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Chapter 5

Advances in Research and Development of Avermectins H. Mrozik

Downloaded by UNIV OF BATH on October 29, 2014 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch005

Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065

Avermectins have potent biocidal activities against a wide spectrum of nematodes, insects, and arachnids. The semisynthetic derivative ivermectin (22,23-dihydro-avermectin B1) has been in wide use as a broad spectrum endectocide in animals for the last decade. The major agricultural pesticidal uses of Abamectin (avermectin B1) currently are as a miticide in crops such as citrus, pear, deciduous tree nuts, ornamental plants, cotton, vegetables and strawberries. Chemical modifications have been carried out with the aim of increasing its insecticidal spectrum, residual activities and chemical stabilities. Emamectin (MK-244, 4"-deoxy-4"-epi-Nmethylaminoavermectin B1), one of many 4"-substituted analogs, has greatly increased potency against lepidoptera larvae. Mode of action studies recently progressed towards the identification of an avermectin binding protein with a molecular weight of approximately 50 kD.

The avermectins are a group of closely related macrocyclic lactones with exceedingly potent activities against helminths and arthropods. They are produced as a mixture of eight components by fermentation of the microbe Streptomyces averminlis. Their chemical structure is related to the milbemycins, which were described first in 1974 by workers at Sankyo as very potent miticides and insecticides for crop protection. In 1975 Merck scientists discovered the structurally closely related avermectins as highly potent endo and ectoparasiticides with a wide spectrum of activities mainly for animal, but also certain human applications. Similar interesting anthelmintic activities were subsequently also described for 13deoxyavermectin aglycones and for the milbemycins by the Merck group. Ivermectin, the 22,23-dihydro derivative of avermectin B1, is a chemically modified derivative of this group of natural products that has found wide use as a systemic antiparasitic agent against endo and ectoparasites of animals. Ivermectin is also used as a treatment for human filarial worm infections ( Onchocerca volvulus, River Blindness ) . The scientific and commercial success of ivermectin 1,2

3

4

5,6

7

8

9

10

n

0097-6156/94/0551-0054$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.

5. MROZIK

Advances in Research and Development of Avermectins

55

as an endectocide for animal health attracted the interest of organic chemists to the total synthesis of the interesting structures, and of biochemists to the investigation of the novel mode of action. It also encouraged many drug companies to search for their own avermectin or milbemycin. 12,13


14,15

Antiparasitic Efficacies Although the concern of this publication is agricultural pest managment, this article will review some of the animal and human health data, since the major impact of the avermectins lies in that area, and crop protection is of secondary importance. Currently two avermectin derivatives, ivermectin and avermectin B l , are commercially available for animal health applications. A third one, doramectin, is under development by Pfizer ( FIGURE 1 ). Avermectin B l , with the generic name abamectin, is the major and most important product of the fermentation of Streptomyces avermitilis. It also serves as the starting material for the chemical conversion to ivermectin via selective hydrogénation of the 22,23 double bond. Milbemycin derivatives have only recently become commercially available. Milbemycin A ^ / A ^ has just been introduced in Japan for crop protection, milbemycin D is used in Japan as an anthelmintic agent, milbemycin A^/A^ 5oxime is marketed by Ciba-Geigy as an anthelmintic exclusively for dogs, and moxidectin is being developed by American Cyanamid as a broad spectrum animal health drug ( FIGURE 2 ). Ivermectin has been on the market since 1980 as an endectocide for animals. Its special advantage over conventional anthelmintic agents is its wide spectrum against gastrointestinal and systemic parasites as well as against many ectoparasitic insects with a single application. Ivermectin is used at the unprecedented low dose of 200 ug/kg in cattle, sheep and horses. The small dose allows for easy formulation in oral, parenteral, and topical applications. It is used in swine at 300 ug/kg, the higher dose being required due to higher metabolism in this species. In dogs it is used under the name Heartgard 30 for the prevention of heartworm infections at the low dose of 6 ug/kg, and is effective with one single monthly application. It has been used by several hundred thousand humans with a single dose every six months at 50 to 200 ug/kg to alleviate the most damaging symptoms of onchocerciasis, or river blindness. It is highly efficacious against a wide spectrum of parasitic nematodes and many ectoparasitic species from the grub, lice, mite, tick and bot families. Representatives of each of these, for instance, are responsible for economic losses in cattle or sheep. No activity, however, has been observed against flatworms, protozoa, bacteria or fungi. Ivermectin has revolutionized the treatment of man for onchocerciasis in Africanriverregions, where a good part of the adult population becomes blind due to irritation of the eys caused by the microfilaria, and where children are seen to guide their blind fathers to the fields for their work. Most parasites have complicated life cycles with obligate intermediate hosts. Onchocerciasis, as an example, is spread by the "Black Fly" which lives in fast flowing rivers and picks up Onchocerca volvulus microfilariae from human blood while feeding. After molting in the insect the infective larvae are then reintroduced into man, where they migrate through the lymph system and finally settle as adults in nodes under the skin. The females deliver microfilaria by life birth, which then spread via the bloodstream over the body. They cause particular damage to the eyes, leading in severe cases of repeat infections to blindness. Eventually the microfilaria are picked up by the flies for a new cycle. It was found that mass treatment of whole villages in Africa so drastically reduces the number of microfilaria in human blood that the subsequent infection rate was greatly reduced. Although ivermectin possesses 16

