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Photoaffinity Labeling of Avermectin Binding Sites from Caenorhabditis Elegans and Drosophila Melanogaster SUSAN P

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Photoaffinity Labeling of Avermectin Binding Sites from Caenorhabditis Elegans and Drosophila Melanogaster SUSAN P Proc. Natl. Acad. Sci. USA Vol. 89, pp. 4168-4172, May 1992 Biochemistry Photoaffinity labeling of avermectin binding sites from Caenorhabditis elegans and Drosophila melanogaster SUSAN P. ROHRER*t, PETER T. MEINKEt, EDWARD C. HAYES*, HELMUT MROZIKT, AND JAMES M. SCHAEFFER* Departments of *Biochemical Parasitology and tBasic Medicinal Chemistry, Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065 Communicated by Edward M. Scolnick, February 19, 1992 ABSTRACT An azido-avermectin analog [4"a-(4-azidosal- A icylamido-e-caproylamido-fi-alanylamido)-4"-deoxyaver- mectin B18; azido-AVMJ was synthesized and used to photo- affinity label avermectin binding sites present in the mem- branes of Caenorhabditis elegans and Drosophila melanogaster. Azido-AVM was biologically active and behaved like a com- petitive inhibitor of [3lHlivermectin binding to C. elegans membranes (K; = 0.2 nM). Radiolabeled azido-AVM bound specifically and with high affinity to C. elegans membranes (Kd = 0.14 nM) and, upon photoactivation, became covalently linked to three C. elegans polypeptides of 53, 47, and 8 kDa. Photoaffinity labeling of a membrane preparation from D. melanogaster heads resulted in labeling of a single major R = HO = Avermectin B18 polypeptide of :47 kDa. The proteins that were covalently OH O HH tagged in these experiments are believed to be associated with R = RI N N avermectin-sensitive chloride channels present in the neuro- Nl' ~ f-~ muscular systems of C. elegans and D. melanogaster. Azido- AVM did not bind to rat brain membranes and therefore was RI = H = Azldo-AVM selective for the nematode and insect receptors. RI = 1251 = 1251IAzido-AVM The avermectins are a family ofmacrocyclic lactones isolated as natural fermentation products from Streptomyces avermi- B tilis (1, 2). Ivermectin (22,23-dihydroavermectin BIa) is a semisynthetic avermectin analog with unprecedented effi- cacy and breadth of spectrum against nematode and arthro- pod parasites. Since its introduction in 1981, it has had a tremendous impact on veterinary health (3-5) and more recently on human health (6, 7). It has been approved for prophylactic use against Dirofilaria immitis, the causative agent ofheartworm disease in dogs, and is widely used for the treatment of livestock against a variety of intestinal nema- todes. It is currently the drug of choice for the control and prevention of human onchocerciasis, commonly known as river blindness, a debilitating tropical disease that affects an estimated 18 million people in Africa, Latin America, and the FIG. 1. Avermectin analogs used in this study. (A) Azido-AVM Middle East. Because the avermectins are without toxic side [4"a-(4-azidosalicylamido-E-caproylamido-p-alanylamido)-4"- effects and are efficacious miticidal and insecticidal com- deoxyavermectin Bia)] and 125I-azido-AVM. (B) Octahydroavermec- pounds (8, 9), they also have been developed for use in crop tin (3,4,8,9,10,11,22,23-octahydroavermectin Bia). protection programs. relatively low mammalian toxicity of this family of com- Although the mode of action of avermectins in target pounds (11). Electrophysiological and biochemical studies species is not completely understood, they cause an increase support a role for the avermectins in the modulation of in membrane permeability to chloride ions that results in paralysis (10). Specific, high-affinity avermectin binding sites y-aminobutyrate-gated chloride channels in vertebrate neu- have been identified and characterized in the free-living ronal tissues (13-16). In contrast, the primary site of action nematode, Caenorhabditis elegans (11, 12). A clear correla- of the avermectins in nematodes and insects appears to be a tion exists between the binding affinities for C. elegans non-y-aminobutyrate-gated chloride channel (17-19). membranes and in vivo efficacy of a series of avermectin A detailed understanding of the mechanism of action of analogs, indicating that the binding site is physiologically ivermectin in nematodes and insects could be obtained important. Specific avermectin binding sites also have been through biochemical isolation and characterization of the identified in mammalian brain tissue; however, the affinity is proteins possessing the drug binding sites. Purification of the lower by a factor of - 100 in brain, which may account for the biologically active receptor is not trivial, due to the low abundance of the receptor in invertebrate tissues and the of ivermectin to the C. The publication costs of this article were defrayed in part by page charge nearly irreversible binding elegans payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 4168 Downloaded by guest on September 24, 2021 