Screening for <em>Thermotoga maritima</em> Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

University of Helsinki

Interactions between the orexin peptides and their cognate OX1 and OX2 receptors remain poorly characterized. Site-directed mutagenesis studies on orexin peptides and receptors have indicated amino acids important for ligand binding and receptor activation. However, a better understanding of specific pairwise interactions would benefit small molecule discovery.

Modeling of the OX1R-orexin-A complex suggests two alternative binding modes.

Small molecule agonists and antagonists of the orexinergic system have key implications for research and therapeutic purposes. We report a pharmacophore model trained on ∼200 antagonists and prospectively validated by screening a collection of ∼137,000 compounds. The resulting hit list, 395 compounds, was tested for OX1 and OX2 receptor activity using calcium mobilization assay in recombinant cell lines. Validation was conducted using both calcium mobilization and [(125)I]-orexin-A competition binding. Compounds 4-7 have weak agonist activity and Ki's in the 1-30 μM range; compounds 8-14 are antagonists with Ki's in the 0.1-10 μM range for OX2 and 1-50 μM for the OX1 receptor. Docking simulations were used to devise a working hypothesis where two subpockets are important for activation, one between TM5 and TM6 lined by Phe5.42, Tyr5.47, and Tyr6.48 and another above the orthosteric pocket lined by Asp2.65 and Tyr7.32. 

Pharmacophore Model To Discover OX1 and OX2 Orexin Receptor Ligands.

Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PP) to the pumping of Na, H, or both these ions across a membrane. Recently solved structures of the Na-pumping  mPPase (TmPPase) and H-pumping  mPPase revealed the basis of ion selectivity between these enzymes and provided evidence for the mechanisms of substrate hydrolysis and ion-pumping. Our atomistic molecular dynamics (MD) simulations of TmPPase demonstrate that loop 5-6 is mobile in the absence of the substrate or substrate-analogue bound to the active site, explaining the lack of electron density for this loop in resting state structures. Furthermore, creating an  model of TmPPase by removing ligands from the TmPPase:IDP:Na structure in MD simulations resulted in increased dynamics in loop 5-6, which results in this loop moving to uncover the active site, suggesting that interactions between loop 5-6 and the imidodiphosphate and its associated Mg are important for holding a loop-closed conformation. We also provide further evidence for the transport-before-hydrolysis mechanism by showing that the non-hydrolyzable substrate analogue, methylene diphosphonate, induces low levels of proton pumping by VrPPase.

Insights into the mechanism of membrane pyrophosphatases by combining experiment and computer simulation.

Two promising lead structures of small molecular orexin receptor agonist have been reported, but without detailed analyses of the pharmacological properties. One of them, 1-(3,4-dichlorophenyl)-2-[2-imino-3-(4-methylbenzyl)-2,3-dihydro-1H-benzo[d]imidazol-1-yl]ethan-1-ol (Yan 7874), is commercially available, and we set out to analyze its properties. As test system we utilized human OX1 and OX2 orexin receptor-expressing Chinese hamster ovary (CHO) K1 cells as well as control CHO-K1 and neuro-2a neuroblastoma cells. Gq-coupling was assessed by measurement of intracellular Ca2+ and phospholipase C activity, and the coupling to Gi and Gs by adenylyl cyclase inhibition and stimulation, respectively. At concentrations above 1 μM, strong Ca2+ and low phospholipase C responses to Yan 7874 were observed in both OX1- and OX2-expressing cells. However, a major fraction of the response was not mediated by orexin receptors, as determined utilizing the non-selective orexin receptor antagonist N-biphenyl-2-yl-1-{[(1-methyl-1H-benzimidazol-2-yl)sulfanyl]acetyl}-L-prolinamide (TCS 1102) as well as control CHO-K1 cells. Yan 7874 did not produce any specific adenylyl cyclase response. Some experiments suggested an effect on cell viability by Yan 7874, and we thus analyzed this. Within a few hours of exposure, Yan 7874 markedly changed cell morphology (shrunken, rich in vacuoles), reduced growth, promoted cell detachment, and induced necrotic cell death. The effect was equal in cells expressing orexin receptors or not. Thus, Yan 7874 is a weak partial agonist of orexin receptors. It also displays strong off-target effects in the same concentration range, culminating in necrotic cell demise. This makes Yan 7874 unsuitable as orexin receptor agonist.

