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Biochemical Pharmacology (v.74, #3)

Editorial Advisory Board (pp. iii).

Allosteric approaches to the targeting of G-protein-coupled receptors for novel drug discovery: A critical assessment by Rita Raddatz; Hervé Schaffhauser; Michael J. Marino (pp. 383-391).

In recent years, the concept of allosteric modulation of G-protein-coupled receptors (GPCRs) has matured and now represents an increasingly viable approach to drug discovery. This is evident in the fact that allosteric modulators have been reported for every class of GPCR, and several are currently in clinical trials with one drug example approved and launched. The allosteric approach has been highlighted for the potential of identifying highly selective compounds with a minimal propensity to produce adverse effect. While much has been written regarding the promises of this approach, important challenges, caveats, and pitfalls exist that are often overlooked. Therefore, a balanced overview of the field that describes both the promises and the challenges of discovering allosteric modulators of GPCRs as novel drugs is presented.

Keywords: Drug discovery; G-protein-coupled receptor; Allosteric; Orthosteric

p53 dependent and independent sensitivity to oxaliplatin of colon cancer cells by Florent Toscano; Béatrice Parmentier; Zineb El Fajoui; Yann Estornes; Jean-Alain Chayvialle; Jean-Christophe Saurin; Jacques Abello (pp. 392-406).

Oxaliplatin is an efficient chemotherapeutic agent used for the treatment of metastatic human colon cancer, but cancer cells are frequently resistant. The aim of this study was to analyse the underlying mechanisms in a panel of 10 human colorectal cancer cell lines submitted to a short (2h) oxaliplatin treatment period, accordingly to the usual therapeutic procedure in humans. Sensitivity to oxaliplatin was a characteristic of p53 wild-type colon cancer cells. In contrast, all p53-mutated cell lines had a high IC50 to oxaliplatin, with the exception of the V9P cell line. Exposure to oxaliplatin resulted in G0/G1 arrest in p53 wild-type cell lines, and in S phase in p53-mutated cell lines. In our treatment conditions, no DNA accumulation in sub G0/G1 phase, no caspase-3 activation nor PARP cleavage were detected after oxaliplatin treatment, except for the V9P cell line. The major role of the p53–p21 pathway in oxaliplatin sensitivity was confirmed in the p53 wild-type HCT116 cell line, using siRNA duplex, and knockdown of the TAp73 protein also enhanced resistance to oxaliplatin in this cell line. Surprisingly, siRNA duplex invalidation revealed a residual effect of the mutant p53 protein in p53-mutated cell lines. Persistent sensitivity to oxaliplatin of the p53-mutated V9P cell line was associated with oxalipatin-induced apoptosis but TAp73 was not the responsible alternative pathway.

Keywords: Oxaliplatin; Colorectal cancer; Chemosensitivity; Cell cycle; Apoptosis; p53

Epothilone-paclitaxel resistant leukemic cells CEM/dEpoB300 are sensitive to albendazole: Involvement of apoptotic pathways by Azita Khalilzadeh; Kiran T. Wangoo; David L. Morris; Mohammad H. Pourgholami (pp. 407-414).

Altered or deficient activation of apoptosis signalling pathways may contribute to drug resistance. Here, we assess the role of apoptotic mediators in eliciting an anti-proliferative response to paclitaxel (PTX) in a T cell acute lymphoblastic leukemia (ALL) cell line CEM and its epothilone-paclitaxel resistant sub-line CEM/dEpoB300. Furthermore, the cellular response to PTX was compared to those elicited by cells in response to treatment with albendazole (ABZ; a microtubule depolymerizing agent). In cell proliferation studies, CEM cells were sensitive to both PTX and ABZ, while the CEM/dEpoB300 cells were highly resistant to PTX (IC₅₀ 2.86nM versus 30.26nM, respectively). In contrast, the resistant cells showed a 2-fold increase in sensitivity to ABZ (0.32μM in CEM compared to 0.16μM in CEM/dEpoB300). Analysis of caspase-3 activity and cytochrome c release in response to PTX or ABZ treatment (24, 48 and 72h) revealed that, compared to the parent cells, the resistant cells have diminished response to PTX and enhanced response to ABZ. A similar pattern was observed for the pro-apoptotic protein Bax. Levels of the anti-apoptotic protein Bcl-2 was highly elevated in CEM/dEpoB300 cells and in these cells, ABZ was more effective in lowering the Bcl-2 levels than PTX. Similarly, ABZ treatment led to profound down regulation of the Mcl-1 protein. These results reveal for the first time, the changes in apoptotic mediators following development of resistance to PTX in an ALL cell and the significantly increased sensitivity of these PTX resistant cells to ABZ.

