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Biochemical Pharmacology (v.83, #12)

Editorial Board (pp. iii).

Apremilast mechanism of action and application to psoriasis and psoriatic arthritis by Peter Schafer (pp. 1583-1590).

Psoriasis and psoriatic arthritis are common clinical conditions that negatively impact health-related quality of life and are linked to serious medical comorbidities. Disease mechanisms involve local and systemic chronic inflammatory processes. Available biologic therapies specifically target single inflammatory mediators, such as tumor necrosis factor-α (TNF-α), in the context of a larger inflammatory signaling cascade. To interrupt this pathological cascade earlier in the response or further upstream, and return pro-inflammatory and anti-inflammatory signaling to a homeostatic balance, the use of a phosphodiesterase4 (PDE4) inhibitor has been explored. PDE4 is the major enzyme class responsible for the hydrolysis of cyclic adenosine monophosphate (cAMP), an intracellular second messenger that controls a network of pro-inflammatory and anti-inflammatory mediators. With PDE4 inhibition, and the resulting increases in cAMP levels in immune and non-immune cell types, expression of a network of pro-inflammatory and anti-inflammatory mediators can be modulated. Apremilast is an orally available targeted PDE4 inhibitor that modulates a wide array of inflammatory mediators involved in psoriasis and psoriatic arthritis, including decreases in the expression of inducible nitric oxide synthase, TNF-α, and interleukin (IL)-23 and increases IL-10. In phase II studies of subjects with psoriasis and psoriatic arthritis, apremilast reversed features of the inflammatory pathophysiology in skin and joints and significantly reduces clinical symptoms. The use of an oral targeted PDE4 inhibitor for chronic inflammatory diseases, like psoriasis and psoriatic arthritis, represents a novel treatment approach that does not target any single mediator, but rather focuses on restoring a balance of pro-inflammatory and anti-inflammatory signals.

Keywords: Abbreviations; ACR20; 20% or greater improvement from baseline in American College of Rheumatology; cAMP; cyclic adenosine monophosphate; CREB; cAMP-response element binding protein; DMARDs; disease-modifying antirheumatic drugs; IC; ₅₀; half maximal inhibitory concentration; IFN-α; interferon-αl; IL; interleukin; iNOS; induciblenitricoxidesynthase; NF-κB; nuclear factor kappa B; PDE; phosphodiesterase; PDE4; phosphodiesterase 4; PKA; protein kinase A; Th1; type 1 helper T cell; Th17; type 17 helper T cell; TNF-α; tumor necrosis factor-α; VEGF; vascular endothelial growth factorPsoriasis; Psoriatic arthritis; Phosphodiesterase 4 inhibitor; Apremilast

MicroRNAs in cancer management and their modulation by dietary agents by Tommy Karius; Michael Schnekenburger; Mario Dicato; Marc Diederich (pp. 1591-1601).

MicroRNAs (miRNAs) represent a class of small (21–23 nucleotides) non-coding RNAs that emerged as key post-transcriptional gene regulators, implicated in numerous physiological and pathological processes. Currently, a main focus of miRNA research is related to the roles of miRNAs in cancer development. The biogenesis and modes of action of miRNAs have not been completely elucidated; however, miRNA-mediated translational repression is involved in the regulation of almost every cellular process. Thus, pathological alterations in miRNA expression signatures are commonly associated with disease development. This review specifically focuses on miRNAs in cancer, with an emphasis on their use as potential biomarkers for cancer diagnosis and prognosis. Then, we discuss the potential use of synthetic antisense or miRNA mimetic oligonucleotides and dietary agents to modulate miRNA expression for chemotherapy and chemoprevention of cancer, respectively.

