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Chapter 8

Metabolites of Induced Fungi: A Potential Chemical Library for Next-Generation Pesticides S. Furutani, M. Ihara, K. Kai, H. Hayashi, and K. Matsuda* Department of Applied Biological Chemistry, Faculty of Agriculture, Kindai University, 3327-204 Nakamachi, Nara 631-8505, Japan Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan *E-mail [email protected].

Although some fungi produce mycotoxins that render food on which they grow inedible, other fungi produce metabolites that, in addition, to human and animal healthcare, are useful in pest control. Penicillium simplicissimum AK-40 produces okaramines as an insecticide when grown on okara, a by-product of soybean curd production. Okaramines selectively activate glutamate-gated chloride channels expressed only in the nervous system of invertebrates. Asperparalines from Aspergillus japonicus JV-23 and chrodrimanins from Talaromyces sp. YO-2, both of which were isolated similarly to okaramines, selectively block insect nicotinic acetylcholine and γ-aminobutyric acid receptors, respectively. These results suggest that fungi induced by certain plant factors have potential for producing next-generation pesticide leads.

Introduction In the late 1980s, Dr. Hideo Hayashi, now Emeritus Professor of Osaka Prefecture University, began to explore environmentally benign, next-generation pesticide leads with new skeletons from fungal metabolites due to their diverse metabolite structures (1). Based on literature, potato-dextrose and Czapek-Dox © 2017 American Chemical Society Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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culture media etc. were found to be suitable for proliferating fungi. However, he did not employ known media but instead used okara, a by-product of soybean curd (tofu) production, since it was very cost-effective, and its use was still unknown. He screened various fungal metabolites for toxicity using larvae of Bombyx mori and discovered two indole alkaloids, okaramine A and B, from the metabolites of Penicillium simplicissimum AK-40 (Figure 1) (2), which act as insecticides with LD50 (dose that kills 50% of insects) values of 8 and 0.2 μg/g diet, respectively. Both compounds possess a unique eight-membered azocine ring structure. Additionally, a searh for other okaramines and was able to further identify 16 okaramines (1). Okaramine B possesses a methoxy and a hydroxy group as well as a four-membered azetidine ring in addition to the azocine and indole rings and shows the highest insecticidal activity against the silkworm larvae among the 18 okaramines (1). Partial hydrogenation of its azocine ring led to reduced activity (3), and okaramine C (4) lacking the azocine ring was less potent than okaramine B. These results indicate the essential role of the azocine ring in okaramine potency.

Figure 1. Insect–active metabolites produced by fungi in okara.

Motivated by the discovery of okaramines, Dr. Hayashi continued to explore insect-active fungal metabolites with distint skeletons from okaramines (1). During this process, meroterpenoids, such as chrodrimanins, as well as other classes of indole alkaloids, such as asperparalines, were discovered (Figure 1) (1). When tested by oral application, chrodrimanin B (5) and E (6) show the highest insecticidal activity among the chrodrimanins tested on the silkworm larvae with an LD50 value of 10 μg/g diet, while asperparaline A induces paralysis at the same dose (7). Their unique and complex structures inspired total synthesis (8–11). However, their mechanism of action remains unknown, and thus the authors have worked to resolve their enigma. 126 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Figure 2. Selective blocking action of asperparaline A on ACh-induced currents in the silkworm larval neurons. Asperparaline A was applied for 1 min and then co-applied with 10 μM ACh (left), 30 μM GABA (center) and 30 μM L-glutamate (right). Applications of ACh, GABA and L-glutamate are indicated by horizondal lines, while those of asperparaline are indicated by dashed horizontal lines. Adapted with permission from ref. (12). Copyright 2011 PLoS.

Establishing a Patch-Clamp Recording Technique for Silkworm Larval Neurons and Clarifying the Mode of Action of Asperparaline All the fungal products shown in Figure 1 exhibited toxicity with a rapid onset of action in the silkworm larvae, indicating possible interactions with the larval nervous system (1). Therefore, we employed patch-clamp electrophysiology to investigate their actions on the ion channels expressed in the silkworm larval neuron. When applied alone, asperparaline A did not induce any currents in the neurons. However, it blocked the responses to acetylcholine (ACh) when co-applied with ACh, while scarcely influencing the responses to γ-aminobutyric acid (GABA) and L-glutamate (Figure 2) (12). The IC50s, half-maximal inhibitory concentrations, of asperparaline A for the peak and steady-state ACh-induced currents were 20 and 40 nM, respectively. By contrast, 10 μM asperparaline A only slightly attenuated the peak current amplitude of the response to ACh of the avian α4β2, α3β4 and α7 nAChRs expressed in Xenopus laevis oocytes (12), suggesting a high selectivity for insect nAChRs.