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

Ivermectin: X = -CH CH Abamectin: X = -CH=CHDoramectin: X = -CH=CH2

2

R25 = CH(CH )CH CH and CH(CH ) R = CH(CH )CH CH and CH(CH ) R = Cyclohexyl 25

3

2

3

3 2

3

2

3

3 2

25

Figure 1. Avermectins for animal and human uses.

0-

Milbemycin D: Milbemycin

! \ 5 / V

R25

=

-CH(CH )

= =N0H

R25

=

CH andC H

=

R23

=

=NOCH

R5

=

5-Oxime:

R

5

Moxidectin:

R

5

— O H

— O H

3

3

2

2

3

R

5

2 5

-YY

Figure 2. Milbemycins for animal health.


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57

considerable activities against the major gastrointestinal worm infections of man, it is judged to be insufficiently efficacious for this application. Agricultural Applications With a good part of development costs and safety testing absorbed by the animal applications, it was possible to introduce abamectin, the natural precurser of ivermectin, for agricultural uses. One derivative of abamectin is currently under development for crop protection, and veryrecentlymilbemycin A^/A^ has become available in Japan for agricultural uses. Avermectin B l , or abamectin, possesses in laboratory assays particularly high toxicity towards mites with an LCQQ of .02 to .03 ppm, while it is generally less toxic against insects. For example lepidoptera larvae such as the corn earworm ( Heliothis zea ) and the southern armyworm ( Spodoptera eridania ) require 1.5 and 6.0 ppm respectively to achieve an LC^Q. The toxic effects are mainly caused by feeding of the insects on treated foliage. Abamectin is a stomach poison, and is less effective on contact. There are no ovicidal activities, and the onset of activity coincides with the beginning of the feeding of the larvae. As suggested by the assay results, the primary commercial use of abamectin is as an acaricide. In combination with paraffinic oil it is used against citrus rust mites at 13.5 to 54 g ai/ha on citrus fruit, spider mites on ornamentals and on cotton at 9 to 22 g ai/ha, the twospotted spider mite, European red mite, pear rust mite on various crops such as tomatoes, canteloupe, strawberries, pears and similar high value crops due to its considerable cost. Leafminers are controlled on ornamentals and vegetables, and psylla are significantly reduced on pears. Varying potency of .02 to 6.0 ppm against lepidoptera allows select uses for tomato pinworm control at 11 to 22 g ai/ha, and for control of the diamondback moth, which is highly susceptible. Abamectin is considered safe for the environment due to a rapid breakdown in UV light through photooxidation. Its halflife in the environment in bright sunlight is 4 to 24 hours. It is tightly bound to the soil, which prevents it from being washed into aquatic bodies. It's halflife in soil is 20 to 40 days. All residue studies in crops point to rapid degradation. It is, however, acutely toxic to fish at 3 to 40 ppb, but accumulation in aquatic organisms is low, so that its concentration is not likely to be multiplied through a food chain. Honeybees are very sensitive, but in practice little effect is seen due to the rapid depletion of abamectin residue.


17,18

19

Structural Modifications of Avermectins Avermectins have been chemically converted by selective hydrolysis of either one or two of the oleandrose glycoside bonds to monosaccharides and aglycones. Deoxygenation of the 13-hydroxy group gave the 13-deoxyaglycones, which are structurally closely related to the milbemycins ( FIGURE 3 ). Conversely, somewhat costly chemical and microbiological procedures exist to introduce a 13-hydroxy group into milbemycins and to attach the sugars to yield avermectins. The modification of the multifunctional molecule was largely guided by chemistry. It was discovered early on that derivatization of the 4"-hydroxy group retains the high anthelmintic activity against the sheep nematode Trichstrongylus colubriformis in a gerbil in vivo assay, but that the free 5-hydroxy group was required for activity. A number of 4"-esters and carbamates were highly active anthelmintics. Selective protection of the 5 and 7 hydroxy groups is possible, and subsequent reaction of the 4"-hydroxy group with the fluorinating reagent diethylaminosulfur trifluoride (DAST), for instance, gives via an internal oxonium ion the expected 4"-deoxy-4 -fluoro analog plus a ring contracted isomer (FIGURE ,,




58

Figure 3. Avermectin aglycones.


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59

4). The fluoro analogs, however, had somewhat reduced biological activities. On the other hand, 4"-epi-hydroxy, oxo, cyano, even deoxy, and particularly methyl analogs have good activities in the twospotted spider mite and southern armyworm assays. One also often observes with a minor modification a shift in spectrum, as seen with the southern army worm activities of the two epimeric 4"-methyl derivatives (EC^Q = 0.5 and 8.0 ppm, respectively). 4'-Amino Substituted Second Generation Avermectins For a number of reasons we were interested in the introduction of a basic amino group into the avermectin molecule. This should change its physical properties, make it more polar, and result in a different tissue distribution. In addition, most of the antibacterially active macrolide antibiotics contain an aminosugar. After suitable protection and deprotection, reductive amination of a 4"oxo intermediate gave the 4 -amino-4"-deoxy-analog as major reaction product ( FIGURE 5 ). When testing these new amino analogs we discovered that they had reduced potency in our brine shrimp assay and also in the twospotted spider mite assay in comparison with abamectin. A substantial increase in efficacy, however, was discovered in the southern army worm assay, where the EC^Q of the amino and epi-amino analogs were 80 and 400 fold lower, respectively, than those of abamectin. Since we had observed earlier better activities in the southern armyworm assay also for certain monosaccharides, we prepared their 4-amino derivatives as well as the 13-amino aglycone, but did not observe further improvement of biological activities. We then looked at alkylamino analogs and found the monomethyl analog the most promising of the group. Further modifications with acylamino, aroylamino, sulfonamido, hydrazino, amidino, cyanamido, azido, carbamino, ureido, or hydrazono substituents represent mostly potent derivatives, and some of these are of considerable interest as endectocides for animal health, but none surpassed the epi-monomethyl derivatives in potency against lepidoptera species ( TABLE 1 ).


,,

H

2 0

4 -Deoxy-4"-epi-N-methylaminoavermectin B l , or M K 244, with the generic name emamectin, is currently under development as an agricultural insecticide, particularly against lepidoptera species ( FIGURE 6 ). It is reserved initially for high value crops, since it will be a rather expensive compound. The foliar ingestion toxicities of MK 244 for a number of important insects such as tobacco hornworm, cabbage looper, beet armyworm, fall armyworm, Colorado potato beetle are from .003 to .03 ppm ( LC^Q ), for Mexican been beetle and twospotted spider mite .2 to .3 ppm, but for bean aphid as high as 20 ppm. The methylamino compound MK-244 showed topical potency superior to the amino analog against the three lepidoptera species of southern armyworm, tobacco budworm, and corn earworm. MK-244 is considerably more potent in a foliar residue test against southern armyworm and tobacco budworm than thiodicarb or fenvalerate. 21

Structural Modifications of the Spiroketal of Avermectins More recently we, as well as others, directed our attention to the modification of the spiroketal ring, in particular the 24 and 25 substitutions. Avermectin B2 is a fermentation product distinguished by an axial hydroxy group at the 23 position from abamectin, which contains a 22,23-double bond. This was a readily available starting material for the desired reaction sequence ( FIGURE 7 ). To this end suitably 4",5-diprotected avermectin B2 was oxidized to the 23-oxo analog, reacted via the 22,23-en-23-ol trimethylsilyl ether, and the 22,23-oxide to




60



OCH,

2

3

H2N,,

1

, .

- ,

o

9CH3

J.

4"-Epiavermectin B1a

N

4"-Amino-4 -Deoxy-3"--Epiavermectin B1a

4"-Amino-4"-Deoxyavermectin B1a

Avermectin B1a

HJU^O^'''O...

Figure 5. Synthesis of 4 —amino—4"—deoxyavermectin derivatives.

M

" 3 °X '

4) p-TsOH - H 0 - MeOH

4"-EPI-Amine-4"-Deoxyavermectin B1a

" χ ι

0CH3

4

3) NH OAC - N a C N B H

2) Oxalyl Chloride - DMSO

1) t-Butyfdimethylsilyl Chloride


62


Table 1 Biological Activities of Aminosubstituted Avermectin Derivatives Avermectin Analogs

Two-spotted spider mite assay E C (ppm)

263 2600 1730

0.03 0.25 0.25 1.25

8.000 0.100 0.020 0.500

4'-epi-amino-4'-deoxy Bj monosaccharide 4'-epi-amino-4'-deoxy22,23-dihydroB monosaccharide

>0.25

>0.500

>0.05

0.100

13-amino- 13-deoxy-22,23dihydro-Bj aglycone

>0.10

> 1.000

1730 1300 1730 >55500

0.25 >.05 0.25 0.25 0.25

0.004 0.020 0.020 0.020 0.100

4"-epi-CH CO-NH4"-epi-CH CaMeN4"-epi-C H CO-NH4"-epi-(Me) NNH-

540 650 28000 430

0.25 0.50

0.500 0.050

0.05

0.100

4"-epi-CH S0 -NH-

430

0.05

0.100

0.25 >.05 >.05

0.100 0.500 2.000

0.05 1.25 0.05

0.100 0.500 0.500

IC


Southern armyworm assay EC (ppm)

Brine Shrimp 100 ng/ml

Bl( ABAMECTIN) 4"-amino-4"-deoxy B^ 4"-epi-amino-4"-deoxy Bj 4"-epi-amino-4"-deoxy22,23-dihydroB

9 0

90

1

1

4"-epi-MeNH4"-epi-(Me)2N4"-epi-(Me) CHNH4"-epi-C H CH NH4"-epi-H C(CH ) NH2

6

5

2

3

2 7

3

3

6

5

2

3

2

4"-epi-(CH ) NCH=N4"-epi-HN-CN 4"-epi-N 3 2

3

4"-epi-CH 0-CO-NH4"-epi-NHCONHCH 4"=Ν-ΝΗΟΟΝΗ 3

3

Λ

430



5. MROZIK


4" -Deoxy-4 -epi-Methylaminoavermectin B1 Benzoate Salt M

Figure 6. MK-244 / Emamectin.


