Angeliki P. Kourounakis

Bio: Angeliki P. Kourounakis is an academic researcher from National and Kapodistrian University of Athens. The author has contributed to research in topics: Lipid peroxidation & Antioxidant. The author has an hindex of 24, co-authored 59 publications receiving 1684 citations. Previous affiliations of Angeliki P. Kourounakis include Utrecht University & Aristotle University of Thessaloniki.

Inverse agonism at G protein‐coupled receptors: (patho)physiological relevance and implications for drug discovery

Abstract: The largest family of cell surface receptors involved in signal transduction, G protein coupled receptors (GPCRs), are one of the major targets for current drugs as well as new drug development. Ligands interacting with for e.g. adrenergic, histamine, adenosine, opioid, dopamine or serotonin receptors, constitute a large portion of currently used therapeutics. A common property of GPCRs is that upon activation (agonist binding) they transmit signals across the plasma membrane via an interaction with heterotrimeric G proteins (Stadel et al., 1997). The corresponding activated G protein subsequently interacts with an intracellular effector system, such as adenylate cyclase or phospholipase C, leading to a wide variety of distinct physiological responses. Recent evidence suggests that GPCRs have the potential to be 'active' even in the absence of an agonist. This exhibition of spontaneous receptor activity has led to the observation that various ligands, previously considered as antagonists with no intrinsic activity, actually can inhibit this spontaneous activity, appearing to possess 'negative intrinsic activity'. This phenomenon has been termed inverse agonism and the corresponding ligands are referred to as inverse agonists. Although intrinsic constitutive receptor activity and inverse agonism have unequivocally been demonstrated in vitro, (patho)physiological consequences are far from self-evident. Thus, in this review we should like to focus on the expression of inverse agonism under more 'physiological conditions', since it appears timely to address the physiological relevance and consequences of this new concept, both in GPCR research and drug discovery. Historical overview Traditional receptor theory has postulated on a single, 'quiescent' receptor state to which agonists bind inducing a conformational change of the receptor to an activated and 'functional' state. This view, initially formed in the early 1950s, was more clearly expressed by Del Castillo & Katz (1957), and stood as the foundation of receptor pharmacology for decades. It was believed that antagonists interact with the receptor, thereby preventing agonist binding, without having an effect on conformational changes of the receptor that remained in its 'quiescent' state. The first evidence of a ligand ('antagonist') producing opposite effects to those of an agonist, and thus not merely inhibiting agonist binding, stems from the GABA-benzodiazepine field. In Braestrup et al. 1982, reported on the discovery of an agent, DMCM (methyl 6,7-dimethoxy-4-ethyl-β-carboline-e-carboxylate), which in contrast to benzodiazepines, not only was a potent convulsant in vivo, but seemed to favour binding to benzodiazepine receptors that were in the non-GABA(agonist)-stimulated conformation. For the first time, it was elaborated that GABA-benzodiazepine receptors may perhaps exist in two conformations which are in equilibrium, an open chloride channel form (activated conformation) and a closed one (inactivated conformation) for which DMCM may have a high affinity and a tendency to stabilize, thereby decreasing binding of GABA to the activated conformation. Interestingly, the above concept of an 'agonist-independent' two-state receptor conformation, introduced initially for ion-channel-coupled receptors, soon found support from those studying the large family of GPCRs. Costa & Herz (1989) were pioneers in setting the grounds for what later would be referred to as 'inverse agonism'. They demonstrated for the first time that some antagonists of the δ opioid receptor had 'negative intrinsic activity' in vitro, in contrast to others that lacked any intrinsic activity. Moreover, such ligands diminished even further the 'basal' or constitutive activity of the receptor defined as the activity of the receptor in the absence of any ligand. It was apparent that the concept of 'negative intrinsic activity' implied a pre-existing equilibrium between (at least) two states of a receptor. These two states could easily be defined as either a G protein-bound or a free form of the receptor, the first one active and the latter inactive. In their studies, basal GTPase activity in NG108-15 cell membranes was suggested to be due to stimulated activity resulting from a spontaneous interaction between empty or free receptors and G proteins. Their data lent support to the receptor model of Wregget & De Lean (1984) which predicted that 'antagonists may be active by hindering the ability of receptors to associate spontaneously with G proteins in membranes'. Since then, and especially over the past few years, disclosure of negative intrinsic activity has corroborated even further this two-state model of GPCR activation. Hence, a large number of publications within this decade are concerned with, and demonstrate with a variety of systems or means, the phenomenon of constitutive receptor activity and its implication for inverse agonism. In this review we will first address some of the more seminal papers, showing that in most cases genetically engineered cell systems were pivotal for the development of this new concept. These and many more studies have been adequately and thoroughly reviewed recently by Milligan et al. (1997) and Leurs et al. (1998). We will then gradually move to more 'physiological' systems, in order to address the central issue of this review whether inverse agonism and spontaneous receptor activity are relevant phenomena in health and disease, and hence for drug discovery. It should be pointed out, however, that a classification of ligands based on their (negative) intrinsic activity is not an easy task. Due to the large influence of receptor systems and experimental conditions (whole cells versus membranes, stoichiometry of receptors/G protein, signalling proteins etc.) the same ligand may behave as an inverse agonist, a neutral antagonist or even a (partial) agonist.

