Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.Peer reviewe

Discovery of Retinoic Acid-Related Orphan Receptor gamma t Inverse Agonists via Docking and Negative Image-Based Screening

Retinoic acid-related orphan receptor gamma t (ROR gamma t) has a vital role in the differentiation of T-helper 17 (TH17) cells. Potent and specific ROR gamma t inverse agonists are sought for treating TH17-related diseases such as psoriasis, rheumatoid arthritis, and type 1 diabetes. Here, the aim was to discover novel ROR gamma t ligands using both standard molecular docking and negative image-based screening. Interestingly, both of these in silico techniques put forward mostly the same compounds for experimental testing. In total, 11 of the 34 molecules purchased for testing were verified as ROR gamma t inverse agonists, thus making the effective hit rate 32%. The pIC(50) values for the compounds varied from 4.9 (11 mu M) to 6.2 (590 nM). Importantly, the fact that the verified hits represent four different cores highlights the structural diversity of the ROR gamma t inverse agonism and the ability of the applied screening methodologies to facilitate much-desired scaffold hopping for drug design

Structure-Activity Relationship Analysis of 3-Phenylcoumarin-Based Monoamine Oxidase B Inhibitors

Monoamine oxidase B (MAO-B) catalyzes deamination of monoamines such as neurotransmitters dopamine and norepinephrine. Accordingly, small-molecule MAO-B inhibitors potentially alleviate the symptoms of dopamine-linked neuropathologies such as depression or Parkinson's disease. Coumarin with a functionalized 3-phenyl ring system is a promising scaffold for building potent MAO-B inhibitors. Here, a vast set of 3-phenylcoumarin derivatives was designed using virtual combinatorial chemistry or rationally de novo and synthesized using microwave chemistry. The derivatives inhibited the MAO-B at 100 nM-1 mu M. The IC50 value of the most potent derivative 1 was 56 nM. A docking-based structure-activity relationship analysis summarizes the atom-level determinants of the MAO-B inhibition by the derivatives. Finally, the cross-reactivity of the derivatives was tested against monoamine oxidase A and a specific subset of enzymes linked to estradiol metabolism, known to have coumarin-based inhibitors. Overall, the results indicate that the 3-phenylcoumarins, especially derivative 1, present unique pharmacological features worth considering in future drug development

