Natural and Engineered Pest Management Agents - ACS Publications

Chapter 2

Natural Products as Sources of Pest Management Agents Koji Nakanishi

Downloaded by UNIV OF AUCKLAND on May 3, 2015 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch002

Department of Chemistry, Columbia University, New York, NY 10027

Terrestrial and marine animals, plants, insects, and microorganisms produce numerous secondary metabolites. Some are for self-defense, symbiosis, sexual attraction, or to fulfill a variety of other purposes while some are simply metabolic degradation products. The structures of most such Natural Products are extremely cleverly designed so that not only are the skeletons constructed but even each functional groups are arranged in a manner to exert maximal effect. The design of natural products are very ingenious and "imaginative" and it is impossible for scientists to cope with Nature in terms of designing unprecedented bioactive structures. In the following are summarized some examples taken from our past and present studies; some were left at the stage of isolation and structure determination without further investigations, while more recent studies are being investigated beyond the stage of mere structural elucidation. No doubt a great number of natural products published in the literature and described with no mention of activity or simply listed to be "antibiotic" have to be reinvestigated. However, the most difficult is how to assay them, and this depends a great deal on a better understanding of the mode of bioactivity.

With the explosive advancement in biology and biochemistry in recent years, it is not surprising that Natural Products lost its glamorous status when structure determination was at its peak up to the 1970's. The situation has, however, changed. Previously, when a new structure was determined, it was simply regarded as providing a lead to more active compounds by synthesizing a myriad of derivatives. After 20 years of rapid advancement, application of modern molecular biology and/or biochemical techniques have now become routine, and it has become possible to start probing into the very difficult multidisciplinary question of what constitutes the basis of bioactivity, i.e., why is a compound active? The solution of this lies in understanding the interaction between a bioactive substrate and its receptor, often membrane proteins, on a truly molecular structural basis. Thus, it is not surprising that natural products chemistry is facing a revival. Natural product structures can now be reinvestigated from a totally different viewpoint which includes a rationalization of their mode of action. However, such an approach has just started, and requires collaborative efforts between all disciplines, chemists, biochemists, biologists, pharmacologists, biophysicists, clinicians, theorists, ethologists, etc. The artificially

0097-6156/94/0551-0011$06.00/0 © 1994 American Chemical Society In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS

12

divided disciplines in the past now have to be recombined to understand life and utilize natural products. It opens a totally new field that is extremely challenging. A Plant Hormone that Promotes Cell Arrest in the G2 Stage of the Cell Cycle.


Gap-2 (G2) refers to the stage in the mitotic cycle after cells have doubled their DNA content and which precedes mitosis (Fig. 1). Cells deprived of energy source will be predominantly arrested in the G l , the stage before DNA synthesis. An understanding and control of the mitotic cell cycle obviously has a direct bearing with cancer research. Natural substances that regulates this cellular proliferation are still poorly characterized glycoproteins called chalones. In contrast to the biopolymeric chalones, Evans and Van't Hoff characterized the physiological parameters of a small molecule in the cotyledons of the garden pea that promotes cell arrest in G2 after germination; many of the responses of this factor resembled those of the chalones (7). 3200 cotyledons from 3-day-old seeds of peas, Pisum

sativum

incubated 2 days in 160 flasks

Mitosls

/

50 μς isolation repeated 5 times: total 250 μQ

stage of cells G1 normal

80%

+trigon.

20%

Gap-2 / (G-2) ' ) I a

2

COCr

G2 20%

ce cell pr

proliferation cycle

Gap-1 (G-1)

Synthesis of DNA (S)

80% Me

trigonelline

Figure 1. Mitotic cycle, isolation and assay of N-methylnicotinic acid (2,3). Aseptic excision of 3200 seedlings of garden peas, incubation in 160 culture flasks for 2 days, and assay-monitored isolation yielded 50 μg of pure G2 factor, λπ^χ 265 nm; 5 repetitions gave a total of 250 μg of a very polar material. The factor turned out to be none other than N-methylnicotonic acid (trigonelline), a coupound isolated in 1895 and synthesized in 1896. After many failures to measure the MS, satisfactory HRMS was obtained with a sample presumably containing a mixture of the zwitterionic and the more volatile protonated form. FTIR, 80 MHz ÏH-NMR and a 9day measurement of its C-NMR clarified the structure (2,3). 13

Seed Germination Inhibitor. Extracts of plants collected in East Africa were tested for seed antigermination activity using a simple lettuce seed assay performed in a Petri dish or test tube. Extracts of the East African herb described in Fig. 2 resulted in the isolation and characterization of 4-hydroxyisoxazole ("triurnferor), a simple but new compound (4).

