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Biochemical Pharmacology (v.83, #2)

Editorial Board (pp. iii).

Combating enterovirus replication: State-of-the-art on antiviral research by Hendrik Jan Thibaut; Armando M. De Palma; Johan Neyts (pp. 185-192).

Enteroviruses form an important genus within the large family of Picornaviridae. They are small, non-enveloped (+)RNA viruses, many of which are important pathogens in human and veterinary science. Despite their huge medical and socio-economical impact, there is still no approved antiviral therapy at hand for the treatment of these infections. Three capsid-targeting molecules (pleconaril, BTA-798 and V-073) are in clinical development. Pleconaril and BTA-798 are in phase II clinical trials for the treatment of enterovirus-induced sepsis syndrome and rhinovirus-induced aggravation of pre-existing asthma or COPD respectively. V-073 is in preclinical development for the treatment of poliovirus infections in the context of the worldwide polio eradication program. The capsid binding molecules have shown good in vitro potency against a number of enterovirus species, but lack activity against others. Another potential drawback of capsid inhibitors in the clinical setting could be the rapid emergence of drug resistance. It will therefore be important to develop inhibitors that affect other stages in the viral replication cycle. Several viral proteins, such as the viral 3C protease, the putative 2C helicase and the 3D RNA-dependent RNA polymerase may be/are excellent targets for inhibition of viral replication. Also host cell factors that are crucial in viral replication may be considered as potential targets for an antiviral approach. Unraveling these complex virus–host interactions will also provide better insights into the replication of enteroviruses. This review aims to summarize and discuss known inhibitors and potential viral and cellular targets for antiviral therapy against enteroviruses.

Keywords: Antiviral; Picornavirus; Poliovirus; Rhinovirus; Enterovirus

Imaging the high-affinity state of the dopamine D₂ receptor in vivo: Fact or fiction? by Mette Skinbjerg; David R. Sibley; Jonathan A. Javitch; Anissa Abi-Dargham (pp. 193-198).

Positron Emission Tomography (PET) has been used for more than three decades to image and quantify dopamine D₂ receptors (D₂R) in vivo with antagonist radioligands but in the recent years agonist radioligands have also been employed. In vitro competition studies have demonstrated that agonists bind to both a high and a low-affinity state of the D₂Rs, of which the high affinity state reflects receptors that are coupled to G-proteins and the low-affinity state reflects receptors uncoupled from G-proteins. In contrast, antagonists bind with uniform affinity to the total pool of receptors. Results of these studies led to the proposal that D₂Rs exist in high and low-affinity states for agonists in vivo and sparked the development and use of agonist radioligands for PET imaging with the primary purpose of measuring the proportion of receptors in the high-affinity (activating) state. Although several lines of research support the presence of high and low-affinity states of D₂Rs and their detection by in vivo imaging paradigms, a growing body of controversial data has now called this into question. These include both in vivo and ex vivo studies of anesthesia effects, rodent models with increased proportions of high-affinity state D₂Rs as well as the molecular evidence for stable receptor–G-protein complexes. In this commentary we review these data and discuss the evidence for the in vivo existence of D₂Rs configured in high and low-affinity states and whether or not the high-affinity state of the D₂R can, in fact, be imaged in vivo with agonist radioligands.

Keywords: D; ₂; receptor; High-affinity state; Agonist binding; PET imaging

Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis by phosphorylation of Mcl-1, promoting its degradation and freeing Bak from sequestration by Rong Chu; David T. Terrano; Timothy C. Chambers (pp. 199-206).

Mcl-1 is one of the major anti-apoptotic members of the Bcl-2 family of apoptotic regulatory proteins. In this study we investigated the role of Mcl-1 in mitotic arrest-induced apoptosis. Vinblastine treatment of KB-3 cells initially resulted in a phosphatase-sensitive mobility shift in Mcl-1 and then subsequent loss of Mcl-1 protein expression which was prevented by MG132, suggesting that phosphorylation triggered proteosome-mediated degradation. Mcl-1 phosphorylation/degradation was a specific response to microtubule inhibition and did not occur in response to lethal concentrations of DNA damaging agents. Vinblastine treatment caused degradation of Mcl-1 in cells in which apoptosis was blocked by Bcl-xL overexpression, indicating that Mcl-1 degradation was not a consequence of apoptosis. A partial reversible phosphorylation of Mcl-1 was observed in synchronized cells traversing mitosis, whereas more extensive phosphorylation and subsequent degradation of Mcl-1 was observed if synchronized cells were treated with vinblastine. Mcl-1 phosphorylation closely paralleled cyclin B expression, and specific cyclin-dependent kinase (Cdk) inhibitors blocked vinblastine-induced Mcl-1 phosphorylation, its subsequent degradation, and improved cell viability after mitotic arrest. Co-immunoprecipitation studies indicated that Mcl-1 was complexed with Bak, but not Bax or Noxa, in untreated cells, and that Bak became activated in concert with loss of Mcl-1 expression. These results suggest that Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis via Mcl-1 phosphorylation, promoting its degradation and subsequently releasing Bak from sequestration.

Keywords: Abbreviations; Cdk; cyclin-dependent kinase; JNK; c-Jun N-terminal kinase; ERK; extracellular response kinase; GSK; glycogen synthase kinase; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; PARP; poly(ADP-ribose) polymerase; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidMcl-1; Cdk1; Microtubule inhibitors; Apoptosis; Bcl-2 proteins

Proteasome-based mechanisms of intrinsic and acquired bortezomib resistance in non-small cell lung cancer by Leonie H.A.M. de Wilt; Gerrit Jansen; Yehuda G. Assaraf; Johan van Meerloo; Jacqueline Cloos; Aaron D. Schimmer; Elena T. Chan; Christopher J. Kirk; Godefridus J. Peters; Frank A.E. Kruyt (pp. 207-217).

The proteasome inhibitor bortezomib, registered for Multiple Myeloma treatment, is currently explored for activity in solid tumors including non-small cell lung cancer (NSCLC). Here we studied the proteasome-based mechanisms underlying intrinsic and acquired bortezomib resistance in NSCLC cells. Various NSCLC cell lines displayed differential intrinsic sensitivities to bortezomib. High basal chymotrypsin- and caspase-like proteasome activities correlated with bortezomib resistance in these cells. Next, via stepwise selection, acquired bortezomib resistant cells were obtained with 8–70-fold increased resistance. Cross-resistance was found to proteasome inhibitors specifically targeting β-subunits, but not to the novel α-subunit-specific proteasome inhibitor (5AHQ). Consistently, bortezomib-resistant cells required higher bortezomib concentrations to induce G2/M arrest and apoptosis. Interestingly, bortezomib concentration-dependent caspase cleavage, Mcl-1 and NOXA accumulation remained intact in resistant H460 and SW1573 cells, while A549 resistant cells displayed different expression profiles suggesting additional and more protein specific adaptations. Furthermore, bortezomib-resistant cells exhibited increased levels of both constitutive and immuno-β-subunits. Sequence analysis of the bortezomib-binding pocket in the β5-subunit revealed Ala49Thr, Met45Val and Cys52Phe substitutions that were not previously described in solid tumors. Bortezomib-resistant cells displayed reduced catalytic proteasome activities and required higher bortezomib concentrations to achieve comparable inhibition of proteasome activity. Taken together, these findings establish that high basal levels of proteasome activity correlate with intrinsic bortezomib resistance. Furthermore, acquired bortezomib resistance in NSCLC is associated with proteasome subunit overexpression and emergence of mutant β5-subunits that likely compromise bortezomib binding. α-Subunit-specific proteasome inhibitors, however, can efficiently bypass this resistance modality.

Keywords: Bortezomib; Resistance; Lung cancer; Mutation; β5 proteasome subunit; Proteasome inhibitors

Inhibition of genotoxic stress induced apoptosis by novel TAT-fused peptides targeting PIDDosome by Tae-Ho Jang; Sung-Jun Lee; Chang-Hoon Woo; Kyung Jin Lee; Ju-Hong Jeon; Dong-Sup Lee; Kihang Choi; In-Gyu Kim; Young Whan Kim; Tae-Jin Lee; Hyun Ho Park (pp. 218-227).

Genotoxic stress induced apoptosis is mediated by the formation of PIDDosome, which is a caspase-2 activating complex composed of three protein components, PIDD, RAIDD, and caspase-2. Here, synthetic TAT-fused peptides designed by the structure of PIDD and RAIDD, TAT-Y814A and TAT-R147E, respectively, were produced and tested for their ability to inhibit PIDDosome formation in vitro as well as to attenuate genotoxic stress-induced apoptosis in human renal cancer cells. The results show that TAT-Y814A and TAT-R147E have the potential to inhibit formation of the PIDDosome in a dose-dependent manner. Furthermore, both peptides partially inhibit genotoxic stress mediated apoptosis and activation of caspase2 and caspase3 in Caki cells. These results suggest that TAT-Y814A (also TAT-R147E) is a novel inhibitor of genotoxic stress-induced apoptosis that may serve as a prototype for anti-apoptotic drug development.

Keywords: Apoptosis; Caspase-2; RAIDD; PIDD; PIDDosome; Cisplatin; TAT-fused peptides

Molecular characterization ofEP6—A novel imidazo[1,2- a]pyridine based direct 5-lipoxygenase inhibitor by Joanna M. Wisniewska; Carmen B. Rödl; Astrid S. Kahnt; Estel.la Buscató; Sandra Ulrich; Yusuf Tanrikulu; Janosch Achenbach; Florian Rörsch; Sabine Grösch; Gisbert Schneider; Jindrich Cinatl Jr.; Ewgenij Proschak; Dieter Steinhilber; Bettina Hofmann (pp. 228-240).

5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic acid (AA) cascade and catalyzes the formation of bioactive leukotrienes (LTs) which are involved in inflammatory diseases and allergic reactions. The pathophysiological effects of LTs are considered to be prevented by 5-LO inhibitors. In this study we present cyclohexyl-[6-methyl-2-(4-morpholin-4-yl-phenyl)-imidazo[1,2- a]pyridin-3-yl]-amine (EP6), a novel imidazo[1,2- a]pyridine based compound and its characterization in several in vitro assays.EP6 suppresses 5-LO activity in intact polymorphonuclear leukocytes with an IC₅₀ value of 0.16μM and exhibits full inhibitory potency in cell free assays (IC₅₀ value of 0.05μM for purified 5-LO). The efficacy ofEP6 was not affected by the redox tone or the concentration of exogenous AA, characteristic drawbacks known for the class of nonredox-type 5-LO inhibitors. Furthermore,EP6 suppressed 5-LO activity independently of the cell stimulus or the activation pathway of 5-LO contrary to what is known for some nonredox-type inhibitors. Using molecular modeling and site-directed mutagenesis studies, we were able to derive a feasible binding region within the C2-like domain of 5-LO that can serve as a new starting point for optimization and development of new 5-LO inhibitors targeting this site.EP6 has promising effects on cell viability of tumor cells without mutagenic activity. Hence the drug may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer including leukemia.

Keywords: Abbreviations; 5-H(p)ETE; 5(; S; )-hydro(pero)xy-6-; trans; -8,11,14-; cis; -eicosatetraenoic acid; 12-H(p)ETE; 12(; S; )-hydro(pero)xy-6-; trans; -8,11,14-; cis; -eicosatetraenoic acid; 15-H(p)ETE; 15(; S; )-hydro(pero)xy-5,8,11-; cis; -13-; trans; -eicosatetraenoic; 5(; S; ),12(; S; )-diHETE; 5(; S; ),12(; S; )-dihydroxy-6,10-; trans; -8,14-; cis; -eicosatetraenoic acid; A; alanine; AA; arachidonic acid; Ada; adenosine deaminase; CML; chronic myeloid leukemia; COX; cyclooxygenase; ERK; extracellular signal-regulated kinase; FCS; fetal calf serum; FLAP; 5-LO activating protein; fMLP; N-formyl-methionine-leucine-phenylalanine; LC–MS/MS; liquid chromatography coupled with tandem mass spectrometry; LDH; lactate dehydrogenase; LO; lipoxygenase; LT; leukotriene; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MBP; maltose binding protein; mPGES-1; microsomal prostaglandin E; ₂; synthase-1; PARP; poly ADP-ribose polymerase; PB; phosphate buffer pH 7.4; PC; phosphatidylcholine; PDB; Protein Data Bank; PG buffer; PBS containing 1; mg/ml glucose; PGC buffer; PBS containing 1; mg/ml glucose and 1; mM CaCl; ₂; PLIF; Protein Ligand Interaction Fingerprints; PMNL; polymorphonuclear leukocytes; PMSF; phenylmethylsulfonyl fluoride; RPE; retinal pigment epithelium; RT; room temperature; S100; 100; 000; ×; g; supernatant; SA; sodium arsenite; SDS-PAGE; SDS-polyacrylamide gel electrophoresis; W; tryptophan; WT; wild-type; Y; tyrosine5-Lipoxygenase; Inflammation; Inhibitor; Leukotrienes; Molecular modeling

A novel long acting DPP-IV inhibitor PKF-275-055 stimulates β-cell proliferation resulting in improved glucose homeostasis in diabetic rats by Atul Sureshrao Akarte; B.P. Srinivasan; Sonia Gandhi (pp. 241-252).

The enzyme dipeptidyl peptidase-IV (DPP-4) inactivates the incretin hormone glucagon-like peptide-1 (GLP-1). GLP-1 has therapeutic effects in patients with type 2 diabetes, but its potential is limited by a short half-life, DPP-4 inhibition is a promising approach to diabetes treatment. This study examined chronic (once-a-day dosing for 8 weeks) effects of the DPP-4 inhibitor PKF-275-055 (1, 3, and 10mg/kg) on β-cell regeneration and plasma DPP-IV activity, intact GLP-1, glucose, and insulin after an oral glucose load in neonatal wistar rats injected with streptozotocin (STZ) (n2-STZ model), a recognized model of type 2 diabetes. In streptozotocin induced diabetic rats, PKF-275-055 (3, and 10mg/kg) significantly reduced glucose excursion during the oral glucose tolerance test conducted 2h and 10h after administration, with increases in plasma insulin and active glucagon-like peptide-1 (GLP-1) levels and significantly inhibited (> 50% inhibition) plasma DPP-IV activity during both the 1st and 2nd OGTT in diabetic rats. In contrast, PKF-275-055 (1–10mg/kg) did not cause hypoglycemia in fasted normal rats. Furthermore, PKF-275-055 significantly inhibited advance glycation end product (HbA1c), HOMA-Index, gastric emptying and small intestinal transit rates, with significance at doses of 1mg/kg or higher. Immunological staining showed PKF-275-055 stimulates β-cell regeneration and reduces pancreatic cell apoptosis in diabetic treated rats. The present preclinical studies indicate that PKF-275-055 is a novel selective DPP-IV inhibitor with long-acting antidiabetic effect that might be a potential agent for type 2 diabetes

Keywords: Dipeptidyl peptidase-IV; Glucagon like peptide -1; Insulin; β-cell regeneration; Diabetes

N, N′-Dicyclohexylsulfamide and N, N′-diphenethylsulfamide are anticonvulsant sulfamides with affinity for the benzodiazepine binding site of the GABA_A receptor and anxiolytic activity in mice by Cristina Wasowski; Luciana Gavernet; Ivana A. Barrios; Maria L. Villalba; Valentina Pastore; Gisela Samaja; Andrea Enrique; Luis E. Bruno-Blanch; Mariel Marder (pp. 253-259).

A set of sulfamides designed, synthesized and evaluated against maximal electroshock seizure (MES) and pentilenetetrazol (PTZ) tests with promising results, were tested for their affinity for the benzodiazepine binding site of the GABA_A receptor. The most active compounds, N, N′-dicyclohexylsulfamide (7) and N, N′-diphenethylsulfamide (10), competitively inhibited the binding of [³H]-flunitrazepam to the benzodiazepine binding site with K_i±SEM values of 27.7±4.5μM ( n=3) and 6.0±1.2μM ( n=3), respectively. The behavioral actions of these sulfamides, i.p. administered in mice, were examined in the plus-maze, hole-board and locomotor activity assays. Compound7 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the hole-board test (at 1 and 3mg/kg) and the plus-maze assay (at 1 and 3mg/kg). Compound10 evidenced anxiolytic activity in the plus-maze and the hole-board tests at 1mg/kg. Locomotor activity of mice was not modified by compound7 or10 at the doses tested. Flumazenil, a non selective benzodiazepine binding site antagonist, was able to completely reverse the anxiolytic-like effects of these sulfamides, proving that the GABA_A receptor is implicated in this action. Anxiety represents a major problem for people with epilepsy. The use of anxiolytic and anticonvulsant sulfamides would be beneficial to individuals who suffer from both disorders.