Keywords: Abbreviations; ACTR1A; ARP1 actin-related protein 1 homolog A centractin alpha; AGTR1; angiotensin II receptor type 1; AICDA; activation-induced cytidine deaminase; AKT; v-akt murine thymoma viral oncogene homolog; ALL; acute lymphocytic leukemia; AML; acute myeloid leukemia; APAF1; apoptotic peptidase activating factor 1; APL; acute promyelocytic leukemia; ARHI; age-related hearing impairment; ASO; antisense oligonucleotides; ATRA; all-trans retinoic acid; BACH1; BTB and CNC homology basic leucine zipper transcription factor; BAK1; BCL2-antagonist/killer 1; BCL2; B-cell CLL/lymphoma 2; BIM; BCL2-interacting mediator of cell death; CAGR; cancer-associated genomic region; CCN; cyclin; CDC2; Cell division control protein 2 homolog; CDC25A; cell division cycle 25A; CDK; cyclin-dependent kinase; CDKN; CDK inhibitor; CEBPB; CCAAT/enhancer binding protein (C/EBP) beta; CLL; chronic lymphoid leukemia; c-Kit; transcription factor; CREB; cAMP responsive element binding protein; DGCR8; DiGeorge syndrome critical region 8; DIM; 3,3′-diindolylmethane; DNMT; DNA methyltransferase; E2F; E2F transcription factor; EGCG; epigallocatechin gallate; EGFR; epidermal growth factor receptor; ERBB/HER; epidermal growth factor receptor class avian erythroblastosis oncogene B; ERK; extracellular signal-regulated kinase; ESF1; ESF1 nucleolar pre-rRNA processing protein homolog; ESR1; estrogen receptor 1; ETS1; v-ets erythroblastosis virus E26 oncogene homolog 1; Fas; tumor necrosis factor receptor superfamily member 6; FDA; food and drug administration; FOX; forkhead box; HAT; histone acetyltransferase; HDAC; histone deacetylase; HMGA2; high mobility group AT-hook 2; HOX; homeobox; I3C; indole-3-carbinol; IGF1R; insulin-like growth factor 1 receptor; IGF2; insulin-like growth factor 1; IL6R; interleukin 6 receptor; IRS1; insulin receptor substrate 1; JUN; jun proto-oncogene; KRAS; v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; LATS2; LATS large tumor suppressor homolog 2; LDOC1; leucine zipper downregulated in cancer; MAFK; v-maf musculoaponeuritic fibrosarcoma oncogene homolog K; MATR3; matrin 3; MCL1; myeloid cell leukemia sequence 1; MCM2; minichromosome maintenance complex component 2; MET; met proto-oncogene tyrosine kinase; miRNA; microRNA; mirtron; pri-miRNA-containing intron; MNT; MAX binding protein; MSH2; mutS homolog 2; mTOR; mammalian target of rapamycin; MYOD; myogenic differentiation; NFIB; nuclear factor I/B; NF-κB; nuclear factor kappa B; NRAS; neuroblastoma RAS viral (v-ras) oncogene homolog; PACT; protein kinase R activating protein; PDCD4; programmed cell death 4; PDCD6IP; programed cell death 6 interacting protein; PML-RARα; promyelocytic-retinoic receptor alpha; PRIM1; primase DNA polypeptide 1; PTEN; phosphatase and tensin homolog; RAS; proto-oncogene; RAB; member of the RAS oncogene family; RAR; retinoic acid receptor; RARE; retinoic acid response elements; Rho; rhodopsin; RISC; RNA-induced silencing complex; RNA Pol; RNA polymerase; RXR; retinoid X receptor; SHIP1; SH2 domain-containing inositol phosphatase 1; SNAI2; snail homolog 2 (Slug); SNP; single nucleotide polymorphism; SOCS; suppressor of cytokine signaling; SP1; specificity protein 1; TCL1; T-cell leukemia/lymphoma 1A; TGFBR; transforming growth factor beta receptor; TIMP3; Tissue inhibitor of metalloproteinases 3; TM6SF1; transmembrane 6 superfamily member 1; TP; tumor protein; TP53INP1; TP53 inducible nuclear protein 1; TPM1; tropomyosin 1; TRBP (TARBP); trans-activator RNA binding protein; TSG; tumor suppressor gene; UTR; untranslated region; VIM; vimentin; WT1; Wilms tumor 1; ZBTB10; zinc finger and BTB domain containing 10; ZEB; zinc finger E-box binding homeoboxmicroRNA; Cancer; Biomarker; Chemotherapy; Chemoprevention; miRNA inhibitors; miRNA mimics; Dietary agents

Activation of DNA damage response pathways as a consequence of anthracycline-DNA adduct formation by Robert A. Forrest; Lonnie P. Swift; Ada Rephaeli; Abraham Nudelman; Ken-Ichi Kimura; Don R. Phillips; Suzanne M. Cutts (pp. 1602-1612).

