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Biochemical Pharmacology (v.76, #5)
The effects of environmental neurotoxicants on the dopaminergic system: A possible role in drug addiction by Douglas C. Jones; Gary W. Miller (pp. 569-581).
Humans are routinely exposed to a vast array of environmental neurotoxicants, including pesticides, endocrine disrupters, and heavy metals. The long-term consequences of exposure have become a major human health concern as research has indicated strong associations between neurotoxicants and a variety of dopamine-related neurological disorders. Developmental exposure to pesticides including paraquat, organochlorines, and rotenone produce alterations in the dopaminergic system and has been linked to neurodegenerative disorders, including Parkinson's disease. Endocrine disrupters such as Bisphenol A, mimic estrogenic activity and impact various dopaminergic processes to enhance mesolimbic dopamine activity resulting in hyperactivity, attention deficits, and a heightened sensitivity to drugs of abuse. A second class of endocrine disrupters, the polychlorinated biphenyls, may act directly on dopaminergic processes to disrupt the dopamine system and produce Parkinson-like symptoms. Exposure to the heavy metal lead enhances dopaminergic activity and has been associated with attention deficits, Alzheimer's disease, and increased drug sensitivity. Manganese exposure, in contrast, results in dopamine deficiencies and Parkinson-like symptoms. Therefore, this commentary will discuss the effects and consequences that exposure to these three classes of environmental neurotoxicants have on the dopamine system and related behaviors and disorders. Finally, the recent hypothesis that exposure to environmental compounds which have effects on dopaminergic neurotransmission, including 2,4-dichlorophenoxyacetic acid, Bisphenol A, and multiple heavy metals, may potentiate drug-induced behaviors and increase the brain's vulnerability to drug addiction will be discussed.
Keywords: Abbreviations; DA; dopamine; DAT; dopamine transporter; VMAT; vesicular monoamine transporter; PD; Parkinson's disease; TH; tyrosine hydroxylase; BPA; Bisphenol A; EDCs; environmental endocrine disrupters; PCBs; polychlorinated biphenyls; MA; methamphetamineEnvironmental; Neurotoxicant; Dopamine; Drug; Addiction
Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells by Hua Zhu; Hao Wu; Xiuping Liu; Brad R. Evans; Daniel J. Medina; Chang-Gong Liu; Jin-Ming Yang (pp. 582-588).
MicroRNAs are short non-coding RNA molecules able to affect stability and/or translation of mRNA, thereby regulating the expression of genes involved in many biological processes. We report here that microRNAs miR-27a and miR-451 are involved in activating the expression of P-glycoprotein, the MDR1 gene product that confers cancer cell resistance to a broad range of chemotherapeutics. We showed that expressions of miR-27a and miR-451 were up-regulated in multidrug resistant (MDR) cancer cell lines A2780DX5 and KB-V1, as compared with their parental lines A2780 and KB-3-1. Treatment of A2780DX5 cells with the antagomirs of miR-27a or miR-451 decreased the expression of P-glycoprotein and MDR1 mRNA. In contrast, the mimics of miR-27a and miR-451 increased MDR1 expression in the parental cells A2780. The sensitivity to and intracellular accumulation of cytotoxic drugs that are transported by P-glycoprotein were enhanced by the treatment with the antagomirs of miR-27a or miR-451. Our results demonstrate for the first time the roles of microRNAs in the regulation of drug resistance mediated by MDR1/P-glycoprotein, and suggest the potential for targeting miR-27a and miR-451 as a therapeutic strategy for modulating MDR in cancer cells.
Keywords: MicroRNA; MDR; 1; P-glycoprotein; Gene expression; Drug resistance; Chemotherapy
Honokiol is a potent scavenger of superoxide and peroxyl radicals by Sergey Dikalov; Tanya Losik; Jack L. Arbiser (pp. 589-596).
Honokiol, a compound extracted from Magnolia officinalis, has antitumor and antiangiogenic properties in several tumor models in vivo. Among the downstream pathways inhibited by honokiol is nuclear factor kappa beta (NFκβ). A prime physiologic stimulus of NFκβ is reactive oxygen species. The chemical structure of honokiol suggests that it may be an effective scavenger of reactive oxygen species. In this work, we have studied the reactions of honokiol with superoxide and peroxyl radicals in cell-free and cellular systems using electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques. Honokiol efficiently scavenged superoxide radicals in xanthine oxidase and cytochrome P-450 cell-free systems with the rate constant 3.2×105M−1s−1, which is similar to reactivity of ascorbic acid but 20-times higher than reactivity of vitamin E analog trolox. Honokiol potently scavenged intracellular superoxide within melanoma cells. In addition, honokiol scavenged peroxyl radicals generated by 2,2′-azo-bis(2-amidinopropane hydrochloride) (AAPH). The rate constant of the reaction of honokiol with peroxyl radicals (1.4×106M−1s−1) was calculated from the competition with spin trap 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO), and was found close to reactivity of trolox (2.5×106M−1s−1). Therefore, honokiol is an effective scavenger of both superoxide and peroxyl radicals, which may be important for physiological activity of honokiol.
Keywords: Abbreviations; AAPH; 2,2′-azo-bis(2-amidinopropane hydrochloride; CM; 3-methoxycarbonyl-proxyl; CMH; 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine; DHE; dihydroethidium; DTPA; diethylenetriaminepentaacetic acid; EMPO; 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide; ESR; electron spin resonance; HO; honokiol; HPLC; high-performance liquid chromatography; MQ; menadione; NFκβ; nuclear factor kappa beta; PKC; protein kinase C; PEG–SOD; polyethylene glycol conjugated superoxide dismutaseAntioxidant; Honokiol; Superoxide; Melanoma; Electron spin resonance
Renal protection by 3 H-1,2-dithiole-3-thione against cisplatin through the Nrf2-antioxidant pathway by Hyun-Min Park; Jeong-Min Cho; Hyang-Rim Lee; Gi-seoung Shim; Mi-Kyoung Kwak (pp. 597-607).
Cisplatin is commonly used for the treatment of several solid tumors. However, its clinical use is often limited by renal toxicity. The indirect antioxidant 3 H-1,2-dithiole-3-thione (D3T) has been known to protect cells from oxidative damage by up-regulating the expression of antioxidative genes through the transcription factor NF-E2-related factor 2 (Nrf2) pathway. We hypothesized that D3T treatment may be protective against cisplatin-induced nephrotoxicity by enhancing the antioxidative capacity of renal cells. In cultured murine tubular epithelial cells, D3T facilitates the nuclear accumulation of Nrf2 and the subsequent expression of its target genes such as glutamate cysteine ligase (GCL). Increased GSH pool in D3T-treated renal cells appears to be associated with amelioration of cisplatin-mediated cell death. Protective effects of D3T were also observed in mice. Oral administration of D3T (0.25mmol/kg) increased the expression of GCL in mouse kidney, which resulted in suppression of cisplatin-mediated increases in blood urea nitrogen and serum creatinine. Histopathological changes representing cisplatin-induced acute renal failure were also effectively ameliorated by D3T treatment. Collectively, these results indicate that pharmacological activation of the Nrf2 pathway might have a beneficial effect on reducing chemotherapy-associated cytotoxic adverse effects.
Keywords: Abbreviations; D3T; 3; H; -1,2-dithiole-3-thione; Nqo1; NAD(P)H:quinone oxidoreductase 1; GCLC; glutamate cysteine ligase catalytic subunit; GCLM; glutamate cysteine ligase modifier subunit; BSO; L; -buthionine-sulfoxamine; DTNB; 5′,5-dithio-bis(2-nitrobenzoic acid); MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BUN; blood urea nitrogen; ARE; antioxidant response element; HO-1; heme oxygenase-1Cisplatin; Nephrotoxicity; Nrf2; D3T
Mutational study of the “catalytic tetrad” of DNA topoisomerase IB from the hemoflagellate Leishmania donovani: Role of Asp-353 and Asn-221 in camptothecin resistance by Rosario Diaz-González; Yolanda Pérez-Pertejo; Yves Pommier; Rafael Balaña-Fouce; Rosa M. Reguera (pp. 608-619).
Leishmania donovani, the causative organism for visceral leishmaniasis, contains a unique bisubunit DNA–topoisomerase IB (LdTopIB). The catalytically active enzyme is a heterodimer constituted by a large subunit (LdTopIL) containing a non-conserved N-terminal end and the phylogenetically conserved core domain, whereas the small subunit (LdTopIS) harbors the C-terminal domain with the characteristic tyrosine residue in the active site. Site-directed mutagenesis was used to substitute the basic amino acid (Arg-314, Lys-352, Arg-410 and His-453) of the LdTopIL subunit by the neutral amino acid alanine. The expression of these mutants in a topoisomerase-free yeast strain produced inactive proteins. Similarly, when the Tyr-222 from small subunit, involved in DNA cleavage, was substituted by Phe no topoisomerase activity was detected in yeast overexpressing extracts. In addition two substitutions involved in camptothecin inhibition were also analyzed. Asp-353 located in the core domain of the large subunit and Asn-221 which heads Tyr-222 in the small subunit, were replaced by Ala and Ser, respectively. These mutants were insensitive to the inhibitor; despite they displayed significant relaxation activity.
Keywords: Abbreviations; TopIB; type IB DNA topoisomerase; TopII; type II DNA topoisomerase; LdTopIB; Leishmanial DNA topoisomerase; LdTopIS; small subunit of leishmanial DNA topoisomeraseIB; LdTopIL; large subunit of leishmanial DNA topoisomerase; yTopIB; yeast DNA topoisomerase; CPT; camptothecin; DMSO; dimethylsulfoxideTopoisomerase IB; Leishmania; Trypanosomatids; Camptothecin; Site-directed mutagenesis; Tropical diseases
Recombinant fibrinogenase from Agkistrodon acutus venom protects against sepsis via direct degradation of fibrin and TNF-α by Rongrong Wang; Pengxin Qiu; Weijian Jiang; Xiaofeng Cai; Yanqiu Ou; Xingwen Su; Jinlian Cai; Jiashu Chen; Wei Yin; Guangmei Yan (pp. 620-630).
Severe sepsis remains a leading cause of death and disability because of less effective therapy available for this disease. A complex interplay between the inflammatory factors and the coagulation pathways seems to be the fundamental mechanisms for the pathogenesis of sepsis. Here we report that recombinant fibrinogenase II (rF II) from Agkistrodon acutus plasmin-independently degraded the thrombi, and inhibited inflammatory responses by direct and specific degradation of tumor necrosis factor alpha (TNF-α) induced by lipopolysaccharide (LPS) without showing proteolytic activities on interleukin-1 (IL-1), cluster of differentiation 68 (CD68) and some other serum proteins. We also report that rF II effectively protected against LPS induced sepsis in a rabbit model. Administration of rF II reduced hepatic and renal damage, decreased the levels of alanine aminotransferase (ALT) and blood urea nitrogen (BUN), and increased survival rate in LPS-induced sepsis rabbits. We further confirmed the rescue effect of rF II on severe sepsis in rat caecal ligation and puncture (CLP) model. Our findings suggest that rF II could effectively protect against sepsis via direct degradation of microthrombi and inflammatory factor TNF-α as well as provide a novel strategy to develop a single proteinase molecule for targeting the main pathological processes of this disease.
Keywords: Sepsis; Lipopolysaccharide; Fibrinogenase; Microthrombi; Tumor necrosis factor-α
Ethyl pyruvate and ethyl lactate down-regulate the production of pro-inflammatory cytokines and modulate expression of immune receptors by Marcus Hollenbach; Anja Hintersdorf; Klaus Huse; Ulrich Sack; Marina Bigl; Marco Groth; Thore Santel; Martin Buchold; Inge Lindner; Andreas Otto; Dieter Sicker; Wolfgang Schellenberger; Johannes Almendinger; Barbara Pustowoit; Claudia Birkemeyer; Mathias Platzer; Ilka Oerlecke; Nasr Hemdan; Gerd Birkenmeier (pp. 631-644).
Esters of α-oxo-carbonic acids such as ethyl pyruvate (EP) have been demonstrated to exert inhibitory effects on the production of anti-inflammatory cytokines. So far, there is no information about effects, if any, of ethyl lactate (EL), an obviously inactive analogue of EP, on inflammatory immune responses. In the present study, we provide evidence that the anti-inflammatory action of α-oxo-carbonic acid esters is mediated by inhibition of glyoxalases (Glo), cytosolic enzymes that catalyse the conversion of α-oxo-aldehydes such as methylglyoxal (MGO) into the corresponding α-hydroxy acids using glutathione as a cofactor. In vitro enzyme activity measurements revealed the inhibition of human Glo1 by α-oxo-carbonic acid esters, whilst α-hydroxy-carbonic acid esters such as EL were not inhibitory. In contrast, both EP and EL were shown to suppress the Lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-8 from human immunocompetent cells, and modulated the expression of the immune receptors HLA-DR, CD14 and CD91 on human monocytes. Here, we show a crossing link between glyoxalases and the immune system. The results described herein introduce glyoxalases as a possible target for therapeutic approaches of immune suppression.
Keywords: Abbreviations; BGCD; p; -bromobenzylglutathione cyclopentyl diester; EL; ethyl lactate; EP; ethyl pyruvate; HAGH; hydroxyacyl glutathione hydrolase (glyoxalase 2); IFN; interferon; Glo1; glyoxalase 1; GSH; l; -glutathione; LDH; lactate dehydrogenase; LPS; lipopolysaccharide; mAb; monoclonal antibody; MFI; mean fluorescence intensity; MGO; methylglyoxal; NAC; N; -acetyl cysteine; PBMC; peripheral blood mononuclear cells; PBS; phosphate-buffered saline; PHA; phytohemagglutinin; PMSF; phenylmethylsulfonyl fluoride; TBS; Tris-buffered salineEthyl lactate; Ethyl pyruvate; Glyoxalase; Immune receptors; Methylglyoxal; Pro-inflammatory cytokines
A comparative study of the effects of quercetin and its glucuronide and sulfate metabolites on human neutrophil function in vitro by Sunita Suri; Moira A. Taylor; Anne Verity; Sandra Tribolo; Paul W. Needs; Paul A. Kroon; David A. Hughes; Vincent G. Wilson (pp. 645-653).
Exposure of neutrophils to either lipopolysaccharide (LPS) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) is associated with changes in the expression of cell adhesion molecules and elevation of intracellular calcium ions. Although dietary flavonoids are reported to possess anti-inflammatory properties, little is known regarding the effect of their metabolites. We have investigated the effects of quercetin and its major metabolites on LPS and fMLP-stimulated human neutrophils using concentrations comparable to those reported in feeding studies on human volunteers. The metabolite quercetin 3-glucuronide caused a significant reduction in fMLP-evoked calcium influx in human neutrophils (approximately 35%), while neither quercetin 3′-sulfate nor quercetin produced a similar change. Acute exposure of human neutrophils to LPS altered cell shape and surface expression of CD16, but neither of these events were significantly altered by quercetin, quercetin 3-glucuronide nor quercetin 3′-sulfate. In addition, LPS caused a fivefold up-regulation in the expression of β2-integrin (CD11b/Mac 1) and a concomitant 70% down-regulation of L-selectin (CD62L) adhesion molecule expression in human neutrophils. Neither effect was altered by quercetin, quercetin 3-glucuronide or quercetin 3′-sulfate. In conclusion, we found that acute exposure to quercetin and quercetin 3′-sulfate does not affect either expression of cell adhesion molecules or the elevation of intracellular calcium ions in response to LPS and fMLP in human neutrophils. However, quercetin 3-glucuronide reduced fMLP-evoked calcium responses. While this study highlights that metabolites of quercetin may possess different biological properties, dietary ingestion of quercetin is unlikely to exert a major effect on neutrophil function in vivo.
Keywords: Abbreviations; [Ca; 2+; ]; i; intracellular calcium; CD11b/Mac 1,β; 2; -integrin/membrane-activated complex-1; CD62L; L-selectin; DMSO; dimethylsulfoxide; FITC; fluorescein isothiocyanate; fMLP; N; -formyl-methionyl-leucyl-phenylalanine; FS; forward scatter; LKS; low potassium salt; LPS; lipopolysaccharide; PBS; phosphate-buffered saline; PE; phycoerthythrin; Q; quercetin; Q3GlcA; quercetin 3-glucuronide; Q3′S; quercetin 3′-sulfateHuman neutrophils; Flavonoids; Quercetin metabolites; Inflammation; Intracellular calcium; Adhesion molecules
Enantioselective effect of 12( S)-hydroxyeicosatetraenoic acid on 3T6 fibroblast growth through ERK 1/2 and p38 MAPK pathways and cyclin D1 activation by Diana Nieves; Juan J. Moreno (pp. 654-661).
Hydroxyeicosatetraenoic acids (HETEs) have numerous physiological effects, including modulation of cell proliferation and differentiation. However, little is known about the selective effects of HETE enantiomers on cell proliferation and cell signalling pathways involved in the regulation of cell growth. Furthermore, information on epithelial and endothelial cells growth is controversial. Recently, we demonstrated that 5-, 12-, and 15-HETE are involved in the control of 3T6 fibroblast growth though serine/treonine Akt/PKB (Akt) pathway. Here we examined the participation of both enantiomers ( S and R) of HETEs in the control of 3T6 fibroblast growth. Our results show that HETEs (5-, 12-, and 15-HETE) are enantioselective on protein and DNA synthesis and 3T6 fibroblast growth. Furthermore, we observed that 12( S)-HETE induces the enhancement of cAMP and intracellular calcium concentration, whereas 12( R)-HETE was uneffective. Our findings also demonstrated that 12( S)-HETE exerts these effects through enantiospecific interactions with a cellular element, probably a plasma membrane receptor coupling to a pertussis toxin-sensitive protein G. Moreover, these elements may be involved in the activation of mitogen-activated protein kinase pathways which induce the enhancement of cyclin D1 levels.
Keywords: Cell proliferation; Cell cycle; Mitogen-activated protein kinase cascade; cAMP; Intracellular calcium
Celecoxib potently inhibits TNFα-induced nuclear translocation and activation of NF-κB by Megumi Funakoshi-Tago; Taeko Shimizu; Kenji Tago; Motohiro Nakamura; Hiroshi Itoh; Yoshiko Sonoda; Tadashi Kasahara (pp. 662-671).
Celecoxib is a specific inhibitor of cyclooxygenase 2 (COX2). While it has been used for the treatment of chronic inflammatory conditions, including rheumatoid arthritis, its detailed anti-inflammatory mechanism has not been clarified. Here, we found that Celecoxib potently inhibited TNFα-induced transcriptional activity and DNA binding activity of NF-κB; however, Celecoxib had no effect on TNFα-induced IKK activation and degradation of IκBα and IκBβ, suggesting that it inhibited NF-κB activation via suppressing downstream of IKK activation and IκBs degradation. Interestingly, it was also found that Celecoxib abrogated TNFα-induced nuclear accumulation of the NF-κB p65 subunit. As a result, TNFα-induced expression of inflammatory cytokines, CXCL1/KC and CCL2/MCP-1, was clearly inhibited by Celecoxib. On the other hand, Celecoxib had no effect on the TNFα-induced nuclear translocation of c-jun and activation of ERK, JNK, p38 and Akt. Taken together, these data indicate that Celecoxib specifically inhibits TNFα-induced NF-κB activation at the level of its nuclear translocation. This negative regulation of NF-κB activation by Celecoxib might be an important mechanism leading to its anti-inflammatory activity.
Keywords: Abbreviations; COX2; cyclooxygenase 2; DMSO; dimethyl sulfoxide; ELISA; enzyme-linked immunosorbent assay; MAP kinase; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinase; FBS; fetal bovine serum; IκBa; inhibitor of NF-κB; IKK; IκB kinase; IL-1; interleukin-1; Jak; Janus kinase; JNK; c-jun N-terminal kinase; MCP-1; monocyte chemotactic protein-1; NF-κB; nuclear factor κB; NSAIDs; non-steroidal anti-inflammatory drugs; PBS; phosphate buffered saline; PVDF; polyvinylidene difluoride; RA; rheumatoid arthritis; RIP; receptor-interacting protein; TNFα; tumor necrosis factor alpha; TRAF2; TNF receptor associated factor 2Celecoxib; NF-κB; TNFα; CXCL1/KC; CCL2/MCP-1
In vivo chronic exposure to heroin or naltrexone selectively inhibits liver microsome formation of estradiol-3-glucuronide in the rat by Letizia Antonilli; Valentina Brusadin; Michele S. Milella; Fabrizia Sobrero; Aldo Badiani; Paolo Nencini (pp. 672-679).
We have previously found that repeated exposure to heroin reduces liver synthesis of morphine-3-glucuronide (M3G) and increases the production of morphine-6-glucuronide (M6G), which normally is not formed in the rat. By contrast repeated exposure to naltrexone does not activate M6G synthesis but increases the Vmax of M3G formation. M3G synthesis depends on the activity of two isoforms of the UDP-glucuronosyltransferase (UGT), UGT1A1 and UGT2B1. These isozymes also activate the formation of estradiol-3-glucuronide (E3G) and estradiol-17-glucuronide (E17G), respectively. The goal of the present study was to investigate the role of UGT1A1 and UGT2B1 in the effects of heroin and naltrexone by determining their influence on the synthesis of E3G and E17G. Estradiol glucuronidation was performed using microsomes of rats treated daily, for 10 days, with saline, heroin (10mg/kg, i.p.), or naltrexone (40mg/kg, i.p.). Moreover, liver expression of both UGT1A1 and UGT2B1 was studied in the same experimental conditions by polymerase chain reaction analysis. Kinetic analysis showed that the Vmax for E3G formation was significantly reduced by both heroin (168.82±9.73nmol/mg/min) and naltrexone (194.60±16.6) relative to saline (624.60±17.6). Moreover, homotropic kinetic of E3G formation (Hill coefficient: 1.8) was transformed in Michaelis–Menten kinetic by both heroin (0.88) and naltrexone (1.15). The synthesis of E17G was not affected by either opioid. The expression of liver UGT1A1 and UGT2B1 did not differ across groups. The present results suggest that heroin and naltrexone can reduce estradiol glucuronidation via a specific interaction with UGT1A1 isoform.
Keywords: Heroin; Naltrexone; Estradiol-3-glucuronide; Estradiol-17-glucuronide; UGT1A1
Application and interpretation of hPXR screening data: Validation of reporter signal requirements for prediction of clinically relevant CYP3A4 inducers by Xiaoming Cui; Ann Thomas; Valerie Gerlach; Ronald E. White; Richard A. Morrison; K.-C. Cheng (pp. 680-689).
A human pregnane X receptor (PXR) reporter-gene assay was established and validated using 19 therapeutic agents known to be clinical CYP3A4 inducers, 5 clinical non-inducers, and 6 known inducers in human hepatocytes. The extent of CYP3A4 induction (measured as RIF ratio in comparison to rifampicin) and EC50 was obtained from the dose–response curve. All of the clinical inducers (19/19) and human hepatocyte inducers (6/6) showed positive responses in the PXR assay. One out of five clinical non-inducers, pioglitazone, also showed a positive response. An additional series of 18 commonly used drugs with no reports of clinical induction was also evaluated as putative negative controls. Sixteen of these were negative (89%), whereas two of these, flutamide and haloperidol showed 16-fold (RIF ratio 0.79) and 10-fold (RIF ratio 0.48) maximal induction, respectively in the reporter-gene system. Flutamide and haloperidol were further demonstrated to cause CYP3A4 induction in human cryopreserved hepatocytes based on testosterone 6β-hydroxylation activity. The induction potential index calculated based on the maximum RIF ratio, EC50, and in vivo maximum plasma concentration was used to predict the likelihood of CYP3A4 induction in humans. When the induction potential index is greater than 0.08, the compound is likely to cause induction in humans. A high-throughput screening strategy was developed based on the validation results at 1μM and 10μM for the same set of drugs. A RIF ratio of 0.4 was set as more practical screening cut-off to minimize the possibility of generating false positives. Thus, a tiered approach was implemented to use the human PXR reporter-gene assay from early lead optimization to late lead characterization in drug discovery.
Keywords: Abbreviations; CAR; constitutive androstane receptor; CYP; cytochrome P450; DEX; dexamethasone; GR; glucocorticoid receptor; NCEs; new chemical entities; PB; phenobarbital; PCN; pregnenolone 16α-carbonitrile; PPAR; peroxisome proliferator activated receptor; PXR; pregnane X receptor; RIF; rifampicin; RXR; retinoic acid X receptor; VDR; vitamin D receptorPXR; CYP3A4; P450 induction; Reporter-gene system; Drug interaction; Human
A point mutation produced a class 3 aldehyde dehydrogenase with increased protective ability against the killing effect of cyclophosphamide by Kwok Ki Ho; Abhijit Mukhopadhyay; Yi Feng Li; Soma Mukhopadhyay; Henry Weiner (pp. 690-696).
Cyclophosphamides are pro-drugs whose killing agent is produced from an aldehyde that is formed by the action of a P450 oxidation step. The mustard from the aldehyde can destroy bone marrow cells as well as the tumor. Aldehyde dehydrogenase (EC 1.2.1.3) can oxidize the aldehyde and hence inactivate the cytotoxic intermediate but bone marrow has little, if any, of the enzyme. Others have shown that over-expression of the enzyme can afford protection of the marrow. A T186S mutant of the human stomach enzyme (ALDH3) that we developed has increased activity against the aldehyde compared to the native enzyme and HeLa cells transformed with the point mutant are better protected against the killing effect of the drug. It took threefold more drug to kill 90% of the cells transformed with the mutant compared to the native enzyme (15.8 compared to 5.1mM of a precursor of the toxic aldehyde). Analysis of molecular models makes it appear that removing the methyl group of threonine in the T186S mutant allows the bulky aldehyde to bind better. The mutant was found to be a poorer enzyme when small substrates such as benzaldehyde derivatives were investigated. Thus, the enzyme appears to be better only with large substrates such as the one produced by cyclophosphamide.
Keywords: Aldehyde dehydrogenase; Cyclophosphamide; HeLa cell protection; Benzaldehyde; Aldophosphamide