There are no previous reports in the scientific literature of the effect of prothioconazole on sterols of treated fungi, and we demonstrated here that it behaves as an active inhibitor of sterol 14 α-demethylation and reduces ergosterol in M. graminicola
cells. It is active against growing M. graminicola
at concentrations equivalent to those of the azole fungicides epoxiconazole, tebuconazole, and triadimenol. The sterol profile of treated cultures showed a similar accumulation of eburicol and, to a lesser extent, lanosterol, which is consistent with the normal metabolic route suggested for filamentous fungi (eburicol rather than lanosterol being the CYP51 substrate). Since no other enzyme in fungi can fulfill the essential function of CYP51, the accumulation of 14 α-methyl sterols and the depletion of ergosterol confirms that CYP51 is the target of the prothioconazole-mediated growth inhibition of M. graminicola
. The confirmation here that the ergosterol isomer observed in M. graminicola
) is ergosta-5,8,22-trienol suggests that C8 isomerization may be less efficient in M. graminicola
than in other fungal species.
Successful heterologous expression of MgCYP51 was achieved with E. coli, and this allowed the interaction of DMIs, including prothioconazole, to be studied for the first time. The interaction of epoxiconazole, tebuconazole, and triadimenol indicated typical type II spectral interaction with high affinity for MgCYP51, confirming the formation of a low-spin complex with the compounds bound as a sixth ligand of the heme. Prothioconazole did not bind in this way, as reflected by the novel spectrum observed and the absence of type II spectral interaction. The binding affinity of MgCYP51 for prothioconazole was 840-fold less than for epoxiconazole. Epoxiconazole bound with greatest affinity to MgCYP51, followed closely by tebuconazole, and triadimenol bound with 18-fold less affinity than epoxiconazole. The ability to compare the affinities of different azole compounds for MgCYP51, as well as comparison to MgCYP51 strains harboring mutations implicated in resistance to azoles and with other CYP51s, may be of use in aiding the further understanding of the resistance to azoles and may aid in drug development.
The nature of the spectrum observed for prothioconazole is not currently clear and does not conform to inhibitor or substrate effects observed previously. The perturbation of prothioconazole on binding to MgCYP51 might be caused by a weak interaction between the electronegative sulfur atom on the azole ring of prothioconazole (Fig. ) and the ferric ion of the heme prosthetic group.
To compare the interaction of prothioconazole with MgCYP51 more closely, the interference with 14α-methyl substrate interaction was investigated. The presence of 0.35 mM prothioconazole caused 4.6- and 7.5-fold increases in the apparent Ks
values for eburicol and lanosterol, respectively, resulting in estimates of the Kei
for prothioconazole of 51 to 104 μM. This suggested that prothioconazole “competes” for the substrate binding site on MgCYP51. It is not surprising that epoxiconazole was a noncompetitive inhibitor of MgCYP51 since azole antifungal agents bind to the CYP51 molecule through direct coordination with the heme prosthetic group and not through interactions with the substrate binding site (8
The treatment of whole cells revealed that prothioconazole has an efficacy comparable to that of epoxiconazole, tebuconazole, and triadimenol. However, the competitive inhibition observed with prothioconazole and the high Kd value for prothioconazole would not alone account for the effectiveness of prothioconazole in vivo as a CYP51 inhibitor if it were binding directly as a sixth ligand of the heme. Prothioconazole may therefore inhibit MgCYP51 activity in an additional capacity that does not result in the perturbation of the heme environment (and hence cannot be directly measured spectrophotometrically).
The possibility exists that in vivo metabolism of prothioconazole to the desthio metabolite (a triazole) in wheat may be important for the efficacy of this compound in the field, but our results show inhibition of sterol biosynthesis in M. graminicola cells grown in broth cultures and therefore indicate that the antifungal activity does not rely on in planta metabolism. Alternatively, it is possible that the in vivo metabolism of prothioconazole occurs in fungal cells and that this is necessary for the antifungal activity of the compound. Further work on prothioconazole-treated M. graminicola cultures will elucidate how prothioconazole itself is inhibiting CYP51 and whether biotransformation resulting in the desthio triazole compound contributes to CYP51 inhibition. In either case, prothioconazole presents a novel antifungal agent. The findings described here suggest an inhibition of CYP51 activity by binding to the enzyme in a manner other than direct coordination to the heme. Additionally, an eventual metabolism to the desthio triazole compound within the fungus would lead to an active antifungal. This mode of antifungal action therefore presents a possible basis for the development of new compounds both for agriculturally important pathogenic species and for the causative agents of human diseases, and we are currently examining other CYP51s of such pathogens for their interaction with this new fungicide.