Antinociceptive Properties of 1,8-Cineole and β-Pinene, from the Essential Oil of Eucalyptus camaldulensis Leaves, in Rodents

Abstract: 1,8-cineole (cineole) and beta-pinene, two monoterpenes isolated from the essential oil obtained from Eucalyptus camaldulensis Dehn leaves were tested for antinociceptive properties Tail-flick and hot-plate methods, reflecting the spinal and supraspinal levels, respectively, were used in mice and/or rats using morphine and naloxone for comparison Cineole exhibited an antinociceptive activity comparable to that of morphine, in both algesic stimuli A significant synergism between cineole and morphine was observed, but naloxone failed to antagonize the effect of cineole Beta-pinene exerted supraspinal antinociceptive actions in rats only and it reversed the antinociceptive effect of morphine in a degree equivalent to naloxone, probably acting as a partial agonist through the mu opioid receptors From structure-activity relationships of the pairs morphine+cineole and naloxone+beta-pinene, it was shown that similarities exist in the stereochemistry and in the respective atomic charges of these molecules Further studies are in progress in order to elucidate the mechanism of action of the two terpenoids

Alkannin and shikonin: effect on free radical processes and on inflammation - a preliminary pharmacochemical investigation

Abstract: Alkannin and shikonin, two natural products from Alkanna tinctoria and Lithospermum erhythrorhizon (Boraginaceae), are used in folk medicine where they are claimed to possess, among other properties, wound healing and anti-inflammatory activity. We investigated, together with the structurally related naphthazarin, their in vitro antioxidant and hydroxyl radical scavenging activity as well as their in vivo antiinflammatory activity. I was found that all examined compounds significantly inhibited in vitro lipid peroxidation of ra hepatic microsomal membranes, competed with DMSO for free hydroxyl radicals, and reduced inflammation (mouse paw edema induced by FCA) very efficiently. The examined compounds proved equal or superior to the common reference compounds for each of these properties. I is concluded that the claimed and/or proven actions of alkannin and shikonin are attributable at least partly to their intervention in free radical processes.

Investigation of the effect of chamazulene on lipid peroxidation and free radical processes.

Abstract: Oxygen toxicity and related free radical reactions are implicated in numerous pathophysiological conditions, like atherosclerosis, inflammation, gastric ulceration, neuronal degeneration, tumour promotion. The flowers of Matricaria chamomilla, Asteraceae, have been used therapeutically for conditions in which oxidative stress is supposed to be implicated. We considered interesting to investigate the effect of Chamazulene, the active substance of chamomile, on free radical processes. Membrane lipid peroxidation was induced by Fe2+/ascorbate and assessed as the 2-thiobarbituric acid reactive material. The hydroxyl radical scavenging activity was studied as the competition of Chamazulene with DMSO for HO. generated by Fe3+/ascorbate. Finally, the interaction of Chamazulene with the N-centered stable free radical DPPH was estimated photometrically (517 nm). It was found that Chamazulene inhibited lipid peroxidation in a concentration and time dependent manner presenting an IC50 of 18 microM after 45 min incubation. It could also inhibit the autoxidation of DMSO (33 mM) by 76% at 25 mM, and had a weak capacity to interact with DPPH. In conclusion, Chamazulene presents interesting properties concerning radical processes.

Morpholine as a privileged structure: A review on the medicinal chemistry and pharmacological activity of morpholine containing bioactive molecules.

