Discovery of DTX3L inhibitors through a homogeneous FRET-based assay that monitors formation and removal of poly-ubiquitin chains.

Ubiquitination is a reversible protein post-translational modification in which consequent enzymatic activity results in the covalent linking of ubiquitin to a target protein. Once ubiquitinated, a protein can undergo multiple rounds of ubiquitination on multiple sites or form poly-ubiquitin chains. Ubiquitination regulates various cellular processes, and dysregulation of ubiquitination has been associated with more than one type of cancer. Therefore, efforts have been carried out to identify modulators of the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor ubiquitination activity of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a good signal window with a robust average Z' factor of 0.76 on 384-well microplates, indicating a good assay for screening inhibitors in a high-throughput setting. From a validatory screening experiment, we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. We also demonstrate that the method can be expanded to study deubiquitinases, such as USP28, that reduce FRET due to hydrolysis of fluorescent poly-ubiquitin chains.

[1,2,4]Triazolo[3,4-b]benzothiazole Scaffold as Versatile Nicotinamide Mimic Allowing Nanomolar Inhibition of Different PARP Enzymes.

We report [1,2,4]triazolo[3,4-b]benzothiazole (TBT) as a new inhibitor scaffold, which competes with nicotinamide in the binding pocket of human poly- and mono-ADP-ribosylating enzymes. The binding mode was studied through analogues and cocrystal structures with TNKS2, PARP2, PARP14, and PARP15. Based on the substitution pattern, we were able to identify 3-amino derivatives 21 (OUL243) and 27 (OUL232) as inhibitors of mono-ARTs PARP7, PARP10, PARP11, PARP12, PARP14, and PARP15 at nM potencies, with 27 being the most potent PARP10 inhibitor described to date (IC50 of 7.8 nM) and the first PARP12 inhibitor ever reported. On the contrary, hydroxy derivative 16 (OUL245) inhibits poly-ARTs with a selectivity toward PARP2. The scaffold does not possess inherent cell toxicity, and the inhibitors can enter cells and engage with the target protein. This, together with favorable ADME properties, demonstrates the potential of TBT scaffold for future drug development efforts toward selective inhibitors against specific enzymes.

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high-affinity heterodimerization and multimeric assembly.

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L-PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L-PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230-510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1-200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions.

FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici.

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized in detail before. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues, we were not able to detect either oxidase or monooxygenase activity when L-lactate was used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

Discovery of Compounds Inhibiting the ADP-Ribosyltransferase Activity of Pertussis Toxin.

The targeted pathogen-selective approach to drug development holds promise to minimize collateral damage to the beneficial microbiome. The AB5-topology pertussis toxin (PtxS1-S5) is a major virulence factor of Bordetella pertussis, the causative agent of the highly contagious respiratory disease whooping cough. Once internalized into the host cell, PtxS1 ADP-ribosylates α-subunits of the heterotrimeric Gαi-superfamily, thereby disrupting G-protein-coupled receptor signaling. Here, we report the discovery of the first small molecules inhibiting the ADP-ribosyltransferase activity of pertussis toxin. We developed protocols to purify milligram-levels of active recombinant B. pertussis PtxS1 from Escherichia coli and an in vitro high throughput-compatible assay to quantify NAD+ consumption during PtxS1-catalyzed ADP-ribosylation of Gαi. Two inhibitory compounds (NSC228155 and NSC29193) with low micromolar IC50-values (3.0 µM and 6.8 µM) were identified in the in vitro NAD+ consumption assay that also were potent in an independent in vitro assay monitoring conjugation of ADP-ribose to Gαi. Docking and molecular dynamics simulations identified plausible binding poses of NSC228155 and in particular of NSC29193, most likely owing to the rigidity of the latter ligand, at the NAD+-binding pocket of PtxS1. NSC228155 inhibited the pertussis AB5 holotoxin-catalyzed ADP-ribosylation of Gαi in living human cells with a low micromolar IC50-value (2.4 µM). NSC228155 and NSC29193 might prove to be useful hit compounds in targeted B. pertussis-selective drug development.

4-(Phenoxy) and 4-(benzyloxy)benzamides as potent and selective inhibitors of mono-ADP-ribosyltransferase PARP10/ARTD10.

Human Diphtheria toxin-like ADP-ribosyltranferases (ARTD) 10 is an enzyme carrying out mono-ADP-ribosylation of a range of cellular proteins and affecting their activities. It shuttles between cytoplasm and nucleus and influences signaling events in both compartments, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and S phase DNA repair. Furthermore, overexpression of ARTD10 induces cell death. We recently reported on the discovery of a hit compound, OUL35 (compound 1), with 330 nM potency and remarkable selectivity towards ARTD10 over other enzymes in the human protein family. Here we aimed at establishing a structure-activity relationship of the OUL35 scaffold, by evaluating an array of 4-phenoxybenzamide derivatives. By exploring modifications on the linker between the aromatic rings, we identified also a 4-(benzyloxy)benzamide derivative, compound 32, which is potent (IC50 = 230 nM) and selective, and like OUL35 was able to rescue HeLa cells from ARTD10-induced cell death. Evaluation of an enlarged series of derivatives produced detailed knowledge on the structural requirements for ARTD10 inhibition and allowed the discovery of further tool compounds with submicromolar cellular potency that will help in understanding the roles of ARTD10 in biological systems.