In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

Natural Products as Sources of Pest Management Agents13

2. NAKANISHI Triumfetta

500

(Tillaceae), East African medicinal herb

rhomboidea

g air-dried

leaves

)H 210

mg

triumferol

(4-hydroxyisoxazole) antigermination

against

lettuce

seeds


Figure 2. Seed antigermination factor from East African plant. Although many 3- and 3,5-disubstituted-4-hydroxyisozoles had been synthesized and tested for antigermination activity (5), the parent compound itself had never been synthesized. This is probably because all conventional syntheses failed and hence it may not have been clear whether it could exist at all. It was isolated, hence we knew it existed; a specific synthesis was therefore devised (4). What is its activity due to? Phytoalexins from Sweet Popato infected by Pathogenic Fungi. Infection of plants by pathogens lead to the rapid production of antifungal compounds for self-defense. The molecular mechanism of this rapid and efficient defense is an important problem but is still obscure. When the sweet potato Ipomea batatas is is invaded by the pathogenic black rot fungus Ceratocystisfimbriataand other fungi, ipomeamarone, a bitter sesquiterpenoid, rapidly accumulates in the infected tissue. The structure of this first phytoalexin was elucidated in 1956 by Kubota and Matsuura (6). As shown in Fig. 3 and 4, inoculation of the roots with fungi or treatment with HgCl2, incubation for 3 days and extraction yielded 7-hydroxycostal, a new class of phytoalexins (7), and ten new farnesol-derived ipomeamarones (8). Inoculation of roots of sweet potato {Ipomea batatas) with spore suspension of black rot fungus Ceratocystis fimbriata and other pathogenic fungi or HgCI . 2

Incubation for 3 days, 30°, extraction with CHCI /MeOH -> many 3

phytoalexins

!

7-hydroxycostata new class) ^CHO

ipomeamarone (Kubota, 1953)

HO 1 week incubation

0.45 g from 15 g CHCI effective germination

3

extract inhibitor

Schneider, Nakanishi, Chem. Comm. 1 9 83, 353

abs. config.: Schneider et al., Chem. Comm. 1 9 8 3 , 352

r|HC HO

biosynth.: Schneider et al., Chem. Comm. 1 9 8 4 , 372 10%

H-^F

Figure 3. 7-Hydroxycostal, a new class of phytoalexin. The absolute configuration of ipomeamarone was in conflict, but this was settled unambiguously by chemical correlations. (9). The 7-deoxyaldehyde was also produced but this was devoid of activity. It was therefore proposed that the antifungal activity of 7-hydroxycostal is due to the production of acrolein; indeed, a one week In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

14


incubation led to a 10% production of the by cyclic ketone. In view of the multitude of phytoalexins produced upon inoculation or injuring, this may provide an attractive and simple means of producing new antifungal agents in general.


Schneider et al., Phytochem., 19 84, 23, 759

Figure 4. New phytoalexins produced by fungal inoculation or HgCl2 injuring. Cabenegrins, Anti-snake Venom Factors from Cabeca ne negra. This problem evolved in a most unusual style, the general picture being still enigmatic and requiring further investigation. Dr. Laszlo Darko whom I had never met walked into my office and produced a bottle containing a colorless solution claiming it was an extract of an Amazonian plant "Cabeca de negrd\ and was used by plantation workers in the jungle as an antidote against snake and spider venoms. He had obtained it from a hunter who did not want to disclose the identity of the plant. Two-years of extraction, monitored by injections into mice the 2.5-fold lethal dose of the venom from a common South American snake Fer de lance (Bothrops atrox) indeed yielded two phytoalexins, cabenegrins A-I and II (Fig. 5). The minimal dosage required for survival was 2.0-2.8 mg/kg of A-I or A-II (70).

cabenegrin

A-I

cabenegrin A-II

Figure 5. Cabenegrins A-I and A-II, anti-snake venom factors.


2. NAKANISHI



Injection of 2.5 mg/kg of the snake venom into a 9 kg male beagle dog led to hypotension, followed by respiratory and cardiac arrest. However, injection of 1 mg/kg of A-I 15 min prior to i.v. injection of the venom restored all three symptoms after 1 hour; converseley, injection of venom followed by injection of cabenegrin also reversed the toxic cardiovascular effects. However, when A-I and -II were tested against some other snake venoms (at a snake venom laboratory in Okinawa) they were ineffective. The cabenegrins must be specific for the Fer de lance venom (and spider venom?). The colorless solution contained a more potent antidote, but the studies were discontinued because Dr. Vick who was performing the assays was retiring. Furthermore, we found that at least ten plants exist in South America called by the name of "Cabeca de negra". Not only would studies of the mode of action be interesting, but from a more pragmatic view point, further investigations to collect more material and characterize the more potent antidote(s) are necessary. The Brassinosteroids, Plant Growth Promoters. Following the isolation of the first steroidal plant growth promoter, brassinolide from rape pollen (77), 24-methylenebrassinolide and castasterone were characterized (Fig. 6). Fractionation of cat-tail pollen (25 kg), monitored by the standard rice lamina joint bending assay, led to the fourth brassinosteroid typhassterol; 1.7 mg from 25 kg of pollen (72). rice

lamina

b r a s s i n o l i d e ( r a p e pollen) Grove et al., Nature, 1 9 7 9, 281, 216 24-methylene: dolicholide{Dolichos lablab seeds) Yokota et al., Tett Lett, 1 9 8 * 2 3 , 4965.

joint

bending

assay

t y p h a s t e r o l (2-deoxycastasterone) cat-tail (Typha latifolia L ) pollen ι 25 kq

S

c

h

n

e

j

d

e

f 1 7 mg typhasterol ^

r

Tett

e t

a |

Lett, 1 9 8 3 , 24, 38559.

Figure 6. Brassinolisteroids, promising plant growth promoters. The contents of these plant growth promoters are minuscule, but all are relatively simple to synthesize in quantities. The low LD50, the potent plant growth activity, the ease in synthesizing analogs, growth promoting activity in some fungi as well as in plants, etc. make them attractive candidates for field use in agriculture. Note that typhasterol retains potent plant-growth activity despite the lack of the 2-hydroxy function. Plant physiology is far behind mammalian physiology, and no receptor for a plant hormone has yet been cloned (to my knowledge); moreover, the mode of action is still very obscure in most cases. Clearly, further research is required.


16



Intracellular Symbionts as a Source of New Antibiotics. Some insects, e.g., planthoppers, leafhoppers, aphids, and beetles, carry intracellular symbiotic microorganisms in the fat body which are transmitted to the next generation through the eggs. Incubation of the eggs at 35°C gives rise to insects with many defects, i.e., covered with mold, fail to reproduce, greatly reduced in size, etc. The chemical basis of these phenomena remained unaccounted for, due to difficulties associated in culturing these anaerobic organisms, until the successful incubation studies carried out by Nasu, Noda and coworkers (73,-75). In the case of the brown planthopper Nilaparvata lugens, it has been found that yeast-like symbionts biosynthesize a cholesterol intermediate from mevalonic acid and provide it to the insects which lack steroid biosynthetic capabilities; the planthopper then converts the intermediate into cholesterol, the precursor to the molting hormone, 20hydroxyecdysone (77). Insects lack immune systems and hence depend on survival by other means such as secretion of defense substances, cuticle formation, and rearing of symbionts. Thus selective killing of symbionts lead to infection by microorganisms and hence nymphs covered with mold. The systematic screening of symbiont cultures from eggs of several insects against crop pest microorganisms led to the characterization of a new antibiotic andrimid from Nilaparvata lugens (brown planthopper, collected in Thailand)(7S-20). As described in Fig. 7, it is highly specific against Xanthomonas campestris pv. oryzae, the white blight pathogen that invades the Thai rice plant; however, against Gram positive and negative bacteria including most plant pathogens it is inactive or only weakly active. The rational for this specificity may be that the