Keywords: Sulfamides; GABA; _A; -receptor; Benzodiazepine binding site; Anticonvulsant; Anxiolytic

The inverse agonist effect of rimonabant on G protein activation is not mediated by the cannabinoid CB1 receptor: Evidence from postmortem human brain by A.M. Erdozain; R. Diez-Alarcia; J.J. Meana; L.F. Callado (pp. 260-268).

Rimonabant (SR141716) was the first potent and selective cannabinoid CB1 receptor antagonist synthesized. Several data support that rimonabant behaves as an inverse agonist. Moreover, there is evidence suggesting that this inverse agonism may be CB1 receptor-independent. The aim of the present study was to elucidate whether the effect of rimonabant over G protein activation in postmortem human brain is CB1 dependent or independent.[³⁵S]GTPγS binding assays and antibody-capture [³⁵S]GTPγS scintillation proximity assays (SPA) were performed in human and mice brain. [³H]SR141716 binding characteristics were also studied.Rimonabant concentration-dependently decreased basal [³⁵S]GTPγS binding to human cortical membranes. This effect did not change in the presence of either the CB1 receptor agonist WIN 55,212-2, the CB1 receptor neutral antagonist O-2050, or the CB1 allosteric modulator Org 27569. [³⁵S]GTPγS binding assays performed in CB1 knockout mice brains revealed that rimonabant inhibited the [³⁵S]GTPγS binding in the same manner as it did in wild-type mice. The SPA combined with the use of specific antibody-capture of G_α specific subunits showed that rimonabant produces its inverse agonist effect through G_i3, G_o and G_z subtypes. This effect was not inhibited by the CB1 receptor antagonist O-2050. Finally, [³H]SR141716 binding assays in human cortical membranes demonstrated that rimonabant recognizes an additional binding site other than the CB1 receptor orthosteric binding site recognized by O-2050.This study provides new data demonstrating that at least the inverse agonist effect observed with >1μM concentrations of rimonabant in [³⁵S]GTPγS binding assays is not mediated by the CB1 receptor in human brain.

Keywords: Abbreviations; BSA; bovine serum albumin; cAMP; cyclic 3′,5′-adenosine monophosphate; DPCPX; 1,3-dipropyl-8-cyclopentyl-1,3-dipropylxanthine; DTT; dithiothreitol; EGTA; etylene glycol-bis ((-aminoethyl ether); GDP; guanosine diphosphate; GPCR; G protein-coupled receptor; GTPγS; guanosine-; 5; ′; -O; -(3-thio)triphosphate; MAPK; mitogen-activated protein kinase; O-2050; (6A; r; ,10a; R; )-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6; H; -dibenzo[; b; ,; d; ]pyran; Org 27569; (5-chloro-3-ethyl-1; H; -indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide); [; ³⁵; S]GTPγS; guanosine-; 5; ′; -O; -(3-[; ³⁵; S]-thio)triphosphate; SEM; standard error of the mean; SR141716; N; -piperidin-; O; -5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-3-carboxamide; Tris–HCl; 2-amino-2-(hydoxymethyl)-1,3-propanediol hydrochloride; WIN 55,212-2; (; R; )-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanoneRimonabant; Inverse agonism; G proteins; Cannabinoid CB1 receptors; Human brain

Ivermectin induces P-glycoprotein expression and function through mRNA stabilization in murine hepatocyte cell line by Cécile Ménez; Laïla Mselli-Lakhal; Magali Foucaud-Vignault; Patrick Balaguer; Michel Alvinerie; Anne Lespine (pp. 269-278).

