Avermectin. 2. Physical data (Avermectin B1a) White powder. C 48 H 72 O 14 ; mol wt Sol. in CHCl 3, acetone, MeOH. Insol. in H 2 O.

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1 Chapter Chapter 1 Avermectin 1. Discovery, producing organism and structures 1,) The discovery of avermectins was the result of a collaboration with Merck harp & Dohm Research Laboratories. Natural products were screened for anthelmentic activity using a direct in vivo test in mice infected with the nematode, Nematospiroides dubus. Avermectins were isolated from the culture broth of the actinomycete strain MA-40 T. Avermectins are a family of four closely related major components, A1a, Aa, B1a and Ba, and four minor components, A1b, Ab, B1b and Bb, which are lower homologs of the corresponding major components. The first total synthesis of avermectin A1a was achieved by Danishefsky et al 3). cince then, the total synthesis of avermectins has been reported by many groups (ee Appendix-I). Avermectins 11 a 1 1 R 1 X Y R C3 treptomyces avermectinius (formerly. avermitilis) MA-40 T Bar: µm Avermectin A1a A1b Aa Ab B1a B1b Ba Bb R 1 R X Y C C C C C=C C=C C C(α-) C C(α-) C=C C=C C C(α-) C C(α-). Physical data (Avermectin B1a) White powder. C 4 14 ; mol wt 3.0. ol. in CCl 3, acetone, Me. Insol. in. 3. Biological activity 1,4) Avermectins have potent anthelmintic and insecticidal activities. In particular, the hydrogenated product of avermectin B1,,-dihydroavermectin B1 (ivermectin), is used as an important anthelmintic in veterinary medicine as well as for the control of onchocerciasis, lymphatic filariasis, strongyloidiasis, mites and scabies in humans. The hydroxyl group at the C position and the disaccharide moiety are essential for the potency of avermectins

2 Numbers of nematodes and anthelmintic efficacy in lambs treated with dihydroavermectin B1a* Group 1 (control) Group ( µg/ml) Group 3 (0 µg/ml) Range Range Range Parasite (Min-max) Mean (Min-max) Mean Efficacy (%) (Min-max) Mean Efficacy (%). contortus 1,0 11,0, > > 99 T. axei > > 99. ciecumcincta 1, > > 99 T. colubriformis 3,,0 4, > > 99 Cooperia spp 0 1, > > 99 Immature (abomasum) 40,90, > > 99 Immature (small intestine) > > 99 *Modified from Ref. 4 Chapter 1 Chapter 4. New metabolites related to avermectin -) The following novel avermectin metabolites were obtained from blocked mutants of the producer. 4'-Deoleandrosyl-,a-seco-,a-deoxy AVM B1a 4'-Deoleandrosyl-,a-seco-,a-deoxy--oxo AVM B1a 4'-Deoleandrosyl-,a-seco-,a-deoxy--oxo AVM Ba 4'-Deoleandrosyl-,a-seco-,a-deoxy AVM A1a,a-seco-,a-deoxy-,- anhydro AVM Ba -xo AVM Ba -xo AVM B1a - 9 -

3 Chapter Chapter 1. Biosynthesis -,-31) Eight kinds of genes involving avermectin biosynthesis, avea1, ave A, ave A3, ave A4 (polyketide synthase), aveb (glycosylation), avec (C-, dehydration?), aved (C- -methylation), avee (C-, a furan ring formation), avef (C- keto reduction), and aver(regulation), were characterized and mapped on the chromosome as shown below. R 1 : or R : or C X Y: C =C or C C() 11 1 aveb mutation a R 1 avee mutation 11 a 1 a 1 R 1 avef mutation R avec mutation Y X R C3 a aved mutation C3 X mutation Characteristics of Mutants kpb Bam I map avea1 avea avea3 avea4 1 kbp 1 kbp 1 kbp aver avef aved avec avee orf1 avebi avebii avebiii avebiv avebv avebvi rganization of the gene cluster for avermectin biosynthesis. The direction of transcription and relative sizes of the RFs deduced from analysis of the nucleotide sequence are indicated. avebvii avebviii - 9 -

4 L-Isoleucine Transaminase (IlvE) or Valine dehydrogenase (Vdh) L-Valine Chapter 1 P CoA AMP PPi 3-methyl--oxovalerate CoA NAD + C NAD+ + Branched-chain -oxo acid dehydrogenase (BkdF, G & ) -methylbutyrate -methylbutyryl-coa Acyl-CoA synthase methylmalonyl-coa AVM-PK module1 C (AVE1) -oxoisovalerate CoA NAD + C NAD+ + C AMP PPi P CoA isobutyryl-coa isobutyrate Acyl-CoA synthase methylmalonyl-coa Chapter 10 C precursor to avermectin "a" AVM-PK module (AVE1) D - C precursor to avermectin "a" C malonyl-coa AVM-PK module (AVE1) D + (AveC?) 10 C precursor to avermectin "1a" x malonyl-coa 4 x methylmalonyl-coa C precursor to avermectin "b" C 9 C precursor to avermectin "1b" AVM-PK module (AVE1) malonyl-coa D - 9 C precursor to avermectin "b" Polyketide synthesis 10 x C AVM-PK module3 - module1 (AVE1,, 3 & 4) Avermectin Ba and Aa Avermectin B1b and A1b Avermectin Bb and Ab,a-eco-,a-deoxy--oxoavermectin "1a" aglycone C-Ca cyclase (AveE) Glucose-1-phosphate dtdp-glucose synthetase (AveBIII) dtdp-glucose dtdp-glucose 4,-dehydratase (AveBII) dtdp-4-oxo--deoxyglucose AveBV, BVI, BVIII & BIV dtdp-3-demethyloleandrose dtdp-3-demethyloleandrose -methyltransferase (AveBVII) -xoavermectin "1a" aglycone NADP + NADP + + C ketoreductase (AveF) 4" C3 3" 1" 4' 3' 1' C3 4' 3' 1' Avermectin B1a aglycone Avermectin B1a monosaccharide Avermectin B1a -Adenosylmethionine C -glycosyltransferase (AveBI) C4' -glycosyltransferase (AveBI) C -methyltransferase (AveD) -Adenosylhomocystein Avermectin A1a monosaccharide Avermectin A1a Avermectin A1a aglycone Proposed biosynthetic pathway of avermectins in. avermitilis. The 1 modules of avermectin PK (AVE1-4) act sequentially to add 1 acyl units (seven acetate and five propionate units). The initial stage of avermectin biosynthesis, designated as initial aglycon, is considered to be up until the formation of,a-seco-,a-deoxy--oxoavermectin aglycon. The middle stage of biosynthesis is a post-polyketide modification from AveE to AveD and the last stage is glycosylation

5 Chapter Chapter 1 BamI 1 NcoI 3 EcoRI BamI BamI 4 BstBI 4 aci 14 BamI 130 aci 44 BglII aci 1 BglII 90 XhoI 34 BamI 40 NcoI 41 EcoRI 49 BamI 9 BamI EcoRI aci 33 avea4 orf-1 avebi avebii avebiii avebiv avebv avebvi avebvii C dttp PPi P AveBIII Glucose-1-phosphate XhoI 30 NcoI 4 BamI 43 cheme for the biosynthesis of L-oleandrose. ClaI 91 XhoI 901 avebviii AveBVI -adenosyl-l-homocysteine -adenosyl-l-methionine NAD(P) + NAD(P)+ + NAD(P) + NAD(P)+ + C3 C3 C 3 C 3 dtdp AveBVII dtdp AveBIV dtdp AveBVIII dtdp TDP-L-oleandrose aglycons or monosaccharides AveBI dtdp C dtdp AveBII C3 R Avermectins or monosaccharides dtdp AveBV The putative gene products believed to be associated with various steps are indicated at respective points in the pathway. C3 avea1 avea avea3 avea4 translate translate translate translate AVE 1 AVE AVE 3 AVE 4 C3 PstI 9990 dtdp load module module 4 module module module 10 module 1 module 1 module 3 module module module 9 module 11 end D D D D D D ACP K ACP K ACP K ACP K ACP K ACP K ACP K ACP K ACP K ACP K ACP K ACP K ACP TE AveE, AveF, AveD, AveBI - AveBVIII AveC? Avermectin A1a,a-seco-,a-deoxy--oxo-avermectin aglycon Model for,a-seco-,a-deoxy--oxoavermectin aglycon formation and predicted domain structure of the avermectin PK. Each circle represents an enzymatic domain in the PK multifunctional polypeptide. Abbreviations:, acyltransferase; D, dehydratase;, β-ketoacyl-acp reductase; K, β-ketoacyl-acp synthase; TE, thioesterase. The reaction order from module to 9, and from 10 to 1 in avea3 and avea4, respectively, is drawn in the direction opposite to the gene order on the genome. The crossed-out domain in module is nonfunctional. The shaded domain in module 10 does not function in polyketide-chain elongation