The cytotoxicity of doxorubicin, a clinically used anti-neoplastic drug, can be enhanced by formaldehyde (either endogenous or exogenous) to promote the formation of doxorubicin-DNA adducts. Formaldehyde supplies the carbon required for the covalent linkage of doxorubicin to one strand of DNA, with hydrogen bonds stabilising the doxorubicin mono-adduct to the other strand of DNA, to act much like an interstrand crosslink. Interstrand crosslinks present a major challenge for cellular repair processes, requiring the activation of numerous DNA damage response proteins for resolution of the resulting DNA intermediates and damage. This work investigates DNA damage response proteins activated by doxorubicin-DNA adducts. Although p53 was phosphorylated at Serine 15 in response to adducts, long term growth inhibition of mammalian cells was not affected by p53 status. Using siRNA technology and kinase inhibitors we observed enhanced cellular sensitivity to doxorubicin-DNA adducts when the activity of the signalling protein kinases ATM and ATR were lost. Cells synchronised using a double thymidine block were sensitised to adduct-initiated cell death upon ATR knockdown, but relatively unaffected by ATM knockdown. Loss of ATR was associated with abrogation of a drug-induced G₂/M block and induction of mitotic catastrophe, while loss of ATM was associated with drug-induced apoptosis in non-synchronised cells. These proteins may therefore be potential drug targets to achieve synergistic cytotoxic responses to doxorubicin-DNA adduct forming therapies. The analysis of these protein kinases with respect to cell cycle progression indicates that ATR is required for G₂/M checkpoint responses while ATM appears to function in G₁ mediated responses to anthracycline adducts.

Keywords: Doxorubicin; Formaldehyde; DNA damage; ATR; ATM

GW583340 and GW2974, human EGFR and HER-2 inhibitors, reverse ABCG2- and ABCB1-mediated drug resistance by Kamlesh Sodani; Amit K. Tiwari; Satyakam Singh; Atish Patel; Zhi-Jie Xiao; Jun-Jiang Chen; Yue-Li Sun; Tanaji T. Talele; Zhe-Sheng Chen (pp. 1613-1622).

The overexpression of ATP binding cassette (ABC) transporters often leads to the development of multidrug resistance (MDR) and results in a suboptimal response to chemotherapy. Previously, we reported that lapatinib (GW572016), a human epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinase inhibitor (TKI), significantly reverses MDR in cancer cells by blocking the efflux function of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2). In the present study, we conducted in vitro experiments to evaluate if GW583340 and GW2974, structural analogues of lapatinib, could reverse ABCB1- and ABCG2-mediated MDR. Our results showed that GW583340 and GW2974 significantly sensitized ABCB1 and ABCG2 overexpressing MDR cells to their anticancer substrates. GW583340 and GW2974 significantly increased the intracellular accumulation of [³H]-paclitaxel in ABCB1 overexpressing cells and [³H]-mitoxantrone in ABCG2 overexpressing cells respectively. In addition, GW583340 and GW2974 significantly inhibited ABCG2-mediated transport of methotrexate in ABCG2 overexpressing membrane vesicles. There was no significant change in the expression levels of ABCB1 and ABCG2 in the cell lines exposed to 5μM of either GW583340 or GW2974 for 3 days. In addition, a docking model predicted the binding conformation of GW583340 and GW2974 to be within the transmembrane region of homology modeled human ABCB1 and ABCG2. We conclude that GW583340 and GW2974, at clinically achievable plasma concentrations, reverse ABCB1- and ABCG2-mediated MDR by blocking the drug efflux function of these transporters. These findings may be useful in developing combination therapy for cancer treatment with EGFR TKIs.

Keywords: Abbreviations; MDR; multidrug resistance; ABC; ATP-binding cassette; ABCB1 (P-gp); P-glycoprotein; ABCG2; also called BCRP (breast cancer resistance protein)/MXR (mitoxantrone resistance protein); ABCC1 (MRP1); multidrug resistance protein 1; EGFR; epidermal growth factor receptor; HER2; human epidermal growth factor receptor 2; TKI; tyrosine kinase inhibitor; PBS; phosphate-buffered saline; FTC; Fumitremorgin CGW583340; GW2974; ABCB1; ABCG2; Multidrug resistance; Tyrosine kinase inhibitor

Impact of terminal dimethylation on the resistance profile of α- N-heterocyclic thiosemicarbazones by Petra Heffeter; Christine Pirker; Christian R. Kowol; Gerrit Herrman; Rita Dornetshuber; Walter Miklos; Ute Jungwirth; Gunda Koellensperger; Bernhard K. Keppler; Walter Berger (pp. 1623-1633).

Triapine is an α- N-heterocyclic thiosemicarbazone with promising anticancer activity against hematologic malignancies but widely ineffective against solid tumor types in clinical trials. The anticancer activity of thiosemicarbazones can be dramatically increased by terminal dimethylation. KP1089 is a gallium compound containing two terminal dimethylated thiosemicarbazone ligands. To gain insights on the vulnerability of this highly active terminal dimethylated thiosemicarbazone to drug resistance mechanisms, a new cell model with acquired resistance against the lead compound KP1089 was established. Subsequent genomic analyses (arrayCGH and FISH) revealed amplification of the ABCC1 gene on double minute chromosomal DNA in KP1089-resistant cells as well as overexpression of ABCC1 and ABCG2 on the protein level. KP1089 was further confirmed as a substrate of ABCC1 and ABCG2 but not of ABCB1 using a panel of ABC transporter-overexpressing cell models as well as ABC transporter inhibitors. Moreover, glutathione depletion strongly enhanced KP1089 activity, although no glutathione conjugate formation by glutathione-S-transferase was observed. Thus, a co-transport of KP1089 together with glutathione is suggested. Finally, a panel of thiosemicarbazone derivatives was tested on the new KP1089-resistant cell line. Notably, KP1089-resistant cells were not cross-resistant against thiosemicarbazones lacking terminal dimethylation (e.g. Triapine) which are less active than KP1089. This suggests that terminal dimethylation of thiosemicarbazones – linked with distinctly enhanced anticancer activity – leads to altered resistance profiles compared to classical thiosemicarbazones making this compound class of interest for further (pre)clinical evaluation.

Keywords: Abbreviations; ABC; ATP-binding cassette; BCRP; breast cancer resistance protein; BSA; bovine serum albumin; CDNB; 1-chloro-2,4-dinitrobenzene; CSA; cyclosporin A; DMF; dimethylformamide; DMSO; dimethyl sulfoxide; ESI-MS; electrospray ionization mass spectrometry; GSH; glutathione; GST; glutathione-S-transferase; KP1089; [bis(2-acetylpyridine 4,4-dimethylthiosemicarbazonato-; N; ,; N; ,; S; )gallium(III)] tetrachloridogallate; KP1550; 2-acetylpyridine 4,4-dimethylthiosemicarbazone; KP1657; [bis(2-acetylpyridine thiosemicarbazonato-; N; ,; N; ,; S; )gallium(III)] nitrate; KP1719; [bis(3-aminopyridine-2-carboxaldehyde 4,4-dimethylthiosemicarbazonato-; N; ,; N; ,; S; )gallium(III)] hexafluorophosphate; KP1740; [bis(2-formylpyridine 4,4-dimethylthiosemicarbazonato-; N; ,; N; ,; S; )gallium(III)] hexafluorophosphate; LRP; lung resistance protein; MDR; multidrug resistance; MRP; multidrug resistance-related protein; PBS; phosphate-buffered saline; P-gp; P-glycoprotein; TD; terminal dimethylation; Triapine; 3-aminopyridine-2-carboxaldehyde thiosemicarbazoneResistance; Thiosemicarbazones; Multidrug resistance; Triapine; ABC transporter; Glutathione

The AKT/NF-κB inhibitor xanthohumol is a potent anti-lymphocytic leukemia drug overcoming chemoresistance and cell infiltration by Roberto Benelli; Roberta Venè; Monica Ciarlo; Sebastiano Carlone; Ottavia Barbieri; Nicoletta Ferrari (pp. 1634-1642).

Although the vast majority of patients with acute lymphocytic leukemia (ALL) attain remission with modern therapies, relapsed leukemia will continue to be a common malignancy both in childhood and in adults, until new treatments are available.Therapeutic options for advanced B-cell acute lymphocytic leukemia are still limited and acquired drug resistance and extramedullary tissue infiltration are two major obstacles during treatment. The prenylflavonoid xanthohumol (XN) has shown in vitro and in vivo therapeutic potential against a range of tumors. In the present study we investigated the effects of XN on B-ALL cells in vitro and in an ALL-like xenograft mouse model.Treatment of ALL cell lines with XN resulted in growth arrest and apoptosis induction. XN retained its cytotoxicity when adriamycin resistant cells were examined while ALL cell clones adapted to long-term exposure to XN resulted highly responsive to cytotoxic drugs. Administration of 50μg XN/mouse (5 days/week) significantly increased animal life span by delaying the insurgence of neurological disorders due to leukemic cells dissemination. In agreement with a less invasive phenotype, cell migration and invasion were impaired by XN and basal levels of FAK, AKT and NF-κB signaling pathways were down-regulated in ALL cells upon XN exposure.Our data indicate that XN has significant antileukemic activity both in vitro and in vivo, which associates with impaired cell migration and invasion. Interestingly, this activity overcomes mechanisms leading to drug-resistance. XN represents a promising agent perspective for ALL therapy and recurrence prevention and would deserve clinical testing in the near future.

Keywords: Xanthohumol; ALL; Migration; Chemoresistance; Chemoprevention

Mitochondrial superoxide mediates doxorubicin-induced keratinocyte apoptosis through oxidative modification of ERK and Bcl-2 ubiquitination by Sudjit Luanpitpong; Pithi Chanvorachote; Ubonthip Nimmannit; Stephen S. Leonard; Christian Stehlik; Liying Wang; Yon Rojanasakul (pp. 1643-1654).

Massive apoptosis of keratinocytes has been implicated in the pathogenesis of chemotherapy-induced skin toxicities, but the underlying mechanisms of action are not well understood. The present study investigated the apoptotic effect of doxorubicin (DOX) on HaCaT keratinocytes and determined the underlying mechanisms. Treatment of the cells with DOX induced reactive oxygen species (ROS) generation and a concomitant increase in apoptotic cell death through the mitochondrial death pathway independent of p53. Electron spin resonance and flow cytometry studies showed that superoxide is the primary oxidative species induced by DOX and responsible for the death inducing effect. Ectopic expression of mitochondrial superoxide scavenging enzyme (MnSOD) or treatment with MnSOD mimetic (MnTBAP) inhibited DOX-induced superoxide generation and apoptosis. The mechanism by which superoxide mediates the apoptotic effect of DOX was shown to involve downregulation of Bcl-2 through ubiquitin–proteasomal degradation. Superoxide induces dephosphorylation of Bcl-2 through MAP kinase ERK1/2 inactivation, which promotes ubiquitination of Bcl-2. We also provide evidence for the oxidative modification of ERK1/2 through cysteine sulfenic acid formation. These findings indicate a novel pathway for redox regulation of apoptosis regulatory proteins, which could be important in the understanding of chemotherapy-induced toxicities and development of preventive treatment strategies which are currently lacking.

Keywords: Abbreviations; DOX; doxorubicin; ROS; reactive oxygen species; zVAD-fmk; benzyloxy-carbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone; zIETD-fmk; benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone; zLEHD-fmk; benzyloxycarbonyl-Leu-Glu(OMe)-His-Asp(OMe)-fluoromethylketone; control; CNTL; MnTBAP; Mn(III)tetrakis(4-benzoic acid) porphyrin chloride; CAT; cell permeable catalase, catalase-polyethylene glycol; DMTU; dimethylthiourea; MnSOD; manganese superoxide dismutase; H; ₂; DCF-DA; dihydrodichlorofluorescein diacetate; DHE; dihydroethidium; ESR; electron spin resonance; DMPO; 5,5-dimethyl-1-pyrroline-N-oxide; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; LAC; lactacystin; CMA; concanamycin A; Bcl-2-Ub; Bcl-2 ubiquitination; Cys-SOH; cysteine sulfenic acidApoptosis; Doxorubicin; Keratinocytes; Reactive oxygen species; ERK; Bcl-2

A non-peptide receptor inhibitor with selectivity for one of the neutrophil formyl peptide receptors, FPR 1 by Hülya Çevik-Aras; Christina Kalderén; Annika Jenmalm Jensen; Tudor Oprea; Claes Dahlgren; Huamei Forsman (pp. 1655-1662).

The neutrophil formyl peptide receptors (FPR1 and FPR2) are members of the G-protein coupled receptor family. The signals generated by occupied FPRs are both pro-inflammatory and anti-inflammatory. Accordingly, these receptors have become a therapeutic target for the development of novel drugs that may be used to reduce injuries in inflammatory diseases including asthma, rheumatoid arthritis, Alzheimer's disease and cardiovascular diseases. To support the basis for a future pharmacological characterization, we have identified a small molecular non-peptide inhibitor with selectivity for FPR1. We used the FPR1 and FPR2 specific ligands fMLF and WKYMVM, respectively, and an earlier described ratio technique, to determine inhibitory activity combined with selectivity. We show that the compound 3,5-dichloro-N-(2-chloro-5-methyl-phenyl)-2-hydroxy-benzamide (BVT173187) fulfills the criteria for an FPR1 inhibitor selective for FPR1 over FPR2, and it inhibits the same functional repertoire in neutrophils as earlier described peptide antagonists. Accordingly, the new inhibitor reduced neutrophil activation with FPR1 agonists, leading to mobilization of adhesion molecules (CR3) and the generation of superoxide anion from the neutrophil NADPH-oxidase. The effects of a number of structural analogs were determined but these were either without activity or less active/specific than BVT173187. The potency of the new inhibitor for reduction of FPR1 activity was the same as that of the earlier described FPR1 antagonist cyclosporine H, but signaling through the C5aR and CXCR (recognizing IL8) was also affected by BVT173187.

Keywords: Neutrophils; Formyl peptide receptors; Receptor selective inhibitor; Signal transduction; NADPH-oxidase; Chemoattractant receptors

Protection of lung epithelial cells from protease-mediated injury by trappin-2 A62L, an engineered inhibitor of neutrophil serine proteases by Annabelle Tanga; Ahlame Saidi; Marie-Lise Jourdan; Sandrine Dallet-Choisy; Marie-Louise Zani; Thierry Moreau (pp. 1663-1673).

Neutrophil serine proteases (NSPs), including elastase, proteinase 3 and cathepsin G, play critical roles in the pathogenesis of chronic inflammatory lung diseases. The release of excess NSPs leads to the destruction of lung tissue and an overexuberant, sustained inflammatory response. Antiproteases could be valuable tools for controlling these NSP-mediated inflammatory events. We have examined the capacity of trappin-2 A62L, a potent engineered inhibitor of all three NSPs, to protect human lung A549 epithelial cells from the deleterious effects of NSPs. Trappin-2 A62L, significantly inhibited the detachment of A549 cells and the degradation of the tight-junction proteins, E-cadherin, β-catenin and ZO-1, induced by each individual NSP and by activated neutrophils. Trappin-2 A62L also decreased the release of the pro-inflammatory cytokines IL-6 and IL-8 from A549 cells that had been stimulated with elastase or LPS. Trappin-2 A62D/M63L, a trappin-2 variant that has no antiprotease activity, has similar properties, suggesting that the anti-inflammatory action of trappin-2 is independent of its antiprotease activity. Interestingly, we present evidence that trappin-2 A62L, as well as wild-type trappin-2, enter A549 cells and move rapidly to the cytoplasm and nucleus, where they are likely to exert their anti-inflammatory effects. We have also demonstrated that trappin-2 A62L inhibits the early apoptosis of A549 cells mediated by NSPs. Thus, our data indicate that trappin-2 A62L is a powerful anti-protease and anti-inflammatory agent that could be used to develop a treatment for patients with inflammatory lung diseases.

