Discovery of OX1 and OX2 orexin receptor ligands

The main objective of this dissertation was to gain an understanding of orexin receptor activation at the molecular level and apply it in discovery of novel orexin receptor ligands. As non-peptide orexin receptor activators were almost completely unknown at the start of this study, attention was focused on them. To accomplish these goals, I utilized a combination of molecular modelling and pharmacological in vitro studies. First, I studied the known orexin receptor ligands by structure- and ligand-based computational methods, and assembled the hypothesized activation features for a pharmacophore model. The model was utilized in a virtual screening, and a hit list of 395 compounds continued to a pharmacological screening phase, wherein I assessed their activities in a functional Ca2+-based screening assay developed particularly for that purpose. I validated the screening hits in the competition binding and Ca2+ elevation assays; six compounds showed weak agonist activity and Ki’s in the 1−30 µM range (Publication I). Antagonists with sub-micromolar binding affinities were also identified. Retrospective docking simulations of these agonistic hits and known non-peptide orexin receptor agonists (Nag26 and Yan7874, the latter of which was pharmacologically characterized in Publication II) were used to devise a working hypothesis of the binding pocket regions important for orexin receptor activation. Interactions in the antagonist binding region and two additional sub-pockets—one between TM5 and TM6, and the other approximately one helical turn above the antagonist binding site close to TM7—would be needed for orexin receptor activation (Publication I). Relying on this, I constructed a targeted azulene-based combinatory compound library accessible to in-house chemistry. The azulene library was virtually screened at the crystal structure of OX2 receptor, and compounds selected from the hit list were synthesized and screened in vitro. I validated the hits as above, and novel antagonists, weak agonists and compounds potentiating the actions of orexin-A were identified (Publications III and IV). The literature review focuses on the concept of GPCR activation and the orexin system: its structure, functions, and pharmaceutical applications thereof.Oreksiinijärjestelmä, kaksi G-proteiinikytkentäistä reseptoria (OX1 ja OX2) ja niitä aktivoivaa neuropeptidiä (oreksiini-A ja -B), on keskeinen tekijä uni-valverytmin säätelyssä. Häiriöt oreksiinijärjestelmän toiminnassa voivat johtaa esimerkiksi narkolepsian syntyyn; narkolepsian taudinkuvaan kuuluu oreksiinineuronien dramaattinen kato ja siksi elimistön oman oreksiinituotannon heikkeneminen. Pienmolekyyleille, jotka aktivoivat oreksiinijärjestelmää, on näin selvä tarve sekä lääkekehityksessä että oreksiinijärjestelmän toiminnan tutkimuksessa. Tutkin väitöskirjatyössäni oreksiinireseptoreja ja niiden kanssa vuorovaikuttavia pienmolekyylejä sekä tietokoneavusteisin menetelmin että solukokein laboratoriossa. Työni alkuvaiheessa oreksiinireseptoreja aktivoivia yhdisteitä ei tunnettu käytännössä lainkaan, ja edelleenkin oreksiinireseptoriaktivaatioon johtavat atomitason mekanismit ovat pääasiassa tuntemattomia. Näin ollen työni tavoite oli ymmärtää oreksiinireseptoriaktivaatiota atomitasolla ja löytää uusia yhdisteitä, jotka sitoutuvat oreksiinireseptoreihin, ja aktivoivat niitä. Kehitimme tutkimukseni alussa seulontamenetelmän, joka yhdistää farmakoforimalliin perustuvan virtuaaliseulonnan solupohjaiseen in vitro -seulontaan. Virtuaaliseuloin n. 140000 yhdisteen molekyylikirjaston, ja seulonnan tulokset, 395 yhdistettä, jatkoivat laboratoriokokeisiin. Laboratoriotulokset osoittivat kuuden yhdisteen olevan heikkoja oreksiinireseptoriagonisteja (eli aktivaattoreita), lisäksi löysimme seitsemän antagonistia (eli oreksiinireseptoreihin sitoutuvaa molekyyliä), joiden sitoutumisaffiniteetit olivat nanomolaarisella tasolla. Seuraavaksi tutkimme löytämiämme yhdisteitä sekä muita tunnettuja oreksiinireseptoriligandeja tietokoneavusteisesti telakoinnin avulla; telakointitulokset viittasivat kolmen sitoutumistaskun tärkeyteen oreksiinireseptoriaktivaation kannalta. Suunnittelimme tähän aktivaatiohypoteesiin perustuen virtuaalisen atsuleeniyhdistekirjaston, jonka synteesi oli mahdollista osastollamme. Virtuaaliseuloin kirjaston n. 70000 yhdistettä OX2-reseptorin kiderakenteeseen ja seulonnan tuloksista valitut yhdisteet syntetisoitiin ja testattiin kuten yllä. Myös atsuleeniseulontamme johti uusien oreksiinireseptorimodulaattorien löytymiseen – antagonistien ja osittaisagonistien lisäksi löysimme yhdisteitä, jotka vahvistavat oreksiini-A-välitteistä reseptoriaktivaatiota. Vaikka agonismi on lääkekehityksen kannalta toivottavin ilmiö, myös oreksiinivasteen potensointi voisi olla mielekästä tilanteissa, joissa elimistön oma oreksiini-A-tuotanto ei ole vielä kokonaan loppunut. Näin ollen löytämämme yhdisteet tarjoavat lupaavan lähtökohdan ja uusia näkökulmia tulevaisuuden lääkeainemolekyylien kehitykselle

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

Background: 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. Results: We constructed a set of three-dimensional models of the orexin 1 receptor based on the 3D-structures of the orexin 2 receptor (released while this manuscript was under review), neurotensin receptor 1 and chemokine receptor CXCR4, conducted an exhaustive docking of orexin-A(16-33) peptide fragment with ZDOCK and RDOCK, and analyzed a total of 4301 complexes through multidimensional scaling and clustering. The best docking poses reveal two alternative binding modes, where the C-terminus of the peptide lies deep in the binding pocket, on average about 5-6 angstrom above Tyr(6.48) and close to Gln(3.32). The binding modes differ in the about 100 degrees rotation of the peptide; the peptide His26 faces either the receptor's fifth transmembrane helix or the seventh helix. Both binding modes are well in line with previous mutation studies and partake in hydrogen bonding similar to suvorexant. Conclusions: We present two binding modes for orexin-A into orexin 1 receptor, which help rationalize previous results from site-directed mutagenesis studies. The binding modes should serve small molecule discovery, and offer insights into the mechanism of receptor activation.Peer reviewe

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

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-dichloropheny1)-242-imino-3-(4-methylbenzy1)-2,3-dihydro-1H-benzo[climidazol-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 G(i) and G(s) by adenylyl cyclase inhibition and stimulation, respectively. At concentrations above 1 pM, strong Ca' 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 nonselective orexin receptor antagonist N-biphenyl-2-y1-1-{[(1-methyl-1H-benzimidazol-2-y1) 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.Peer reviewe

Azulene-based compounds for targeting orexin receptors

A library of 70 000 synthetically accessible azulene-based compounds was virtually screened at the OX2 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 mu M range and two other compounds showed weak OX2 receptor agonism. Furthermore, three compounds exhibited a concentration-dependent potentiation of the response to orexin-A at the OX1 but not the OX2 receptors. Altogether this data opens new approaches for further development of antagonists, agonists, and potentiators of orexin response based on the azulene scaffold. (C) 2018 Elsevier Masson SAS. All rights reserved.Peer reviewe

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

Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PPi) to the pumping of Na+, H+, or both these ions across a membrane. Recently solved structures of the Na+-pumping Thermotoga maritima mPPase (TmPPase) and H+-pumping Vigna radiata 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 apo 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 Mg2+ 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. (C) 2017 Author(s).Peer reviewe

Discovery of Membrane-Bound Pyrophosphatase Inhibitors Derived from an Isoxazole Fragment