63


64


C-22 Aldehyde TBDMS = tert-butyldimethylsilyl TMS = trimethylsilyl MCPBA = metachloroperbenzoic acid DMSO = dimethylsulfoxide (COCI)o = oxalyl chloride Pb(OAc) = lead tetraacetate 4

T.L SHIH et al., Tel Letters 1990, 31,3525-3528

Figure 7A. Degradation of avermectin B2 to the starting materials for C24/25 modified avermectins. (Reprinted with permission from reference 22. Copyright 1990 Pergamon Press Ltd.)


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H


Wittig. React.

HÇ'

J 9^

65

OMe

+

'"

Ο Μ θ

l(Ph) PCH CHR CHR OTMS 3

2

24

25

'"H


C-22 Aldehyde C-21 Epimeric Mixture

H

HÇ'

Deprotection C - 24 / 25 Substituted Avermectins R

24

R

25

= H, ALKYL, ARYL = H, ALKYL, ALKENYL, ALKINYL, ARYL, OXO-, THICK AMINO-ALKYL,

Figure 7B. Synthesis of new 24 and 25 substituted avermectins.


66


1) BOM-CI/Et N 2) LiAIH 3) SWERN 3

CH J,

3

CH j,

4

OCH.

3

Ο

BOM-O

BOM-O


CUO "V^^Cu' '

nr

n r iO

u

»H

Ο

BOM-O Ratio 73:27

1) 2) 3) 4) 5)

Separate Isomers Na / NH / EtOH TsCI / Et N Nal PH P 3

3

3

6) TMS - NMe 7) C-21-Aldehyde 2

8) Cyclization 9) Deprotection l Ph3P© G

OTMS


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67

the rearranged 22-hydroxy-23-ketone. This is then cleaved to the 22,23-seco compound, which gave upon methanolysis the desired C-22 aldehyde intermediate for further synthetic modifications. The epimeric mixture of this aldehyde is now used for a new carbon-carbon bond formation with a Wittig reagent containing the desired C-24 and C-25 substituents. The C-21 epimeric mixture of avermectin B l precursors is then equilibrated with pyridinium tosylate to the thermodynamically more stable natural avermectin B l analog. After deprotection a wide variety of C-24 and C-25 substituted analogs with natural and epimeric stereochemistry are obtained. This approach lends itself also to the chiral syntheses of analogs with predetermined stereochemistry at C-24 and C-25. Starting with methyl (S)-(+)-3hydroxy-2-methylpropionate and reaction with a cyclohexylcuprate synthon gives the chiral Wittig reagent needed for the synthesis of 25-des-(2-butyl)-25 cyclohexylavermectin B l . This compound was also obtained via directed biosynthesis and is currently under development by Pfizer. The activities of the new compounds in the brine shrimp assay suggest that none of them are more active than avermectin B l . Additional testing in an in vivo anthelmintic model assay confirmed anthelmintic activities for these compounds. In a definitive sheep test against a spectrum of gastrointestinal nematodes again little difference from avermectin B l was shown in potency and spectrum. In a twospotted spider mite laboratory assay the 25-phenyl analog, for instance, had activity comparable to that of abamectin. Although many of these compounds are potent avermectin analogs, they did not show any significant improvement over the parent compound. We used a related synthetic scheme to prepare derivatives where the 6,6-spiroketal of natural avermectins was modified to a 6,5-system ( FIGURE 8 ). A series of reactions starting with a C-22-aldehyde and a stabilized Wittig or Horner-Emmons olefination reagent, subsequent reduction of the double bond, reduction of the carbonyl to an epimeric mixture of alcohols, cyclization and deprotection gave a series of analogs as epimeric mixtures at C-24, which were separated chromatographically. Either one of these stereoisomers could also be prepared by stereospecific reduction of the carbonyl group with a chiral oxazaborolidine-borohydride complex. A wide variety of these derivatives were prepared in yields from 30 to 76 % from the avermectin B2 intermediate ( not optimized ). These 6,5-spiroketal compounds are 22