Orexin receptor agonist Yan 7874 is a weak agonist of orexin/hypocretin receptors and shows orexin receptor-independent cytotoxicity.

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain-targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C.

A library of 70 000 synthetically accessible azulene-based compounds was virtually screened at the OX receptor. Based on the results, a series of azulene derivatives was synthesized and the binding to and activation of both orexin receptor subtypes were assessed. Two most promising binders were determined to have inhibition constants in the 3-9 μM range and two other compounds showed weak OX receptor agonism. Furthermore, three compounds exhibited a concentration-dependent potentiation of the response to orexin-A at the OX but not the OX receptors. Altogether this data opens new approaches for further development of antagonists, agonists, and potentiators of orexin response based on the azulene scaffold.

Azulene-based compounds for targeting orexin receptors.

Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.

Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET.

One promising series of small-molecule orexin receptor agonists has been described, but the molecular pharmacological properties, i.e. ability and potency to activate the different orexin receptor-regulated signal pathways have not been reported for any of these ligands. We have thus here assessed these properties for the most potent ligand of the series, 4'-methoxy-N,N-dimethyl-3'-[N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl sulfamoyl]-(1,1'-biphenyl)-3-carboxamide (Nag 26). Chinese hamster ovary-K1 cells expressing human orexin receptor subtypes OX and OX were used. Ca elevation and cell viability and death were assessed by fluorescent methods, the extracellular signal-regulated kinase pathway by a luminescent Elk-1 reporter assay, and phospholipase C and adenylyl cyclase activities by radioactive methods. The data suggest that for the G-dependent responses, Ca, phospholipase C and Elk-1, Nag 26 is a full agonist for both receptors, though of much lower potency. However, saturation was not always reached for OX, partially due to Nag 26's low solubility and partially because the response decreased at high concentrations. The latter occurs in the same range as some reduction of cell viability, which is independent of orexin receptors. Based on the EC, Nag 26 was OX-selective by 20-200 fold in different assays, with some indication of biased agonism (as compared to orexin-A). Nag 26 is a potent orexin receptor agonist with a largely similar pharmacological profile as orexin-A. However, its weaker potency (low-mid micromolar) and low water solubility as well as the non-specific effect in the mid-micromolar range may limit its usefulness under physiological conditions.

Pharmacological characterization of the orexin/hypocretin receptor agonist Nag 26.

We previously demonstrated the potential of di- or trisubstituted azulenes as ligands (potentiators, weak agonists, and antagonists) of the orexin receptors. In this study we investigated 27 1-benzoylazulene derivatives, uncovering seven potentiators of the orexin response on OX and two weak dual orexin receptor agonists. For potentiators, replacement of the azulene scaffold by indole retained the activity of four out of six compounds. The structure-activity relationships for agonism and potentiation can be summarized into a bicyclic aromatic ring system substituted with two hydrogen-bond acceptors (1-position, benzoyl; 6-position, carboxyl/ester) within 7-8 Å of each other; a third acceptor at the 3-position is also well tolerated. The same pharmacophoric signature is found in the preferred conformations of the orexin receptor agonist Nag26 from molecular dynamics simulations. Subtle changes switch the activity between weak agonism and potentiation, suggesting overlapping binding sites.

Structure-Activity Relationships of 1-Benzoylazulenes at the OX and OX Orexin Receptors.

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium  membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1-2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

Ainoleena Turku

Drug Research Program,
Division of Pharmaceutical Chemistry and Technology,
Faculty of Pharmacy,
Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy

University of Helsinki

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Ainoleena Turku

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Drug Research Program,Division of Pharmaceutical Chemistry and Technology,Faculty of Pharmacy,Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy

University of Helsinki

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Drug Research Program,
Division of Pharmaceutical Chemistry and Technology,
Faculty of Pharmacy,
Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy