Sulfa drugs inhibit sepiapterin reduction and chemical redox cycling by sepiapterin reductase.

Sepiapterin reductase (SPR) catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), the precursor for tetrahydrobiopterin (BH4), a cofactor critical for nitric oxide biosynthesis and alkylglycerol and aromatic amino acid metabolism. SPR also mediates chemical redox cycling, catalyzing one-electron reduction of redox-active chemicals, including quinones and bipyridinium herbicides (e.g., menadione, 9,10-phenanthrenequinone, and diquat); rapid reaction of the reduced radicals with molecular oxygen generates reactive oxygen species (ROS). Using recombinant human SPR, sulfonamide- and sulfonylurea-based sulfa drugs were found to be potent noncompetitive inhibitors of both sepiapterin reduction and redox cycling. The most potent inhibitors of sepiapterin reduction (IC50s = 31-180 nM) were sulfasalazine, sulfathiazole, sulfapyridine, sulfamethoxazole, and chlorpropamide. Higher concentrations of the sulfa drugs (IC50s = 0.37-19.4 µM) were required to inhibit redox cycling, presumably because of distinct mechanisms of sepiapterin reduction and redox cycling. In PC12 cells, which generate catecholamine and monoamine neurotransmitters via BH4-dependent amino acid hydroxylases, sulfa drugs inhibited both BH2/BH4 biosynthesis and redox cycling mediated by SPR. Inhibition of BH2/BH4 resulted in decreased production of dopamine and dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 5-hydroxytryptamine. Sulfathiazole (200 µM) markedly suppressed neurotransmitter production, an effect reversed by BH4. These data suggest that SPR and BH4-dependent enzymes, are "off-targets" of sulfa drugs, which may underlie their untoward effects. The ability of the sulfa drugs to inhibit redox cycling may ameliorate ROS-mediated toxicity generated by redox active drugs and chemicals, contributing to their anti-inflammatory activity.

Pterin interactions with distinct reductase activities of NO synthase.

Besides oxidizing L-arginine, neuronal NO synthase (NOS) NADPH-dependently reduces various electron acceptors, including cytochrome c and tetrazolium salts. The latter NADPH diaphorase reaction is used as a NOS-specific histochemical stain. Both reductase activities have been utilized to analyse electron transfer mechanisms within NOS. Basal L-arginine turnover by homodimeric NOS is enhanced by exogenous tetrahydrobiopterin, and the intra-subunit electron flow may include intermediate trihydrobiopterin. In the present work we have investigated the possible role of the tetrahydrobiopterin binding site of NOS in its reductase activities by examining the effects of anti-pterin type (PHS) NOS inhibitors. Although the type I anti-pterin, PHS-32, which does not affect basal dimeric NOS activity, also had no effect on either reductase activity, the type II anti-pterin, PHS-72, which inhibits basal NOS activity, inhibited both reductase activities and the NADPH diaphorase histochemical stain. Pterin-free NOS monomers catalysed both cytochrome c and tetrazolium salt reduction. Our data suggest that both NOS reductase activities are independent of tetrahydrobiopterin. However, occupation of an exosite near the pterin site in NOS by type II anti-pterins may interfere with the electron flow within the active centre, suggesting that steric perturbation of the pterin binding pocket or reductase interaction contribute to the mechanism of inhibition by this class of NOS inhibitors.

The transport of pteridines in CCRF-CEM human lymphoblastic cells.

The transport routes used by CCRF-CEM human lymphoblastoid cells for the influx and efflux of unconjugated pteridines were analyzed using [3H]6-hydroxymethylpterin as a model compound. Influx proceeds by a mechanism that exhibits a Km of 66.7 microM and a Vmax of 0.077 nmol/min per mg cellular protein. The process is somewhat sensitive to metabolic inhibitors, particularly uncouplers of oxidative phosphorylation, and is significantly affected by the presence of other pteridines in the extracellular medium. The results suggest that pterins with either no 6-substituent (pterin) or those with methyl, hydroxyl, or formyl groups in this position, which exhibit Ki values between 25 and 77 microM, may share the same pathway for uptake. 6-Carboxypterin exhibits low affinity for the system (Ki greater than 500 microM), as do 7-substituted and 6,7-di-substituted derivatives and compounds with larger groups at the 6-position, such as neopterin and biopterin (Ki = 250-300 microM). Efflux of [3H]6-hydroxymethylpterin occurs rapidly and can proceed by at least two routes. The first, comprising approximately 50% of total efflux, is inhibited by extracellular pterins and exhibits similar properties to the uptake system in both its pattern of sensitivity to metabolic inhibitors and its specificity for pteridine structure. The route by which the remaining efflux occurs is relatively insensitive to metabolic inhibition. Adenine significantly inhibits 6-hydroxymethylpterin influx and efflux (Ki = 10.6 microM for uptake) but does not appear to share the same transport system. Similarly, methotrexate and folic acid exhibit little affinity for the unconjugated pteridine transport routes.