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Determination of metabolic properties of a new chemical entity (NCE) is one of the most important steps during the drug discovery and development process. Nowadays, in vitro methods are used for early estimation and prediction of in vivo metabolism of NCEs. Using in vitro methods, it is possible to determine the metabolic stability of NCEs as well as the risk for drug-drug interactions (DDIs) related to inhibition and induction of drug metabolic enzymes. Metabolic stability is defined as the susceptibility of a chemical compound to biotransformation, and is expressed as in vitro half-life (t(1/2)) and intrinsic clearance (CL(int)). Based on these values, in vivo pharmacokinetic parameters such as bioavailability and in vivo half-life can be calculated. The drug metabolic enzymes possess broad substrate specificity and can metabolize multiple compounds. Therefore, the risk for metabolism-based DDIs is always a potential problem during the drug development process. For this reason, inhibition and induction in vitro screens are used early, before selection of a candidate drug (CD), to estimate the risk for clinically significant DDIs. At present, most pharmaceutical companies perform in vitro drug metabolism studies together with in silico prediction software and automated high-throughput screens (HTS). Available data suggest that in vitro methods are useful tools for identification and elimination of NCEs with unappreciated metabolic properties. However, the quantitative output of the methods has to be improved. The aim of this review is to highlight the practical and theoretical basis of the in vitro metabolic methods and the recent progress in the development of these assays.

Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent. The measured mass of the homotrimer is 53 kDa, allowing for the mass of three MGST molecules in complex with one glutathione molecule. Collision-induced dissociation of the trimer complex resulted in the formation of monomer and homodimer ion species. Two distinct species of homodimer were observed, one unliganded and one identified as a homodimer.glutathione complex. Activation of the enzyme by N-ethylmaleimide through modification of Cys(49) (Svensson, R., Rinaldi, R., Swedmark, S., and Morgenstern, R. (2000) Biochemistry 39, 15144-15149) was monitored by the observation of an appropriate increase in mass in both the denatured monomeric and native trimeric forms of MGST1. Together, the data correspond well with the proposed functional organization of MGST1. These results also represent the first example of direct electrospray mass spectrometry analysis of a detergent-solubilized multimeric membrane protein complex in its native state.

Microsomal glutathione transferase 1 (MGST1) can become activated up to 30-fold by several mechanisms in vitro (e.g. covalent modification by reactive electrophiles such as N-ethylmaleimide (NEM)). Activation has also been observed in vivo during oxidative stress. It has been noted that an NADPH generating system (g.s.) can activate MGST1 (up to 2-fold) in microsomal incubations, but the mechanism was unclear. We show here that NADPH g.s treatment impaired N-ethylmaleimide activation, indicating a shared target (identified as cysteine-49 in the latter case). Furthermore, NADPH activation was prevented by sulfhydryl compounds (glutathione and dithiothreitol). A well established candidate for activation would be oxidative stress, however we could exclude that oxidation mediated by cytochrome P450 2E1 (or flavine monooxygenase) was responsible for activation under a defined set of experimental conditions since superoxide or hydrogen peroxide alone did not activate the enzyme (in microsomes prepared by our routine procedure). Actually, the ability of MGST1 to become activated by hydrogen peroxide is critically dependent on the microsome preparation method (which influences hydrogen peroxide decomposition rate as shown here), explaining variable results in the literature. NADPH g.s. dependent activation of MGST1 could instead be explained, at least partly, by a direct effect observed also with purified enzyme (up to 1.4-fold activation). This activation was inhibited by sulfhydryl compounds and thus displays the same characteristics as that of the microsomal system. Whereas NADPH, and also ATP, activated purified MGST1, several nucleotide analogues did not, demonstrating specificity. It is thus an intriguing possibility that MGST1 function could be modulated by ligands (as well as reactive oxygen species) during oxidative stress when sulfhydryls are depleted.

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 11 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. (C) 2002 Lippincott Williams Wilkins.

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

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.

Microsomal glutathione transferase 1 is a homotrimeric detoxication enzyme protecting against electrophiles. The enzyme can also react with electrophiles, and when modification occurs at a unique Cys49 the reaction often results in activation. Here we describe the characterization of the chemical properties of this sulfhydryl (kinetic pK(a) was 8.8 +/- 0.3 and 9.0 +/- 0.1 with two different reagents) and we conclude that the protein environment does not lower the pK(a). Upon a direct comparison of the reactivity of Cys49 and low molecular weight thiols [L-Cys and glutathione (GSH)], the protein sulfhydryl displayed a 10-fold lower reactivity. The reactivity was correlated to reagent concentration in a linear fashion with a polar reagent, whereas the reactivity toward a hydrophobic reagent displayed saturation behavior (at low concentrations). This finding indicates that Cys49 is situated in a hydrophobic binding pocket. In a series of related quinones, activation occurs with the more reactive and less sterically hindered compounds. Thus, activation can be used to detect reactive intermediates during the metabolism of foreign compounds but certain intermediates can (and will) escape undetected. The reactivities of the three cysteines in the homotrimer were shown not to differ dramatically as the reaction of the protein with 4, 4'-dithiodipyridine could be fitted to a single exponential. On the basis of this result, a probabilistic expression could be used to relate the overall degree of modification to fractional activation. When N-ethylmaleimide activation (determined by the 1-chloro-2, 4-dinitrobenzene assay) was plotted against modification (determined with 4,4'-dithiodipyridine), a nonlinear relation was obtained, clearly showing that subunits do not function independently. The contribution to activation by single-, double-, and triple-modified trimers, were 0 +/- 0.06, 0.74 +/- 0.09, and 0.97 +/- 0.06, respectively. The double-modified enzyme appears partly activated, but this conclusion is more uncertain due to the possibility of independent modification of the purified enzyme upon storage. It is, however, clear that the single-modified enzyme is not activated whereas the triple-modified enzyme is fully activated. These observations together with the fact that MGST1 homotrimers bind only one substrate molecule (GSH) strongly support the view that subunits must interact in a functional manner.

SummaryGlioblastoma multiforme (GBM) is the most aggressive form of brain cancer with marginal life expectancy. Based on the assumption that GBM cells gain functions not necessarily involved in the cancerous process, patient-derived glioblastoma cells (GCs) were screened to identify cellular processes amenable for development of targeted treatments. The quinine-derivative NSC13316 reliably and selectively compromised viability. Synthetic chemical expansion reveals delicate structure-activity relationship and analogs with increased potency, termed Vacquinols. Vacquinols stimulate death by membrane ruffling, cell rounding, massive macropinocytic vacuole accumulation, ATP depletion, and cytoplasmic membrane rupture of GCs. The MAP kinase MKK4, identified by a shRNA screen, represents a critical signaling node. Vacquinol-1 displays excellent in vivo pharmacokinetics and brain exposure, attenuates disease progression, and prolongs survival in a GBM animal model. These results identify a vulnerability to massive vacuolization that can be targeted by small molecules and point to the possible exploitation of this process in the design of anticancer therapies.