Ivermectin is widely used in human and veterinary medicine for the control of helminth infections. Ivermectin is known to interact with P-glycoprotein (P-gp/ MDR1), being a good substrate and a potent inhibitor, however, the influence of ivermectin on the expression of the transporter has not been investigated. Expression of P-glycoprotein was investigated in cultured mouse hepatocytes acutely exposed to ivermectin. The two P-glycoprotein murine isoforms, Mdr1a and Mdr1b, mRNA levels were assessed by real-time RT-PCR. Ivermectin induced a clear time- and concentration-dependent up-regulation of Mdr1a and Mdr1b mRNA levels (as early as a 12-h exposure and up to 2.5-fold at 10μM). Moreover, ivermectin-treated cells displayed enhanced cellular efflux of the P-glycoprotein substrate calcein that was inhibited by the P-glycoprotein blocker valspodar, providing evidence that the ivermectin-induced P-glycoprotein was functional. The mechanisms underlying these effects were investigated. Ivermectin-mediated Mdr1 mRNA induction was independent of the two nuclear receptors CAR and PXR, which are known to be involved in drug transporters regulation. Moreover, by using reporter cell lines that detects specific ligand-activated transcription factors, we showed that ivermectin did not displayed CAR, PXR or AhR ligand activities. However, studies with actinomycin D revealed that the half-life of Mdr1a and Mdr1b mRNA were significantly prolonged by two-fold in ivermectin-treated cells suggesting a post-transcriptional mode of ivermectin regulation. This study demonstrates for the first time that ivermectin induces P-glycoprotein overexpression through post-transcriptional mRNA stabilization, thus offering insight into the mechanism of reduced therapeutic efficacy and development of ivermectin-resistant parasites.

Keywords: Abbreviations; ABC; ATP-binding cassette; AhR; aryl-hydrocarbon receptor; CAR; constitutive androstane receptor; IVM; ivermectin; MLs; macrocyclic lactones; P-gp; P-glycoprotein; PXR; pregnane X receptorIvermectin; Macrocyclic lactones; P-glycoprotein; Mdr1; Hepatocytes

Absolute immunoquantification of the expression of ABC transporters P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein 2 in human liver and duodenum by Theodora G.H.A. Tucker; Alison M. Milne; Sylvie Fournel-Gigleux; Katherine S. Fenner; Michael W.H. Coughtrie (pp. 279-285).

The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2), and P-glycoprotein (Pgp) are important in the distribution and elimination of many drugs and endogenous metabolites. Due to their membrane location and hydrophobicity it is difficult to generate purified protein standards to quantify these transporters in human tissues. The present study generated transporter proteins fused with the S-peptide of ribonuclease for use as standards in immunoquantification in human liver and small intestine. Quantification of the S•tag™, a 15 amino acid peptide, is based on the formation of a functional ribonuclease activity upon its high affinity reconstitution with ribonuclease S-protein. S-tagged transporters were used as full-length protein standards in the immunoquantification of endogenous BCRP, MRP2, and Pgp levels in 14 duodenum and 13 liver human tissue samples. Expression levels in the duodenum were 305±248 (BCRP), 66±70 (MRP2), and 275±205 (Pgp) fmoles per cm². Hepatic levels were 2.6±0.9 (BCRP), 19.8±10.5 (MRP2), and 26.1±10.1 (total Pgp) pmoles per g of liver. The mean hepatic scaling factor was 35.8mg crude membrane per g of liver, and the mean duodenal scaling factor was 1.3mg crude membrane per cm² mucosal lining. Interindividual variability was greater in duodenal samples than liver samples. It is hoped that this innovative method of quantifying these transporters (and other membrane proteins) will improve in vivo– in vitro extrapolation and in silico prediction of drug absorption and elimination, thus supporting drug development.

Keywords: Abbreviations; BCRP; breast cancer resistance protein; MRP2; multidrug resistance-associated protein 2; Pgp; P-glycoproteinDrug transporters; Immunoquantification; BCRP; MRP2; P-glycoprotein

Cyclosporin A and tacrolimus induce renal Erk1/2 pathway via ROS-induced and metalloproteinase-dependent EGF-receptor signaling by El-Sayed Akool; Stefan Gauer; Bashier Osman; Anke Doller; Sebastian Schulz; Helmut Geiger; Josef Pfeilschifter; Wolfgang Eberhardt (pp. 286-295).

