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

Molecular docking and oxidation kinetics of 3-phenyl coumarin derivatives by human CYP2A13

CYP2A13 enzyme is expressed in human extrahepatic tissues, while CYP2A6 is a hepatic enzyme. Reactions catalysed by CYP2A13 activate tobacco-specific nitrosamines and some other toxic xenobiotics in lungs. To compare oxidation characteristics and substrate-enzyme active site interactions in CYP2A13 vs CYP2A6, we evaluated CYP2A13 mediated oxidation characteristics of 23 coumarin derivatives and modelled their interactions at the enzyme active site. CYP2A13 did not oxidise six coumarin derivatives to corresponding fluorescent 7-hydroxycoumarins. The K-m-values of the other coumarins varied 0.85-97 mu M, V-max-values of the oxidation reaction varied 0.25-60 min(-1), and intrinsic clearance varied 26-6190 kL/min*mol CYP2A13). K-m of 6-chloro-3-(3-hydroxyphenyl)-coumarin was 0.85 (0.55-1.15 95% confidence limit) mu M and V-max 0.25 (0.23-0.26) min(-1), whereas K-m of 6-hydroxy-3-(3-hydroxyphenyl)-coumarin was 10.9 (9.9-11.8) mu M and V-max 60 (58-63) min(-1). Docking analyses demonstrated that 6-chloro or 6-methoxy and 3-(3-hydroxyphenyl) or 3-(4-trifluoromethylphenyl) substituents of coumarin increased affinity to CYP2A13, whereas 3-triazole or 3-(3-acetate phenyl) or 3-(4-acetate phenyl) substituents decreased it. The active site of CYP2A13 accepts more diversified types of coumarin substrates than the hepatic CYP2A6 enzyme. New sensitive and convenient profluorescent CYP2A13 substrates were identified, such as 6-chloro-3-(3-hydroxyphenyl)-coumarin having high affinity and 6-hydroxy-3-(3-hydroxyphenyl)-coumarin with high intrinsic clearance

Substrate Selectivity of Coumarin Derivatives by Human CYP1 Enzymes: In Vitro Enzyme Kinetics and In Silico Modeling

Of the three enzymes in the human cytochrome P450 family 1, CYP1A2 is an important enzyme mediating metabolism of xenobiotics including drugs in the liver, while CYP1A1 and CYP1B1 are expressed in extrahepatic tissues. Currently used CYP substrates, such as 7-ethoxycoumarin and 7-ethoxyresorufin, are oxidized by all individual CYP1 forms. The main aim of this study was to find profluorescent coumarin substrates that are more selective for the individual CYP1 forms. Eleven 3-phenylcoumarin derivatives were synthetized, their enzyme kinetic parameters were determined, and their interactions in the active sites of CYP1 enzymes were analyzed by docking and molecular dynamic simulations. All coumarin derivatives and 7-ethoxyresorufin and 7-pentoxyresorufin were oxidized by at least one CYP1 enzyme. 3-(3-Methoxyphenyl)-6-methoxycoumarin (19) was 7-O-demethylated by similar high efficiency [21-30 ML/(min.mol CYP)] by all CYP1 forms and displayed similar binding in the enzyme active sites. 3-(3-Fluoro-4-acetoxyphenyl)coumarin (14) was selectively 7-O-demethylated by CYP1A1, but with low efficiency [0.16 ML/(min mol)]. This was explained by better orientation and stronger H-bond interactions in the active site of CYP1A1 than that of CYP1A2 and CYP1B1. 3-(4-Acetoxyphenyl)-6-chlorocoumarin (20) was 7-O-demethylated most efficiently by CYP1B1 [53 ML/(min.mol CYP)], followed by CYP1A1 [16 ML/(min.mol CYP)] and CYP1A2 [0.6 ML/(min.mol CYP)]. Variations in stabilities of complexes between 20 and the individual CYP enzymes explained these differences. Compounds 14, 19, and 20 are candidates to replace traditional substrates in measuring activity of human CYP1 enzymes

Ilmastonmuutoksen vaikutuksia Suomen huoltovarmuudelle

Huoltovarmuuden keskiössä ovat yhteiskunnalle vakavat häiriötilanteet ja poikkeusolot, joihin yhteiskunta pyrkii varautumaan ja niissä toimimaan. Ajankohtaisia esimerkkejä ovat COVID-pandemia sekä meneillään oleva energiakriisi, joiden osalta myös Suomen huoltovarmuus on ollut vahvasti esillä. Myös sääilmiöt voivat aiheuttaa häiriötilanteita: Suomessa näitä ovat esimerkiksi myrskyt, rajuilmat, tulvat ja kuivuus, jotka voivat ainakin periaatteessa lamaannuttaa yhteiskuntaa. Sääilmiöiden vaikutuksiin varautuminen on mahdollista, mutta asiaa hankaloittaa meneillään oleva ilmaston nopea muuttuminen. Vaikutuksia aiheuttavista sääilmiöistä osa on nopeita ja rajuja (esim. voimakas rajuilma) ja osa puolestaan muodostuu hitaammin (esim. pitkät hellejaksot), ja ilmastonmuutos vaikuttaa ilmiöihin eri tavoin. Tässä työssä perehdyttiin siihen, millaisia arvioituja vaikutuksia ilmastonmuutoksella on Suomen huoltovarmuuteen. Lähtökohtana oli Huoltovarmuuskeskuksen kanssa järjestettyjen työpajojen sekä asiantuntijahaastattelujen avulla saada käsitys siitä, mitkä sääilmiöt ja säätilanteet ovat huoltovarmuuden kannalta keskeisiä ja mitkä huoltovarmuuden toimialat ovat haavoittuvimmat. Haastatteluiden ohella työssä käytiin läpi menneitä merkittäviä säätilanteita, joilla tiedetään olleen merkittäviä yhteiskunnallisia vaikutuksia. Myös viimeaikaisesta kirjallisuudesta poimittiin tietoa mm. eri toimialojen haavoittuvuuksista sekä sopeutumiskyvystä. Koottuun tietoon yhdistettiin arviot ilmastonmuutoksen vaikutuksista tunnistettuihin ilmiöihin, jolloin saatiin ensikäsitys siitä, miten ilmastonmuutos vaikuttaa Suomen huoltovarmuuteen. Tulosten pohjalta voidaan todeta, että ilmastonmuutoksen vaikutukset huoltovarmuuteen ovat varsin monisyisiä, johtuen etenkin sääilmiöiden laajasta kirjosta ja erilaisista vaikutusmekanismeista. Lisäksi asiaa hankaloittaa se, ettei ole olemassa selvää rajanvetoa siitä, mikä itse asiassa on huoltovarmuuskriittinen sääilmiö ja mikä ei. Voivatko esimerkiksi yleistyvien helteiden johdosta kasvavat haittavaikutukset terveydenhuollolle saavuttaa jossain vaiheessa vakavan yhteiskunnallisen häiriötilanteen, jos asiaan ei varauduta riittävästi etukäteen? Toinen keskeinen tulos on, että huoltovarmuuden kannalta ilmastonmuutoksen suorat vaikutukset ovat Suomessa hyvin pieniä verrattuna muualle maailmaan. Vaikka Suomessakin ilmasto on jo muuttunut huomattavasti ja muuttuu tulevaisuudessa edelleen, suurimmat huoltovarmuusvaikutukset näyttävät heijastuvan muualta: pahimpana tilanteena Huoltovarmuuskeskuksen asiantuntijat pitävät ruoan, veden ja elintilan puutetta maailmalla, jonka vaikutukset heijastuisivat myös Suomeen. Tätä huomiota tukevat myös Suomen ilmastonmuutosarviot. Toimialoista haavoittuvimmiksi koetaan elintarvike- ja energiahuolto sekä logistiikka. Työssä keskityttiin pääasiassa ilmastonmuutoksen suoriin vaikutuksiin eli ilmastonmuutoksen vaikutuksiin sääilmiöiden esiintymiseen. Työssä sivutaan kuitenkin myös epäsuoria eli muualta maailmalta heijastuvia vaikutuksia ja siirtymävaikutuksia, jotka johtuvat ilmastonmuutoksen hillintätoimista, etenkin nopeasta energiamurroksesta.I centrum för försörjningsberedskapen i samhället står beredskap för allvarliga störningar och exceptionella förhållanden, som samhället försöker förbereda sig för och agera i dem. Aktuella exempel är COVID-pandemin och den pågående energikrisen, där Finlands försörjningsberedskap också har lyfts fram. Även väderfenomen kan orsaka störningar: i Finland handlar det till exempel om stormar, hårda åskväder, översvämningar och torka, som åtminstone i princip kan förlama samhället. Det är möjligt att förbereda sig mot effekter av väderfenomen, men saken kompliceras av de pågående snabba klimatförändringarna. Vissa av väderfenomenen som orsakar effekter är snabba och våldsamma (t.ex. hård åska) medan en del utvecklas långsammare (t.ex. långa värmeböljor), och klimatförändringen påverkar fenomenen på olika sätt. Detta arbete fördjupade sig i att uppskatta effekterna av klimatförändringen på Finlands försörjningsberedskap. Utgångspunkten var att få en förståelse för vilka väderfenomen och vädersituationer som är centrala för försörjningsberedskapen och vilka sektorer inom försörjningsberedskapen som är mest utsatta, genom arbetsseminarium och expertintervjuer som anordnas tillsammans med Försörjningsberedskapscentralen. Utöver intervjuerna omfattade arbetet genomgång av tidigare signifikanta vädersituationer som man vet har haft betydande effekter i samhället. Information hämtades också från nyare litteratur, t.ex. om olika branschers sårbarhet och anpassningsförmåga. Klimatförändringens uppskattade effekter på de identifierade fenomenen kombinerades med den insamlade informationen, vilket resulterade i en första uppfattning om hur klimatförändringarna påverkar Finlands försörjningsberedskap. Baserat på resultaten kan man dra slutsatsen att klimatförändringens effekter på försörjningsberedskap är ganska komplexa, särskilt på grund av det breda spektrumet av väderfenomen och olika påverkningsmekanismer. Dessutom kompliceras saken av att det inte finns någon tydlig avgränsning av vad som egentligen är och inte är ett väderfenomen som är kritiskt för försörjningsberedskap. Kan till exempelvis de ökande negativa effekterna på hälso- och sjukvården på grund av de allt vanliga värmeförhållandena någon gång orsaka en allvarlig social störningssituation, om ärendet inte är tillräckligt förberett i förväg? Ett annat betydelsefullt resultat är att de direkta effekterna av klimatförändringen i fråga om försörjningsberedskap är mycket små i Finland jämfört med i resten av världen. Även om klimatet i Finland redan har förändrats avsevärt och kommer att fortsätta förändras i framtiden, tycks de största effekterna av försörjningsberedskapen återspeglas på annat håll: experterna på Försörjningsberedskapscentralen anser att den värsta situationen är brist på mat, vatten och livsrum i världen, vars effekter även skulle återspeglas i Finland. Bland industrierna upplevs livsmedel och energiförsörjning och logistik som de mest utsatta. Arbetet fokuserade främst på klimatförändringens direkta effekter, det vill säga klimatförändringens effekter på förekomsten av väderfenomen. Arbetet berör dock även indirekta effekter, det vill säga effekter som reflekteras från resten av världen och övergångseffekter som är ett resultat av åtgärder för att minska klimatförändringen, särskilt från den snabba energiomställningen.Serious disruptions and exceptional circumstances for society, that the society tries to prepare for and act in them are at the center of security of supply. Current examples are the COVID pandemic and the ongoing energy crisis for which Finland's security of supply has also been strongly highlighted. Disturbances can also be caused by weather phenomena: in Finland, such examples are windstorms, severe thunderstorms, floods, and droughts, which can, at least in principle, paralyze the society. It is possible to prepare for the impacts of weather phenomena, but the ongoing rapid climate change makes it more complicated. Some of the weather phenomena that cause impacts are fast and violent (e.g. intense thunderstorms) and some occur more slowly (e.g. long heat waves), and climate change affects the phenomena in different ways. In this work, the estimated impacts of climate change on Finland's security of supply were investigated. The starting point was to gain an understanding of which weather phenomena and weather situations are central to security of supply and which sectors of security of supply are the most vulnerable. The work constituted of workshops and expert interviews organized with the National Emergency Supply Agency. In addition to the interviews, the work covered past significant weather situations in Finland that are known to have had significant societal impacts. Information was also extracted from recent literature, especially regarding the vulnerabilities and adaptability of different sectors in Finland. Estimates of the climate change impacts on the identified phenomena were combined with the collected information, resulting in a first understanding of how climate change affects Finland's security of supply. Based on the results, it can be concluded that the impacts of climate change on security of supply are quite complex, especially due to the wide spectrum of weather phenomena and their different impact mechanisms. In addition, the matter becomes more complicated by the fact that there is no clear distinction of what weather phenomenon actually is critical to security of supply and what is not. For example, could the increasing adverse impacts on health care due to the increasingly common heat conditions reach a serious societal disturbance situation at some point, if it is not sufficiently prepared in advance? Another key result is that in terms of security of supply, the direct effects of climate change are very small in Finland compared to many other countries. Although the climate in Finland has already changed considerably and will continue to change in the future, the biggest impacts to security of supply seem to be reflected from elsewhere: the experts of the National Emergency Supply Agency consider the worst situation to be a lack of food, water and habitable living environment in the world, which would also be reflected to Finland. Among the sectors, food/water and energy supply and logistics are perceived as the most vulnerable. The work mainly focused on the direct effects of climate change, i.e. the effects of climate change on the occurrence of various weather phenomena. However, the work also considers to some extent indirect effects, i.e. those reflected from other parts of the world, and transitional effects that result from climate change mitigation measures, especially from the rapid energy transition

