An improved cell-permeable fluorogenic substrate as the basis for a highly sensitive test for NAD(P)H quinone oxidoreductase 1 (NQO1) in living cells.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme upregulated in response to oxidative stress and in some cancers. Its upregulation by compounds has been used as an indicator of their potential anti-cancer properties. In this study we have designed, produced and tested a fluorogenic coumarin conjugate which selectively releases highly fluorescent 4-methylumbelliferone (4-MU) in the presence of NQO1.

Fatty acid ethyl ester synthase inhibition ameliorates ethanol-induced Ca2+-dependent mitochondrial dysfunction and acute pancreatitis.

Objective: Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable.

Design: Intracellular calcium ([Ca(2+)](C)), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism.

Chemical synthesis and biological evaluation of a NAD(P)H:quinone oxidoreductase-1 targeted tripartite quinone drug delivery system.

NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone "trigger" by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N-{4-[bis-(2-chloroethyl)-amino]-phenyl}-beta,beta,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1).

The 1,4-naphthoquinone scaffold in the design of cysteine protease inhibitors.

A series of 1,4-naphthoquinone derivatives diversely substituted at C-2, C-3, C-5 and C-8, prepared by reaction of amines, amino acids and alcohols with commercial 1,4-naphthoquinones, has been evaluated against papain and bovine spleen cathepsin B. These 1,4-naphthoquinone derivatives were found to be irreversible inhibitors for both cysteine proteases, with second-order rate constants, k(2), ranging from 0.67 to 35.

Design, synthesis and enzymatic evaluation of 6-bridged imidazolyluracil derivatives as inhibitors of human thymidine phosphorylase.

A series of novel imidazolyluracil conjugates were rationally designed and synthesised to probe the active site constraints of the angiogenic enzyme, thymidine phosphorylase (TP, E.C. 2.

Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells.

Oxidative stress may be an important determinant of the severity of acute pancreatitis. One-electron reduction of oxidants generates reactive oxygen species (ROS) via redox cycling, whereas two-electron detoxification, e.g.

Thymidine phosphorylase from Escherichia coli: tight-binding inhibitors as enzyme active-site titrants.

Thymidine phosphorylase (EC 2.4.2.

Quinone bioreductive prodrugs as delivery agents.

Quinone bioreductive prodrugs were developed to target the hypoxic or the reductase- rich population of solid tumours. The mechanism of their selective activation is based on their ability to convert the quinone sub-structure to their activated semiquinone or hydroquinone species affording the active species. Recent studies on their biochemical activation process have resulted in their development as delivery agents that can effectively release a potent (but not necessarily a cytotoxic) agent under hypoxic/reductive conditions.

Effects of dietary salt loading on the responses of isolated rat mesenteric arteries to leptin.

Background: Leptin induces relaxation of vascular smooth muscle through an endothelium-dependent release of nitric oxide (EDNO) and administration of a high-salt diet reduces the relaxation of vessels to EDNO. We would, therefore, predict that salt loading would reduce the leptin-induced dilatation. However, in salt-loaded animals the relaxation to acetylcholine is maintained through an endothelial-dependent hypopolarizing factor instead of EDNO.

Aminoimidazolylmethyluracil analogues as potent inhibitors of thymidine phosphorylase and their bioreductive nitroimidazolyl prodrugs.

Thymidine phosphorylase (TP) is an important target enzyme for cancer chemotherapy because it is expressed at high levels in the hypoxic regions of many tumors and inhibitors of TP have been shown in animal model studies to inhibit angiogenesis and metastasis, and to promote tumor cell apoptosis. The 5-halo-6-[(2'-aminoimidazol-1'-yl)methyl]uracils (3, X = Cl, Br) are very potent inhibitors of E. coli and human TP with IC(50) values of approximately 20 nM when the enzyme concentration is approximately 40 nM.

A novel design strategy for stable metal complexes of nitrogen mustards as bioreductive prodrugs.

Tumor hypoxia provides a key difference between healthy and cancerous cells. It can be exploited to produce drug selectivity, offering a reductase-rich environment for prodrug activation. Nitrogen mustard drugs are cytotoxic, but usually unselective.

Pharmacological and biological evaluation of a series of substituted 1,4-naphthoquinone bioreductive drugs.

The indolequinone compound EO9 has good pharmacodynamic properties in terms of bioreductive activation and selectivity for either NAD(P)H:quinone oxidoreductase-1 (NQO1)-rich aerobic or NQO1-deficient hypoxic cells. However, its pharmacokinetic properties are poor and this fact is believed to be a major reason for EO9's lack of clinical efficacy. The purpose of this study was to develop quinone-based bioreductive drugs that retained EO9's good properties, in terms of bioreductive activation, but have improved pharmacokinetic properties.

Hypoxia targeted gene therapy to increase the efficacy of tirapazamine as an adjuvant to radiotherapy: reversing tumor radioresistance and effecting cure.

Solid tumors are characterized by regions of hypoxia that are inherently resistant to both radiotherapy and some chemotherapy. To target this resistant population, bioreductive drugs that are preferentially toxic to tumor cells in a hypoxic environment are being evaluated in clinical trials; the lead compound, tirapazamine (TPZ), is being used in combination with cisplatin and/or with radiotherapy. Crucially, tumor response to TPZ is also dependent on the cellular complement of reductases.