/ octatrlenoyl

/ " / / / D-p-phenylalanlne

/ /

"

\

u

\ L-vallne

\~ \

Me-succinimide stereochem. important methyl necessary

egg of brown planthopper/tf/aparvafa /ugens(Thalland). Intramolecular bacterial symbiont

very 3 Agrobacterlum*

Enterobactor

sp

andrimid (from culture broth) specific antimicrobial spectrum CorynebacterlumQ

Erwinla,34

Out of 21 Xanthomonap o n l y active againstX. campestrlspv. oryzae and a few others; alsoP rot eu s vulgarl^MlO

Pseudomonas

Inactive

(blight pathogen) at MIC 0.2 μ9/ηιΙ

0.^μg/m\

Figure 7. Andrimid, a potent and specific antibiotic from an intracellular symbiont. symbiont kills the pathogen invading the rice-plant in order to preserve its food source. Several other symbionts were also examined but so far they have only given rise to known antibiotics: Sogatella furcifera (planthopper) tested against Corynebacterium (canker of tomato) gave pyoluteorin and 2,4-diacetylphloroglucinol; P. stali (stinkbug) tested aginst Salmonella gave polymixin E i ; and S. micado (ambrosia beetle) tested against Corynbacterium yielded monoacetyl phloroglucinol In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

2. NAKANISHI


and 2,4-ddiacetyl phloroglucinol. Symbiont cultures should provide an unexplored source for the discovery of new bioactive products. Insect Antifeedants.


An obvious defense mechanism for plants against insects, birds, and animals is to produce feeding deterrents or antifeedants. Furthermore, it is not surprising that plants growing in tropical rain forests are exposed to many more natural hazards and hence are better developed in producing antifeedants. During my connection with the International Centre of Insect Physiology and Ecology, Nairobi, Kenya (1969-1977), we carried out searches for insect antifeedants, which was developed into a systematic and intensive survey by Isao Kubo (27, 22). Of the close to 30 antifeedants that were isolated, warburganal and azadirachtin are the two most potent (Fig. 8). Insect a n t i f e e d a n t s l

i ° H

^ ^ \ ^ \ ^

OH C

H

η

/

/\

\

0

warburganal Warburgia ugandensis

^ ^ ^ ^ Kubo et al., Chem. Comm., 1 9 7 6 , 1013. Nakanishi, Kubo, Israel J. Chem., 1 977, 16, 28.

azadirachtin:Azadlrachta /no7ca(lndlan neem tree) Mella azedarach(Chinaberry tree)

Zanno et al., 1 9 7 5 , 9 5 , 1975 Isolation: Schroeder, Nakanishi, J. Nat. Prod., 1 987, 5 a 241

Kraus 1986; Ley 1987; (Nakanishi 1987) cf. Taylor, Tetrahedron, 1987, 12, 2779 & following 3 papers: - 2830

Figure 8. Warburganal and azadirachtin, two potent insect antifeedants. Warburganal was isolated from the leaves and bark of Warburgia stuhlmannii and W. ugandensis, plants that are widely used as food spice and folk medicine in East Africa. Electrophysiological studies show that it irreversibly blocks the taste buds of Spodoptera exempta (African army-worm)(23, also7.sr. / . Chem.. quoted in Figure 8) so that when the army-worm is first placed onto a corn leaf treated with warburganal for 1 hour and then transferred to an untreated leaf, it starves to death. Warburganal exhibits potent molluscidal and antibiotic activity (e.g., Candida utilis 3.1 μg/ml (27). It is contained in "TADE", a spice traditionally accompanying sashimi, but upon injection exhibits acute toxicity (LD50 20.4 mg/kg against mice) and cytotoxicity (KB 0.01 μg/ml). Many syntheses of warburganal have been published. Electrophysiology tests for insect antifeedants are easy to set up and readily give impulse signals that reflect the responses visually and semiquantitatively. Azadirachtin was first isolated as a potent antifeedant against the desert locust by Morgan (24) who also elucidated partial structures (25). A structure was proposed in 1975 by us (Zanno et al., Figure 8) which was subsequently revised by Kraus (26), Ley (27) and later ourselves (28). Azadirachtin, isolated from the Indian neem tree In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

