Inhibition of Pneumocystis carinii dihydropteroate synthetase by sulfa drugs

Abstract

A new reversed-phase high-pressure liquid chromatography assay procedure for dihydropteroate synthetase (DHPS) that involves the elution of the enzyme incubation solution with a series of three solvents of decreasing polarity (ammonium phosphate buffer, 10% methanol, and 50% methanol) was designed. By this procedure DHPS was detected in Escherichia coli and Pneumocystis carinii with specific activities of 450 and 14 U/mg, respectively. A comparison of the effects of five sulfa drugs on P. carinii DHPS activity revealed that dapsone is the most potent of these drugs.Pneumocystis carinii is the major life-threatening opportunistic agent in patients with acquired immunodeficiency syndrome (AIDS) and affects other immunocompromised patients. Approximately 60% of all patients with AIDS suffer from P. carinii pneumonia, and up to 40% of the episodes prove fatal (10).Patients with P. carinii pneumonia are frequently treated with antifolates (inhibitors of tetrahydrofolate biosynthesis) such as bactrim (a combination of trimethoprim and sulfamethoxazole) (6, 7), dapsone (6, 13), and trimetrexate (1). The ability of P. carinii to synthesize tetrahydrofolate de novo has been reported recently (9).Two enzymes involved in the biosynthesis of tetrahydrofolate that are important as targets of antifolates are dihydropteroate synthetase (DHPS) and dihydrofolate reductase. DHPS catalyzes the formation of dihydropteroate (DHP) from p-aminobenzoic acid (pAB) and 6-hydroxymethyldihydropterin pyrophosphate (MDHP-PP). Dihydrofolate reductase catalyzes the formation of tetrahydrofolate from dihydrofolate. Sulfones and sulfonamides act as competitive inhibitors of DHPS substrates, while drugs such as trimethoprim and pyrimethamine act as inhibitors of dihydrofolate reductase substrates. In this study we measured the inhibitory actions of several sulfonamides and sulfones on the parasite associated with DHPS using a new high-pressure liquid chromatography (HPLC) enzyme assay. MATERIALS AND METHODSGrowth and isolation of organisms. Male Sprague-Dawley rats (weight, 150 to 200 g) were fed ad libitum and administered tetracycline in their drinking water (1 g/liter). Subcutaneous injections of cortisone acetate (25 mg) were given twice a week. After 4 to 6 weeks of injections, those rats which showed signs of rapid breathing, blood in their noses, weight loss, and hair loss were used in the experiments.P. carinii was isolated by a modification of the method of Gradus and Ivey (4). Each rat was sacrificed by cervical dislocation. Their lungs were then quickly removed and kept at 4°C. Touch preparations were made from the lungs and stained with toluidine blue (3) bacteria were evident. Subsequent inoculation of the lung homogenate onto Luria broth agar plates confirmed the absence of contaminants. The lungs were homogenized by repeated passage through a 60-gauge wire screen. The resulting suspension was centrifuged at 2,000 x g for 10 min. The supernatant was discarded, and the pellet was suspended in phosphate-buffered saline. The suspensio...

Inhibition of recombinant Pneumocystis carinii dihydropteroate synthetase by sulfa drugs

“…At least four of them inhibited folate metabolism in intact organisms to an extent similar to that of sulfamethoxazole. The K m s reported here for PABA (0.34 Ϯ 0.02 M) and H 2 PtCH 2 OPP (2.50 Ϯ 0.71 M) are lower than those previously reported with P. carinii (9,14). The K m for H 2 PtCH 2 OPP is similar to that found in one study (3.0 M) (14) but 40-fold lower than that found in the other (81 M) (9); the K m for PABA is Ͼ25-fold lower than those found in either study (8.7 M [14] and 71 M [9]).…”

“…Thus, it is possible that other sulfa drugs might be as effective as sulfamethoxazole without eliciting adverse effects. Very few of the approximately 15,000 sulfa drugs that have been synthesized (8) have ever been tested against P. carinii in vitro (9,14) or in vivo (5,6,20). This low rate of testing is due, in part, to the inability to culture these organisms continuously in vitro (12) and to the complexities of the various animal models (19).…”

“…Therefore, an enzymebased screen represents a more practical approach to the identification of effective sulfa drugs. Two small enzyme-based screens measuring DHPS activity in homogenates of isolated organisms have been carried out (9,14). Both studies were limited by difficulties in obtaining large amounts of organisms.…”

The identification, analysis and structure-based development of novel inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

“…These agents should also be suitable for use in patients with sulfa-allergies and in patient populations with high incidence of sulfa allergies such as those with HIV undergoing prophylaxis to prevent pneumocystis pneumonia. 32 To obtain such therapeutics, it is clear that these compounds must be further optimized to enhance target affinity and microbial penetration, and to maximize exposure in the host. We believe that the structural and biochemical data presented in this study have provided an excellent basis to enable such efforts to move forward rapidly.…”

Catalysis and Sulfa Drug Resistance in Dihydropteroate Synthase

“…Co-trimoxazole has proven to be effective against several emerging threats including community acquired multi-drug resistant Staphylococcus aureus (MRSA) (6, 7) and Pneumocystis jiroveci infections in immune-compromised patients (8). DHPS therefore remains an important drug target, and we are developing new inhibitors that target the DHPP-binding pocket of the enzyme (9–11).…”