The hypolipidemic drug metabolites nafenopin-CoA and ciprofibroyl-CoA are competitive P2Y1 receptor antagonists.

Coenzyme A (CoA-SH), endogenous and drug-derived CoA-derivatives were tested as putative antagonists of P2Y receptors expressed in Xenopus laevis oocytes, a method used to determine calcium-activated chloride current, an indicator of the activation of these receptors. CoA-SH antagonized reversibly and in a concentration-dependent manner the ATP-gated currents evoked by the human P2Y(1) but not the P2Y(2) receptor. Palmitoyl-CoA was four-fold more potent than CoA-SH as an antagonist while palmitoyl-carnitine was inactive, highlighting the role of the CoA-SH moiety in the antagonism. The CoA derivatives of nafenopin and ciprofibrate, two clinically relevant hypolipidemic drugs, increased 13 and three-fold the potency of CoA-SH, respectively. The K(B)s of nafenopin-CoA and ciprofibroyl-CoA were 58 and 148 nM, respectively; the slopes of the Schild plots were unitary. Neither 100 microM nafenopin nor ciprofibrate alone altered the P2Y(1) receptor activity. Neither CoA-SH nor ciprofibroyl-CoA antagonized the rat P2X(2) or the P2X(4) nucleotide receptors nor interacted with the 5-HT(2A/C) receptors. The bulky drug CoA-SH derivatives identify a hydrophobic pocket, which may serve as a potential target for novel selective P2Y(1) antagonists.

In vitro covalent binding of nafenopin-CoA to human liver proteins.

Endogenous fatty acyl-CoAs play an important role in the acylation of proteins. A number of xenobiotic carboxylic acids are able to mimic fatty acids, forming CoA conjugates and acting as substrates in pathways of lipid metabolism. In this study nafenopin, a substrate for human hepatic fatty acid-CoA ligases, was chosen as a model compound to study xenobiotic acylation of human liver proteins. (3)H-nafenopin (+/- unlabeled palmitate) or (14)C-palmitate (+/- unlabeled nafenopin) were incubated for up to 120 min at 37 degrees C with ATP, CoA, and homogenate protein (1 mg/ml) from four individual human livers. Nafenopin covalently bound to proteins was detectable in all human livers and increased with time. Nafenopin adduct formation was directly proportional to nafenopin-CoA formation (r = 0.985, p < 0.05). Attachment of nafenopin to proteins involved both thioester and amide linkages with 76 and 24% of adducts formed with proteins > 100 and 50-100 kDa, respectively. Protein acylation by palmitate was also demonstrated. Palmitate significantly inhibited nafenopin-CoA formation by 29% but had no effect on nafenopin-CoA-mediated protein acylation. In contrast, nafenopin significantly inhibited protein palmitoylation by palmitoyl-CoA. This is the first study to demonstrate a direct relationship between xenobiotic-CoA formation, acylation of human liver proteins, and inhibition of endogenous palmitoylation. The ability of xenobiotics to acylate tissue proteins may have important biological consequences including perturbation of endogenous regulation of protein localization and function.

Saturable binding sites for the coenzyme A ester of nafenopin, a peroxisome proliferator, in rat liver cytosol.

1. At least three different molecular weight binding sites exist in rat liver cytosol for nafenopin-CoA, the coenzyme A ester and metabolic product of the carcinogenic peroxisome proliferator nafenopin. No binding sites for the free drug were observed. 2. Polypeptides of 35-40 kDa molecular weight range where no acyl-CoA binding proteins have been previously described bind the highest proportion of nafenopin-CoA (60-70%). Binding is displaceable by the CoA esters of other peroxisome proliferators (ciprofibrate and tibric acid) and also by oleoyl-CoA but by palmitoyl-CoA. Direct binding studies show that 35-40-kDa polypeptides bind oleoyl-CoA but not oleic or palmitic acid, or palmitoyl-CoA. 3. Polypeptides of 10-14 and 65-70 kDa also bind nafenopin-CoA. However, in contrast with 35-40-kDa polypeptides they also bind oleic and palmitic acid as well as their correspondent acyl-CoA thioesters.

Xenobiotic acyl-CoA formation: evidence of kinetically distinct hepatic microsomal long-chain fatty acid and nafenopin-CoA ligases.

Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids. In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase. The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin (Ki 53 microM) and ciprofibrate (Ki 1000 microM). Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform (Km 6 +/- 2.5 microM, Vmax 0.33 +/- 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean Ki 1.7 microM, n = 5) and R-ibuprofen (mean Ki 10.8 microM, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors. The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (Vmax/Km 0.057 +/- 0.011 nmol/mg/min/ +/- M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes. Evidence of both a substantial difference between the Km and Ki for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct. Thus until the current 'long-chain like' xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates.