18


Azadirachta indica A. Juss is more than an antifeedant and exerts profound physiological effects on insects, the details of which are under study in various laboratories. It is by far the best known "antifeedant", and because ot its ready availability from the plant, it is attracting great interest; several companies dealing with azadirachtin have been formed in India and the US.


CAPE, an Antiinflammatory and Preferential Cytotoxic Agent from Propolis, Propolis, the brown gum made by honeybees on beehives has long been a popular folk medicine in Eastern Europe, the Near East and other countries, and is alleged to possess antibiotic, antiinflammatory and tumor-growth-arresting properties. Fractionation monitored by cytotoxicity assays yielded the simple but unknown caffeic acid phenethyl ester, which can be synthesized in a one-step reaction without protection/deptrotection (Fig. 9)(29). CAPE exhibits preferential cytotoxicity on tumor cells and is a potent antiinflammatory agent. Pharmaceutical studies are still ongoing. CAPE should be an intriguing subject for structure/activity and mechanistic studies. Is the role of the phenethyl moiety simply for increasing the permeability of caffeic acid, which is a well-documented antibiotic? What is the basis of its antiinflammatory activity? It should be noted that since propolis is collected by honeybees, the ingredients are not constant and would differ depending on the season, the region of collection, etc.

from p r o p o l i s dark-brown wax of beehives differential cytotoxicity observed in melanoma and breast carcinoma of: normal rat/human versus transformed rat/human cell lines potent anti-inflammatory agent S y n t h e s i s caffeic acid + phenethyl alcohol TsOH I reflux in benzene, 3 days Τ water removed by Dean-Stark trap CAPE 40% YIELD Figure 9. CAPE from propolis Phytoecdysteroids. At the time of the structure elucidation of ecdysone 1 (J0)(Fig. 10), the precursor of the insect molting hormone which Butenandt and Karlson had isolated (25 mg from 500 kg of silkworm pupae, a brilliant and exciting achievement), and Horn and coworkers had isolated 2 mg of 20-hydroxyecdysone 2 from 1 ton of crayfish waste (37), our group and that of Takemoto's had isolated large quantities of similar compounds which exhibited potent molting activity, the ponasterones 3 and inokosterone 4 (32, 33)(Fig. 10). 20-Hydroxyecdysone 2 is the representative and universal molting hormone of insecta and crustaceans, although other indogenous In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


2. NAKANISHI

Natural Products as Sources ofPest Management Agents19

ecdysteroid molting hormones have recently been found. Approximately 70 ecdysteroids have since been isolated from plants. The discovery of phytoecdysteroids obviously had an impact on insect physiology because it made these hormones readily accessible. Compared to 20-hydroxyecdysone 2, muristerone (5 in Fig. 10 with 14-β-ΟΗ) and ponasterone A 3 have 10-fold increased affinities to the ecdysteroid receptor of cultured Drosophila Kc-H cells (34). Treatment of muristerone with Nal-TMSCl in acetonitrile under argon resulted in removal of the 14-OH to yield 5, which exhibited an unexpected 5-8 fold further increase in the affinity for the ecdysteroid receptor, the strongest of all known ecdysteroids so far; this 14-deoxymuristerone 5 is also active in chromosome puffing and imaginai disk binding (34). 14-Dehydroxylation of various other ecdysteroids have resulted in a clearcut structure/activity relationship in the affinity for the ecdysteroid receptor; in Fig. 10, 6, the number η by parenthesized OH indicates an η-fold increase in affinity upon removal of that OH, whereas η by nonparenthisized OH indicates an η-fold increase with introduction of that OH (Stonard, R.; Trainor, D. Α.; Nakanishi, K.; Cherbas, P. unpublished).

