Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.72, #2)

Editorial Advisory Board (pp. iii).

Insulin sensitizers may prevent metabolic inflammation by Jerry R. Colca (pp. 125-131).

The relative decreased response of peripheral tissues to insulin (insulin resistance) is a key metabolic disturbance that predisposes a large percentage of individuals to the development of type 2 diabetes and to cardiovascular disease. As detailed in an extensive literature over the last two decades, insulin resistance co-exists in varying degrees with a variety of other key risk factors, including dyslipidemia, hypertension, and vascular inflammation, that contribute to poor cardiovascular outcomes of individuals with type 2 diabetes and metabolic syndrome. Whereas insulin resistance is generally thought of as pathology unto itself, this commentary suggests that insulin resistance is a physiological compensation to inappropriate oxidative metabolism that induces a metabolic inflammatory response. Via signaling of this inflammatory response, the protective compensation to excessive oxidative metabolism dampens metabolism by reducing insulin action, fatty acid oxidation, and eventually mitochondrial function and numbers. Such a scenario could explain the coexistence of these phenomena with obesity and reduced mitochondrial function. Recent evidence suggests that thiazolidinediones exert pharmacology through modifications of mitochondrial metabolism, preventing the metabolic inflammation and allowing the up regulation of mitochondrial biogenesis. A further understanding of these mechanisms, which are likely to involve key redox signaling events emanating from mitochondrial biochemistry, is needed to fuel new therapeutic advances for the treatment of metabolic syndrome.

Keywords: Metabolic syndrome; Insulin resistance; Cardiovascular disease prevention; mitoNEET; Metabolic inflammation; Thiazolidinediones

Critical role of pro-apoptotic Bcl-2 family members in andrographolide-induced apoptosis in human cancer cells by Jing Zhou; Siyuan Zhang; Choon-Nam Ong; Han-Ming Shen (pp. 132-144).

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess potent anti-inflammatory activity. In this study, Andro induced apoptosis in human cancer cells via activation of caspase 8 in the extrinsic death receptor pathway and subsequently with the participation of mitochondria. Andro triggered a caspase 8-dependent Bid cleavage, followed by a series of sequential events including Bax conformational change and mitochondrial translocation, cytochrome c release from mitochondria, and activation of caspase 9 and 3. Inhibition of caspase 8 blocked Bid cleavage and Bax conformational change. Consistently, knockdown of Bid protein using small interfering RNA (siRNA) technique suppressed Andro-induced Bax conformational change and apoptosis. In conclusion, the pro-apoptotic Bcl-2 family members (Bid and Bax) are the key mediators in relaying the cell death signaling initiated by Andro from caspase 8 to mitochondria and then to downstream effector caspases, and eventually leading to apoptotic cell death.

Keywords: Abbreviations; Andro; andrographolide; CHX; cycloheximide; DAPI; 4′,6-diamidino-2-phenylindole; DMSO; dimethyl sulfoxide; PARP; poly(ADP-ribose)polymerase; PI; propidium iodide; TNFα; tumor necrosis factor α; z-DEVD-CHO; N; -acetyl-Asp-Glu-Val-Asp-CHO (aldehyde); z-IETD-FMK; benzyloxycarbonyl-Ile-Glu-Thr-Asp-(OMe) fluoromethyl ketone; z-LEHD-CHO; N; -acetyl-Leu-Glu-His-Asp-CHO (aldehyde); z-VAD-FMK; benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketoneAndrographolide; Bid; Bax; Bcl-2 family; Mitochondria; Apoptosis

Biochemical and pharmacological properties of an allosteric modulator site of the human P-glycoprotein (ABCB1) by Nazli Maki; Saibal Dey (pp. 145-155).