Resolving the Enigma of Okaramines Having confirmed the effectiveness of the patch-clamp electrophysiology in elucidating the mode of action of asperparaline A, we next investigated the mechanism of action of okaramines. Unlike asperparaline A, okaramine B was capable of inducing inward currents in a concentration-dependent manner. In the current-voltage relationship, the peak current amplitude of the okaramine-induced current crossed the X-axis at a potential close to the equilibrium potential for the Cl- ion. Furthermore, the reversal potential shifted to a more positive potential when the extracellular Cl- ion concentration was reduced, suggesting that the okaramine-evoked currents were mediated by Cl- ions. Fipronil, a blocker for 127 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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ligand-gated chloride channels, attenuated the peak response of okaramine B, whereas mechamylamine, an antagonist of nicotinic acetylcholine receptors, had no such effect, suggesting that interactions at the ligand-gated chloride channels dictate the reversal potential for the okaramine-induced current (13). Glutamate-gated chloride channels (GluCls) as well as GABA-gated chloride channels (GABACls) are widely expressed in distinct regions of the nervous system of insects (14). Both GABACls (15) and GluCls (16) have variants that occur due to splicing and RNA editing. For the resistant to dieldrin (RDL) GABACls, 4 variants results from alternative splicing at exon 3 and 6. The RDL diversity affects the half-maximal concentration EC50 of GABA (17). In contrast, Bombyx GluCl creates variants by splicing at the exon 3 and 9 sites, and the exon 3 splicing-induced amino acid change affects the receptor density in the membrane (18). To determine the okaramines targets, we isolated cDNAs of the exon 3b/3d variants of Bombyx RDL (BmRDL) and exon 3b/9 (full length) variants of Bombyx GluCl (BmGluCl), since they were expressed most abundantly in the brain and the third thoracic ganglion of the larvae (13). Okaramine B activated only GluCl when tested on the exon 3b/3d variant of BmRDL and exon 3b/9 (full length) variant of BmGluCl expressed in Xenopus oocytes (Figure 3) (13). The BmGluCl-activating potential of okaramines were correlated with both the chloride current-inducing potential in the neurons (r2 = 0.964) (13). Similarly, a strong correlation was observed between the in vivo and in vitro activities (Insecticidal activity vs chloride current inducing activity, r2 = 0.936; insecticidal activity vs GluCl channel-opening activity, r2 = 0.914), suggesting that okaramines selectively activate BmGluCl and thereby induce toxicity in the larvae. Okaramine B was inactive for the human α1β2γ2 GABACl and α1β glycine-gated chloride channel, which suggested its selectivity for insects (13).

Figure 3. Okaramine selectively activates Bombyx GluCl expressed in Xenopus oocytes. Okaramine had no effect on the membrane currents in oocytes expressing the BmRDL, while inducing inward currents in a concentration dependent manner in oocytes expressing the BmGluCl. Applications of GABA and L-glutamate are indicated by horizondal lines, while those of okaramine B are indicated by dashed horizontal lines. Adapted with permission from ref. (13). Copyright 2014 Nature Publishing Group. 128 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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GluCls are the primary target of the macrocyclic compound ivermectin, which persistently activates the channel in a similar manner to okaramines (19). Hence, we investigated okaramine’s ability to displace [3H]ivermectin at exon 3c/9 (full length) variant expressed in HEK293 cells. Okaramine B reduced [3H]ivermectin binding to the GluCls in a non-competitive manner (20). Thus, the okaramines appear to interact with a different site than ivermectin in the GluCls. However, further studies are required to confirm this mechanism of action by using different GluCls as well as testing with radio-labeled okaramines. Furthermore, it is important to examine, not only the effects of mutations that reduce ivermectin sensitivity of GluCl on the action of okaramines, but also coapplication effects of the two compounds on the ivermectin- and the okaramine-induced response of the GluCls, for understanding the mode of action of okaramines.

Figure 4. Chrodriman B selectively blocks Bombyx RDL GABACl expressed in the silkworm larval neurons. The whole-cell patch-clamp electrophysiology was employed to record the membrane currents in the larval neurons. After recording the response to 30 μM GABA, 1 μM chrodrimanin B was applied for 1 min and then co-applied with 30 μM GABA. Applications of GABA are indicated by horizondal lines, while application of okaramine B is indicated by a dashed horizontal line. Adapted with permission from ref. (21). Copyright 2011 PLoS.

Chrodrimanins: Meroterpenoids Targeting GABACls Chrodrimanins are meroterpenoid compounds that are produced in okara as insecticides by YO-2 of the Talaromyces sp (1). The whole-cell patch-clamp electrophysiology was employed to show that chrodrimanin B had no effect on the membrane current in the silkworm larval neuron when applied alone, but the coapplication of 1 μM chrodrimanin B with 30 μM GABA completely blocked the GABA-induced currents (Figure 4) (21). Furthermore, it blocked the response to 30 μM of BmRDL with an IC50 value of 1.13 nM. The order of the IC50 values of 129 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

chrodrimanin A (143 nM), B and D (6.01 nM) for BmRDL were in accordance with the order of LD50 values of >100, 10 and 20 μg/g diet, respectively, for the silkworm larvae. Chrodrimanin B also blocked the GABA-induced response of human α1β2γ2 GABACl with an IC50 of 1.5 μM, which was approximately 1,000-fold higher than BmRDL (21).

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Concluding Remark Insect-active metabolites of various fungi are being continuously isolated from okara, a soybean-derived medium. Surprisingly, most metabolites have high selectivity for insect ligand-gated ion channels. The discovery of “ifungi”, or induced fungi formed by plant stimulants, may be a potential chemical library for next-generation pesticides.

Acknowledgments Kazuhiko Matsuda (KM) and Makoto Ihara (MI) was supported by KAKENHI (KM, grant number: 17H01472; MI, grant number: 16K21507) from the Japan Society for the Promotion of Science.

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