Membrane-bound pyrophosphatases (mPPases) regulate energy homeostasis in pathogenic protozoan parasites and lack human homologues, which makes them promising targets in e.g. malaria. Yet only few nonphosphorus inhibitors have been reported so far. Here, we explore an isoxazole fragment hit, leading to the discovery of small mPPase inhibitors with 6-10 mu M IC50 values in the Thermotoga maritima test system. Promisingly, the compounds retained activity against Plasmodium falciparum mPPase in membranes and inhibited parasite growth.Peer reviewe

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

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 PKC delta 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 PKC alpha 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 PKC alpha 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.Peer reviewe

Comparison of the active sites of DNMT2 and COMT and virtual screening for finding new inhibitors

The aims of this work were (1) to compare the three dimensional structures of different S- adenosylmethionine (SAM)-dependent methyltrasferases and (2) to screen in silico a commercial library for potential methyltransferase inhibitors. In this work we decided to focus on DNA methyltransferase-like enzyme (DNMT2) and catechol-O-methyltransferase(COMT). There were two different parts in my work. The first part was to analyze the 3Dstructures of DNMT2 and COMT in relation with their amino acid sequences. The structures of DNMT2 and COMT were compared together by means of superimposition with Sybyl 8. The ligand binding properties were studied by manual and automatic docking of known inhibitors in order to understand the binding specificity of these methyltransferases. The softwares I used for docking were Autodock 4.2 and Gold 4.0. The sequence alignments and superimposition of the known crystal structures showed that the structures of DNMT2 and COMT share a similar fold. Furthermore the main similarities between the structures of these enzymes are in the co-enzyme binding sites. The only significant difference in the binding sites is the place of one tyrosine residue, which causes a slight change in the conformation of the bound co-enzyme. Unlike co- enzyme binding sites, the substrate binding sites of DNMT2 and COMT are different. There is indeed a bound magnesium ion in the substrate binding site of COMT but not in the substrate binding site of DNMT2. Because the substrate binding sites are more different than the co-enzyme binding sites, we decided to screen the potential active ligands only at the substrate binding sites. The second part of the work was virtual screening. I used a subset of 20.000 molecules of ChemBridge DIVER Set that can be purchased commercially. The softwares I used for library preparation were CONCORD and Balloon, from which Balloon created more reasonable 3D structures for the docking. I did two parallel screenings to the crystal structure of COMT (PDB code 3BWM) with docking program GOLD 4.0, which is the only program that can take account metal coordination. To DNMT2 I did two sets of screenings, one with GOLD 4.0 and another with Autodock 4.2. I used known COMT inhibitors as control in the COMT run and known DNA methyltransferase inhibitors as control in DNMT2 run. Before docking to the three dimensional structure of DNMT2, one loop near the substrate binding site had to be modeled. I used Swiss-Modeler and Modeller softwares for that. Docking to COMT was successful according to the rank of the known COMT inhibitors compared to the subset of the FIMM library that was screened. I created the hitlist of 60 compounds based on the scores of these compounds, pharmacophore search and visual examination. 