Trihydroxynaphthalene reductase of Curvularia lunata--a target for flavonoid action?

Melanin protects dark-pigmented fungi from environmental stresses and serves as an important virulence factor in plant and human pathogenic fungi. The enzymes of melanin biosynthesis thus represent interesting targets for the development of fungicides and new selective antimycotics. In Curvularia lunata, a facultative plant and human pathogen, melanin is produced from 1,8-dihydroxynaphthalene via the pentaketide pathway. Recently, the melanin biosynthetic enzyme trihydroxynaphthalene reductase (3HNR) of C. lunata was cloned and expressed in Escherichia coli, enabling further inhibition studies. Here, we have examined structurally different flavonoids (flavones, flavonols, isoflavones and flavanones) as inhibitors of recombinant 3HNR by following the NADP(+)-dependant oxidation of a non-physiological substrate, 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one. At 40 microM substrate concentration the most potent inhibitors were five flavones that are hydroxylated at positions 5 and 7: apigenin (IC(50), 3.1 microM), acacetin (IC(50), 4.9 microM), diosmetin (IC(50), 5.7 microM), 5,7-dihydroxyflavone (IC(50), 5.8 microM) and luteolin (IC(50), 6.8 microM). Flavonol (kaempferol; IC(50), 7.9 microM), isoflavone (genistein; IC(50), >50 microM) and flavanone (naringenin; IC(50), 26 microM) derivates were less potent than their corresponding flavone analogue apigenin. Among the isoflavones and flavanones, biochanin A was the most active (IC(50), 12 microM). Kinetic studies confirmed that apigenin and biochanin A, the best inhibitors among the flavones and isoflavones, act as competitive inhibitors of 3HNR, with K(i) values of 1.2 microM and 6.5 microM, respectively. Docking of apigenin and biochanin A into the active site of C. lunata 3HNR revealed their possible binding modes, in which they are stacked between the phenol ring of Tyr208 and the coenzyme nicotinamide moiety, forming two H-bonds with Ser149 and Ser228, and Ser149 and Tyr163, respectively. In vivo inhibition study showed that apigenin and one of the less potent inhibitors, baicalein affect fungal pigmentation and growth. Knowing that the flavonoids are formed in plants in response to fungal attack, they can be considered as potential physiological inhibitors of 3HNR.