In vitro evidence for involvement of CoA thioesters in peroxisome proliferation and hypolipidaemia.

The mechanisms of peroxisomal induction and hypolipidaemia caused by treatment with peroxisome proliferators, such as nafenopin and clofibrate, remain to be elucidated. Proposed mechanisms include receptor-mediated processes or adaptations resulting from disruption of hepatic lipid metabolism. The latter mechanism was investigated in a series of in vitro studies. Incubation of primary rat hepatocytes with various carboxyl-containing compounds revealed no clear common factor which imparted potency as a peroxisomal inducer. Inhibitors of fatty acyl-CoA synthetase, norepinephrine and desulpho-CoA, however, decreased the level of peroxisomal induction by nafenopin in rat hepatocytes, suggesting that activation of carboxyl-containing compounds to their CoA thioesters may be a necessary step in initiating peroxisome proliferation. Coenzyme A thioesters of nafenopin, clofibric acid and other carboxyl-containing chemicals were synthesised and found to inhibit the activity of acetyl-CoA carboxylase to varying degrees. The CoA thioester of nafenopin was the most potent inhibitor among this group (Ki = 1.45 x 10(-5) M), but weaker than palmitoyl-CoA (Ki = 2.22 x 10(-6) M), the feedback inhibitor of acetyl-CoA carboxylase. Hypolipidaemia caused by treatment with peroxisome proliferators may, therefore, be related to inhibition of fatty-acid synthesis by the corresponding CoA thioester derivative.

Hypolipidaemic drugs are activated to acyl-CoA esters in isolated rat hepatocytes. Detection of drug activation by human liver homogenates and by human platelets.

The formation of acyl-CoA esters of the hypolipidaemic peroxisome proliferators clofibric acid, ciprofibrate and nafenopin was studied in isolated rat hepatocytes. The concentration of ciprofibroyl-CoA in the liver of ciprofibrate-treated rats was in the range of 10-30 microM. The three drugs formed acyl-CoA esters when incubated with isolated hepatocytes. Their formation was saturable and reached a plateau after 30 min incubation. Maximal intracellular concentrations of ciprofibroyl-CoA and clofibroyl-CoA (100 microM and 55 microM respectively) were attained at 0.5 mM of the free drugs in the incubation medium, whereas for nafenopin-CoA, the maximal intracellular concentration (9 microM) was reached at 1 mM-nafenopin. At low concentrations of the hypolipidaemic compounds in the incubation medium a significant proportion of the total intracellular drug was present as its acyl-CoA ester (25-35% for ciprofibrate). When isolated hepatocytes were incubated with a ciprofibrate concentration comparable with that observed in the blood of drug-treated rats (0.1 mM), ciprofibroyl-CoA attained an intracellular concentration similar to that previously observed in the liver of treated rats. The formation of ciprofibroyl-CoA by isolated rat hepatocytes was stimulated by the addition of carnitine and partially inhibited by the addition of palmitate. Further, it was shown that human liver homogenates synthesized ciprofibroyl-CoA at a rate similar to that observed for rat liver homogenates. Solubilized human platelets also formed ciprofibroyl-CoA, although at a rate two orders of magnitude lower than that of liver. The results support the view that acyl-CoA esters of hypolipidaemic peroxisome proliferators may be the pharmacologically active species of the drugs.

Activation of hypolipidaemic drugs to acyl-coenzyme A thioesters.

Compounds possessing the characteristics of CoA thioesters of the hypolipidaemic peroxisome proliferators clofibric acid, nafenopin and ciprofibrate were formed on incubation of the drugs with rat liver microsomal fractions, ATP and CoA. The reactivity of the drugs correlated with their pharmacological potency. It is proposed that the active species of these compounds are their acyl-CoA thioesters.

New alpha-substituted arylthioacetic derivatives forming analogues of clofibrate.

Synthesis of some alpha-substituted arylthioacetic acids and esters forming analogues of clofibrate and of nafenopin (SU-13437) is reported and discussed. With a view to evaluating the influence of the introduction of a sulphur atom on the blood-lipid-reducing activity, preliminary pharmacological tests have been carried out on alpha-[4-(1-tetralyl)phenylthio] isobutyric acid (XIV) in parallel with its well-known phenoxy isologue. The results obtained so far, do not show any significant difference between the two compounds.