Keywords: ALL; Albendazole; Apoptosis; Paclitaxel; Resistance; Leukemia

Effects of Salviae Mitiorrhizae and Cortex Moutan extract on the rat heart after myocardial infarction: A proteomic study by Yi Wang; Li Liu; Chuchu Hu; Yiyu Cheng (pp. 415-424).

In this study, we characterized the therapeutical effects of Salviae Mitiorrhizae (Danshen) and Cortex Moutan (Danpi) extract (SDD) on Sprague-Dawley rats subjected to coronary artery ligation, and applied proteomic approach to investigate its potential mechanism of action. The chemical composition of SDD was investigated by HPLC/MSⁿ analysis. Measurement for serum levels of creatine kinase (CK), creatine kinase-MB (CK-MB), nitrite and histological study for infarct area of heart were performed. Moreover, protein abundance profiles of myocardium were compared by two-dimensional gel electrophoresis and altered proteins were identified by MALDI-TOF–MS. The results showed SDD significantly decreased CK, CK-MB concentration in serum and infarct area of heart, while increased the release of nitrite in rats with coronary occlusion. Increased concentration of ATP and total adenine nucleotide indicated the energy metabolism has been improved in ischemic heart induced by SDD. Proteomic data revealed that 23 proteins associated with energy metabolism, oxidative stress and cytoskeleton were modulated in SDD treated rats.

Keywords: Abbreviations; ADP; adenosine diphosphate; AMP; adenosine monophosphate; ATP; adenosine triphosphate; CK; creatine kinase; CK-MB; creatine kinase-MB; NO; nitric oxide; SDD; Salviae Mitiorrhizae; and; Cortex Moutan; extractCoronary heart disease; Energy metabolism; Herbal medicine; Ischemia; Protein profiling; Drug treatment

Inhibition of the HERG potassium channel by the tricyclic antidepressant doxepin by R.S. Duncan; M.J. McPate; J.M. Ridley; Z. Gao; A.F. James; D.J. Leishman; J.L. Leaney; H.J. Witchel; J.C. Hancox (pp. 425-437).

HERG (human ether-à-go-go-related gene) encodes channels responsible for the cardiac rapid delayed rectifier potassium current, I_Kr. This study investigated the effects on HERG channels of doxepin, a tricyclic antidepressant linked to QT interval prolongation and cardiac arrhythmia. Whole-cell patch-clamp recordings were made at 37°C of recombinant HERG channel current ( I_HERG), and of native I_Kr ‘tails’ from rabbit ventricular myocytes. Doxepin inhibited I_HERG with an IC₅₀ value of 6.5±1.4μM and native I_Kr with an IC₅₀ of 4.4±0.6μM. The inhibitory effect on I_HERG developed rapidly upon membrane depolarization, but with no significant dependence on voltage and with little alteration to the voltage-dependent kinetics of I_HERG. Neither the S631A nor N588K inactivation-attenuating mutations (of residues located in the channel pore and external S5-Pore linker, respectively) significantly reduced the potency of inhibition. The S6 point mutation Y652A increased the IC₅₀ for I_HERG blockade by ∼4.2-fold; the F656A mutant also attenuated doxepin's action at some concentrations. HERG channel blockade is likely to underpin reported cases of QT interval prolongation with doxepin. Notably, this study also establishes doxepin as an effective inhibitor of mutant (N588K) HERG channels responsible for variant 1 of the short QT syndrome.

Keywords: Antidepressant; Arrhythmia; Doxepin; HERG; I; _Kr; Long QT syndrome; Potassium channel; QT interval; QT-prolongation; Rapid delayed rectifier; Short QT syndrome; Torsade de pointes

The calcium-sensing receptor (CaR) is involved in strontium ranelate-induced osteoblast proliferation by Naibedya Chattopadhyay; Stephen J. Quinn; Olga Kifor; Chianping Ye; Edward M. Brown (pp. 438-447).