Structure and characterization of a novel chicken biotin-binding protein A (BBP-A)

Background. The chicken genome contains a BBP-A gene showing similar characteristics to avidin family genes. In a previous study we reported that the BBP-A gene may encode a biotin-binding protein due to the high sequence similarity with chicken avidin, especially at regions encoding residues known to be located at the ligand-binding site of avidin. Results. Here, we expand the repertoire of known macromolecular biotin binders by reporting a novel biotin-binding protein A (BBP-A) from chicken. The BBP-A recombinant protein was expressed using two different expression systems and purified with affinity chromatography, biochemically characterized and two X-ray structures were solved – in complex with D-biotin (BTN) and in complex with D-biotin D-sulfoxide (BSO). The BBP-A protein binds free biotin with high, "streptavidin-like" affinity (Kd ~ 10-¹³ M), which is about 50 times lower than that of chicken avidin. Surprisingly, the affinity of BBP-A for BSO is even higher than the affinity for BTN. Furthermore, the solved structures of the BBP-A – BTN and BBP-A – BSO complexes, which share the fold with the members of the avidin and lipocalin protein families, are extremely similar to each other. Conclusion. BBP-A is an avidin-like protein having a β-barrel fold and high affinity towards BTN. However, BBP-A differs from the other known members of the avidin protein family in thermal stability and immunological properties. BBP-A also has a unique ligand-binding property, the ability to bind BTN and BSO at comparable affinities. BBP-A may have use as a novel material in, e.g. modern bio(nano)technological applications.peerReviewe

Structure and characterization of a novel chicken biotin-binding protein A (BBP-A)

BACKGROUND: The chicken genome contains a BBP-A gene showing similar characteristics to avidin family genes. In a previous study we reported that the BBP-A gene may encode a biotin-binding protein due to the high sequence similarity with chicken avidin, especially at regions encoding residues known to be located at the ligand-binding site of avidin. RESULTS: Here, we expand the repertoire of known macromolecular biotin binders by reporting a novel biotin-binding protein A (BBP-A) from chicken. The BBP-A recombinant protein was expressed using two different expression systems and purified with affinity chromatography, biochemically characterized and two X-ray structures were solved – in complex with D-biotin (BTN) and in complex with D-biotin D-sulfoxide (BSO). The BBP-A protein binds free biotin with high, "streptavidin-like" affinity (K(d )~ 10(-13 )M), which is about 50 times lower than that of chicken avidin. Surprisingly, the affinity of BBP-A for BSO is even higher than the affinity for BTN. Furthermore, the solved structures of the BBP-A – BTN and BBP-A – BSO complexes, which share the fold with the members of the avidin and lipocalin protein families, are extremely similar to each other. CONCLUSION: BBP-A is an avidin-like protein having a β-barrel fold and high affinity towards BTN. However, BBP-A differs from the other known members of the avidin protein family in thermal stability and immunological properties. BBP-A also has a unique ligand-binding property, the ability to bind BTN and BSO at comparable affinities. BBP-A may have use as a novel material in, e.g. modern bio(nano)technological applications

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

Data-to-music sonification and user engagement

The process of transforming data into sounds for auditory display provides unique user experiences and new perspectives for analyzing and interpreting data. A research study for data transformation to sounds based on musical elements, called data-to-music sonification, reveals how musical characteristics can serve analytical purposes with enhanced user engagement. An existing user engagement scale has been applied to measure engagement levels in three conditions within melodic, rhythmic, and chordal contexts. This article reports findings from a user engagement study with musical traits and states the benefits and challenges of using musical characteristics in sonifications. The results can guide the design of future sonifications of multivariable data

Synthesis and structural studies of some supramolecular compounds

Acidic complexing ligand and neutral macrocyclic compounds were synthesised as receptors for either ions or uncharged organic molecules. The compounds were purified and characterised using normal methods and the X-ray structures of 14 compounds were determined. The acidic ligand (TEATA) formed a Na complex with two ligand molecules, both with one Na+ ion interaction and both co-ordinated to a third, central, Na+ ion (established in solid state by X-ray structure determination) (VII, VIII). Inside this extraordinary dimeric assembly (a pseudo-cryptate) the three sodium ions are encapsulated, as in cryptates. The macrocyclic compounds were synthesised in two ways, one-step and twostep reactions, using piperazine as a building block. These piperazine macrocycles show 4 different types of structural properties in solid state: metal-complexing (II), self-assembly / self-complementary (IV, V, VI), crystal lattice inclusion (clathrate) (IX) and molecular inclusion (X, XI, XII, XIII, XIV) compounds. In addition, azobenzene units have been incorporated into some of these piperazine macrocycles (XIII, XIV) enable variation of the shape and size of the cavity by UV-light. A short review of the literature concerning the place of the supramolecular chemistry in the field of chemistry precedes the summary of experimental results. The reader is recommended to preview the author's original publications (papers I-VI, end of this thesis) before the summary of experimental results. The summary of experimental results contains additions to the publications, especially to that of the packing structures