30 of these compounds were purchased and are currently being tested. The results of the DNMT2 run were not as reliable as the results of COMT run mentioned before, since the DNMT2 run was unable to retrieve known inhibitors better than random. The reason for that can be the quality of the model of the missing loop or the chosen controls. Furthermore only one of the ten small molecules that we used as controls is proved to be DNMT2 inhibitor, the others are DNMT1 and DNMT3 inhibitors and while the binding sites of DNMT1, DNMT2 and DNMT3 are very similar, they are, however, not completely identical.Työn tavoitteena oli (1) vertailla S-adenosyylimetioniini (SAM)-riippuvaisten metyylitransferaasien kolmiuloitteisia rakenteita ja (2) etsiä potentiaalisia metyylitransferaasiinhibiittoreita tekemällä in silico -seulonta eräälle kaupalliselle molekyylikirjastolle. Tässä työssä päätimme keskittyä DNA-metyylitransferaasin kaltaiseen entsyymiin (DNMT2) ja katekoli-O-metyylitransferaasiin (COMT). Työssäni oli kaksi erillistä vaihetta. Ensimmäinen vaihe oli analysoida DNMT2:n ja COMT:n 3D-rakenteita suhteessa niiden aminohapposekvensseihin. Tämä tapahtui vertailemalla entsyymien aminohapposekvenssejä ja kiderakenteita päällekkäinasettelemalla ne ClustalX- ja Sybyl 8-ohjelmien avulla. Tutkin ligandien sitoutumisominaisuuksia sekä käsin tehtävän että automaattisen telakoinnin avulla telakoimalla tunnettuja inhibiittoreita molempien edellä mainittujen entsyymien aktiivisiin keskuksiin. Tähän käytin Autodock 4.2- ja GOLD 4.0 -ohjelmia. DNMT2 ja COMT ovat kolmiuloitteiselta rakenteeltaan samankaltaisia. Lisäksi koentsyymien sitoutumispaikat ovat hyvin samanlaisia näiden kahden entsyymin välillä. Ainoa merkittävä ero on erään tyrosiini aminohappotähteen paikka, mikä aiheuttaa lievän konformaatioeron sitoutuneiden koentsyymien välillä. Toisin kuin DNMT2:n ja COMT:n koentsyymien sitoutumispaikat, substraattien sitoutumispaikat ovat hyvin erilaisia keskenään. Suurin ero on magnesium-ioni, joka on sitoutunut COMT:n substraatin sitoutumispaikan läheisyyteen, mutta jota ei löydy DNMT2:n rakenteesta. Koska näiden entsyymien substraattien sitoutumispaikat ovat hyvin erilaisia, päätimme seuloa potentiaalisia aktiivisia ligandeja vain näihin sitoutumispaikkoihin. Työn toisessa vaiheessa tein virtuaaliseulonnan DNMT2:n ja COMT:n substraattien sitoutumispaikkoihin. Käytin seulontaan kaupallisen ChemBridge DIVER Setmolekyylikirjaston 20000 molekyylin osajoukkoa. Esikäsittelin kirjaston CONCORD- ja Balloon-ohjelmilla, joista Balloon osoittautui tässä tapauksessa paremmaksi luoden käyttökelpoisempia 3D-rakenteita telakointia varten. Tein kaksi rinnakkaista telakointia COMT:n kiderakenteeseen (PDB: 3BWM) GOLD:illa, joka on ainoa metallikoordinaation huomioiva telakointiohjelma. DNMT2:een tein kaksi erillistä telakointia, toisen Autodock- ja toisen GOLD-ohjelmalla. Käytin tunnettuja COMT-inhibiittoreita COMT-ajon kontrolleina ja tunnettuja DNA metyylitransferaasi-inhibiittoreita DNMT2-ajon kontrolleina. DNMT2:n kiderakenteesta (PDB: 1G55) puuttuu silmukka läheltä substraatin sitoutumiskohtaa, mikä piti näin ollen mallintaa ennen telakointia. Mallinsin tämän silmukan Swiss-Modeller- ja Modelerohjelmilla. COMT-ajot olivat onnistuneita, sillä tunnetut COMT-inhibiittorit pisteytyivät korkealle seulotun kirjaston molekyylien joukosta. Tein listan molempien ajojen 60 parhaasta molekyylistä perustuen yhdisteiden saamiin pisteisiin, farmakoforitarkasteluun ja visuaalisiin huomioihin. 30 näistä yhdisteistä on hankittu testattavaksi in vitro. DNMT2-ajojen tulokset eivät olleet yhtä luotettavia kuin COMT-ajojen, sillä DNMT2-ajoissa tunnetut inhibiittorit jakautuivat lähes satunnaisesti muiden seulottujen molekyylien joukkoon. Syitä tähän voivat olla esimerkiksi käytetyn mallin laatu tai valitut kontrollimolekyylit

Pharmacological characterization of the orexin/hypocretin receptor agonist Nag 26

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-3'4N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl sulfamoy1]-(1,1'-biphenyl)-3-carboxamide (Nag 26). Chinese hamster ovary-K1 cells expressing human orexin receptor subtypes OX1 and OX2 were used. Ca2+ 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(q)-dependent responses, Ca2+, 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 OX1, partially due to Nag 26s 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 EC50, Nag 26 was OX2 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.Peer reviewe