Strontium ranelate has several beneficial effects on bone and reduces the risk of vertebral and hip fractures in women with postmenopausal osteoporosis. We investigated whether Sr²⁺ acts via a cell surface calcium-sensing receptor (CaR) in HEK293 cells stably transfected with the bovine CaR (HEK-CaR) and rat primary osteoblasts (POBs) expressing the CaR endogenously. Elevating Ca_o²⁺ or Sr²⁺ concentration-dependently activated the CaR in HEK-CaR but not in non-transfected cells, but the potency of Sr²⁺ varied depending on the biological response tested. Sr²⁺ was less potent than Ca_o²⁺ in stimulating inositol phosphate accumulation and in increasing Ca_i²⁺, but was comparable to Ca_o²⁺ in stimulating ERK phosphorylation and a non-selective cation channel, suggesting that Ca²⁺ and Sr²⁺ have differential effects on specific cellular processes. With physiological concentrations of Ca_o²⁺, Sr²⁺-induced further CaR activation. Neither Sr²⁺ nor Ca_o²⁺ affected the four parameters just described in non-transfected cells. In POB, Sr²⁺ stimulated cellular proliferation. This effect was CaR-mediated, as transfecting the cells with a dominant negative bovine CaR significantly attenuated Ca_o²⁺-stimulated POB proliferation. Finally, Sr²⁺ significantly increased the mRNA levels of the immediate early genes, c-fos and egr-1, which are involved in POB proliferation, and this effect was attenuated by overexpressing the dominant negative CaR. In conclusion, Sr²⁺ is a full CaR agonist in HEK-CaR and POB, and, therefore, the anabolic effect of Sr²⁺ on bone in vivo could be mediated, in part, by the CaR.

Keywords: Inositol phosphates; Cytosolic calcium; Non-selective cation channel; c-fos; Egr-1 HEK293 cells

Effect of diadenosine polyphosphates in achondroplasic chondrocytes: Inhibitory effect of Ap₄A on FGF9 induced MAPK cascade by Ana Guzmán-Aránguez; Marta Irazu; Avner Yayon; Jesús Pintor (pp. 448-456).

Achondroplasia is characterised by a mutation in the gene that encodes for the FGF receptor type 3 (FGFR3), producing a hyperactivation of this receptor and a subsequent increase in MAPK activity. We have tested the ability of nucleotides to decrease the activation of MAPK in chondrocytes with achondroplasic FGFR3 receptor. Diadenosine tetraphosphate, Ap₄A, reduced the phosphorylation of pERK1/2 triggered by FGF9 (38% reduction). Ap₄A diminished the expression of achondroplasic FGFR3 receptor (65% reduction), stimulating FGFR3 receptor degradation. The action of Ap₄A seems to be mediated by a dinucleotide receptor rather than by any other ATP receptor.

Keywords: Abbreviations; MAPK; mitogen-activated protein kinase; ERK1/2; extracellular signal-regulated kinases 1 and 2; FGFR3; fibroblast growth factor receptor 3; FGF9; basic fibroblast growth factor 9; Ap; ₃; A; diadenosine triphosphate; Ap; ₄; A; diadenosine tetraphosphate; Ap; ₅; A; diadenosine pentaphosphate; ATPγS; adenosine 5′-3-; O; -thiotriphosphate; 2MeSADP; 2-(methylthio)-ADP; 2MeSATP; 2-(methylthio)-ATP; AMP-PCP; adenyl 5′-(beta, gamma-methylene)-diphosphonate; IP3; inositol triphosphate; cGMP; cyclic guanosine monophosphate; PLC; phospholipase C; PKC; protein kinase CAchondroplasia; Chondrocytes; FGFR3; ERK1/2; Diadenosine polyphosphates; Dinucleotide receptor

The reactivity of ortho-methoxy-substituted catechol radicals with sulfhydryl groups: Contribution for the comprehension of the mechanism of inhibition of NADPH oxidase by apocynin by Marília P.P. Kanegae; Luiz Marcos da Fonseca; Iguatemy L. Brunetti; Sueli de Oliveira Silva; Valdecir F. Ximenes (pp. 457-464).