Small-Molecule Chemical Probe Rescues Cells from Mono-ADP-Ribosyltransferase ARTD10/PARP10-Induced Apoptosis and Sensitizes Cancer Cells to DNA Damage.

Members of the human diphtheria toxin-like ADP-ribosyltransferase (ARTD or PARP) family play important roles in regulating biological activities by mediating either a mono-ADP-ribosylation (MARylation) of a substrate or a poly-ADP-ribosylation (PARylation). ARTD10/PARP10 belongs to the MARylating ARTDs (mARTDs) subfamily, and plays important roles in biological processes that range from cellular signaling, DNA repair, and cell proliferation to immune response. Despite their biological and disease relevance, no selective inhibitors for mARTDs are available. Here we describe a small-molecule ARTD10 inhibitor, OUL35, a selective and potent inhibitor for this enzyme. We characterize its selectivity profile, model its binding, and demonstrate activity in HeLa cells where OUL35 rescued cells from ARTD10 induced cell death. Using OUL35 as a cell biology tool we show that ARTD10 inhibition sensitizes the cells to the hydroxyurea-induced genotoxic stress. Our study supports the proposed role of ARTD10 in DNA-damage repair and provides a tool compound for selective inhibition of ARTD10-mediated MARylation.

Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay.

Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins.•Adaptable in high throughput format (≥96 well format).•Stability measurement in near-native lipid environment and lipid dependent melting temperatures.

Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1.

Many nucleoside transport inhibitors are in clinical use as anti-cancer, vasodilator and cardioprotective drugs. However, little is known about the binding energetics of these inhibitors to nucleoside transporters (NTs) due to their low endogenous expression levels and difficulties in the biophysical characterization of purified protein with ligands. Here, we present kinetics and thermodynamic analyses of inhibitor binding to the human equilibrative nucleoside transporter-1 (hENT1), also known as SLC29A1. Using a radioligand binding assay, we obtained equilibrium binding and kinetic rate constants of well-known NT inhibitors--[(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR), dilazep, and dipyridamole--and the native permeant, adenosine, to hENT1. We observed that the equilibrium binding affinities for all inhibitors decreased whereas, the kinetic rate constants increased with increasing temperature. Furthermore, we found that binding is enthalpy driven and thus, an exothermic reaction, implying that the transporter does not discriminate between its inhibitors and substrates thermodynamically. This predominantly enthalpy-driven binding by four chemically distinct ligands suggests that the transporter may not tolerate diversity in the type of interactions that lead to high affinity binding. Consistent with this, the measured activation energy of [(3)H]NBMPR association was relatively large (20 kcal mol(-1)) suggesting a conformational change upon inhibitor binding. For all three inhibitors the enthalpy (ΔH°) and entropy (ΔS°) contributions to the reaction energetics were determined by van't Hoff analysis to be roughly similar (25-75% ΔG°). Gains in enthalpy with increasing polar surface area of inhibitors suggest that the binding is favored by electrostatic or polar interactions between the ligands and the transporter.

Crystallogenesis of adenosine A(2A) receptor-T4 lysozyme fusion protein: a practical route for the structure.

G-protein-coupled receptors (GPCRs) represent a major class of receptors through which a number of signals ranging from photons to large glycoprotein hormones are recognized. Human genome encodes about 800 GPCRs, yet very little structural information is available on this class of receptors. Structural studies provide a wealth of information about not only the activation mechanism of the receptor but also the crucial information about the ligand-binding pocket which could lead to the development of subtype-specific ligands. The crystal structure of human adenosine A(2A) receptor was solved in complex with a high-affinity antagonist ZM241385 at 2.6Å resolution. Here, we describe the methods that were undertaken to solve the fusion protein structure.

Structural features of adenosine receptors: from crystal to function.

The important role that extracellular adenosine plays in many physiological processes is mediated by the adenosine class of G protein-coupled receptors, a class of receptors that also responds to the antagonist caffeine, the most widely used pharmacological agent in the world. The crystallographic model of the human adenosine A(2A) receptor was recently solved to 2.6Å in complex with the antagonist ZM241385, which is also referred to as "super-caffeine" because of its strong antagonistic effect on adenosine receptors. The crystallographic model revealed some unexpected and unusual features of the adenosine A(2A) receptor structure that have led to new studies on the receptor and the re-examination of pre-existing data. Compared to other known GPCR structures, the adenosine A(2A) receptor has a unique ligand binding pocket that is nearly perpendicular to the membrane plane. The ligand binding site highlights the integral role of the helical core together with the extracellular loops and the four disulfide bridges in the extracellular domain, in ligand recognition by the adenosine class of GPCRs.

Drosophila toll pathway: the new model.

In Drosophila, recognition of microbe-specific molecules (such as bacterial peptidoglycans) activates serine protease cascades that converge to activate the Toll pathway. Recent data show that the serine protease Grass, which is activated downstream of pattern recognition receptors and was initially thought to be a component only of the Gram-positive bacteria-induced signaling cascade, is also required for the induction of the Toll pathway after fungal infection. Persephone, a serine protease known to be specifically activated by fungal proteases, was also found to be required for sensing Gram-positive bacterial proteases. Thus, Persephone serves as a sensor for microbial activities from both fungi and Gram-positive bacteria. With these new discoveries, a new model has been proposed for activation of the Drosophila Toll pathway.