Viral delivery of P450 reductase recapitulates the ability of constitutive overexpression of reductase enzymes to potentiate the activity of mitomycin C in human breast cancer xenografts.

Indolequinones such as mitomycin C (MMC) require enzymatic bioreduction to yield cytotoxic moieties. An attractive approach to overcome the potential variability in reductive bioactivation between tumors is to exploit specific enzyme-bioreductive drug combinations in an enzyme-directed gene therapy (GDEPT) approach. To this end, human breast cancer cell lines (T47D, MDA468, and MDA231) that overexpress either DT-diaphorase (DTD) or NADPH:cytochrome P450 reductase (P450R) have been developed.

Time-dependence and preliminary SAR studies in inhibition of nitric oxide synthase isoforms by homologues of thiocitrulline.

Treatment of N(alpha)-Cbz-N(epsilon)-(2-hydroxyethylaminothiocarbonyl)-L-lysine N-(2-hydroxyethyl)amide with boiling hydrochloric acid gave N(epsilon)-(4,5-dihydrothiazol-2-yl)-L-lysine. This was a weak and non-isoform selective inhibitor of NOS, whereas N(epsilon)-aminothiocarbonyl-L-lysine and its methyl ester were potent, with IC(50)=13 and 18 microM, respectively, against human iNOS and IC(50)=3 and 8 microM, respectively, against rat nNOS. Time dependence was observed for inhibition of nNOS by the ester.

3-substituted-5-aziridinyl-1-methylindole-4,7-diones as NQO1-directed antitumour agents: mechanism of activation and cytotoxicity in vitro.

Indolequinone agents are a unique class of bioreductive cytotoxins that can function as dual substrates for both one- and two-electron reductases. This endows them with the potential to be either hypoxia-selective cytotoxins or NAD(P)H:quinone oxidoreductase 1 (NQO1)-directed prodrugs, respectively. We have studied the structure-activity relationships of four novel indolequinone analogues with regard to one- and/or two-electron activation.

Synthesis of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas and evaluation as modulators of the isoforms of nitric oxide synthase.

Inhibition of the isoforms of nitric oxide synthase (NOS) has important applications in therapy of several diseases, including cancer. Using 1400 W [N-(3-aminomethylbenzyl)acetamidine], thiocitrulline and N(delta)-(4,5-dihydrothiazol-2-yl)ornithine as lead compounds, series of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas were designed as inhibitors of NOS. Ring-substituted benzyl and phenyl isothiocyanates were synthesised by condensation of the corresponding amines with thiophosgene and addition of ammonia gave the corresponding thioureas in high yields.

Combining bioreductive drugs and radiation for the treatment of solid tumors.

Methods now exist for the identification of human tumors that contain significant numbers of hypoxic cells and are thereby suitable for treatment with bioreductive drugs to eliminate this refractory cell population. However, to fully exploit the potential of bioreductive drugs, they will need to be used in combination with other modalities likely to target the proliferating aerobic cells in the tumor. Radiation is the treatment that is most effective in killing aerobic cells; therefore, the present report reviews the available preclinical data on combined radiation/bioreductive drug treatments.

Potential tumor-selective nitroimidazolylmethyluracil prodrug derivatives: inhibitors of the angiogenic enzyme thymidine phosphorylase.

Thymidine phosphorylase (TP) is an angiogenic growth factor and a target for anticancer drug design. Molecular modeling suggested that 2'-aminoimidazolylmethyluracils would be potent inhibitors of TP. The novel 5-halo-2-aminoimidazolylmethyluracils (4b/4c) were very potent inhibitors of E.

Antiangiogenic, bioreductive and gene therapy approaches to the treatment of hypoxic tumours.

Quinone based bioreductive drugs have, potentially, a very versatile use in cancer chemotherapy. They can be activated by DT-diaphorase and hence can be used to target tumour types rich in this (O2)-independent reductase enzyme. Small molecular modifications can substantially reduce specificity for DT-diaphorase and under these circumstances the quinones become much less toxic in air but retain their potent cytotoxic effects under hypoxic conditions.

Comparative inhibitory effects of niflumic acid and novel synthetic derivatives on the rat isolated stomach fundus.

Novel derivatives of 2-[3-(trifluoromethyl)-analino]nicotinic acid (niflumic acid) were synthesized. The compounds were compared for their inhibitory effects on 5-hydroxytryptamine (5-HT)- and KCI-induced contraction of the rat fundus. The aim was to assess structure-activity relationships regarding the selectivity and potency of these compounds.

Synthesis and enzymatic evaluation of pyridinium-substituted uracil derivatives as novel inhibitors of thymidine phosphorylase.

A series of water soluble N(1)- and C(6)-substituted uracil pyridinium compounds were prepared as potential inhibitors of thymidine phosphorylase (TP). The C(6)-uracil substituted derivatives were the most active. 1-[(5-Chloro-2,4-dihydroxypyrimidin-6-yl)methyl]pyridinium chloride, was identified as the best inhibitor being 5-fold more potent than the known inhibitor, 6-amino-5-bromouracil.