Redox processes are involved in the mechanism of action of NADPH oxidase inhibitors such as diphenyleneiodonium and apocynin. Here, we studied the structure-activity relationship for apocynin and analogous ortho-methoxy-substituted catechols as inhibitors of the NADPH oxidase in neutrophils and their reactivity with peroxidase. Aiming to alter the reduction potential, the ortho-methoxy-catechol moiety was kept constant and the substituents at para position related to the hydroxyl group were varied. Two series of compounds were employed: methoxy-catechols bearing electron-withdrawing groups (MC-W) such as apocynin, vanillin, 4-nitroguaiacol, 4-cyanoguaiacol, and methoxy-catechol bearing electron-donating groups (MC-D) such as 4-methylguaiacol and 4-ethylguaiacol. We found that MC-D were weaker inhibitors compared to MD-W. Furthermore, the radicals generated by oxidation of MC-W via MPO/H₂O₂, but not for MC-D, were able to oxidize glutathione (GSH) as verified by the formation of thiyl radicals, depletion of GSH, and recycling of the ortho-methoxy-catechols during their oxidations. The capacity of oxidizing sulfhydryl (SH) groups was also verified when ovalbumin was incubated with MC-W, but not for MC-D. Since the effect of apocynin has been correlated with inactivation of the cytosolic fractions of the NADPH oxidase complex and its oxidation during the inhibitory process develops a special role in this process, we suggest that the close relationship between the reactivity of the radicals of MC-W compounds with thiol groups and their efficacy as NADPH oxidase inhibitor could be the chemical pathway behind the mechanism of action of apocynin and should be taken into account in the design of new and specific NADPH oxidase inhibitors.

Keywords: Neutrophyl; NADPH oxidase; Myeloperoxidase; Apocynin; Methoxy-catechols; Sulfhydryl residues

Disruption of HDAC4/N-CoR complex by histone deacetylase inhibitors leads to inhibition of IL-2 gene expression by Hideaki Matsuoka; Takao Fujimura; Masako Hayashi; Kaori Matsuda; Yoshinori Ishii; Ichiro Aramori; Seitaro Mutoh (pp. 465-476).

Previous studies have shown that HDAC inhibitors selectively inhibit IL-2 gene expression, but the mechanism of this inhibition remains to be elucidated. It was recently reported that HDAC4, a component of the nuclear hormone receptor corepressor (N-CoR) complex, associates with the IL-2 promoter via the transcription factor myocyte enhancer factor 2 (MEF2). We therefore focused on the role of HDAC4/N-CoR complex in the transcriptional regulation of IL-2. Four approaches were used to characterize this role and to investigate the relation between the regulatory function of HDAC4/N-CoR complex and HDAC4-enzymatic activity: (i) HDAC4 silencing by RNA interference, (ii) overexpression of N-CoR repression domain 3 (RD3), (iii) overexpression of HDAC4 point mutants, and (iv) treatment with HDAC inhibitors. Here, we report that HDAC4 plays an essential role in IL-2 promoter activation, and that the formation of the HDAC4/N-CoR complex, which is closely related to HDAC4-enzymatic activity, might be involved in HDAC inhibitor-mediated inhibition of IL-2 gene expression. These observations indicate that the selective inhibition of HDAC4 or the interaction of HDAC4 with N-CoR is likely a potential target for the development of novel immunosuppressants.

Keywords: Abbreviations; HDAC; histone deacetylase; N-CoR; nuclear hormone receptor corepressor; MEF2; myocyte enhancer factor 2; RD3; repression domain 3; HAT; histone acetyltransferase; SMRT; silencing mediator for retinoid and thyroid receptors; RPD3; reduced potassium dependency 3; HDA1; histone deacetylase-A 1; SIR2; silent information regulator 2; DBD; DNA-binding domain; HRP; horseradish peroxidase; SANT; SWI3/ADA2/NCoR/TFIIIB; PMA; phorbol 12-myristate 13-acetate; TBS-T; tris-buffered saline containing 0.1% Tween-20; siRNA; short interfering RNA; TSA; trichostatin AHDAC4; N-CoR; IL-2; Histone deacetylase; Inhibitor; Immunosuppressants

Differential contribution of organic cation transporters, OCT2 and MATE1, in platinum agent-induced nephrotoxicity by Sachiko Yokoo; Atsushi Yonezawa; Satohiro Masuda; Atsushi Fukatsu; Toshiya Katsura; Ken-Ichi Inui (pp. 477-487).

