Amiloride.HCl is clinically used as an oral potassium-sparing diuretic, but multiple studies in biochemical, cellular and animal models have shown that the drug also possesses anti-tumour and anti-metastasis activities. The additional effects appear to arise through inhibition of two discrete targets: (i) the sodium-hydrogen exchanger 1 (NHE1), a membrane protein responsible for the characteristically low extracellular pH of tumours and (ii) the urokinase-type plasminogen activator (uPA), a serine protease mediator of cell migration, invasion and metastasis and well-known marker of poor prognosis in cancer. This minireview summarises for the first time the reported anti-tumour/metastasis effects of amiloride in experimental models, discusses the putative molecular mechanisms responsible for these effects and concludes by commenting on the pros and cons of trialling amiloride or one of its structural analogues as potential new anti-tumour/metastasis drugs.Amiloride.HCl is an orally administered potassium-sparing diuretic that has been used clinically for more than three decades. Ironically, its principal clinical use is not as a diuretic per se, since its diuretic effects are relatively mild. It is instead more commonly used in combination with thiazide (e.g., hydrochlorothiazide) or loop diuretics (e.g., frusemide) as an antikaliuretic in patients at risk of hypokalaemia during longterm treatment of hepatic cirrhosis and heart failure. It is also frequently used in combination with other diuretics to control K þ -levels when treating hypertension. 1 The drug is usually well tolerated at normal doses, shows low incidence of side effects and is contraindicated only in patients with impaired renal function, hyperkalaemia or acidosis. Hyperkalaemia and associated arrhythmias are the drug's most serious side effects but when used in combination with thiazide diuretics, the incidence of these is <1-2%. 2 Soon after its discovery, amiloride found additional nonclinical uses as a pharmacological tool for probing sodium transport in many types of tissues and cells-uses which continue to this day. 3 Multiple studies over the past three decades have demonstrated that amiloride also exhibits significant anti-tumour and anti-metastasis activities in biochemical, cellular and animal tumour models. This mini-review begins by summarising the reported anti-cancer activities of amiloride in experimental models, discusses its purported anti-cancer mechanisms of action and concludes by commenting on the possibility that amiloride or a close structural analogue might be worth trialling clinically as an orally administered anticancer drug, either as a stand-alone agent or in combination with current chemotherapeutics. Anti-Tumour and Anti-Metastasis Effects of Amiloride in Experimental ModelsReports of in vivo anti-tumour properties for amiloride first appeared in 1983 when it was shown to inhibit H6 hepatoma growth and DMA/J mammary adenocarcinoma growth in a dose-dependent fashion in Male A/J mice. Multiple injections (1 mg/kg) of ...

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, K(i)=7 μM), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride's structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately twofold more potent than amiloride as uPA inhibitors.

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The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride 1 and 5-(N,N-hexamethylene)amiloride 2 (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A2A receptor (hA2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5'-substituted amiloride derivatives as hA2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [(3)H]4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol ([(3)H]ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA2AAR. 4-Ethoxyphenethyl-substituted amiloride 12l was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site.

Recombinant formate dehydrogenase from the acetogen Clostridium carboxidivorans strain P7 T , expressed in Escherichia coli, shows particular activity towards NADH-dependent carbon dioxide reduction to formate due to the relative binding affinities of the substrates and products. The enzyme retains activity over 2 days at 4°C under oxic conditions. F ormate dehydrogenases (FDHs) catalyze the interconversion of CO 2 and formic acid through an oxidoreductive process ( Fig. 1) (1). Consequently, when catalyzing CO 2 reduction, they are of interest for the sequestration of CO 2 and for the production of formic acid as a stabilized form of hydrogen fuel and as a source of commodity chemicals. In many bacteria and eukaryotes, FDHs catalyze the final step of catabolic processes in which formate is oxidized to CO 2 (2). The ability of certain members of this class, such as FDH from Candida boidinii in particular, to efficiently regenerate NADH in conjunction with formate oxidation has been a research focus (3).Acetogens are known to possess a number of pathways distinct from those found in the other species. FDHs present in acetogens are known to take part in a carbon fixation metabolic pathway producing acetate (the Eastern branch of the Wood-Ljungdahl pathway), in which the first step involves reduction of CO 2 to formate (4). Several FDHs are known to catalyze CO 2 reduction under appropriate conditions (5-9). Those enzymes from acetogenic and related anaerobes, such as Moorella thermoacetica and Clostridium pasteurianum, are better than other FDHs as reduction catalysts but also show similar catalytic efficiency toward formate oxidation and are considered highly oxygen labile, requiring anaerobic expression and purification as well as anoxic assay conditions (10, 11). Clostridium carboxidivorans strain P7 T (equivalent to ATCC BAA-624 T and DSM 15243 T ) was isolated from the sediment of an agricultural settling lagoon after enrichment with CO as the substrate and is an obligate anaerobe that can grow autotrophically with H 2 and CO 2 or CO (fixing carbon via the Wood-Ljungdahl pathway) (12, 13). Therefore, when the gene of a selenocysteine-containing formate dehydrogenase H (FDH H ) from the acetogen Clostridium carboxidivorans strain P7T was first identified, it was suggested that FDH H would catalyze the conversion of CO 2 to formate (14, 15). Here we report the first production of FDH H and its catalytic preference for CO 2 reduction, as well as its tolerance for oxic conditions. Cloning, expression, and purification of FDHs. The overexpression and purification of recombinant FDH H from the Clostridium carboxidivorans strain P7 T (FDH H _CloCa) was carried out, along with that of NAD ϩ -dependent recombinant FDH from Candida boidinii (FDH_CanBo), in order to compare expression and activity of formate dehydrogenases that take part in distinct metabolic pathways. The DNA sequences for the FDH H _CloCa (UniProt E2IQB0) and FDH_CanBo (UniProt O13437) genes were codon optimized for expression in Escherichia coli (com...

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E. coli wild-type translational machinery utilizes a range of nonproteinogenic amino acids for protein synthesis with incorporation levels greater than 95%.

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC(50)1=0.8 nM, IC(50)2=33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC(50) >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA.