Identification of Poly(ADP-Ribose) Polymerase Macrodomain Inhibitors Using an AlphaScreen Protocol.

Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay. Several mono-ADP-ribosylation-dependent interactions were identified, and they were found to be in the micromolar affinity range using surface plasmon resonance (SPR). We then focused on the interaction between PARP14 macrodomain-2 and the mono-ADP-ribosylated PARP10 catalytic domain, and probed a ~1500-compound diverse library for inhibitors of this interaction using AlphaScreen. Initial hit compounds were verified by concentration-response experiments using AlphaScreen and SPR, and they were tested against PARP14 macrodomain-2 and -3. Two initial hit compounds and one chemical analog each were further characterized using SPR and microscale thermophoresis. In conclusion, our results reveal novel macrodomain interactions and establish protocols for identification of inhibitors of such interactions.

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.

Sirtuins are Unaffected by PARP Inhibitors Containing Planar Nicotinamide Bioisosteres.

PARP-family ADP-ribosyltransferases (PARPs) and sirtuin deacetylases all use NAD(+) as cosubstrate for ADP-ribosyl transfer. PARP inhibitors are important research tools and several are being evaluated in cancer treatment. With the exception of a few tankyrase inhibitors, all current PARP inhibitors mimic the nicotinamide moiety in NAD(+) and block the nicotinamide binding pocket. 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.

Tankyrase 1 Inhibitors with Drug-like Properties Identified by Screening a DNA-Encoded Chemical Library.

We describe the synthesis and screening of a DNA-encoded chemical library containing 76230 compounds. In this library, sets of amines and carboxylic acids are directly linked producing encoded compounds with compact structures and drug-like properties. Affinity screening of this library yielded inhibitors of the potential pharmaceutical target tankyrase 1, a poly(ADP-ribose) polymerase. These compounds have drug-like characteristics, and the most potent hit compound (X066/Y469) inhibited tankyrase 1 with an IC50 value of 250 nM.

Towards small molecule inhibitors of mono-ADP-ribosyltransferases.

Protein ADP-ribosylation is a post-translational modification involved in DNA repair, protein degradation, transcription regulation, and epigenetic events. Intracellular ADP-ribosylation is catalyzed predominantly by ADP-ribosyltransferases with diphtheria toxin homology (ARTDs). The most prominent member of the ARTD family, poly(ADP-ribose) polymerase-1 (ARTD1/PARP1) has been a target for cancer drug development for decades. Current PARP inhibitors are generally non-selective, and inhibit the mono-ADP-ribosyltransferases with low potency. Here we describe the synthesis of acylated amino benzamides and screening against the mono-ADP-ribosyltransferases ARTD7/PARP15, ARTD8/PARP14, ARTD10/PARP10, and the poly-ADP-ribosyltransferase ARTD1/PARP1. The most potent compound inhibits ARTD10 with sub-micromolar IC50.

Identification of structure-activity relationships from screening a structurally compact DNA-encoded chemical library.

Methods for the rapid and inexpensive discovery of hit compounds are essential for pharmaceutical research and DNA-encoded chemical libraries represent promising tools for this purpose. We here report on the design and synthesis of DAL-100K, a DNA-encoded chemical library containing 103 200 structurally compact compounds. Affinity screening experiments and DNA-sequencing analysis provided ligands with nanomolar affinities to several proteins, including prostate-specific membrane antigen and tankyrase 1. Correlations of sequence counts with binding affinities and potencies of enzyme inhibition were observed and enabled the identification of structural features critical for activity. These results indicate that libraries of this type represent a useful source of small-molecule binders for target proteins of pharmaceutical interest and information on structural features important for binding.

Chemical probes to study ADP-ribosylation: synthesis and biochemical evaluation of inhibitors of the human ADP-ribosyltransferase ARTD3/PARP3.

The racemic 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-N-[1-(pyridin-2-yl)ethyl]propanamide, 1, has previously been identified as a potent but unselective inhibitor of diphtheria toxin-like ADP-ribosyltransferase 3 (ARTD3). Herein we describe synthesis and evaluation of 55 compounds in this class. It was found that the stereochemistry is of great importance for both selectivity and potency and that substituents on the phenyl ring resulted in poor solubility. Certain variations at the meso position were tolerated and caused a large shift in the binding pose. Changes to the ethylene linker that connects the quinazolinone to the amide were also investigated but proved detrimental to binding. By combination of synthetic organic chemistry and structure-based design, two selective inhibitors of ARTD3 were discovered.

PARP inhibitor with selectivity toward ADP-ribosyltransferase ARTD3/PARP3.

Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.

PARP inhibitors: polypharmacology versus selective inhibition.