Biochemistry: Rohrer et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4169 receptor. Covalent modification of the receptor proteins with (Pierce) supplied as a 10% (vol/vol) solution was added to a photoactive analog of the drug would facilitate purification give a final detergent concentration of 0.5%. This mixture of these proteins under denaturing conditions. In this report was stirred for 1 hr on ice and then centrifuged for 1 hr in a we describe the identification of specific avermectin-binding Ti 65 rotor (Beckman) at 100,000 x g. The 100,000 x g proteins from a nematode (C. elegans) and an insect (Droso- supernatant was filtered through a 0.22-,tm GV-X Millipore phila melanogaster) by photoaffinity labeling with a radio- filter prior to use in binding assays or labeling experiments. iodinated azidoavermectin analog. [31H]Ivermectin Binding Assay. For measurement of binding of [3H]ivermectin to C. elegans membranes or to a Triton X-100-soluble membrane protein preparation, =200 pug of MATERIALS AND METHODS protein was used per 1-ml assay. The standard binding assay Chemicals. Ivermectin and 3,4,8,9,10,11,22,23-octahy- (11) was used without modification for both the membrane- droavermectin Bia (Fig. 1) were supplied by Helmut Mrozik bound and detergent-soluble C. elegans tissue preparations. and Tom Shih (Merck Sharp and Dohme Research Labora- '251-Azido-AVM Binding and Crosslinking. Triton- tories). [3H]Ivermectin was labeled at the 22,23-position by solubilized C. elegans membrane proteins were diluted with catalytic hydrogenation with tritium gas to a specific activity 50 mM Hepes (pH 7) to a final protein concentration of 200 of 60 Ci/mmol (1 Ci = 37 GBq). ,ug/ml in 0.1% Triton X-100. This extract was then incubated Azido-AVM was synthesized by acylation of 4"-a-amino- with 0.8 nM 125I-azido-AVM in the dark for 1 hr at 220C. To 5-O-tert-butyldimethylsilyl-4"-deoxyavermectin Bla with flu- remove the unbound avermectin, dextran-coated activated oren-9-ylmethoxycarbonyl (Fmoc)-,3-AlaOH. Both protect- charcoal (final concentration, 0.3%) was added. After 10 min ing groups were removed and the resultant amine was acy- the charcoal was removed by centrifugation at 1000 X g. The lated with Fmoc-E-aminocaproic acid. The Fmoc group was supernatant was filtered through a 0.22-,um GV-X Millipore removed and the amine was acylated with N-hydroxysuc- filter. Triton X-100 was added to a final concentration of 1.0% cinimidyl 4-azidosalicylate. All compounds were purified to and the mixture was maintained in the dark at 22°C for 30 min. homogeneity prior to use. Details of the synthesis of azido- Aliquots (2 ml) of the mixture contained in 20-ml glass AVM, will be described elsewhere (P.T.M., S.P.R., E.C.H., scintillation vials were placed in an ice bath on a rotary J.M.S., M. Fisher, and H.M., unpublished work). shaker. The distance from the surface of the sample to the Azido-AVM was radioiodinated by employing chloramine lamp was 6.5 cm. Crosslinking of 125I-azido-AVM to the C. T (20). Two micrograms of azido-AVM in 1 ,1 of dimethyl elegans receptor was achieved by photolysis for 5 min under sulfoxide was added to 50 pl of freshly prepared chloramine a Spectroline model XX-1SB medium-wave UV lamp (30-W T (3 ug/IA1 in acetone). Carrier-free Na1251 (5 mCi in 15 Al output). Comparable results were obtained with UV expo- of 0.01 M sodium phosphate, pH 8.5) was then added and the sure times of 1-10 min (data not shown). C. elegans proteins reaction was maintained at room temperature for 5 min. The were precipitated in 1.5-ml microcentrifuge tubes by addition acetone was removed under a stream of N2 and replaced with of 4 volumes of methanol, storage at -20°C for 1 hr, and 50 ,ul of methanol. The azido-AVM and the 1251I-azido-AVM centrifugation for 10 min at 14,000 rpm in an Eppendorf were resolved by reverse-phase HPLC. The reaction mixture microcentrifuge. was applied to a C18 column (Vydac, 4.6 mm x 25 cm) and Drosophila Head Membrane Preparation and Affinity La- eluted under isocratic conditions with 84% methanol. Azido- beling. Drosophila melanogaster adults of the Oregon-R AVM and 125I-azido-AVM had retention times of9.4 and 10.5 strain were collected and frozen at -70°C for at least 1 hr but min, respectively. 125I-azido-AVM was obtained in 25% yield up to 2 weeks. Immediately upon removal from the freezer, and was essentially carrier-free with a specific activity of the flies, contained either in 100-ml glass culture bottles or 1700 Ci/mmol. 50-ml plastic conical culture tubes, were shaken vigorously in All other compounds were supplied by commercial order to break offtheir heads and then poured through a wire sources. mesh screen in order to separate heads from bodies. The C. elegans Cultures. All studies described in this paper were heads were homogenized in a Dounce homogenizer with 50 performed with tissue extracts prepared from the wild-type mM Hepes (pH 7) and centrifuged for 30 min at 28,000 x g.
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