The mechanism of severe nephrotoxicity caused by cisplatin, but not carboplatin, oxaliplatin, and nedaplatin, is not fully understood. The renal accumulation and subsequent nephrotoxicity of platinum agents were examined in rats. Among these four drugs, only cisplatin induced nephrotoxicity at 2 days after its intraperitoneal administration. The urinary activity of N-acetyl-β-d-glucosaminidase and expression of kidney injury molecule-1 mRNA and osteopontin were markedly enhanced in the cisplatin-treated rats. Although some markers were affected in the rats administered nedaplatin, only minor histological change was observed. The renal accumulation of cisplatin was much greater than that of the other drugs. In the in vitro study, the cellular accumulation of cisplatin and oxaliplatin was stimulated by the expression of rat (r) OCT2. Oxaliplatin was also transported by rOCT3. A luminal H⁺/organic cation antiporter, rMATE1 (multidrug and toxin extrusion) as well as human (h) MATE1 and hMATE2-K, stimulated the H⁺-gradient-dependent antiport of oxaliplatin, but not of cisplatin. Carboplatin and nedaplatin were not transported by these transporters. In conclusion, the nephrotoxicity of platinum agents was closely associated with their renal accumulation, which is determined by the substrate specificity of the OCT and MATE families.

Keywords: Cisplatin; MATE1; Nephrotoxicity; OCT2; Oxaliplatin; Renal accumulation

Change of drug excretory pathway by CCl₄-induced liver dysfunction in rat by Hirotoshi Okumura; Miki Katoh; Keiichi Minami; Miki Nakajima; Tsuyoshi Yokoi (pp. 488-495).

Liver dysfunction affects the pharmacokinetics of drugs. The liver plays an important role in drug excretion as well as drug metabolism and pharmacokinetics. In the present study, the relationship between changes in the cefmetazole (CMZ) excretory pathway and the degree of liver dysfunction induced by CCl₄ treatment was investigated. CMZ is mainly excreted as an unchanged form in feces in control rats. Depending on the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), urinary CMZ excretion was increased, whereas fecal CMZ excretion was decreased in rat with liver dysfunction. The AUC of CMZ in rats with severe liver dysfunction was approximately 2-fold higher than that in control rats. Since drug transporters could be involved in drug excretion, changes in the expression of representative hepatic drug transporters in liver dysfunction were investigated by rat DNA microarray. Basolateral solute carrier transporters such as Ntcp, Oct1, and Oatp2 were decreased and basolateral ATP-binding cassette transporters such as Mrp3 and Mrp4 were increased by the CCl₄ treatment. On the other hand, canalicular Mrp2 and Bsep were decreased, but Mdr1 was increased. However, the transporter system for CMZ has not been identified yet. In conclusion, we clarified that the fecal and urinary excretory profiles of CMZ were changed clearly depending on the serum AST and ALT levels in liver dysfunction. The changes in the CMZ excretory pathway might be responsible for the changes in the expression of drug transporters.

Keywords: Abbreviations; ABC; ATP-binding cassette; ALT; alanine aminotransferase; AST; aspartate aminotransferase; AUC; area under the serum concentration–time curve; Bcrp; breast cancer resistance protein; Bsep; bile-salt export pump; CCl; ₄; carbon tetrachloride; CL; _tot; total clearance; CMZ; cefmetazole; K; elimination rate constant; Mdr; multidrug resistance; Mrp; multidrug resistance associated protein; MRT; mean residence time; Ntcp; Na; ⁺; -taurocholate co-transporting polypeptide; Oat; organic anion transporter; Oatp; organic anion transporting polypeptide; Oct; organic cation transporter; PCR; polymerase chain reaction; RT; reverse transcription; SLC; solute carrier; VRT; variance of residence time; V; volume of distributionLiver dysfunction; CMZ; DNA microarray; Excretion; Transporter

Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells by Monica Ek; Therese Söderdahl; Barbara Küppers-Munther; Josefina Edsbagge; Tommy B. Andersson; Petter Björquist; Ian Cotgreave; Bengt Jernström; Magnus Ingelman-Sundberg; Inger Johansson (pp. 496-503).