Schematic representation of mitogenic HB-EGF-EGFR-ERK signaling cascade induced by CsA and FK506. CsA and FK506 via a rapid generation of mitochondrial ROS can induce proteolytic activity of metalloproteinases, like ADAM17, which cause shedding of the membrane-bound growth factor HB-EGF. HB-EGF itself transactivates the EGF-receptor and activates signaling events downstream of the EGFR including activation of ERK1/2. The increase in ERK1/2 activity presumably will lead to transcriptional induction mainly of those genes which mediate proliferative responses in renal mesangial cells.We previously demonstrated that the widely used immunosuppressive drugs cyclosporin A (CsA) and tacrolimus (FK506), independent of immunophilin binding, can activate profibrogenic transforming growth factor β (TGFβ)/Smad signaling cascades in rat renal mesangial cells (MC). Here we report that both peptidyl-prolyl cis/trans isomerase (PPIase) inhibitors activate the extracellular-signaling regulated kinase (ERK) a member of the mitogen activated protein kinase (MAPK) and induce a rapid and transient increase in ERK phosphorylation. The MEK inhibitor U0126, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), a cell-permeant superoxide dismutase (SOD) and stigmatellin, an inhibitor of mitochondrial cytochrome bc1 complex strongly attenuated the increase in ERK1/2 phosphorylation triggered by PPIase inhibitors. Moreover, neutralizing antibodies against heparin binding-epidermal growth factor (HB-EGF), and inhibition of the EGF receptor by either small interfering (si)RNA or AG1478, demonstrate that ERK activation by both PPIase inhibitors is mediated via HB-EGF-induced EGF receptor (EGFR) tyrosine kinase activation. The strong inhibitory effects achieved by GM6001 and TAPI-2 furthermore implicate the involvement of a desintegrin and metalloproteinase 17 (ADAM17). Concomitantly, the PPIase inhibitor-induced ADAM17 secretase activity was significantly reduced by SOD and stigmatellin thus suggesting that mitochondrial ROS play a primary role in PPIase inhibitor-induced and ADAM17-mediated HB-EGF shedding. Functionally, both immunosuppressants caused a strong increase in MC proliferation which was similarly impeded when cells were treated in the presence of NAC, TAPI-2 or AG1478, respectively. Our data suggest that CsA and FK506, via ROS-dependent and ADAM17-catalyzed HB-EGF shedding induce the mitogenic ERK1/2 signaling cascade in renal MC.

Keywords: Abbreviations; ADAM-17; a disintegrin and metalloproteinase 17; PPIase; peptidyl-prolyl; cis/trans; isomerase; CsA; cyclosporin A; ERK; extracellular signal-regulated kinase; MAPK; mitogen activated protein kinase; MC; mesangial cells; NAC; N; -acetyl cysteine; ROS; reactive oxygen species; siRNA; small interfering RNACalcineurin inhibitor; Heparin binding-epidermal growth factor; Metalloproteinases; Reactive oxygen species; Extracellular-regulated kinase

Multifunctional Ebselen drug functions through the activation of DNA damage response and alterations in nuclear proteins by Gajendra K. Azad; Shah Jaimin Balkrishna; Narayanan Sathish; Sangit Kumar; Raghuvir S. Tomar (pp. 296-303).

Several studies have demonstrated that Ebselen is an anti-inflammatory and anti-oxidative agent. Contrary to this, studies have also shown a high degree of cellular toxicity associated with Ebselen usage, the underlying mechanism of which remains less understood. In this study we have attempted to identify a possible molecular mechanism behind the above by investigating the effects of Ebselen on Saccharomyces cerevisiae. Significant growth arrest was documented in yeast cells exposed to Ebselen similar to that seen in presence of DNA damaging agents (including methyl methane sulfonate [MMS] and hydroxy urea [HU]). Furthermore, mutations in specific lysine residues in the histone H3 tail (H3 K56R) resulted in increased sensitivity of yeast cells to Ebselen presumably due to alterations in post-translational modifications of histone proteins towards regulating replication and DNA damage repair. Our findings suggest that Ebselen functions through activation of DNA damage response, alterations in histone modifications, activation of checkpoint kinase pathway and derepression of ribonucleotide reductases (DNA repair genes) which to the best of our knowledge is being reported for the first time. Interestingly subsequent to Ebselen exposure there were changes in global yeast protein expression and specific histone modifications, identification of which is expected to reveal a fundamental cellular mechanism underlying the action of Ebselen. Taken together these observations will help to redesign Ebselen-based therapy in clinical trials.

Keywords: Ebselen; DNA damage response; Chromatin; Post translational modifications

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Biochemical Pharmacology (v.83, #2)