Immune tolerance disruption by human parvovirus B19 viral infection mechanisms

Virusinfektioita pidetään kasvavissa määrin suurimpana ympäristöperäisenä syynä, joka vaikuttaa autoimmuunisairauksien kehitykseen ja autoimmuniteetin muodostukseen. Esimerkiksi parvovirus B19 (B19V) on yhdistetty punahukkaan sekä nivelreumaan. On todettu, että B19V:n ei-rakenteellinen proteiini 1 (NS1) aiheuttaa apoptoosia soluissa, jotka eivät tavallisesti ole B19V infektion kohteena. Lisäksi hiljattain on osoitettu, että apoptoosin seurauksena muodostuvat apoptoottiset kappaleet kykenevät esittelemään antigeenejä niitä esittelemään erikoistuneille soluille, kuten makrofageille tai dendriittisoluille. Tämän tutkimuksen tarkoituksena on löytää B19V infektioon liittyviä autoantigeenejä sekä tarkastella NS1:n aiheuttamaa immuunivastetta. Tätä varten NS1 on yhdistetty vihreään fluoresoivaan proteiiniin (EGFP) ja kloonattu bakulovirusvektoriin sytomegaloviruksen välittömän aikaisen promoottorin alle. Tämän jälkeen näitä yhdistelmä-bakuloviruksia lisättiin Sf9-hyönteissolulinjan avulla. Tällä tavoin tuotettua virusta käytettiin ihmisen maksasta peräisin olevien HepG2-solujen transduktiossa. Kyseisen transduktion tehokkuus mitattiin virtaussytometrillä, hyödyntäen EGFP-signaalia merkkinä onnistuneesta transduktiosta. Transduktion tehokkuuden perusteella määritettiin viruskannasta sellainen tilavuus, mikä tuottaa 70 prosentin transduktion. Tätä tilavuutta käytettiin apoptoottisten kappaleiden tuotannossa. Apoptoottiset kappaleet puhdistettiin ensin suodattamalla ja koottiin sitten pelleteiksi ultrasentrifugilla. Kootuista apoptoottisista kappaleista immunoleimattiin tumaperäiset antigeenit apolipoproteiini H (ApoH), histoni 4 (H4), histoni 2B (H2B), lysosomiin liittyvä kalvoproteiini 2 (Lamp2), Ku80 ja Smith. Kaikki tumaperäiset antigeenit, poissulkien H2B, löydettiin apoptoottisista kappaleista yhdessä NS1 kanssa. Tumaperäisten antigeenien löytyminen apoptoottisista kappaleista saattaa aiheuttaa häiriön immunitoleranssissa, mitä tutkittiin seuraavaksi hyödyntäen antigeenejä esitteleviä soluja. Tähän tarkoitukseen valittiin akuutista monosyyttisesta leukemiasta peräisin olevat ihmisen monosyytit (THP-1), jotka erilaistettiin forboli-12-myristaatti-13-asetaatilla (PMA). Soluja viljeltiin PMA:a sisältävällä kasvatusalustalla ensin kolme päivää ja viljelyä jatkettiin vielä erilaistumisen varmistamiseksi puhtaalla alustalla viiden päivän ajan. Seuraavaksi THP-1 soluja stimuloitiiin apoptoottisilla kappaleilla ja niiden päätyminen solujen sisään varmistettiin konfokaalimikroskopialla. Koska erilaistetut THP-1 solut pystyivät ottamaan apoptoottisia kappaleita sisäänsä, tästä johtuvaa sytokiiniprofiilia tarkasteltiin vielä alustavasti tarkoitukseen sopivan kaupallisen tuotteen avulla. Mahdollisia immunologisia vaikutuksia tarkasteltaessa tultiin lopputulokseen, jossa ainoastaan yhden proteiinin, γ-interferonin indusoima proteiini 10:n (IP-10), läsnäolo erotti EGFP- ja EGFP-NS1-fuusioproteiinien avulla tuotetut näytteet kontrollinäytteistä. Vaikka B19V:n infektiomekanismien aiheuttamat häiriöt immunitoleranssiin vaativatkin vielä lisätutkimuksia, tässä tutkimuksessa saadut tulokset tukevat alkuperäistä hypoteesia. B19V NS1:n avulla tuotetut apoptoottiset kappaleet, jotka ovat peräisin soluissa, joita B19V ei tavallisesti infektoi, sisältävät autoantigeenejä ja nämä autoantigeenit voidaan esitellä immuunijärjestelmälle antigeenejä esittelevien solujen kautta.There are several factors, of which viral infections are increasingly regarded as the greatest environmental cause of autoimmunity, influencing the development of an autoimmune disease. For example human parvovirus B19 (B19V) has been previously connected to systemic lupus erythematosus and rheumatic arthritis. It has also been shown that nonstructural protein 1 (NS1) of B19V induces apoptosis in cells not normally permissive for B19V infections. In addition, it has been recently demonstrated that apoptotic bodies resulting from apoptosis can present antigens to antigen presenting cells (APCs) such as macrophages or dendritic cells. The aim of this study is to detect self antigens related to B19V infections and study the immune response caused by NS1. For that, the NS1 has been fused with enhanced green fluorescent protein (EGFP) and incorporated into baculovirus vector under cytomegalovirus immediate early promoter. These recombinant baculovirus particles were amplified in Sf9 insect cell line. The resulting virus was then used to transduce HepG2 human hepatocytic cells. The transduction efficiency of the virus was measured with flow cytometry, employing the presence of the EGFP as a sign of transduction. Based on the transduction efficiency, a volume providing 70 % transduction efficiency of the virus stock was used to produce apoptotic bodies. These apoptotic bodies were first purified by filtering and then pelleted by ultracentrifugation. Collected apoptotic bodies were immunolabeled using nuclear antigens apolipoprotein H (ApoH), histone 4 (H4), histone 2B (H2B), lysosomal-associated membrane protein 2 (Lamp2), Ku80 and Smith. The selected nuclear antigens as well as NS1 were detected from the apoptotic bodies, except for H2B. The presence of nuclear antigens in the apoptotic bodies is a potential method for immune tolerance disruption which was then addressed by utilizing APCs. The APCs of choice were acute monocytic leukemia derived human monocytes (THP-1) due to their close resemblance to peripheral blood mononuclear cells. THP-1 cells were differentiated using phorbol-12-myristate-13-acetate (PMA). The cells were incubated in medium containing PMA for three days and then five more days in fresh medium to ensure proper differentiation. Differentiated THP-1 cells (dTHP-1) were next fed with purified apoptotic bodies. Internalization of B19V NS1 induced apoptotic bodies was observed using confocal microscopy. Since the APCs were capable of internalizing apoptotic bodies, the following cytokine profile was tentatively studied. The examination of possible immunological effects derived to a conclusion that only the presence of interferon γ inducible protein 10 (IP-10) separated the EGFP or EGFP-NS1 fusion protein containing samples from the control conditions. Although the underlying mechanisms leading into immune tolerance disruption by B19V infection mechanisms warrants further investigation, the data here supports the hypothesis that B19V NS1 induced apoptotic bodies from nonpermissive cells contain self-antigens which could be introduced to the immune system though APCs

Small molecule modulators of amine oxidation, nuclear receptor signaling and glucuronidation : 3-phenylcoumarin as a scaffold of interest

The costs of the drug development process are moderated as computer-aided drug design methods are able to expedite the steps required for lead identification. In fact, computational tools are nowadays virtually indispensable from target identification and validation to preclinical tests due to exponential growth of available information regarding both potential targets and small molecules. One such small molecule with growing number of variations is coumarin. Coumarin scaffold and its various derivatives continue to interest researchers for their vast application potential. Since naturally occurring coumarins are known for example for their antioxidant and anti-inflammatory properties, those molecules are used to guide research endeavors toward similar molecules but with enhanced or newly directed activities. In this doctoral thesis, coumarin derivatives are used to gain novel details regarding monoamine oxidase and nuclear receptor modulation in context relevant for example in neurological conditions and cancer. In order to achieve this, diverse collection of coumarin derivatives is investigated in these targets and corresponding antitargets using both computational and experimental methods. As a result, novel coumarin derivatives with activity in nanomolar range are identified in case of monoamine oxidase B and estrogen receptor ǂ and comparable activity is reached for retinoid-acid-receptor-related orphan receptor DŽt with novel core. In addition, the usability of coumarin derivatives as assay development tools is put to test by designing selective ligands for glucuronidation. Consequently, the metabolic fate of the coumarins is investigated as they are allocated to metabolizing target using homology models, computational methods and experimental techniques. Subsequently, two coumarin derivatives selective for human uridine 5'-diphospho-glucuronosyltransferase 1A10 are identified

