Infoscience

Thesis

Inhibition of Sepiapterin Reductase through Formation of a Ternary Complex with Sulfasalazine or other Sulfa Drugs and NADP+

The mechanism of action of sulfasalazine, an anti-inflammatory drug used against inflammatory bowel diseases and rheumatoid arthritis is, despite its medical importance, still not well understood. Recently, it was shown that sulfasalazine and its metabolite sulfapyridine are potent inhibitors of sepiapterin reductase (SPR). Additionally, it has been proposed that inhibition of tetrahydrobiopterin (BH4) biosynthesis contributes to the anti-inflammatory activity of sulfasalazine. The final step in BH4 biosynthesis from GTP is a NADPH dependent reduction of 6-pyruvoyl-5,6,7,8-tetrahydropterin by SPR. BH4 acts as cofactor of aromatic hydroxylases such as phenylalanine, tyrosine and tryptophan hydroxylases, all isoforms of nitric oxide synthases and glyceryl-ether mono-oxygenase. A screen of therapeutically approved drugs identified several antibacterial sulfa drugs, amongst those sulfamethoxazole, as inhibitors of SPR. Sulfamethoxazole is used in the treatment of bacterial infections and Pneumocystis pneumonia, a prevalent opportunistic infection affecting immunocompromised individuals. Potent inhibition of SPR in vitro as well as of the BH4 biosynthetic pathway in various cell lines was then confirmed. Dose dependant interference of SPR inhibition with BH4-dependent catecholamine biosynthesis in cell culture was observed in further experiments and attributed to cofactor depletion. Decreased neurotransmitter levels and resulting adverse neurological effects are components of clinical symptoms of genetically inherited SPR deficiency and provide a rationale for the central nervous system-related side effects observed in sulfonamide treatment. The finding that sulfa drugs interfere with BH4-dependent neurotransmitter biosynthesis consequently proposes for potential improvement of sulfa drug therapy. These findings encouraged the achievement of crystal structures of human SPR in complex with sulfasalazine and relevant sulfa drugs. The structure of SPR with bound drug revealed how structurally diverse sulfonamides can achieve specific inhibition of the enzyme. An enzymatic, biochemical and thermodynamic analysis combined with high-resolution crystal structures provide valuable knowledge about the enzyme mechanism and binding of drug. The formation of a ternary complex, of drug with human SPR and NADP+, in which the interaction between the drug and the cofactor is crucial for the stability of the complex is demonstrated and described in detail. Collectively, the results obtained improve the understanding of the mechanism of action of sulfasalazine and open up new avenues for improved therapeutic use of sulfasalazine and related sulfa drugs.

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