(Achyrenthêt faurl*) Takemoto et al.: 1967

cherbas et al · 1982

structure/activity relations for affinity to ecdysteroid receptor of Drosophila Kc-H ce

Figure 10. Ecdysteroids. Preecdysone and Ecdysteroid Radioligands. The prothoracid glands (PTG) of insects produce ecdysone which is then oxidized to 20hydroxyecdysone in peripheral tissues. Gilbert and coworkers found that incubation of tobacco hornworm (Manduca sexta) PTG in the presence of a hemolymph protein fraction (HPF) increased the ecdysone cohntent 8-fold, thus an unknown substance X which is converted into ecdysone must be present (Fig. 11). Usage of RIA antisera H-2 and H-22, which specifically recognize side-chain and ring A modified ecdysteroids, respectively, showed substance X to be ring A modified. Microscale chemical and spectroscopic methods, including CD measurements that showed the presence of an extra sdaturated carbonyl group (Cotton effect at 287 nm), showed substance X t be a 2:1 mixture of 3-dehydrœcdysone 1 and 2-dehydroecdysone. This led to the intriguing question as to whether these were preecdysones or their storage forms (35). It has recently been shown that indeed in crustaceans, 3-dehydrœcdysone is a precursor to ecdysone (Y. Naya et al., in press; see below). A 4 step derivatization of inokosterone (Fig. 10, 4) yielded 26-iodoponasterone which turned out to be an ecdysteroid with the strongest affinity to the ecdysteroid receptor (Kd 3.8xl0 M , 20-fold of 20-hydroecdysone) after 14-deoxymuristerone -10


20


(50-80 fold, Figure 9, 5) (56, 57); the radioactive analog 2 (56) has been used widely in locating the ecdysteroid receptor and used in its receent cloning (38). For the purpose of investigating the tertiary structure of the ecdysteroid receptor the potent photoaffinity labeled ecdysteroids 3 and 4 linked to a new phenyl acetate moiety, which readily reacts with hindered secondary OH groups (i.e., 26-OH in inokosterone) have also been prepared (59)(Fig. 11). Present in prothoradc gland. Preecdysone or storage form?

Manduca

sexta "Substance X"

a) Incubation of PTG with HPF -> ecd. titer X 8


b) RIA with H-2 and H-22 antisera -> ring A modified 1 ) 3-dehydroecdysone

c) Extra CD at 287 nm -» η,π* of satd. ketone

[ HJAPA:

1 9

C j / m m o l

OH Ο

APApH]

2175 Ci/mmol 20-hydroxyecdysone

2)

26(27)-[

125

l]lodoponasterone

A

2-f H]azldophenyl

acetate

4 ) Inokosterone

26(27)

[ H ] a z l d o p h e n y l acetate 3

Figure 11. Preecdysone and ecdysteroid radioligands. Ecdysone Biosynthesis Inhibitor (EBI) of Crustaceans. Since the early 1900's it had been known that removal of eyestalks from crustaceans leads to precocious ecdysis, thus suggesting the presence of a "molt-inhibiting hormone (MIH)". Using 4,000 blue crabs Callinectes sapidus , Nay a and coworkers finally succeeded in identifying this endogenous compound contained in the X organ of theeyestalk as 3-hydroxy-l-kynurenine (3-OH-K)(Fig. 12), a key metabolite of Ltryptophan (40). It was further found that during the flow of 3-OH-K to the Y-organ complex (YOC) located at the stem of the eyestalk, the site of ecdysone biosynthesis, it was converted into xanthurenic acid, the real ecdysone biosynthesis inhibitor. Xanthurenic acid interferes with the hydroxylation of cholestrol to ecdysosne by interacting with the P-450 system, but does not block the hydroxylation step leading from ecdysone to 20-hydroxyecdysone (41, 42). More specifically, the 8-OH of xanthurenic acid coordinates with the P-450 central iron (42). Extensive physiological studies performed recently on the American crayfish (Procambarus clarkii) have clarified the following (Naya, Y.; Ikeda, M . Pure & Appl. Chem., in press): (i) 3-dehydroecdysone (Fig. 11, 1) is the precursor of ecdysone in crustaceans; (ii) 3-dehydroecdysone is not produced upon incubation of the Y organ homogenate, presumably due to destruction of membranes involved in its biosynthesis, but is formed when the total Y organ complex is incubated; (iii) xanthurenic acid also inhibits the ecdysone biosynthesis in the silkworm upon addition to the PTG culture with a detergent. Oligopeptides with molt inhibitory activities have recently been reported for crustaceans (43, 44); there clearly must be a close relation between these putative MIHs and EBI but currently this is not clear.



