientists, 11

National and International Activities

Visiting Researchers in 2017 (over two weeks)
Sudeep Karki, University of Helsinki, Finland
Awlokita Tiwari, University of Turku, Finland
Laura Kevorkian, University of Buenos Aires, Argentina (CIMO)

Last updated: 6.7.2018