The drug-transport function of the human P-glycoprotein (Pgp or ABCB1) is inhibited by a number of structurally unrelated compounds, known as modulators or reversing agents. Among them, the thioxanthene derivative flupentixol inhibits Pgp-mediated drug transport by an allosteric mechanism. Unlike most other Pgp modulators, the cis isomer of flupentixol [ cis-( Z)-flupentixol] facilitates interaction of Pgp with its transport-substrate [¹²⁵I]iodoarylazidoprazosin (or [¹²⁵I]IAAP), yet inhibits transport. In this study, we show that the flupentixol site acts as a common site of interaction for the tricyclic ring-containing modulators thioxanthenes and phenothiazines. The allosteric stimulation of [¹²⁵I]IAAP binding to Pgp occurs independent of the phosphorylation status of the transporter. Stimulation is retained in purified Pgp reconstituted into proteoliposomes, suggesting no involvement of any other cellular protein in the phenomenon. However, perturbation of the lipid environment of the reconstituted Pgp by nonionic detergent octylglucoside abolishes stimulation by cis-( Z)-flupentixol of [¹²⁵I]IAAP binding. Extensive trypsin digestion of the [¹²⁵I]IAAP-labeled Pgp generates a 5.5kDa fragment with 80% of the stimulated level of labeling associated with it. Sensitivity to inhibition by transport-substrate vinblastine and competitive modulator cyclosporin A suggests that the elevated level of [¹²⁵I]IAAP binding to the fragment represents a functionally relevant interaction with the substrate site of Pgp. In summary, we demonstrate that allosteric modulation by cis-( Z)-flupentixol is mediated through its interaction with Pgp at a site specific for tricyclic ring-containing Pgp modulators of thioxanthene and phenothiazine backbone, independent of other cellular components and the phosphorylation status of the protein.

Keywords: Flupentixol; Thioxanthene; Phenothiazine; [; ¹²⁵; I]iodoarylazidoprazosin (or [; ¹²⁵; I]IAAP); Multidrug resistance

Dual-acting diamine antiplasmodial and chloroquine resistance modulating agents by Susan Yeh; Peter J. Smith; Kelly Chibale (pp. 156-165).

On the basis of structural features known to be critical for the antimalarial activity, accumulation and uptake of chloroquine (CQ), as well as chemosensitization of CQ resistant Plasmodium falciparum, an exploratory novel series of potential dual acting antiplasmodial and chemosensitizing agents was designed and synthesized for biological evaluation. All four compounds contain a common alkyl side chain with two amino groups and differ only in the chemical nature of the hydrophobic aromatic moieties. Among them, N′-[4-(biphenyl-2-ylmethoxy)-benzyl]- N, N-dimethyl-propane-1,3-diamine (P7) displayed the greatest potential as a dual-acting antiplasmodial agent against CQ-resistant strains(IC50K1/RSA11<0.6μM) and chemosensitizer (RMI_K1=0.67; RMI_RSA11=0.82) while displaying low in vitro cytotoxicity against a mammalian cell line (CHO). At 1μM, P7 caused a 8.5 and 4-fold potentiation in CQ accumulation in resistant P. falciparum K1 and RSA11 strains, respectively. In a parallel experiment, 1μM verapamil showed a 6.5 (K1) and 2 (RSA11)-fold increase in CQ accumulation. The preliminary studies point to structural features that may determine antiplasmodial and/or CQ resistance modulating activity in this new series of compounds. An additive effect was observed against both CQ^S (D10) and CQ^R (RSA11) strains when CQ and P7 were used at their corresponding IC₅₀ concentrations in isobologram analysis.

Keywords: Malaria; Antimalarial agents; Chloroquine; Drug resistance; Privileged structures; Chemosensitizers

XPA versus ERCC1 as chemosensitising agents to cisplatin and mitomycin C in prostate cancer cells: Role of ERCC1 in homologous recombination repair by Michele Cummings; Karen Higginbottom; Claire J. McGurk; Oscar Gee-Wang Wong; Beate Köberle; R. Timothy D. Oliver; John R. Masters (pp. 166-175).

Nucleotide excision repair is the principal mechanism for the removal of bulky DNA adducts caused by a range of chemotherapeutic drugs, and contributes to cisplatin resistance. In this study, we used synthetic siRNAs targeted to XPA and ERCC1 and compared their effectiveness in sensitising mismatch repair deficient prostate cancer cell lines to cisplatin and mitomycin C. Downregulation of ERCC1 sensitised DU145 and PC3 cells to cisplatin and mitomycin C. In contrast, XPA downregulation did not sensitise either cell line to mitomycin C, and only sensitised DU145 cells to cisplatin. The effects of ERCC1 downregulation may be due to its role in homologous recombination repair. Excision repair of cisplatin adducts in PC3 cells was attenuated to a similar extent by XPA and ERCC1 downregulation. Downregulation of XPA but not ERCC1 caused an increase in the number of cisplatin-induced RAD51 foci in PC3 cells, suggesting that HRR is able to substitute for NER in these cells. We observed co-localisation of ERCC1 and RAD51 in cisplatin treated PC3 cells by immunofluorescence and co-immunoprecipitation, which may represent recruitment of ERCC1/XPF to sites of recombination repair. These results indicate that ERCC1 is a broader therapeutic target than XPA with which to sensitise cancer cells to chemotherapy because of its additional role in recombination repair.

Keywords: Abbreviations; SiRNA; small interfering RNA; MMC; mitomycin C; ICL; interstrand crosslink; HRR; homologous recombinational repairSiRNA; XPA; ERCC1; Cisplatin; Mitomycin C; DNA repair

Metformin reduces angiotensin-mediated intracellular production of reactive oxygen species in endothelial cells through the inhibition of protein kinase C by M. Mahrouf; N. Ouslimani; J. Peynet; R. Djelidi; M. Couturier; P. Therond; A. Legrand; J.-L. Beaudeux (pp. 176-183).

Oxidative stress plays a major role in the pathogenesis and in the onset of macrovascular complications of diabetes. We previously reported that the antihyperglycaemic drug metformin was able to decrease significantly intracellular reactive oxygen species (ROS) production of bovine aortic endothelial cells (BAEC) activated by high levels of glucose and angiotensin II (ANG). The aim of the present study was to investigate whether the antioxidant effect of metformin on BAEC could be mediated through a modulation of protein kinase C (PKC) activity, which plays a key role in the pathophysiology of diabetes. The effects of metformin on intracellular ROS production, PKC translocation and activity were studied on endothelial cells stimulated by PMA (a direct PKC activator), ANG or high levels of glucose as pathophysiological stimuli of endothelial dysfunction in diabetes. We showed that metformin decreased ROS production on PMA-, ANG- and glucose-stimulated BAEC in a similar manner to that obtained by PKC specific inhibitors (calphostin C, chelerythrine) alone. On the other hand, metformin reduced both PKC membrane translocation and kinase activity in ANG-stimulated cells. In PMA-activated cells, metformin reduced membrane PKC activity but we did not observe any alteration of PKC membrane translocation. Finally, in vitro incubation with purified PKC indicated that metformin had no direct effect on PKC activity. Taken together, our results suggest that metformin exerted intracellular antioxidant properties by decreasing ROS production through the inhibition of PKC activity.

Keywords: Diabetes; ROS; Metformin; Endothelial cells; Angiotensin; PKC

Attenuation of osteoclastogenesis and osteoclast function by apigenin by Sanghamitra Bandyopadhyay; Jean-Marc Lion; Romuald Mentaverri; Dennis A. Ricupero; Said Kamel; Jose R. Romero; Naibedya Chattopadhyay (pp. 184-197).

The physiological effects of the flavone, apigenin on bone cells were studied. We first show that apigenin inhibits tumor necrosis factor alpha (TNFα)- and interferon gamma (IFNγ)-induced secretion of several osteoclastogenic cytokines from MC3T3-E1 mouse calvarial osteoblast cell line. Ligands of the TNF receptor family constitute the most potent osteoclastic cytokines. In MC3T3-E1 cells, apigenin dose-dependently (from 5 to 20μM) inhibits TNFα-induced production of the osteoclastogenic cytokines, IL-6 (interleukin-6), RANTES (regulated upon activation, normal T cell-expressed and -secreted), monocyte chemoattractant protein-1 (MCP-1) and MCP-3. In addition, apigenin inhibits IFNγ-stimulated secretion of monokines, CXCL-9, and -10 in MC3T3-E1 cells. Next, we show that apigenin strongly inhibits differentiation of 3T3-L1 preadipocytes to adipocytes with attendant inhibition of adipocyte differentiation-induced IL-6, MCP-1, and leptin production. Inhibition of adipogenic differentiation by apigenin could be due to induction of osteogensis as it robustly upregulates mRNA levels of bone morphogenetic protein-6 (BMP-6). Finally, the presence of apigenin inhibited osteoclast differentiation from the RAW 264.7 cell line by reducing receptor activator of nuclear factor kappa ligand (RANKL)-induced expression of tartrate-resistant acid phosphatase (TRAP), RANK, and calcitonin receptor but not CCR1, resulting in the inhibition of multinucleated osteoclast formation. Similarly, apigenin inhibited expression of the osteoclast differentiation markers TRAP, RANK, and c-Fms in osteoclast precursor cells obtained from mouse bone marrow following treatment with RANKL and macrophage colony stimulating factor (MCSF). Furthermore, apigenin induced apoptosis of mature osteoclasts obtained from rabbit long bone and inhibited bone resorption. In all instances, a structurally related compound, flavone had no significant effect. These data suggest that apigenin has multiple effects on all three bone cells that could prevent bone loss in vivo.

Keywords: Flavonoids; Bone resorption; Osteoporosis; Rheumatic arthritis; Tumor necrosis factor; Interferon gamma

Glucose utilization is suppressed in the gut of insulin-resistant high fat-fed rats and is restored by metformin by Gilles Mithieux; Fabienne Rajas; Carine Zitoun (pp. 198-203).

It has been recently suggested that the small intestine (SI) has the capacity to contribute to endogenous glucose production (EGP), in addition to the liver and kidney. The aim of this work was: (1) to estimate the role of SI glucose fluxes in glucose homeostasis in insulin resistance states (induced by high-fat (HF) feeding); (2) to assess the effect of metformin, an anti-diabetic molecule, on these fluxes. Rats were fed for 6 weeks on a HF-diet, supplemented or not with metformin (HF-Met) at a daily dosage of 50mg/kg during the last week. We combined arterio-venous glucose balance measurements and isotopic dilution techniques to separate basal intestinal glucose uptake (IGU) and release (IGR). Contrary to what was observed in control starch-fed rats, IGU was negligible in HF-fed rats: 0.6±2.4μmol/kg/min (mean±S.E.M., n=9). It was restored to a level close to that of control rats in the HF-Met group: 13.0±6.7μmol/kg/min (mean±S.E.M., n=9, p<0.05 compared to the non-treated group). Similarly, IGR was close to zero in HF-fed rats (−3.8±2.6μmol/kg/min), but was significant in HF-Met rats (7.4±4.4μmol/kg/min, p<0.05 compared to non-treated rats). These data strongly suggest that the impairment of glucose uptake in the SI might be a crucial feature of insulin resistance states and that a key beneficial effect of metformin in these situations might be to restore a normal glucose metabolism in this tissue.

Keywords: Metformin; Glucose metabolism; Small intestine; Glucose tracers; Endogenous Glucose production; Glucose-6 phosphatase

Salicylate modulates oxidative stress in the rat colon: A proteomic approach by Janice E. Drew; Sara Padidar; Graham Horgan; Garry G. Duthie; Wendy R. Russell; Martin Reid; Gary Duncan; Garry J. Rucklidge (pp. 204-216).

The dietary phenolic compound, salicylic acid, decreases oxidative stress and pro-inflammatory and potentially neo-plastic prostaglandins with a concomitant increase in glutathione peroxidase activity. Salicylic acid, a dietary plant-based phenolic compound and also the main metabolite of aspirin, may contribute to the colon protective effects of plant-based diets. Oxidative stress is a characteristic of pre-cancerous and cancerous colon and inflammatory bowel diseases (IBD) that increase colon cancer risk. The mechanism(s) whereby salicylic acid modulates potentially pro-cancerous activity associated with oxidative stress is further investigated here using a proteomic approach. A rat model of oxidative stress was supplemented with salicylic acid (1mg/kg diet, mean plasma levels 310±32μmol/l). Soluble colon protein extracts were subjected to 2D PAGE. Salicylic acid modulated proteins, identified using MALDI-TOF and LC/MS/MS, are involved in protein folding, transport, redox, energy metabolism and cytoskeletal regulation. A partial least squares (PLS) analysis approach was used to assist biological interpretation of the altered protein profiles via their associations between previously published biochemical measurements of oxidative stress, prostaglandin levels and increased glutathione peroxidase activity. Early detection of altered homeostasis in colon may assist in identifying pre-pathological features preceding colon tumorigenesis and contribute to an understanding of epidemiological evidence supporting a protective effect of plant-based diets.

Keywords: Abbreviations; CRC; colorectal cancer; cyGpx; cytosolic glutathione peroxidase; IBD; inflammatory bowel disease; NSAIDs non-steroidal anti-inflammatory drugs; PCA; principal component analysis; PDI; protein disulphide isomerase; PLS; partial least squares; ROS; reactive oxygen species; SDS; sodium dodecyl sulphate; TBARS; thibarbituric acid reactive substancesMitochondria; Redox status; Phenolic compound; Partial least squares

Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity by Anne-Marie J.F. Boerboom; Martijn Vermeulen; Hester van der Woude; Birgit I. Bremer; Yee Y. Lee-Hilz; Ellen Kampman; Peter J. van Bladeren; Ivonne M.C.M. Rietjens; Jac M.M.J.G. Aarts (pp. 217-226).

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from the human NAD(P)H:quinone oxidoreductase (hNQO1) and the mouse glutathione-S-transferase Ya (mGST-Ya) gene, containing one and two tandem EpRE core sequences, respectively. The standard inducer tert-butylhydroquinone (tBHQ), the electrophile benzyl isothiocyanate (BITC), and the antioxidant flavonoid quercetin were found to induce luciferase expression, thereby validating these newly developed reporter cell lines. For tBHQ and BITC, but not for quercetin, higher maximum luciferase induction was found under control of the mGST-Ya EpRE as compared to the hNQO1 EpRE, pointing at different induction mechanisms. Furthermore, we investigated the structure-activity relationship for induction of luciferase expression by flavonoids in EpRE(mGST-Ya)-LUX cells, and also the relation between luciferase induction and flavonoid antioxidant potency. Five different flavonoids with a planar molecular structure were found to induce various levels of luciferase activity, whereas taxifolin, a non-planar flavonoid, did not induce luciferase activity. This suggests that a stereospecific molecular interaction may be important for EpRE-mediated gene activation, possibly with Keap1, a regulator of EpRE-controlled transcription, or with another effector or receptor protein. No consistent relation between luciferase induction level and flavonoid antioxidant potential was observed. Altogether, these results point to differences in induction mechanism between the various chemoprotective compounds tested. The newly developed stably transfected reporter cell lines provide a validated tool for future screening and mechanistic studies of EpRE-mediated gene transcription.

Keywords: Abbreviations; BITC; benzyl isothiocyanate; DMSO; dimethyl sulfoxide; EpRE; electrophile-responsive element; FCS; foetal calf serum; GSH; glutathione; hNQO1; human NAD(P)H:quinone oxidoreductase 1; Keap1; Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associating protein 1; mGST-Ya; mouse glutathione S-transferase Ya; Nrf2; nuclear factor erythroid 2-related factor 2; PKC; protein kinase C; ROS; reactive oxygen species; tBHQ; tert; -butylhydroquinoneElectrophile-responsive element (EpRE); Stable luciferase reporter cell lines; Flavonoids; Benzyl isothiocyanate; Transient transfection; Hepa-1c1c7 cells

The phyto-chemical (−)-epigallocatechin gallate suppresses gene expression of epidermal growth factor receptor in rat hepatic stellate cells in vitro by reducing the activity of Egr-1 by Yumei Fu; Anping Chen (pp. 227-238).

Hepatic stellate cells (HSC) are the major effectors in hepatic fibrogenesis. During liver injury, HSC become activated and proliferative. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are the potent mitogens for many cell types. We previously demonstrated that (−)-epigallocatechin gallate (EGCG), the major and active component in green tea extracts, inhibited HSC growth, including reducing cell proliferation, and inducing apoptosis. We have reported that EGCG interrupts PDGF signaling by reducing receptor tyrosine phosphorylation and gene expression of PDGF-β receptor. Additional experiments are necessary to elucidate the effect of EGCG on EGF signaling in activated HSC. The aims of this study are to evaluate the effect of EGCG on the expression of EGFR and to elucidate the underlying molecular mechanisms in activated HSC. We hypothesize that EGCG might interrupt EGF signaling by suppressing gene expression of EGF receptor (EGFR) in activated HSC, which, together with the interruption of PDGF signaling, might collectively result in the inhibition of HSC growth. The present report demonstrates that the phyto-chemical dose-dependently suppresses gene expression of EGFR in activated HSC in vitro. The Egr-1 binding site located in the egfr promoter is found to be cis-activating element in regulating the promoter activity of the gene. EGCG inhibits the trans-activation activity of Egr-1 in activated HSC by suppressing gene expression of the transcription factor. The interruption of the ERK signaling pathway by EGCG reduces the trans-activation activity of Egr-1 and the promoter activity of EGFR gene in HSC. Taken together, our results demonstrate that EGCG suppresses gene expression of EGFR in rat activated HSC in vitro mediated by reducing the trans-activation activity of Egr-1.

Keywords: Abbreviations; DMEM; Dulbecco's modified Eagle's medium; EBS; Egr-1 binding sites; ECM; extracellular matrix; EGCG; (−)-epigallocatechin gallate; EGFR; epidermal growth factor receptor; EMSA; electrophoretic mobility shift assays; ERK; extracellular signal-regulated kinase; FBS; fetal bovine serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HSC; hepatic stellate cells; LDH; lactate dehydrogenase; MAPK; the mitogen-activated protein kinases; PDGF; platelet-derived growth factorCollagen; Fibrogenesis; Green tea; Gene expression; Signal transduction; Receptors

3′-Azido-3′-deoxythymidine (AZT) is a competitive inhibitor of thymidine phosphorylation in isolated rat heart and liver mitochondria by Matthew D. Lynx; Edward E. McKee (pp. 239-243).

Long-term use of 3′-azido-3′-deoxythymidine (AZT) is associated with various tissue toxicities, including hepatotoxicity and cardiomyopathy, and with mitochondrial DNA depletion. AZT-5′-triphosphate (AZTTP) is a known inhibitor of the mitochondrial DNA polymerase γ and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart and liver mitochondria, AZT itself was shown to be a more potent inhibitor of thymidine phosphorylation (IC₅₀ of 7.0±1.0μM AZT in heart mitochondria and of 14.4±2.6μM AZT in liver mitochondria) than AZTTP is of polymerase γ (IC₅₀ of >100μM AZTTP), suggesting that depletion of mitochondrial stores of TTP may limit replication and could be the cause of the mitochondrial DNA depletion observed in tissues affected by AZT toxicity. The purpose of this work is to characterize the nature of AZT inhibition of thymidine phosphorylation in isolated rat heart and rat liver mitochondria. In both of these tissues, AZT was found to be a competitive inhibitor of the phosphorylation of thymidine to TMP, catalyzed by thymidine kinase 2. The inhibition constant ( K_i) for heart mitochondria is 10.6±4.5μM AZT, and for liver mitochondria K_i is 14.0±2.5μM AZT. Since AZT is functioning as a competitive inhibitor, increasing thymidine concentrations may be one mechanism to overcome the inhibition and decrease AZT-related toxicity in these tissues.

Keywords: Abbreviations; AIC; Akaike's information criterion; AIDS; acquired immunodeficiency syndrome; AZT; 3′-azido-3′-deoxythymidine; AZTMP; 3′-azido-3′-deoxythymidine-5′-monophosphate; AZTTP; 3′-azido-3′-deoxythymidine-5′-triphosphate; IC; ₅₀; 50% inhibitory concentration; HAART; highly active anti-retroviral therapy; HIV; human immunodeficiency virus; NRTI; nucleoside analog reverse transcriptase inhibitorAZT; Competitive inhibition; Mitochondrial toxicity; Nucleoside reverse transcriptase inhibitors (NRTIs); Thymidine; Thymidine kinase 2

Single amino acid residue determinants of non-peptide antagonist binding to the corticotropin-releasing factor₁ (CRF₁) receptor by Sam R.J. Hoare; Brock T. Brown; Mark A. Santos; Siobhan Malany; Stephen F. Betz; Dimitri E. Grigoriadis (pp. 244-255).

The molecular interactions between non-peptide antagonists and the corticotropin-releasing factor type 1 (CRF₁) receptor are poorly understood. A CRF₁ receptor mutation has been identified that reduces binding affinity of the non-peptide antagonist NBI 27914 (M276I in transmembrane domain 5). We have investigated the mechanism of the mutation's effect using a combination of peptide and non-peptide ligands and receptor mutations. The M276I mutation reduced binding affinity of standard non-peptide antagonists 5–75-fold while having no effect on peptide ligand binding. We hypothesized that the side chain of isoleucine, β-branched and so rotationally constrained when within an α-helix, introduces a barrier to non-peptide antagonist binding. In agreement with this hypothesis, mutation of M276 to the rotationally constrained valine produced similar reductions of affinity as M276I mutation, whereas mutation to leucine (with an unbranched β-carbon) minimally affected non-peptide antagonist affinity. Mutation to alanine did not appreciably affect non-peptide antagonist affinity, implying the methionine side chain does not contribute directly to binding. Three observations suggested M276I/V mutations interfere with binding of the heterocyclic core of the compounds: (1) all compounds affected by M276I/V mutations possess a planar heterocyclic core. (2) None of the M276 mutations affected binding of an acylic compound. (3) The mutations differentially affected affinity of two compounds that differ only by core methylation. These findings imply that non-peptide antagonists, and specifically the heterocyclic core of such molecules, bind in the vicinity of M276 of the CRF₁ receptor. M276 mutations did not affect peptide ligand binding and this residue is distant from determinants of peptide binding (predominantly in the extracellular regions), providing molecular evidence for non-overlapping (allosteric) binding sites for peptide and non-peptide ligands within the CRF₁ receptor.

Keywords: Corticotropin releasing factor; Non-peptide antagonist; Mutagenesis; Allosteric; Depression; Ligand binding

Leucovorin-induced resistance against FDH growth suppressor effects occurs through DHFR up-regulation by Natalia V. Oleinik; Natalia I. Krupenko; Steven N. Reuland; Sergey A. Krupenko (pp. 256-266).

10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate to tetrahydrofolate (THF). Expression of the enzyme in FDH-deficient cancer cells induces cytotoxicity that can be reversed by supplementation with high concentrations of a reduced folate, 5-formyl-THF (leucovorin). In contrast, non-tumor cells are resistant to FDH. The present study was undertaken to investigate mechanisms that could protect cells against FDH suppressor effects. Using 10μM leucovorin supplementation of FDH-sensitive A549 cells transfected for FDH expression, we selected clones that have acquired resistance against FDH. Resistant cells expressed high levels of FDH and were capable of growing after withdrawal of leucovorin. These cells, however, have increased doubling time due to prolonged S phase. They also have significantly increased levels of total folate pool and THF/5,10-methylene-THF pool while the level of 10-formyl-THF was two-fold lower than in parental FDH-sensitive cells. We have shown that the FDH-catalyzed reaction proceeds at about a three-fold slower rate at the ratio of 10-formyl-THF/THF corresponding to the resistant cells than at the ratio corresponding to parental sensitive cells, due to product inhibition ( K_I is 2.35μM). FDH-resistant cells have strongly up-regulated dihydrofolate reductase (DHFR) that is proposed to be a mechanism for the alteration of folate pools and a key component of the acquired resistance. Elevation of DHFR in A549 cells by transient transfection decreased sensitivity to FDH toxicity and allowed selection of FDH-resistant clones. DHFR-induced repression of FDH catalysis could be an S phase-related metabolic adjustment that provides protection against FDH suppressor effects.

Keywords: Abbreviations; AICARFT; 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase; DDF; 5,8-dideazafolate; DHFR; dihydrofolate reductase; FDH; 10-formyltetrahydrofolate dehydrogenase; GARFT; glycinamide ribonucleotide formyltransferase; MTX; methotrexate; PI; propidium iodide; THF; tetrahydrofolateFDH; Folate; DHFR; Leucovorin; Enzyme product inhibition; Growth suppressor; A549 cells; Resistance mechanism

Sections

Personal tools

Biochemical Pharmacology (v.72, #2)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.72, #2)