2.

NAKANISHI


Figure 12. Ecdysone Biosynthesis Inhibitor of Crustaceans (Adapted from ref. 42). Philanthotoxin, Noncompetitive Antagonist of Glutamate and nACh Receptors. The glutamate receptors (Glu-R) are implicated in neurological disorders such as Alzheimer's, Huntington's, Parkinson's diseases, epilepsy, ishemic damage due to strokes, as well as in learning and memory. In recent years many subtypes of the GluRs are being rapidly cloned and are attracting great interest in various fields. The venom of the Egyptian solitary digger wasp Philanthus triangulum, first noted by T. Piek (45), is an antagonist of transmission at the quisqualate-sensitive glutamate synapses (QUIS-R) of locust leg muscles. The major component is a polyamine with the butyryl / tyrosyl / thermospermine sequence, philanthotoxin-433 (PhTX-433)(46, 47). In attempts to obtain radioactive (^H or ^l) photoaffinity-labeled analogs with higher biological activity, close to 100 analogs have been synthesized systematically by dividing the molecule into 4 zones and by making single to multiple changes in respective zones (48, 49). Although it was initially hoped that the philanthotoxins be selective for the QUIS-R, it was found that they inhibit both N-methyl-D-aspartate (NMDA)(50, 57) and kainate receptors (52) as well; in addition, they also inhibited nicotinic acetylcholine (nACh) receptors noncompetitively(55). Certain types of analogs were more selective for QUIS-R while others were more selective for nACh-R (54) . The results of electrophysiological assays using locust muscle for the QUIS-R (55) and [ H]perhydrohistrionicotoxin displacement assays usingTorpedo electric organ for the nACh-R (54) are summarized below; the NMDA-R assays were performed by binding studies with [ H]MK-801 using rat brain cortex (54) and injection of rat or chick brain RNA into Xenopus oocytes (50, 51) but results are not given in Fig. 13 because they were similar to those of nACh-R (54). The nonspecificity of the PhTX analogs make them poor ligands for receptor identification. However, the availability of many radioligands with photoaffinity labels at various sites make them suitable probes for isolating receptors from relatively pure receptor sources. More importantly, the distribution of photoaffinity groups along a long molecule make the analogs unique "rulers" for probing the tertiary structures and the mode of action of receptor channels (Fig. 14). Since no Glu-R protein has as yet been secured even in a minuscule amount for structural studies, our current effort is focused on the much more readily available nACh-R (Fig. 14), composed of 5 subunits forminga channel, i.e., α, α, β, γ, and δ. 3

3


22

NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS PhTX-343

ICso

QUIS-R

locust

nACh-R QUIS-R i) hydrophobic ii)

I > Br > 2

[ H]perH-histrlonicotoxln

nACh-R enhanced

anchor

muscle contraction

activity

:

2

2

2.6

χ

M

10

M

iodination Tyr->

CI >F

: 2.3 χ 10 :

3

S, R configurations

Leu, Ala, Phe, Gly, Trp

: similar

polyamine

chain

activity essential chain

enhances

activity


I I

η-Bu here enhances hydrophobicity, hydrophilic

aromaticity

groups

kill

enhances

activity

activity

activity

(nACh-Fj

Figure 13. PhTX-433 Structure-activity relationships. nACh-R

30-40 A

synapse

cytoplasm (Hucho & Hilgenfeld,

FEBS Lett.,

Natural and Engineered Pest Management Agents - ACS Publications

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