Richard Svensson

Synthesis and characterization of 6-chloroacetyl-2-dimethylaminonaphthalene as a fluorogenic substrate and a mechanistic probe for glutathione transferases☆

Here we demonstrate that the thiol-reactive, environmentally sensitive fluorogenic molecules 6-bromoacetyl-2-dimethylaminonaphthalene and 6-acryloyl-2-dimethylaminonaphthalene are substrates for glutathione transferases (GSTs). Product formation can be measured by strong increase in fluorescence of the glutathione conjugate. As these substances display a high nonenzymatic background reaction rate, we have synthesized and characterized 6-chloroacetyl-2-dimethylaminonaphthalene, which is less reactive, favoring the enzyme-catalyzed reaction. 6-Chloroacetyl-2-dimethylaminonaphthalene was found to be a substrate for all GSTs tested. Apparent k(cat)/K(m) values (ranging between 10 and 500 mM(-1)s(-1)) revealed a strong preference for soluble GSTP1-1, GSTA1-1, and activated MGST1. Thus, 6-chloroacetyl-2-dimethylaminonaphthalene can be used in a highly sensitive assay of these GSTs. 6-Acetyl-2-dimethylaminonaphthalene derivatives are very sensitive toward solvent polarity and potentially also toward properties of binding sites in proteins. Upon binding of the conjugate to GSTs the fluorescence intensity decreased and the emission maximum was blue-shifted. Therefore the interaction of the conjugate with GSTs can be characterized with regard to both binding affinity and kinetics by stopped-flow measurements, and 6-chloroacetyl-2-dimethylaminonaphthalene can be a valuable aid in mechanistic investigations of GSTs, especially those which possess low intrinsic fluorescence.

Repeated blood pressure measurements in a sample of Swedish twins: heritabilities and associations with polymorphisms in the renin-angiotensin-aldosterone system.

Background Twin and family studies have shown that genetic effects explain a relatively high amount of the phenotypic variation in blood pressure. However, many studies have not been able to replicate findings of association between specific polymorphisms and diastolic and systolic blood pressure. Methods In a structural equation-modelling framework the authors investigated longitudinal changes in repeated measures of blood pressures in a sample of 298 like-sexed twin pairs from the population-based Swedish Twin Registry. Also examined was the association between blood pressure and polymorphisms in the angiotensin-I converting enzyme and the angiotensin II receptor type 1 with the ‘Fulker’ test. Both linkage and association were tested simultaneously revealing whether the polymorphism is a Quantitative Trait Locus (QTL) or in linkage disequilibrium with the QTL. Results Genetic influences explained up to 46% of the phenotypic variance in diastolic and 63% of the phenotypic variance in systolic blood pressure. Genetic influences were stable over time and contributed up to 78% of the phenotypic correlation in both diastolic and systolic blood pressure. Non-shared environmental effects were characterised by time specific influences and little transmission from one time point to the next. There was no significant linkage and association between the polymorphisms and blood pressure. Conclusions There is a considerable genetic stability in both diastolic and systolic blood pressure for a 6-year period of time in adult life. Non-shared environmental influences have a small long-term effect. Although associations with the polymorphisms could not be replicated, results should be interpreted with caution due to power considerations.

Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione

The trimeric membrane protein microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activity. Previous data indicated one active site/trimer whereas structural data suggests three GSH-binding sites. Here we have determined ligand interactions of MGST1 by several techniques. Nanoelectrospray mass spectrometry of native MGST1 revealed binding of three GSH molecules/trimer and equilibrium dialysis showed three product molecules/trimer (K(d)=320+/-50 microM). All three product molecules could be competed out with GSH. Reinvestigation of GSH-binding showed one high affinity site per trimer, consistent with earlier data. Using single turnover stopped flow kinetic measurements, K(d) could be determined for a low affinity GSH-binding site (2.5+/-0.5 mM). Thus we can reconcile previous observations and show here that MGST1 contains three active sites with different affinities for GSH and that only the high affinity site is catalytically competent.

Characterisation of polymeric surfactants that are glutathione transferase mimics

Catalysts that can detoxify reactive organic chemicals (electrophiles) could be of potential beneficial use. Electrophilic compounds are common toxic agents that are conjugated to endogenous nucleophiles (i.e. glutathione) in an enzyme catalysed reaction (by glutathione transferases). Here, the properties of newly synthesised polymeric surfactant catalysts, which are glutathione transferase mimics, are described (which are not limited to the glutathione thiol donor). Reactions studied were nucleophilic aromatic substitution with 1-chloro-2,4-dinitrobenzene (CDNB) and thiolysis of p-nitrophenyl acetate. Polymeric quaternary ammonium salts synthesised starting from 2-(dimethyl-amino)ethylmethacrylate or 1,3-bis(dimethylamino)isopropylmethacrylate were used as surfactants. Five polysoaps were studied possessing different charge density and different density of hydrophobic chains. In comparison with cetyltrimethylammonium bromide, the polymeric surfactants were clearly more efficient catalysts (i.e. 4.9 vs. 150 (10(3) per M(2)/s) with benzyl hydrosulfide and CDNB). Polymers with high charge and hydrophobic density were most efficient. With a given catalyst, increasing hydrophobicity of the thiol substrate parallels increasing reaction rates (e.g. 0.7- > or = 37 (10(3) per M(2)/s) with CDNB). Concentration of the substrate in the micellar pseudophase together with solvent shielding is suggested as the underlying rate enhancement mechanism. Dead-end Meisenheimer complex stabilisation, where an extremely electrophilic compound (1,3,5-trinitrobenzene) reversibly interacts with glutathione is seen both with glutathione transferases and the polymeric surfactant catalysts. The degree of stabilisation follows catalytic efficiency and thus supports the above structure activity relationships. In conclusion, polymeric materials that can perform biological functions in detoxication are described, as well as their optimal properties.

Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound

Rift Valley fever virus (RVFV) is a mosquito-borne hemorrhagic fever virus affecting both humans and animals with severe morbidity and mortality and is classified as a potential bioterror agent due to the possible aerosol transmission. At present there is no human vaccine or antiviral therapy available. Thus, there is a great need to develop new antivirals for treatment of RVFV infections. Benzavir-2 was previously identified as potent inhibitor of human adenovirus, herpes simplex virus type 1, and type 2. Here we assess the anti-RVFV activity of benzavir-2 together with four structural analogs and determine pre-clinical pharmacokinetic parameters of benzavir-2. In vitro, benzavir-2 efficiently inhibited RVFV infection, viral RNA production and production of progeny viruses. In vitro, benzavir-2 displayed satisfactory solubility, good permeability and metabolic stability. In mice, benzavir-2 displayed oral bioavailability with adequate maximum serum concentration. Oral administration of benzavir-2 formulated in peanut butter pellets gave high systemic exposure without any observed toxicity in mice. To summarize, our data demonstrated potent anti-RVFV activity of benzavir-2 in vitro together with a promising pre-clinical pharmacokinetic profile. This data support further exploration of the antiviral activity of benzavir-2 in in vivo efficacy models that may lead to further drug development for human use.

Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation.

Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.