How Innovation Service Systems Support the Needs of Research Teams

Innovaatio ymmärretään perinteisesti teknologisena muutoksena. Koska teknologiset edistysaskeleet tarkoittivat kilpailuetua, innovaation tutkimus nousi merkitykselliseksi tutkimusteemaksi. Innovaatiota onkin yritetty ymmärtää useiden sukupolvien ajan, mutta sitä ei ole onnistuttu sovittamaan yhden, kaikenkattavan teorian tai määritelmän alle. Nykyään monimutkaisten pisteytysmenetelmien ja tilastoanalyysien avulla kerätään innovaatioihin liittyvää dataa ja valtioiden, instituutioiden, teollisuuden, ja jopa rahoituselementtien innovaatioaktiviteetit ovat suurennuslasin, yritettäessä tunnistaa niitä tekijöitä, jotka johtavat seuraavaan suureen läpimurtoon. Eikä kaikkea tätä tehdä turhaan – ihmiskuntaa kohtaa ennennäkemättömät haasteet, joiden ratkaisemiseksi tarvitaan kipeästi uusia innovaatioita. Innovaatiopolitiikka on asettanut globaaleja, todella kunnianhimoisia tavoitteita ja rahoitusjärjestöt tarjoavat kohdennettuja ohjelmia ratkaisujen löytämiseksi. On kuitenkin epäselvää, kuinka innovaatiopalvelujärjestelmät, eli järjestelmät, jotka ajavat, seuraavat, ja mittaavat innovaatioita, onnistuvat tukemaan tutkimusryhmien tarpeita. Pohjimmiltaan innovatiivisena aktiviteettina, tutkimus on avain ihmiskunnan suurimpiin haasteisiin, mutta innovaatiopalvelujärjestelmien osallisuus on pääasiassa jätetty huomiotta. Joten tämä tutkimus pyrkii vastaamaan tähän kysymykseen tarkastelemalla kolmea innovaatiopalvelujärjestelmää, jotka ovat: Euroopan komission SEDIA, Suomen akatemian SARA ja Business Finlandin asiointipalvelu. Lisäksi tutkimuksessa haastateltiin kahta eri ikäpolvia edustavaa tutkijaa, jotta mukaan saatiin kokemusasiantuntijoiden mielipide koskien innovaatiopalvelujärjestelmien tukea tutkimusryhmille. Tutkimuskirjallisuuden perusteella todettiin, että tutkimusryhmille ei ole tarjolla yksinomaan heille tarkoitettuja innovaation tunnusmerkkejä. Lisäksi tunnistettiin neljä tarvetta: pitkäjänteinen ja kohdennettu rahoitus, translationaalinen tiede, monitieteelliset tutkimusryhmät, ja tarkoituksen löytäminen. Näiden neljän tarpeen perusteella muodostettiin tutkimushypoteesi, jota testattiin valittuja innovaatiopalvelujärjestelmiä vastaan, ja tutkijoiden mielipiteet kerättiin käyttäen avoimia kysymyksiä. Kaikkinensa todettiin, että vaikka innovaatiopalvelujärjestelmät vastaavat melko hyvin tutkimusryhmien rahoitustarpeisiin, tarvitaan merkittäviä parannuksia ennen kuin järjestelmien voidaan katsoa tukevan tutkimusryhmien tarpeita.Innovation has been traditionally understood as technological change. Since technological advancements meant competitive advantage, it soon became of interest to study innovation. Accordingly, innovation has been poked and probed by generations of researchers, yet it has not been tamed under all-encompassing theory or definition. Nowadays complicated scoring methods and statistical analyses are used to extract innovation data and the innovation activities of nations, institutions, industries, even funding elements alike are under scrutiny when trying to identify the factors leading into big breakthroughs. And it is not all in vain – humanity is facing unprecedented challenges and there is an urgent need for novel innovations. Innovation policies have set ambitious targets all over the globe and funding agencies offer target-ed pro-grams for finding solutions. However, it is unclear how the innovation service systems, the systems that drive, follow, and measure innovation, are able to support the needs of researcher teams in this equation. As a fundamentally innovative activity, research is the key to solving the humanity’s grand challenges, but the contribution of innovation service systems has been largely ignored. Thus, this study sets out to answer this question by looking into three innovation service systems, SEDIA by European Commission, SARA by Academy of Finland, and Business Finland Online service by Business Finland. In addition, two researchers, representing different levels of seniority were inter-viewed to gain experienced expert opinions regarding the support innovation service systems offer to research teams. Based on research literature, it was noticed that research teams lack dedicated innovation indicators. In addition, four needs were identified; sustained and directed funding, translational science, interdisciplinary research teams and finding purpose. These four needs formulated the hypothesis that innovation service systems are in their support. The hypothesis was then tested against the selected innovation service systems, and the opinions of the researchers were collected using open-ended questions. Ultimately, it was concluded that, although the innovation service systems answer rather well to the funding needs of research teams, there are significant improvements that must occur before innovation service systems support the needs of research teams

Identification of estrogen receptor α ligands with virtual screening techniques

Utilization of computer-aided molecular discovery methods in virtual screening (VS) is a cost-effective approach to identify novel bioactive small molecules. Unfortunately, no universal VS strategy can guarantee high hit rates for all biological targets, but each target requires distinct, fine-tuned solutions. Here, we have studied in retrospective manner the effectiveness and usefulness of common pharmacophore hypothesis, molecular docking and negative image-based screening as potential VS tools for a widely applied drug discovery target, estrogen receptor α (ERα). The comparison of the methods helps to demonstrate the differences in their ability to identify active molecules. For example, structure-based methods identified an already known active ligand from the widely-used bechmarking decoy molecule set. Although prospective VS against one commercially available database with around 100,000 drug-like molecules did not retrieve many testworthy hits, one novel hit molecule with pIC50 value of 6.6, was identified. Furthermore, our small in-house compound collection of easy-to-synthesize molecules was virtually screened against ERα, yielding to five hit candidates, which were found to be active in vitro having pIC50 values from 5.5 to 6.5.peerReviewe

Human Parvovirus B19 Induced Apoptotic Bodies Contain Altered Self-Antigens that are Phagocytosed by Antigen Presenting Cells.

Human parvovirus B19 (B19V) from the erythrovirus genus is known to be a pathogenic virus in humans. Prevalence of B19V infection has been reported worldwide in all seasons, with a high incidence in the spring. B19V is responsible for erythema infectiosum (fifth disease) commonly seen in children. Its other clinical presentations include arthralgia, arthritis, transient aplastic crisis, chronic anemia, congenital anemia, and hydrops fetalis. In addition, B19V infection has been reported to trigger autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. However, the mechanisms of B19V participation in autoimmunity are not fully understood. B19V induced chronic disease and persistent infection suggests B19V can serve as a model for viral host interactions and the role of viruses in the pathogenesis of autoimmune diseases. Here we investigate the involvement of B19V in the breakdown of immune tolerance. Previously, we demonstrated that the non-structural protein 1 (NS 1) of B19V induces apoptosis in non-permissive cells lines and that this protein can cleave host DNA as well as form NS1-DNA adducts. Here we provide evidence that through programmed cell death, apoptotic bodies (ApoBods) are generated by B19V NS1 expression in a nonpermissive cell line. Characterization of purified ApoBods identified potential self-antigens within them. In particular, signature self-antigens such as Smith, ApoH, DNA, histone H4 and phosphatidylserine associated with autoimmunity were present in these ApoBods. In addition, when purified ApoBods were introduced to differentiated macrophages, recognition, engulfment and uptake occurred. This suggests that B19V can produce a source of self-antigens for immune cell processing. The results support our hypothesis that B19V NS1-DNA adducts, and nucleosomal and lysosomal antigens present in ApoBods created in non-permissive cell lines, are a source of self-antigens.peerReviewe

Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives

A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-b-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase pathway. Five analogues produced 62% HSD1 inhibition at 5 mM and, furthermore, three of them produced 68% inhibition at 1 mM. A docking-based structure-activity relationship analysis was done to determine the molecular basis of the inhibition and the cross-reactivity of the analogues was tested against oestrogen receptor, aromatase, cytochrome P450 1A2, and monoamine oxidases. Most of the analogues are only modestly active with 17-b-hydroxysteroid dehydrogenase 2 – a requirement for lowering effective oestradiol levels in vivo. Moreover, the analysis led to the synthesis and discovery of 3-imidazolecoumarin as a potent aromatase inhibitor. In short, coumarin core can be tailored with specific ring and polar moiety substitutions to block either the sulphatase pathway or the aromatase pathway for treating breast cancer and endometriosis.peerReviewe

Lupus specific antigens DNA and Smith are seen in purified ApoBods.

<p>Confocal microscopy images of purified ApoBods produced from with (<b>A</b>) <i>Ac</i>EGFP, and (<b>B</b>) <i>Ac</i>EGFP-NS1 transduced cells and (<b>C</b>) Staurosporine treated HepG2 cells. The visualized for EGFP, DNA, H2B and Smith antigens are presented as green, blue, red and violet. Morphology of the ApoBods are seen in the DIC image. Bars 5 µm. Antigen markers of ApoBods from each group was seen in (<b>D</b>) by showing as mean ± SEM, N = 30. *<i>P</i> < 0.05.</p