Pharmacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: structure/activity relationships and functional activity in tumour cells.

NAD(P)H quinone oxidoreductase (NQO1) has multiple functions in the cell including an ability to act as a detoxifying enzyme and as a protein chaperone. The latter property is particularly important in oncology as one of the client proteins of NQO1 is p53. The inhibitor, dicoumarol, is classically used to probe the biological properties of NQO1, but interpretation of enzyme function is compromised by the multiple "off-target" effects of this agent. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of recombinant human NQO1 have been identified (Nolan et al., J Med Chem 2009;52:7142-56) The purpose of the work reported here is to demonstrate the functional activity of these agents for inhibiting NQO1 in cells. To do this, advantage was taken of the NQO1-mediated toxicity of the chemotherapeutic drug EO9 (Apaziquone). The toxicity of this drug is substantially reduced when the function of NQO1 is inhibited and many of the coumarin-based compounds are more efficient than dicoumarol for inhibiting EO9 toxicity. The ability to do this appears to be related to their capacity to inhibit NQO1 in cell free systems. In conclusion, agents have been identified that may be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues.

Induction of diaphorase-1 by dicoumarol in Drosophila virilis larvae.

Drosophila diaphorase-1 (DIA-1) is an enzyme similar to mammalian DT-diaphorase and is inhibited in vitro by dicoumarol. However, a ten-fold increase in DIA-1 activity was observed when third instar Drosophila virilis larvae were fed on a diet containing 0.1 M dicoumarol for 48 h. This induction was shown to be dose dependent and immunoprecipitation experiments with DIA-1 anti-serum demonstrated an increase in the DIA-1 protein level in dicoumarol-treated larvae. The induction of DIA1 by dicoumarol was found to be blocked by actinomycin D, which suggests a transcriptional mechanism of regulation. The opposite effect of dicoumarol on DIA-1 in vitro vs. in vivo suggests that a metabolic conversion takes place after the ingestion of this compound by D. virilis larvae.

Evaluation of p-amidinophenyl esters as potential antithrombotic agents.

Three p-amidinophenyl esters have been synthesized and characterized as irreversible inhibitors of the vitamin-K dependent proteinases; factors IXa, Xa and thrombin (Turner et al. [4]).+ In the present report we describe the in vitro and in vivo effects of these agents on standard coagulation tests in vitro and in blood from animals treated with the compounds. At a concentration of 500 microM, the three esters increased the activated partial thromboplastin time (PTT) of pooled human plasma 3 to 5-fold. The prothrombin time increased 1.4 to 3.7-fold under similar conditions. The p-amidinophenyl ester of cinnamic acid (CINN) showed the most pronounced effect on both assays. This ester also is the best inhibitor of human factors IXa and Xa, while the p-amidinophenyl ester of benzoic acid (BENZ) is a slightly better alpha-thrombin inhibitor (4). The effect of these esters on the thrombin clotting time correlated with in vitro kinetic measurements of alpha-thrombin inhibition rates. Both BENZ and CINN increased the assay endpoint more than 6-fold. The three esters also were studied using mouse plasma. A comparable effect on the PTT was noted. Intravenous administration of 300 microliter of 1 mM CINN as a single bolus in mice caused a 2.3-fold increase in the PTT which remained 1.2-fold normal 2 h later. The BENZ and alpha-methyl-cinnamic acid (MECINN) esters were somewhat less effective as predicted from their in vitro effect on the PTT. This investigation and previous studies indicate that these compounds demonstrate low toxicity at therapeutic levels. It is concluded that the p-amidinophenyl esters may be useful in antithrombotic therapy.

Chloride-dependent restoration of coupling by oligomycin in rat liver mitochondria.

In rat liver mitochondria suspended in KC1 medium, oligomycin interfered with the effect of uncouplers on energy conservation. It antagonized the effect of uncouplers that are weak acids (2,4-dinitrophenol etc.), but enhanced that of the lipid-penetrating cation NN-dimethyl-N'N'-dibenzylammonium. Oligomycin caused none of the above effects when Br- or NO-/3 was substituted for C1- as the major anionic species in the assay medium. The concentration of oligomycin that exerted the above-mentioned effects was lower than that necessary for the inhibition of energy transfer, but was in the range that induced C1- permeation through the cristae membrane. The possible connexion between the effect of oligomycin on C1- permeation and its interference with the action of uncouplers is discussed.

Don't use the wrong vitamin K.

The emergency use of vitamin K is essentially limited to the reversal of drug-induced hypoprothrombinemia. In patients with adequate liver function, phytonadione acts promptly and predictably in this capacity whereas the derivatives of menadione counteract coumarin drugs only slightly or not at all. It is dangerous to rely on menadione analogues, and these drugs should be removed from emergency room drug stores.