Homogeneous assay technologies in drug screening: Quenching Resonance Energy Transfer (QRET) technique

Information gained from the human genome project and improvements in compound synthesizing have increased the number of both therapeutic targets and potential lead compounds. This has evolved a need for better screening techniques to have a capacity to screen number of compound libraries against increasing amount of targets. Radioactivity based assays have been traditionally used in drug screening but the fluorescence based assays have become more popular in high throughput screening (HTS) as they avoid safety and waste problems confronted with radioactivity. In comparison to conventional fluorescence more sensitive detection is obtained with time-resolved luminescence which has increased the popularity of time-resolved fluorescence resonance energy transfer (TR-FRET) based assays. To simplify the current TR-FRET based assay concept the luminometric homogeneous single-label utilizing assay technique, Quenching Resonance Energy Transfer (QRET), was developed. The technique utilizes soluble quencher to quench non-specifically the signal of unbound fraction of lanthanide labeled ligand. One labeling procedure and fewer manipulation steps in the assay concept are saving resources. The QRET technique is suitable for both biochemical and cell-based assays as indicated in four studies:1) ligand screening study of β2 -adrenergic receptor (cell-based), 2) activation study of Gs-/Gi-protein coupled receptors by measuring intracellular concentration of cyclic adenosine monophosphate (cell-based), 3) activation study of G-protein coupled receptors by observing the binding of guanosine-5’-triphosphate (cell membranes), and 4) activation study of small GTP binding protein Ras (biochemical). Signal-to-background ratios were between 2.4 to 10 and coefficient of variation varied from 0.5 to 17% indicating their suitability to HTS use.

Alpha- and gamma-melanocyte stimulating hormones in obesity. A study of the key central areas of metabolic regulation

The melanocortin system is an important regulator of feeding, energy metabolism,and cardiovascular function and it consists of the pro-opiomelanocortin (POMC) derived melanocyte stimulating hormones (α-, β- and γ-MSH) and their endogenous melanocortin receptors, MC1R to MC5R. In the hypothalamus, α-MSH reduces food intake, and increases energy expenditure and sympathetic tone by binding to MC4R. Mutations affecting the MC4R gene lead to obesity in mammals. On the other hand, the metabolic effects of MC3R stimulation using agonists such as the endogenously expressed γ-MSH have been less extensively explored. The main objective of this study was to investigate the long-term effects of increased melanocortin tone in key areas of metabolic regulation in the central nervous system (CNS) in order to investigate the sitespecific roles of both α-MSH and γ-MSH. The aim was to stereotaxically induce local overexpression of single melanocortin peptides using lentiviral vectors expressing α-MSH (LVi-α-MSH-EGFP) and γ-MSH (LVi-γ-MSH-EGFP). The lentiviral vectors were shown to produce a long-term overexpression and biologically active peptides in cell-based assays. The LVi-α-MSHEGFP was targeted to the arcuate nucleus in the hypothalamus of diet induced obese mice where it reduced weight gain and adiposity independently of food intake. When the nucleus tractus solitarus in the brainstem was targeted, the LVi-α-MSH-EGFP treatment was shown to cause a small decrease in adiposity, which did not impact weight development. However, the α-MSH treatment increased heart rate, which was attenuated by adrenergic receptor blockade indicative of increased sympathetic activity. The LVi-γ-MSH-EGFP was targeted to the hypothalamus where it decreased fat mass in mice eating the standard diet, but the effect was abated if animals consumed a high-fat Western type diet. When the diet induced obese mice were subjected again to the standard diet, the LVi-γ-MSH-EGFP treated animals displayed increased weight loss and reduced adiposity. These results indicate that the long-term central anti-obesity effects of α-MSH are independent of food intake. In addition, overexpression of α-MSH in the brain stem efficiently blocked the development of adiposity, but increased sympathetic tone. The evidence presented in this thesis also indicates that selective MC3R agonists such as γ-MSH could be potential therapeutics in combination with low fat diets.

The effects of selective estrogen receptor modulators on the death of breast cancer cells and osteoblastic cells