Abstract: Morpholine is a heterocycle featured in numerous approved and experimental drugs as well as bioactive molecules. It is often employed in the field of medicinal chemistry for its advantageous physicochemical, biological, and metabolic properties, as well as its facile synthetic routes. The morpholine ring is a versatile and readily accessible synthetic building block, it is easily introduced as an amine reagent or can be built according to a variety of available synthetic methodologies. This versatile scaffold, appropriately substituted, possesses a wide range of biological activities. There are many examples of molecular targets of morpholine bioactive in which the significant contribution of the morpholine moiety has been demonstrated; it is an integral component of the pharmacophore for certain enzyme active-site inhibitors whereas it bestows selective affinity for a wide range of receptors. A large body of in vivo studies has demonstrated morpholine's potential to not only increase potency but also provide compounds with desirable drug-like properties and improved pharamacokinetics. In this review we describe the medicinal chemistry/pharmacological activity of morpholine derivatives on various therapeutically related molecular targets, attempting to highlight the importance of the morpholine ring in drug design and development as well as to justify its classification as a privileged structure.

Multicomponent Reactions with Isocyanides.

Abstract: Multicomponent reactions (MCRs) are fundamentally different from two-component reactions in several aspects. Among the MCRs, those with isocyanides have developed into popular organic-chemical reactions in the pharmaceutical industry for the preparation of compound libraries of low-molecular druglike compounds. With a small set of starting materials, very large libraries can be built up within a short time, which can then be used for research on medicinal substances. Due to the intensive research of the last few years, many new backbone types have become accessible. MCRs are also increasingly being employed in the total synthesis of natural products. MCRs and especially MCRs with isocyanides offer many opportunities to attain new reactions and basic structures. However, this requires that the chemist learns the “language” of MCRs, something that this review wishes to stimulate.

Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method

Abstract: Brazilian plant extracts belonging to 16 species of 5 different families (71 extracts) were tested against the stable DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free-radical. The ability to scavenge DPPH radical was measured in these experiments by the discoloration of the solution. Ginkgo biloba and rutin, commonly used as antioxidants for medical purposes, were used as standards. Based on our results, we can say that as a general rule the ethanol extracts of plants belonging to the Verbenaceae family showed lower EC50 values than the other plant extracts. Among the partitions, the more polar ones (ethyl acetate and n-butanol) are those that generally have higher antioxidant activity (AA). Copyright © 2001 John Wiley & Sons, Ltd.

Constitutive activity of G-protein-coupled receptors: cause of disease and common property of wild-type receptors

Abstract: The aim of this review is to provide a systematic overview on constitutively active G-protein-coupled receptors (GPCRs), a rapidly evolving area in signal transduction research. We will discuss mechanisms, pharmacological tools and methodological approaches to analyze constitutive activity. The two-state model defines constitutive activity as the ability of a GPCR to undergo agonist-independent isomerization from an inactive (R) state to an active (R*) state. While the two-state model explains basic concepts of constitutive GPCR activity and inverse agonism, there is increasing evidence for multiple active GPCR conformations with distinct biological activities. As a result of constitutive GPCR activity, basal G-protein activity increases. Until now, constitutive activity has been observed for more than 60 wild-type GPCRs from the families 1–3 and from different species including humans and commonly used laboratory animal species. Additionally, several naturally occurring and disease-causing GPCR mutants with increased constitutive activity relative to wild-type GPCRs have been identified. Alternative splicing, RNA editing, polymorphisms within a given species, species variants and coupling to specific G-proteins all modulate the constitutive activity of GPCRs, providing multiple regulatory switches to fine-tune basal cellular activities. The most important pharmacological tools to analyze constitutive activity are inverse agonists and Na+ that stabilize the R state, and pertussis toxin that uncouples GPCRs from Gi/Go-proteins. Constitutive activity is observed at low and high GPCR expression levels, in native systems and in recombinant systems, and has been reported for GPCRs coupled to Gs-, Gi- and Gq-proteins. Constitutive activity of neurotransmitter GPCRs may provide a tonic support for basal neuronal activity. For the majority of GPCRs known to be constitutively active, inverse agonists have already been identified. Inverse agonists may be useful in the treatment of neuropsychiatric and cardiovascular diseases and of diseases caused by constitutively active GPCR mutants.