Keywords: Serine proteases; Protease inhibitor; Trappin-2; Elafin; Inflammatory lung diseases

Investigation of imatinib and other approved drugs as starting points for antidiabetic drug discovery with FXR modulating activity by Ramona Steri; Janosch Achenbach; Dieter Steinhilber; Manfred Schubert-Zsilavecz; Ewgenij Proschak (pp. 1674-1681).

A self-organizing map (SOM) is a virtual screening method used for correlation of molecular structure and potential biological activity on a certain target and offers a way to represent multi-dimensional data of large databases in a two-dimensional space. Large databases, for example the DrugBank database, provide information about biological activity and chemical structure of small molecules and are widely used in drug development for identification of new lead structures.The farnesoid X receptor (FXR) is a ligand activated transcription factor involved in key regulation mechanisms within glucose and lipid homeostasis. Although FXR became an established target in drug development for diseases associated with lipid, glucose or hepatic disorders during the last decade, none of the developed compounds have reached later phases of clinical development so far.We used a SOM trained with known FXR ligands to screen the DrugBank database for potential ligands for FXR. In this article, we report the successful identification of six approved drugs out of the Drugbank as FXR modulators (ketoconazole, pentamidine, dobutamine, imatinib, papaverine and montelukast) by using a SOM for screening of the DrugBank database. We show FXR modulation by selected compounds in a full length FXR transactivation assay and modulation of a FXR target gene by imatinib.

Keywords: Abbreviations; CATS2D; chemically advanced template search 2D; CDCA; chenodeoxycholic acid; CHF; congestive heart failure; CML; chronic myeloid leukemia; COPD; chronic obstructive pulmonary disease; DMEM; Dulbeccós modified eagle medium; DMSO; dimethylsulfoxide; FCS; fetal calf serum; FXR; farnesoid X receptor; GIST; gastrointestinal stromal tumors; PCR; polymerase chain reaction; PPAR; peroxisome-proliferator activated receptor; PS; penicillin/streptomycine; qPCR; quantitative PCR; SOM; self-organizing map; SHP; small heterodimer partner; SP; sodium pyruvate; SSRI; selective serotonin reuptake inhibitor; PDE; phosphodiesterase; SERM; selective estrogen receptor modulatorFarnesoid X receptor; Self-organizing maps; Imatinib; Virtual screening; Antidiabetic compounds; Drug repurposing

Murine collagen antibody induced arthritis (CAIA) and primary mouse hepatocyte culture as models to study cytochrome P450 suppression by Leslie J. Dickmann; Helen J. McBride; Sonal K. Patel; Kent Miner; Larry C. Wienkers; J. Greg Slatter (pp. 1682-1689).

Changes in cytochrome P450 expression incurred by inflammatory disease were studied in a murine collagen antibody induced arthritis (CAIA) model and compared to bacterial lipopolysaccharide-treated mice and to cytochrome P450 changes in primary mouse hepatocytes following combination treatments with cytokines IL-1β, IL-6, or TNFα. CAIA in female mice increased serum IL-1β, IL-6 and hepatic serum amyloid A (SAA) mRNA and significantly altered cytochrome P450 mRNA and activity levels. Most cytochrome P450 isoforms were down-regulated, although some, such as Cyp3a13, were up-regulated. Cytokine effects on cytochrome P450 levels in mouse hepatocytes were compared at in vitro cytokine concentrations similar to those measured in CAIA mouse serum in vivo. In vivo and in vitro cytochrome P450 suppression by cytokines was congruent for some cytochrome P450 isoforms (Cyp1a2, Cyp2c29, and Cyp3a11) but not for others (cytochrome P450 oxidoreductase (POR) and Cyp2e1). In mouse hepatocytes, IL-6 and IL-1β in combination in vitro caused a synergistic increase in SAA mRNA expression, but not in cytochrome P450 suppression. IL-1β and IL-6 were equipotent in the suppression of cytochrome P450 gene expression, while TNFα caused mild suppression only at the highest concentrations used. TNFα in combination with IL-1β, IL-6, or both had a protective effect against IL-1β and IL-6-mediated cytochrome P450 suppression. When IL-1β or IL-6 was combined with low concentrations of TNFα, several P450 isoforms were induced rather than suppressed. These data highlight the complexities of performing in vitro–in vivo comparisons using disease models for cytochrome P450 regulation by cytokines.

Keywords: Cytochrome P450; Inflammation; Interleukin-1β; Interleukin-6; Tumor necrosis factor α; Hepatocyte

Structure-based development of bacterial nitroreductase against nitrobenzodiazepine-induced hypnosis by Shiuan-Woei LinWu; Che-An Wu; Fu-Chuo Peng; Andrew H.-J. Wang (pp. 1690-1699).

Rational design of E. coli nitroreductase NfsB for the in vivo disposition of nitrobenzodiazepine (NBDZ) and shortening the duration of hypnosis.Nitrobenzodiazepine (NBDZ) is an addictive drug of the abused substances that causes severe neurological effects and even death. Bacterial type I nitroreductase NfsB (EC 1.5.1.34) has been reported to catalyze NBDZ into inactive metabolite 7-amino-benzodiazepine (7ABDZ) with promising activity, so as to become an attractive candidate for treatment of NBDZ overdose and addiction. Here, we investigate the nitroreduction of an NBDZ, flunitrazepam (FZ), by various mutants of NfsB designed from the solved crystal structure and characterize their in vitro and in vivo potency. Conformational changes occurred in the active site of N71S/F124W in contrast to the wild-type, including the flipping on the aromatic rings of W124 and F70 as well as the extension on the hydrogen bond network between flavin mononucleotide (FMN) and S71, which allow the significant enlargement in the active site pocket. In the complex structure of N71S/F124W and nicotinamide (NIA), stacking sandwich attractions of W124-FMN-NIA were also found, implying the importance of W124 in substrate accessibility. Consequently, N71S/F124W exhibited increased 7AFZ production in vitro with nearly no toxicity and reduced 50% sleeping time (hypnosis) in vivo. Taken together, we demonstrate for the first time that N71S/F124W can serve as an effective antidote for NBDZ-induced hypnosis and provide the molecular basis for designing NfsB and the like in the future.

Keywords: Nitrobenzodiazepine; Escherichia coli; type I nitroreductase NfsB; Flunitrazepam; Rational protein design; Antidote

Reactivation kinetics of a series of related bispyridinium oximes with organophosphate-inhibited human acetylcholinesterase—Structure–activity relationships by Franz Worek; Timo Wille; Marianne Koller; Horst Thiermann (pp. 1700-1706).

Structure–activity-relationship of reactivation kinetics of structurally different oximes with organophosphate-inhibited human acetylcholinesterase.Despite extensive research in the last six decades, oximes are the only available drugs which enable a causal treatment of poisoning by organophosphorus compounds (OP). However, numerous in vitro and in vivo studies demonstrated a limited ability of these oximes to reactivate acetylcholinesterase (AChE) inhibited by different OP pesticides and nerve agents. New oximes were mostly tested for their therapeutic efficacy by using different animal models and for their reactivating potency with AChE from different species. Due to the use of different experimental protocols a comparison of data from the various studies is hardly possible. Now, we found it tempting to determine the reactivation kinetics of a series of bispyridinium oximes bearing one or two oxime groups at different positions and having an oxybismethylene or a trimethylene linker under identical conditions with human AChE inhibited by structurally different OP. The data indicate that the position of the oxime group(s) is decisive for the reactivating potency and that different positions of the oxime groups are important for different OP inhibitors while the nature of the linker, oxybismethylene or trimethylene, is obviously of minor importance. Hence, these and previous data emphasize the necessity for thorough kinetic investigations of OP-oxime-AChE interactions and underline the difficulty to develop a broad spectrum oxime reactivator which is efficient against structurally different OP inhibitors.

Keywords: Acetylcholinesterase; Organophosphorus compounds; Oximes; Kinetics; Structure–activity relationship

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Biochemical Pharmacology (v.83, #12)