Studies on specific inhibitors for mycotoxin production

Sakuda, S.,Yoshinari, T.,Nakamura, K., Akiyama, T., Takahashi, Y.,

Muraoka, Y., Nonomura, Y., and Nagasawa, H.

1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo

(Bunkyo-ku, Tokyo 113-8657, Japan)

2 Microbial Chemistry Research Center

(3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan)

Abstract

Specific inhibitors for mycotoxin production are useful not only to prevent

mycotoxin contamination in foods and feeds without incurring rapid spread of resistant strains, but also to know the regulatory mechanism of mycotoxin production by fungi at the molecular level. We have been studying some aflatoxin production inhibitors, aflastatins A and B, and blasticidin A, this past decade. All these

compounds are metabolites of Streptomyces and have similar unique structures. In

this proceedings, we describe our recent work on the stereochemistry of these

compounds and a fluorescence probe of blasticidin A to investigate its localization in the fungal hypha. Furthermore, we present detailed biological activity of dioctatin A as a strong inhibitor of aflatoxin production by Aspergillus parasiticus. Our results indicate that dioctatin A has pleiotropic effects on regulatory mechanisms of

fungal secondary metabolite production and differentiation, which lead to inhibition

of aflatoxin production.

Key words: aflastatin A, blasticidin A, dioctatin A

Introduction

Mycotoxins are produced as secondary metabolites by fungi. Their physiological roles in

the fungi are not clear, but they are not necessary for the fungal growth. Therefore, specific

inhibitors for mycotoxin production with no fungicidal activity may be useful to prevent

mycotoxin contamination in foods and feeds without incurring rapid spread of resistant strains.

Since there is little information concerning a regulatory mechanism for production of

secondary metabolites including mycotoxins in fungi, inhibitors affecting the mechanism may

be very important probes to know it at the molecular level. Novel knowledge on the

mechanism for mycotoxin production can provide promising targets for developing more

effective and practical mycotoxin production inhibitors.

We have been studying specific inhibitors of aflatoxin production by Aspergillus

parasiticus this past decade. We found aflastatins A and B (AsA and AsB) and blasticidin A

(BcA) as inhibitors of aflatoxin production and determined their structures. They are

produced by Streptomyces and have similar unique structures. AsA and BcA strongly

inhibited aflatoxin production of A. parasiticus with the IC50 values of 0.07 and 0.04ƒÊM,

respectively. They also inhibited production of norsolorinic acid and sterigmatocystin,

biosynthetic intermediates of aflatoxin, and reduced the mRNA levels of genes encoding

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aflatoxin biosynthetic enzymes and a key regulatory protein AflR. Therefore, their target

was suggested to be resent in a regulatory system leading to expression of aflatoxin 5 biosynthetic enzymes Since AsA and BcA changed carbon metabolism in the fungus

drastically, their target may be involved in a primary metabolism. However, detailed

molecular mechanism of inhibition of aflatoxin production by these compounds has not yet been clarified.

In this proceedings, we describe our recent work on the absolute configuration of AsA and

BcA and a probe to investigate their mode of action. In addition, discovery of the strong aflatoxin production inhibitory activity of dioctatin A and analysis of its detailed biological

activities are described.

Absolute configuration of AsA and BcA

AsA and BcA are tetramic acid derivatives with a highly oxygenated long alkyl chain. Both compounds have a number of chiral centers in their molecules. With respect to the stereochemistry of AsA, we had proposed its whole absolute configuration, but Kishi et al. recently pointed out that stereochemistry at the pentaol moiety (C27-C31) of AsA should be revised. On the other hand, with respect to the stereochemistry of BcA, we have determined all absolute configurations except for those at the diol (C8, C9) and pentaol (C25-C29) moieties .

To clarify the configurations at the diol and pentaol moieties of BcA, we analyzed the corresponding parts of methyl glycoside of its long polyol fragment 1. Since the absolute configuration at C8 of 1 was confirmed, determination of the relative stereochemistry from C6 to C8 was enough for assignment of the absolute configurations at C6 and C7 of 1. To assign the relative stereochemistry from C6 to C8, we synthesized four model stereoisomers, 2a, 2b, 2'a and 2'b, which could cover all possible stereochemistry for C6-C8 of 1, in expectation that one of them shows similar 3JH,H values to those observed in!. Among them, only the values observed in 2b (3JH2,H3=5.0Hz, 3JH3,H4=4.3Hz) were comparable to those in the C6-C8 moiety of 1 (3JH6,H7=3JH7,H8=4.2 Hz), indicating the both relative configurations for both C6-C7 and C7-C8 were assigned as threo. Based on the absolute configuration at C8, the absolute stereochemistry at C6 and C7 of 1 was determined.

To assign the absolute stereochemistry at the pentaol moiety based on the absolute configuration at C21 of 1, the relative configuration from C21 to C23 of 1 was assigned as syn by the J-based method. On the other hand, the NMR database method clearly showed that the relative configuration from C23 to C27 was erythro/threo/threo/threo from the profile of the observed 3JH,H values (3JH23,H24=7.6Hz, 3JH24,H25=<2Hz, 3JH25,H26=4.4Hz, 3JH26,H27=3.4Hz). This relative stereochemistry from C21 to C27 afforded the absolute configuration at the pentaol moiety of 1 based on the configuration at C21. From the results obtained, the absolute configuration of BcA was completely assigned as shown in Fig. 1.

Since the NMR data around the pentaol moiety of 1 were well identical with those of the corresponding polyol fragment of AsA, the pentaol moiety in both BcA and AsA should have the same relative stereochemisty. The NMR data at C4-C7 of 1 were also well identical with those of the AsA polyol fragment. From these observations, we revised the absolute stereochemistry of AsA as shown in Fig. 1. AsA and BcA have very similar stereochemistry overall through the structures.

Preparation of a fluorescence probe of BcA

We attempted to prepare a fluorescence probe maintaining the biological activity of

BcA to analyze the localization of BcA in the fungal hypha, which is important to investigate

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Fig. 1. Structures of aflastatin A (AsA), blasticidin A (BcA), BcA-FITC, dioctatin A (DotA) and compounds 1 and 2.

the mode of action of BcA. Our previous study on the structure-activity relationship of BcA

showed that the methyl glycoside of BcA maintained high activity. Thus, the FITC

derivative of BcA (BcA-FITC, Figure 1) was prepared. FITC-BcA showed inhibitory

activity toward aflatoxin production of A. parasiticus in a PD medium with the IC50 value of

8.5ƒÊM.

Confocal microscopic study with BcA-FITC was carried out as follows. Spores of A.

parasiticus were inoculated into a liquid medium dropped on a cover glass. After incubating

for 20 h at 27•Ž statically, BcA-FITC and/or FM4-64, a tracer of endocytosis11 , were added

into the culture and cultivation continued for 0-120 min under the same culture conditions.

Mycelia on the cover glass obtained at each cultivation time was washed with a fresh liquid

Fig. 2. Time course of BcA-FITC internalization within the hyphal tip of A. parasiticus. Confocal fluorescence images 30 min, 60 min and 120 min after dye application were shown in (a), (b) and (c), respectively. Dye-staining moieties appear white in the images. Scale bar, 20ƒÊm.

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medium twice and subjected to observation under microscope. Fig. 2 shows confocal images of the hypha cultured in the PD medium for 30, 60 or 120 min after addition of BcA-FITC, in which internalization of BcA-FITC from the fungal tip is observed at 30 min and it is spread over the hypha gradually. When BcA-FITC and FM4-64 were added to the culture at the same time, localization of the fluorescence of BcA-FITC in the hypha was well identical to that of FM4-64. These observations indicated that the BcA molecule can enter the inside of the fungal hypha through endocytosis.

Discovery of aflatoxin production inhibitory activity of dioctatin A

BcA can inhibit growth of a yeast, Saccharomyces cerevisiae. To obtain a clue to investigate the mode of action of BcA, we analyzed metabolites in yeast cells cultured with or without BcA. As a result, it was found that the amount of a diketopiperazine drastically decreased in the presence of BcA. Since the diketopiperazine, which was composed of methionine and proline, was thought to be synthesized through a dipeptide produced by the action of a dipeptidyl peptidase (DPP) from a certain protein or peptide, it was assumed that there might be a relationship between BcA's action and DPP. Thus, inhibitory activity of BcA toward DPP and that of several known DPP inhibitors toward aflatoxin production was tested. The results showed that BcA exhibited no inhibition toward human DPP II or DPP IV, but it was found that one of the DPP inhibitors, dioctatin A (DotA, Fig. 1), strongly inhibited aflatoxin production of A. parasiticus without affecting the fungal growth. Although it is not clear why production of the diketopiperazine was reduced by BcA, we discovered a new biological activity of DotA.