Inhibition of ADP-ribosyltransferases with diphtheria toxin homology (ARTD), widely known as the poly(ADP-ribose) polymerase (PARP) family, is a strategy under development for treatment of various conditions, including cancers and ischemia. Here, we give a brief summary of ARTD enzyme functions and the implications for their potential as therapeutic targets. We present an overview of the PARP inhibitors that have been used in clinical trials. Finally, we summarize recent insights from structural biology, and discuss the molecular aspects of PARP inhibitors in terms of broad-range versus selective inhibition of ARTD family enzymes.

Discovery of ligands for ADP-ribosyltransferases via docking-based virtual screening.

The diphtheria toxin-like ADP-ribosyltransferases (ARTDs) are an enzyme family that catalyzes the transfer of ADP-ribose units onto substrate proteins by using nicotinamide adenine dinucleotide (NAD(+)) as a cosubstrate. They have a documented role in chromatin remodelling and DNA repair, and inhibitors of ARTD1 and 2 (PARP1 and 2) are currently in clinical trials for the treatment of cancer. The detailed function of most other ARTDs is still unknown. By using virtual screening, we identified small ligands of ARTD7 (PARP15/BAL3) and ARTD8 (PARP14/BAL2). Thermal-shift assays confirmed that 16 compounds, belonging to eight structural classes, bound to ARTD7/ARTD8. Affinity measurements with isothermal titration calorimetry for two isomers of the most promising hit compound confirmed binding in the low micromolar range to ARTD8. Crystal structures showed anchoring of the hits in the nicotinamide pocket. These results form a starting point in the development of chemical tools for the study of the role and function of ARTD7 and ARTD8.

Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors.

Inhibitors of poly-ADP-ribose polymerase (PARP) family proteins are currently in clinical trials as cancer therapeutics, yet the specificity of many of these compounds is unknown. Here we evaluated a series of 185 small-molecule inhibitors, including research reagents and compounds being tested clinically, for the ability to bind to the catalytic domains of 13 of the 17 human PARP family members including the tankyrases, TNKS1 and TNKS2. Many of the best-known inhibitors, including TIQ-A, 6(5H)-phenanthridinone, olaparib, ABT-888 and rucaparib, bound to several PARP family members, suggesting that these molecules lack specificity and have promiscuous inhibitory activity. We also determined X-ray crystal structures for five TNKS2 ligand complexes and four PARP14 ligand complexes. In addition to showing that the majority of PARP inhibitors bind multiple targets, these results provide insight into the design of new inhibitors.

(186)Re-maSGS-Z (HER2:342), a potential Affibody conjugate for systemic therapy of HER2-expressing tumours.

PURPOSE: Affibody molecules are a novel class of tumour-targeting proteins, which combine small size (7 kDa) and picomolar affinities. The Affibody molecule Z(HER2:342) has been suggested for imaging of HER2 expression in order to select patients for trastuzumab therapy. When optimizing chelators for (99m)Tc-labelling, we have found that synthetic Z(HER2:342) conjugated with mercaptoacetyl-glycyl-glycyl-glycyl (maGGG) and mercaptoacetyl-glycyl-seryl-glycyl (maGSG) chelators provides relatively low renal uptake of radioactivity and could be suitable for therapy. METHODS: maGGG-Z(HER2:342) and maGSG-Z(HER2:342) were labelled with (186)Re and their biodistribution was studied in normal mice. Dosimetric evaluation and tumour targeting to HER2-overexpressed xenografts (SKOV-3) by (186)Re-maGSG-Z(HER2:342) were studied. RESULTS: Gluconate-mediated labelling of maGGG-Z(HER2:342) and maGSG-Z(HER2:342) with (186)Re provided a yield of more than 95% within 60 min. The conjugates were stable and demonstrated specific binding to HER2-expressing SKOV-3 cells. Biodistribution in normal mice demonstrated rapid blood clearance, low accumulation of radioactivity in the kidney and other organs, accumulating free perrhenate. Both (186)Re-maGGG-Z(HER2:342) and (186)Re-maGSG-Z(HER2:342) demonstrated lower renal uptake than their (99m)Tc-labelled counterparts. (186)Re-maGSG-Z(HER2:342) provided the lowest uptake in healthy tissues. Biodistribution of (186)Re-maGSG-Z(HER2:342) in nude mice bearing SKOV-3 xenografts showed specific targeting of tumours. Tumour uptake 24 h after injection (5.84+/-0.54%ID/g) exceeded the concentration in blood by more than 500-fold, and uptake in kidneys by about 8-fold. Preliminary dosimetric evaluation showed that dose-to-tumour should exceed dose-to-kidney by approximately 5-fold. CONCLUSION: Optimization of chelators improves biodistribution properties of rhenium-labelled small scaffold proteins and enables selection of promising radiotherapeutic agents based on the Affibody molecule.

Positioning of 99mTc-chelators influences radiolabeling, stability and biodistribution of Affibody molecules.