23

24

potent anthelmintic and insecticidal compounds, but no significant advantages over abamectin could be found in subsequent tests. Additional C-25-substituted avermectins were also obtained by directed biosynthesis. Incorporation studies revealed that natural avermectins are produced in the fermentation medium from 7 acetate and 5 propionate building blocks, which account for all the carbon atoms except for C-25 and the attached C-25substituent. These atoms are not labelled by acetate or propionate. Instead it was found that isoleucine or 2-methylbutyrate are incorporated into the 2-butyl group of avermectin Β la. Subsequently it was shown that addition of 2-methylpentanoate and 2-methylhexanoate to the avermectin fermentation gave additional products corresponding to mono and bis homologs of avermectin Β l a . A systematic approach by scientists at Pfizer using a mutant devoid of branched chain 2-oxo acid dehydrogenase, which blocks the formation of 2-methylbutyrate, and addition of a wide variety of carboxylic acids lead to avermectin derivatives with modified C-2525

26

27

28 20

side chains. ' Mode of Action and Avermectin Binding Site Many pharmacologic effects of avermectin B l in a number of different animals and tissue preparations have been described. Nematodes are paralyzed rapidly without causing hypercontraction or flaccid paralysis. Signal transmission from ventral interneurons to excitatory motorneurons of Ascaris are blocked. A 14

30

30



68


0Μ·

4", 5-bisOTBDMS7-OTMSAVM 4



-AVM 7

a) Pb(OAc) , MeOH, pyr; b) (MeO) P(0)CH C(0)R, LiCI, DIEA; c) Ph PCHC(0)R; d) 9 eq Na S 0 ,18 eq NaHC0 ;1:1 PhH:H 0, reflux; e) Pd(PPh ) , nBu SnH; f) NaBH ; g) RMgBr; h) BH SMe , oxazaborolidine, 0° C; i) 4:1 PPTS:TsOH; j) HF.pyr 4

2

3

2

2

3 4

3

3

4

2

3

2

4

2

OMe

R

Yield (%, 3 — » » 7)

H Me i-Pr f-Bu n-C H c-C H^ CH OH

62 42 68 76 41 59 48

8

6

2

1 7

R CH OMe CH OPh Ph (p-F)Ph (/>MeO)Ph 2-Furyl OMe 2

2

Yield (%, 3

—**7)

57 43 38 30 42 25 56

Figure 8. Top: 25-NOR-6,5-spiroketal avermectins. Bottom: 25-NOR-24substituted avermectin analogs. (Table reprinted with permission from reference 24. Copyright 1992 Pergamon Press Ltd.)


70



1 2 3 4 5

« 5 3 KD - « 4 7 KD

« 8KD

Ivermectin Concentration Lane 1 Lane 2 Lane 3 Lane 4 Lane 5

0 2x10- M 8x10- M 2x10" M 2x10" M 10

10

9

8

Figure 9. Photoaffinity labeling of the C. Elegans avermectin receptor. (Reprinted with permission from reference 37. Copyright 1992 National Academy of Sciences.)


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69


reversible increase of chloride ion permeability of GABA sensitive fibers of the extensor tibiae muscle of the locust Schistocerca gregaria is observed at nanomolar concentrations. In contrast an irreversible inhibition of GABA sensitive and insensitive muscle fibers of Schistocerca gregaria is observed at micromolar concentrations. A reversible opening of crayfish stomach chloride channels is observed at subpicomolar concentrations, and irreversible opening of crayfish stomach chloride channels at 10 pmol or higher. Avermectin binds specifically to a number of chloride channel proteins but its binding site is distinct from that of all other effector molecules. A high affinity binding site was described for a membrane preparation of C. elegans, a free living nematode, which is known to be very sensitive to avermectins. Recently the protein containing the binding site was solubilized without much destruction as suggested by the almost identical dissociation constants for ivermectin of 0.14 and 0.18 nM, respectively. The binding affinity of several avermectin derivatives closely correlates to their biological activities. The highly potent ivermectin, the water soluble avermectin Β1 phosphate, and avermectin B2 have the lowest Kj values. In contrast the biologically inactive octahydroavermectin Β ^ does not inhibit the binding of tritiated ivermectin. Aglycones and monosaccharides with intermediate activities have intermediate inhibition constants. 31