Biological activities of dioctatin A

DotA strongly inhibited aflatoxin production by A. parasiticus dose-dependently with

the IC50 value of 4.0ƒÊM. On the other hand, mycelial weight of the fungus was not affected

by DotA even at the concentration of 50ƒÊM. DotA inhibited production of norsolorinic acid

(NA), an early biosynthetic intermediate of aflatoxin (Fig. 3), more strongly than the case of

aflatoxin production with the IC50 value of 0.8ƒÊM. Furthermore, DotA reduced the mRNA

levels of pksA and omtA encoding enzyme proteins involved in the aflatoxin biosynthetic

pathway and of lR encoding a key regulatory gene whose product regulates transcription of

some enzyme genes for aflatoxin production including the former two genes (Fig. 3)

These facts may indicate that DotA affects a pathway leading to expression of AflR.

When A. parasiticus was cultured on a solid medium, DotA inhibited conidia formation

of the fungus and induced fluffy morphology. The mRNA level of brlA encoding a

conidiation-specific transcription factor was significantly reduced by addition of DotA.

Phenomena suggesting a close relationship between conidiation and mycotoxin production

have been known in Aspergillus species. DotA may affect a regulatory system which

controls aflatoxin production and conidiation in common.

We noticed that unidentified yellow pigment and kojic acid production was clearly

increased by addition of DotA on a solid culture. The effect of DotA on increase of kojic

acid production was very dramatic. The amount of kojic acid in the culture with 3.2ƒÊM of

DotA reached 35 times higher than that of control. This may suggest that DotA is useful to

change the secondary metabolite pathway of a fungus, which leads to production of useful or

novel compounds.

Figure 3 summarizes the pleiotropic effects of DotA on secondary metabolism and

differentiation of A. parasiticus. DotA can inhibit aflatoxin production and conidiation by

reducing expression of regulatory proteins, AflR and BrlA, respectively. The target

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Fig. 3. Pleiotropic effects of DotA on secondary metabolism and differentiation of A. parasiticus. DotA inihibits production of aflatoxin and norsolorinic acid (NA), and suppresses expression of genes responsible for aflatoxin biosynthesis, pksA, omtA and aflR . It also reduces the mRNA of brlA and inhibits conidiation. On the other hand, production of a yellow pigment and kojic acid is dramatically increased by DotA.

molecule of DotA may also regulate production of secondary metabolites other than aflatoxin. Since DotA is a strong inhibitor of human DPP II, it may be possible to speculate that these activities of DotA are attributed to inhibition of a function of a DPP II-like protein present in A. parasiticus. Until now, there is no biological information on fungal DPP II like proteins. From the homology search based on known fungal genome sequences of Aspergillus oryzae, Aspergillus nidulans and Aspergillus fumigatus, no gene encoding a protein with high similarity to human DPP II is present in all these fungal genomes, although a gene encoding an unknown serine protease-like protein with about 25% similarity to DPP II is present in each fungal genome. We tested the effects of known synthetic DPP II inhibitors on aflatoxin production, but they did not inhibit it. At the present, it is not clear if the mode of action of DotA in A. parasiticus is related to a function of a DPP II-like protein.

DotA strongly inhibits aflatoxin production and conidiation without affecting the fungal

growth. These features are very advantageous to prevent aflatoxin contamination of foods and feeds without incurring not only a rapid spread of resistant strains but also wide diffusion of conidia. Furthermore, DotA has a relatively simple structure and shows no toxicity to mammals. Therefore, DotA may be a good lead compound for developing practically effective drugs. From the viewpoint of basic research, DotA is a very important probe to investigate the regulatory mechanism of fungal secondary metabolite production and differentiation. Work to clarify the mode of action of DotA and to develop more effective DotA derivatives is now in progress.

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