6 . Mode of action -4,3-3) The molecular target of avermectins is a glutamate-gated chloride channel -4). Cully et al. reported in nematodes and in insects to be a target of the antiparasitic agent avermectins and revealed the molecular basis underlying it 3). Recently, Althoff et al., Ghosh et al., and ibbs and Gouaux reported an understanding mechanism of avermectins-binding site and atomic interaction of the target molecule by X-ray crystallography analysis 3-34).. Avermectin and Ivermectin are commercially available as anthelmintic agents for both human and animals as well as being widely used for agriculture purposes. Ivermectin has become an invaluable tool for two global elimination initiatives, against human onchocerciasis and lymphatic filariasis,39-44). Chapter 1 Chapter. References 1. [1] R. W. Burg et al., Antimicrob. Agents Chemother. 1, 31-3 (199). [] Y. Takahashi et al., Int. J. yst. Evol. Microbiol., 3- (00) 3.. J. Danishefsky et al., J. Am. Chem. oc. 111, 9-90 (199) 4. K.. Todd et al., Am. J. Vet. Res. 4, 9-9 (194). [4] C.. Pang et al., J. Antibiot. 4, 9- (199). [] C.. Pang et al., J. Antibiot. 4, 9-94 (199). []. Ikeda et al., J. Antibiot. 4, 9-9 (199). [31]. Ikeda et al., J. Bacteriol. 19, 1- (19) 9. [3]. Ikeda et al., Antimicrob., Agents Chemother. 3, -4 (19) 10. [4]. Ōmura et al., J. Antibiot. 44, 0-3 (1991) 11. [4]. Ikeda &. Ōmura, Kitasato Arch. Exp. Med. 3, (1990) 1. [4]. Ikeda &. Ōmura, Actinomycetol., -99 (1991). [04]. Ikeda et al., J. Bacteriol., 0-0 (1993) 14. []. Ikeda &. Ōmura, Actinomycetol., (1993) 1. []. Ikeda et al., J. Antibiot. 4, 3-34 (199) 1. []. Ikeda &. Ōmura, J. Antibiot. 4, 49- (199). []. Ikeda &. Ōmura, Chem. Rev. 9, (199) 1. [93]. Ikeda et al., Gene 0,-10 (199) 19. []. Ikeda et al., Proc. Natl. Acad. ci. UA 9, (1999) 0.. Ikeda et al., Actinomycetol., (1999). [90]. Ikeda et al., J. Ind. Microbiol. Biotechnol., 0- (001). A. Pomes et al., Biochim. Biophys. Acta 39, 3- (199). D. F. Cully et al., J. Biol. Chem. 1, (199) 4. [11]. Ōmura, In Macrolide Antibiotics (nd Edition) pp. 1-, Academic Press (00) A. Crump & K. toguro, Trends Parasitol., 1- (00). [111]. Ikeda et al., J. Ind. Microbiol. Biotechnol. 41, 3-0 (014). [111] M. Komatsu et al., AC ynth. Biol., (0). [91]. Ōmura et al., Proc. Natl. Acad. ci. UA 9, 1-10 (001) 9. [109]. Kitani et al., Proc. Natl. Acad. ci. UA 10, (011) 30. K. T. Miyamoto et al., Microbiology, - (011) 31.. Kitani et al., Appl. Microbiol. Biotechnol., (009)

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