32

33

34


35

34

For the further identification of the binding proteins a photoaffinity probe was constructed from suitably protected 4"-aminoavermectin B l . 4-Azidosalicylic acid was attached to the aminogroup through a beta-alanyl-omega-aminocaproyl spacer. After iodination to the 4-Azido-3- iodosalicylic acid derivative it was found suitable for the radioactive labelling of avermectin binding proteins. The 36

12s

specific binding of I-azido-AVM in the dark to C. elegans membranes occurs with a = 0.136 nM, which is comparable to that of ivermectin. It also is inhibited by ivermectin. Frozen C. elegans membrane bound receptor proteins are obtained from the free living nematode grown in liquid culture with E. coli as food source. After flotation on 35 % sucrose, washing with 0.1 M NaCl the worms were homogenized in HEPES buffer in the presence of protease inhibitors. Centrifugation at 1000 χ G and subsequent centrifugation of the supernatent at 28000 χ G gave a pellet containing the membrane bound receptor proteins, which was resuspended in HEPES plus protease inhibitors in a concentration of 5 mg/ml protein. The solution was dialysed and frozen. The frozen membranes were thawed, diluted, and stirred with TRITON X for 1 hour at 0° C for solubilization of the proteins. Centrifugation and filtration gave the TRITON X solubilized protein solution containing the avermectin binding site. 37

125

After further dilution the protein mixture was incubated with excess Iazido-AVM in the dark, and the unbound I-azido-AVM was removed with charcoal. Photolysis with UV light and precipitation of the proteins with methanol gave a mixture containing the I labelled avermectin binding proteins. The Coomassie stained gel after electrophoresis shows the bands of C. elegans proteins after incubation and photolabelling in the presence of 0.0,0.2,0.8, 2.0, and 20 nM 125

25

37

125

unlabelled ivermectin. The I labelled avermectin binding proteins are, however, not visible in the Coomassie stained gel due to their very low concentrations, and all lanes are identical in this gel. The autoradiogram of the same gel ( FIGURE 9 ) shows three protein bands labelled by I-azido-AVM with molecular weights of 53,47 and 8 kDa. With increasing concentrations of unlabelled ivermectin from left 125


5. MROZIK


71

torightthe binding of I-azido-AVM decreases. The autoradiogram of a similar gel shows the binding experiment in lane 1 with I-azido-AVM only, in lane 2 and 3 after addition of high level cold ivermectin, which inhibits the binding of 125


I-azido-AVM and thus the labelling of the binding proteins. Lane 4 and 5 show addition of high concentrations of 3,4,8,9,10,11,22,23-octahydro avermectin B p a close analog of ivermectin, which is known to be completely inactive and is not binding to the avermectin receptor. Consequently it does not interfere with the labelling of the avermectin binding proteins. A single major avermectin binding protein with a molecular weight of approximately 47 kDA was subsequently detected in Drosophila head membranes. The cloning and structure determination of these binding proteins, which are presumably part of an avermectin sensitive chloride channel, should enhance our understanding of the mode of action of avermectins and milbemycins. REFERENCES (1) Burg, R. W; Miller, B. M; Baker, Ε. E.; Bimbaum, J; Currie, S. Α.; Hartman, R; Kong, Y-L; Monaghan, R. L.; Olsen, G; Putter, I; Tunac, J. B.; Wallick, H.; Stapley, E. O.; Oiwa, R.; Omura, S. Avermectins, New Family of Potent Anthelmintic Agents: Producing Organism and Fermentation. Antimicrob. Agents Chemother. 1979, 15, 361-67. (2) Egerton, J. R.; Ostlind, D. Α.; Blair, L. S.; Eary, C. H.; Suhayda, D.; Cifelli, S.; Fiek, R. F.; Campbell, W. C. Avermectins, New Family of Potent Anthelmintic Agents: Efficacy of the B Component. Antimicrob. Agents Chemother. 1979, 15, 372-00. 1a

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