Affibody molecules represent a novel class of affinity proteins with a high potential as tracers for radionuclide molecular imaging. In this comparative structure-property study, a series of Affibody molecules with the (99m)Tc-chelators maGGG, maSSS, or maESE attached to the epsilon-amine of the internally positioned K49 was prepared by peptide synthesis, for comparison to molecules with similar chelators positioned at the N-terminus. The conjugates were labeled with (99m)Tc and evaluated in vitro and in vivo. It was found that both composition and position of the chelating moiety influence the label stability, biodistribution and targeting properties of HER2-binding Affibody molecules.

Synthesis and chemoselective intramolecular crosslinking of a HER2-binding affibody.

The human epidermal growth factor receptor HER2 has emerged as an important target for molecular imaging of breast cancer. This article presents the design and synthesis of a HER2-targeting affibody molecule with improved stability and tumor targeting capacity, and with potential use as an imaging agent. The 58 aa three-helix bundle protein was assembled using solid-phase peptide synthesis, and a chemoselective ligation strategy was used to establish an intramolecular thioether bond between the side chain thiol group of a cysteine residue, positioned in the loop between helices I and II, and a chloroacetyl group on the side chain amino group of the C-terminal lysine residue. The tethered protein offered an increased thermal stability, with a melting temperature of 64 degrees C, compared to 54 degrees C for the linear control. The ligation did not have a major influence on the HER2 binding affinity, which was 320 and 380 pM for the crosslinked and linear molecules, respectively. Biodistribution studies were performed both in normal and tumor-bearing mice to evaluate the impact of the crosslinking on the in vivo behavior and on the tumor targeting performance. The distribution pattern was characterized by a low uptake in all organs except kidney, and rapid clearance from blood and normal tissue. Crosslinking of the protein resulted in a significantly increased tumor accumulation, rendering the tethered HER2-binding affibody molecule a valuable lead in the development of superior HER2 imaging agents.

Effects of lysine-containing mercaptoacetyl-based chelators on the biodistribution of 99mTc-labeled anti-HER2 Affibody molecules.

The effects of polar (mercaptoacetyl-triseryl) and negatively charged (mercaptoacetyl-triglumatyl) chelators on the biodistribution of 99mTc-labeled anti-HER2 Affibody molecules were previously investigated. With glycine, serine, and glutamate, we demonstrated that substitution with a single amino acid in the chelator can significantly influence the biodistribution properties and the excretion pathways. Here, we have taken this investigation further, by analyzing the effects of introduction of a positive amino acid residue on the in vivo properties of the 99mTc-labeled Affibody molecule. The Affibody molecules with mercaptoacetyl-seryl-lysyl-seryl (maSKS) and mercaptoacetyl-trilysyl (maKKK) extensions were produced by peptide synthesis and labeled with 99mTc in alkaline conditions. A comparative biodistribution was performed in normal mice to evaluate the excretion pathway. A shift toward renal excretion was obtained when serine was substituted with lysine in the chelating sequence. The radioactivity in the gastrointestinal tract was reduced 3-fold for the 99mTc-maSKS-Z(HER2:342) and 99mTc-maKKK-Z(HER2: 342) in comparison with the 99mTc-maSSS-Z(HER2:342) conjugate 4 h post injection (p.i.). The radioactivity in the liver was elevated when a triple substitution of positively charged lysine was used. The tumor targeting properties of 99mTc-maSKS-Z(HER2:342) were further investigated in SKOV-3 xenografts. The tumor uptake of 99mTc-maSKS-Z(HER2: 342) was 17+/-7% IA/g 4 h p.i. Tumor xenografts were well-visualized by gamma scintigraphy. In conclusion, the substitution with one single lysine in the chelator results in better tumor imaging properties of the Affibody molecule Z(HER2:342) and is favorable for imaging of tumors and metastases in the abdominal area. Multiple lysine residues in the chelator are, however, undesirable due to elevated uptake both in the liver and kidneys.

Development and preclinical characterisation of 99mTc-labelled Affibody molecules with reduced renal uptake.

PURPOSE: Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, (99m)Tc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering. MATERIALS AND METHODS: Anti-HER2 Z(HER2:342) Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with (99m)Tc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, (99m)Tc-maEEE-Z(HER2:342,) in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, (99m)Tc-maESE-Z(HER2:342,) was compared with radioiodinated Z(HER2:342). RESULTS: All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 +/- 5, 68 +/- 21 and 71 +/- 10%IA/g, for(99m)Tc-maESE-Z(HER2:342), (99m)Tc-maEES-Z(HER2:342) and (99m)Tc-maSEE-Z(HER2:342), respectively, were significantly reduced in comparison with (99m)Tc-maEEE-Z(HER2:342) (102 +/- 13%IA/g). For (99m)Tc-maESE-Z(HER2:342), a tumour uptake of 9.6 +/- 1.8%IA/g and a tumour-to-blood ratio of 58 +/- 6 were reached at 4 h p.i. CONCLUSIONS: A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of (99m)Tc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.