Human embryonic stem cells (hESC) offer a potential unlimited source for functional human hepatocytes, since they can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing several hepatic markers. Such cells could be used for, e.g. studies of drug metabolism and hepatotoxicity, which however would require a significant expression of drug metabolising enzymes. Thus, we have investigated the expression of cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs), drug transporters, transcription factors and other liver specific genes in hepatocyte-like cells derived from hESC using a simple direct differentiation protocol. The mRNA and protein expression of several important CYPs were determined using low density arrays, real time PCR and Western blotting. Significant CYP expression on the mRNA level was detected in hepatocyte-like cells derived from one out of two different hESC lines tested, which was much higher than in undifferentiated hESC and generally higher than in HepG2 cells. CYP1A2, CYP3A4/7 and low levels of CYP1A1 and CYP2C8/9/19 protein were detected in both lines. The mRNAs for a variety of CYPs and liver specific factors were shown to be inducible in both cell lines, and this was reflected in induced levels of CYP1A2 and CYP3A4/7 protein. This first report on expression of all major CYPs in hepatocyte-like cells derived from hESC represents an important step towards functional hepatocytes, but efforts to further differentiate the cells using optimized protocols are needed before they exhibit similar levels of drug metabolizing enzymes as primary human hepatocytes and liver.

Keywords: Hepatocyte; Human embryonic stem cell; Drug metabolism; Enzyme; Cytochrome P450; Gene expression

Regioselective sulfonation of dopamine by SULT1A3 in vitro provides a molecular explanation for the preponderance of dopamine-3- O-sulfate in human blood circulation by Katriina Itäaho; Sami Alakurtti; Jari Yli-Kauhaluoma; Jyrki Taskinen; Michael W.H. Coughtrie; Risto Kostiainen (pp. 504-510).

SULT1A3 is an enzyme that catalyzes the sulfonation of many endogenous and exogenous phenols and catechols. The most important endogenous substrate is dopamine (DA), which is often used as a probe substrate for SULT1A3. We developed a new method for analyzing the SULT1A3 reaction products by high-performance liquid chromatography (HPLC) with electrochemical detection. The sulfonate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS), DA and the two dopamine sulfates, DA-3- O-sulfate and DA-4- O-sulfate, can be separated within 3min. This enables quantitation of the sulfates without radioactive PAPS or the precipitation of unreacted PAPS. Both sulfates were synthesized as reference substances and characterized by¹H and¹³C nuclear magnetic resonance (NMR), mass spectrometry (MS) and tandem mass spectrometry (MS/MS). The purity of the dopamine sulfates was estimated by HPLC using a diode array detector. We determined the enzyme kinetic parameters for formation of DA-3- O-sulfate and DA-4- O-sulfate using purified recombinant human SULT1A3. The reactions followed Michaelis–Menten kinetics up to 50μM DA concentration, and strong substrate inhibition was observed at higher concentrations. The apparent K_m values for sulfonation at both hydroxy groups were similar (2.21±0.764 and 2.59±1.06μM for DA-4- O-sulfate and DA-3- O-sulfate, respectively), but the V_max was approximately six times higher for the formation of the 3- O-sulfate (344±139nmol/min/mg protein) than the 4- O-sulfate (45.4±16.5nmol/min/mg protein). These results are in accordance with the observation that DA-3- O-sulfate is more abundant in human blood than DA-4- O-sulfate and that in the crystal structure of SULT1A3 with dopamine bound to the active site, the 3-hydroxy group is aligned to form hydrogen bonds with catalytic residues of the enzyme.

Keywords: Abbreviations; DA; dopamine; DA-3S; dopamine-3-; O; -sulfate; DA-4S; dopamine-4-; O; -sulfate; SULT; sulfotransferase; PAPS; 3′-phosphoadenosine-5′-phosphosulfate; PAP; 3′-phosphoadenosine 5′-phosphate; MS/MS; tandem mass spectrometry; HPLC; high-performance liquid chromatography; NMR; nuclear magnetic resonanceDopamine-3-; O; -sulfate; Dopamine-4-; O; -sulfate; SULT1A3; Regioselectivity; Enzyme kinetics; HPLC

Biotechnological synthesis of drug metabolites using human cytochrome P450 2D6 heterologously expressed in fission yeast exemplified for the designer drug metabolite 4′-hydroxymethyl-α-pyrrolidinobutyrophenone by Frank T. Peters; Calin-A. Dragan; Désirée R. Wilde; Markus R. Meyer; Josef Zapp; Matthias Bureik; Hans H. Maurer (pp. 511-520).