Selektiivisten estrogeenireseptorin muuntelijoiden (serm) vaikutus rintasyöpäsolujen ja luun solujen kuolemaan Selektiiviset estrogeenireseptorin muuntelijat (SERMit) ovat ryhmä kemialliselta rakenteeltaan erilaisia yhdisteitä jotka sitoutuvat solunsisäisiin estrogeenireseptoreihin toimien joko estrogeenin kaltaisina yhdisteinä tai estrogeenin vastavaikuttajina. Tamoksifeeni on SERM –yhdiste, jota on jo pitkään käytetty estrogeenireseptoreita (ER) ilmentävän rintasyövän lääkehoidossa. Tamoksifeeni sekä estää rintasyöpäsolujen jakaantumista että toisaalta aikaansaa niiden apoptoosin eli ohjelmoidun solukuoleman muuntelemalla ER-välitteisesti kohdesolun geenien ilmentymistä. Viimeaikaiset tutkimustulokset ovat kuitenkin osoittaneet tamoksifeenilla olevan myös nopeampia, nongenomisia vaikutusmekanismeja. Tässä väitöskirjatyössä tutkimme niitä nopeita vaikutusmekanismeja joiden avulla tamoksifeeni vaikuttaa rintasyöpäsolujen elinkykyyn. Osoitamme että tamoksifeeni farmakologisina pitoisuuksina aikaansaa nopean mitokondriaalisen solukuolemaan johtavan signallointireitin aktivoitumisen rintasyöpäsoluissa. Tämän lisäksi tutkimme myös tamoksifeenin aiheuttamaan mitokondriovaurioon johtavia tekijöitä. Tutkimustuloksemme osoittavat että ER-positiivisissa rintasyöpäsoluissa tamoksifeeni indusoi pitkäkestoisen ERK-kinaasiaktivaation, joka voidaan estää 17-beta-estradiolilla. Tamoksifeenin aikaansaama nopea solukuolema on pääosin ER:sta riippumaton tapahtuma, mutta siihen voidaan vaikuttaa myös ER-välitteisin mekanismein. Sen sijaan epidermaalisen kasvutekijäreseptorin (EGFR) voitiin osoittaa osallistuvan tamoksifeenin nopeiden vaikutusten välittämiseen. Tämän lisäksi vertailimme myös estradiolin ja eri SERM-yhdisteiden kykyä suojata apoptoosilta käyttämällä osteoblastiperäisiä soluja. Pytyäksemme vertailemaan ER-isotyyppien roolia eri yhdisteiden suojavaikutuksissa, transfektoimme U2OS osteosarkoomasolulinjan ilmentämään pysyvästi joko ERalfaa tai ERbetaa. Tulostemme mukaan sekä estradioli että uusi SERM-yhdiste ospemifeeni suojaavat osteoblastin kaltaisia soluja etoposidi-indusoidulta apoptoosilta. Sekä ERalfa että ERbeta pystyivät välittämään suojavaikutusta, joskin vaikutukset erosivat toisistaan. Lisäksi havaitsimme edellä mainitun suojavaikutuksen olevan yhteydessä muutoksiin solujen sytokiiniekspressiossa. Tietoa SERM-yhdisteiden anti-ja proapoptoottisten vaikutusmekanismeista eri kohdekudoksissa voidaan mahdollisesti hyödyntää kehiteltäessä uusia kudosspesifisiä SERM-yhdisteitä.

Alpha2-Adrenoceptor-mediated vascular responses induced by dexmedetomidine

Alpha2-Adrenoceptors are cell-surface G protein coupled receptors that mediate many of the effects of the catecholamines noradrenaline and adrenaline. The three human α2-adrenoceptor subtypes are widely expressed in different tissues and organs, and they mediate many different physiological and pharmacological effects in the central and peripheral nervous system and as postsynaptic receptors in target organs. Previous studies have demonstrated that α2-adrenoceptors mediate both vascular constriction and dilatation in humans. Large inter-individual variation has been observed in the vascular responses to α2-adrenoceptor activation in clinical studies. All three receptor subtypes are potential drug targets. It was therefore considered important to further elucidate the details of adrenergic vascular regulation and its genetic variation, since such knowledge may help to improve the development of future cardiovascular drugs and intensive care therapies. Dexmedetomidine is the most selective and potent α2-adrenoceptor agonist currently available for clinical use. When given systemically, dexmedetomidine induces nearly complete sympatholysis already at low concentrations, and postsynaptic effects, such vasoconstriction, can be observed with increasing concentrations. Thus, local infusions of small doses of dexmedetomidine into dorsal hand veins and the application of pharmacological sympathectomy with brachial plexus block provide a means to assess drug-induced peripheral vascular responses without interference from systemic pharmacological effects and autonomic nervous system regulation. Dexmedetomidine was observed to have biphasic effects on haemodynamics, with an initial decrease in blood pressure at low concentrations followed by substantial increases in blood pressure and coronary vascular resistance at high concentrations. Plasma concentrations of dexmedetomidine that significantly exceeded the recommended therapeutic level did not reduce myocardial blood flow below the level that is observed with the usual therapeutic concentrations and did not induce any evident myocardial ischaemia in healthy subjects. Further, it was demonstrated that dexmedetomidine also had significant vasodilatory effects through activation of endothelial nitric oxide synthesis, and thus when the endothelial component of the blood vessel response to dexmedetomidine was inhibited, peripheral vasoconstriction was augmented. Hand vein constriction responses to α2-adrenoceptor activation by dexmedetomidine were only weakly associated with the constriction responses to α1-adrenoceptor activation, pointing to independent cellular regulation by these two adrenoceptor classes. Substantial inter-individual variation was noted in the venous constriction elicited by activation of α2-adrenoceptors by dexmedetomidine. In two study populations from two different continents, a single nucleotide polymorphism in the PRKCB gene was found to be associated with the dorsal hand vein constriction response to dexmedetomidine, suggesting that protein kinase C beta may have an important role in the vascular α2-adrenoceptor signalling pathways activated by dexmedetomidine.