The aim of this study was evaluating the principle feasibility of biotechnological synthesis of drug metabolites using heterologously expressed human cytochrome P450 (CYP) enzymes. Human CYP2D6 expressed in fission yeast ( Schizosaccharomyces pombe) strain CAD58 was used as model enzyme and the designer drug 4′-methyl-α-pyrrolidinobutyrophenone (MPBP) as model drug. For synthesis of 4′-hydroxmethyl-α-pyrrolidinobutyrophenone (HO-MPBP), 250μmol of MPBP·HNO₃ were incubated with one litre of CAD58 culture (10⁸cells/mL, pH 9, 48h, 30°C). HO-MPBP was isolated by liquid–liquid extraction and precipitated as its hydrochloride salt. Identity and purity of the product were tested by HPLC with ultraviolet (UV) detection, GC-MS, and¹H-NMR. CAD58 was further characterized regarding the influence of incubation pH (5–10), cell density (10⁷–10⁸cells/mL), and incubation time (0–120h) on metabolite formation using the substrates dextromethorphan and MPBP. The preparative experiment yielded 40mg (141μmol) of HO-MPBP·HCl with a purity of >98%. In the characterization experiments, the metabolite formation rate peaked at pH 8. A linear relationship was observed between cell density and metabolite formation ( R²>0.996). The rate of metabolite formation was slower in the earlier stages of incubation but then increased. For HO-MPBP, it became constant in the time interval of 2.5–34h ( R²>998).

Keywords: MPBP; 4′-methyl-α-pyrrolidinobutyrophenone; Drug; Synthesis; Schizosaccharomyces pombe; Cytochrome P450

Alcohol-induced S-adenosylhomocysteine accumulation in the liver sensitizes to TNF hepatotoxicity: Possible involvement of mitochondrial S-adenosylmethionine transport by Zhenyuan Song; Zhanxiang Zhou; Ming Song; Silvia Uriarte; Theresa Chen; Ion Deaciuc; Craig J. McClain (pp. 521-531).

Hepatocytes are resistant to tumor necrosis factor-α- (TNF) induced killing/apoptosis under normal circumstances, but primary hepatocytes from rats chronically fed alcohol have increased TNF cytotoxicity. Therefore, there must be mechanism(s) by which alcohol exposure “sensitizes” to TNF hepatotoxicity. Abnormal metabolism of methionine and S-adenosylmethionine (SAM) are well-documented acquired metabolic abnormalities in ALD. S-adenosylhomocysteine (SAH) is the product of SAM in hepatic transmethylation reactions, and SAH hydrolase (SAHH) is the only enzyme to metabolize SAH to homocysteine and adenosine. Our previous studies demonstrated that chronic intracellular accumulation of SAH sensitized hepatocytes to TNF cytotoxicity in vitro. In the current study, we extended our previous observations by further characterizing the effects of chronic alcohol intake on mitochondrial SAM levels in liver and examining its possible involvement in SAH sensitization to TNF hepatotoxicity. Chronic alcohol consumption in mice not only increased cytosolic SAH levels, but also decreased mitochondrial SAM concentration, leading to decreased mitochondrial SAM to SAH ratio. Moreover, accumulation of hepatic SAH induced by administration of 3-deaza-adenosine (DZA-a potent inhibitor of SAHH) enhanced lipopolysaccharide (LPS)/TNF hepatotoxicity in mice in vivo. Inhibition of SAHH by DZA resulted not only in accumulation of cytoplasmic SAH, but also in depletion of the mitochondrial SAM pool. Further studies using mitochondrial SAM transporter inhibitors showed that inhibition of SAM transport into mitochondria sensitized HepG2 cells to TNF cytotoxicity. In conclusion, our results demonstrate that depletion of the mitochondrial SAM pool by SAH, which is elevated during chronic alcohol consumption, plays a critical role in SAH induced sensitization to TNF hepatotoxicity.

Keywords: Abbreviations; SAH; S-adenosylhomocysteine; SAM; S-adenosylmethionine; MAT; methionine adenosyltranserase; DZA; 3′-deazaadenosine; PLP; pyridoxal 5-phosphate; SAHH; s-adenosylhomocysteine hydrolyse; TUNEL; Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling; GSH; glutathione; ALD; alcoholic liver disease; TNF; tumor necrosis factor α; HPLC; high performance liquid chromatography; LPS; lipopolysaccharide; ELISA; enzyme-linked immunosorbent assayLiver; S-adenosylhomocysteine; S-adenosylmethionine; Tumor necrosis factor; Mitochondria

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Biochemical Pharmacology (v.74, #3)