Avermectin ©

1. Discovery, producing organism and structures1,2)
The discovery of avermectins was the result of a collaboration with Merck Sharp & Dohm Research Laboratories. Natural products were screened for anthelmentic activity using a direct invivo test in mice infected with the nematode, Nematospiroides dubus.
Avermectins were isolated from the culture broth of the actinomycete strain MA-4680T. Avermectins are a family of four closely related major components, A1a, A2a, B1a and B2a, and fourminor components, A1b, A2b, B1b and B2b, which are lower homologs of the corresponding
major components. The first total synthesis of avermectin A1a was achieved by Danishefsky etal3). Scince then, the total synthesis of avermectins has been reported by many groups (See Appendix-I).
OCH3
HOH3C

OCH3O

O
H
H3C

CH3O

O
H
H3C

Streptomyces avermectinius(formerly S. avermitilis) MA-4680T
Bar: 2 µm

X

23Y

118

CH3R2H
H
CH325OO 1 OOH H
2O

Avermectins

O

138aAvermectin A1aA1bA2aA2bB1aB1bB2aB2bH

15225HCH3OR1R1R2X–Y

CH3CH3CH3CH3HH
HH

C2H5CH3C2H5CH3C2H5CH3C2H5CH3CH=CH
CH=CH
CH2–CH(α-OH)
CH2–CH(α-OH)
CH=CH
CH=CH
CH2–CH(α-OH)
CH2–CH(α-OH)

2. Physical data (Avermectin B1a)White powder. C48H72O14; mol wt 873.08. Sol. in CHCl3, acetone, MeOH. Insol. in H2O.
3. Biological activity1,4)
Avermectins have potent anthelmintic and insecticidal activities. In particular, the hydrogenatedproduct of avermectin B1, 22,23-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 C5 position and thedisaccharide moiety are essential for the potency of avermectins.

Numbers of nematodes and anthelmintic efficacy in lambs treated with dihydroavermectin B1a*Group 1 (control)
Range

Parasite(Min-max)

H. contortus
1,550–11,605
T. axei
0–110
O. ciecumcincta
135–1,340
T. colubriformis
3,555–6,130
Cooperia spp
250–1,685
Immature (abomasum)
420–7,590
Immature (small intestine) 160–945

Group 2 (25 µg/ml)
Range

Mean(Min-max)

7,216
41
699
4,843
714
2,463
470

0–45
0–5
0–30
0–5
0–1000Group 3 (50 µg/ml)

Mean Efficacy (%)

1027
1200

Range(Min-max) Mean> 99
> 99
> 99
> 99
> 99
> 99
> 99

0000000

Efficacy (%)

0000000

> 99
> 99
> 99
> 99
> 99
> 99
> 99

*Modified from Ref. 4

4. New metabolites related to avermectin5-7)
The following novel avermectin metabolites were obtained from blocked mutants of the producer.
OCH3H 3C

HOCH3CH3O

OCH3OCH3HOO

OO

H3C

CH3H3COOO
O

H3C

CH3CH3O

CH3O

4'-Deoleandrosyl-6,8a-seco6,8a-deoxy-5-oxo AVM B2aOCH3H 3C

OO

CH3O

CH3O

O
O

6,8a-seco-6,8a-deoxy-2,5anhydro AVM B2a

OCH3CH3OO

CH3OO
O

H3C

CH3O

OO

H3C

CH3CH3O
OHO

H3C
CH3O

5-Oxo AVM B1aCH3CH3O

H3COH

OCH3H3C

H3C

CH3CH34'-Deoleandrosyl-6,8a-seco6,8a-deoxy AVM A1aOOHOO
OH

H3C
OCH3O

OO

CH3H 3C

CH3CH3O

H3C

H3C

O

H3C

CH3OCH3OH

O
H3C

CH3H3C

HOOCH3OO
OHO

H3C

OCH3HOCH3CH3CH3O
OH

4'-Deoleandrosyl-6,8a-seco6,8a-deoxy-5-oxo AVM B1aOCH3HOO
H3C

O

4'-Deoleandrosyl-6,8a-seco6,8a-deoxy AVM B1aO

O

CH3OH

H 3C

O

H3C
CH3HOO

O

O
OH

H3C

H3C

CH3CH3CH3CH3O
O
OH

OH

HOCH3CH3CH3O

5-Oxo AVM B2a5. Biosynthesis8-21,26-31)
Eight kinds of genes involving avermectin biosynthesis, aveA1, ave A2, ave A3, ave A4 (polyketidesynthase), aveB (glycosylation), aveC (C-22,23 dehydration?), aveD (C-5 O-methylation), aveE(C-6, 8a furan ring formation), aveF (C-5 keto reduction), and aveR(regulation), were characterized and mapped on the chromosome as shown below.
OH

R1: H or CH 3
R2: CH 3 or C2H5
X–Y: –CH 2=CH2– or –CH2CH(OH)–O 25

OCH3HOO

O

CH3H
H3CO

CH3HOO
H
H3C

H3C

88CH3R2H
H
CH3258CH3CH3OR1OH8aO

5H

CH3OH

aveD mutation

H5H

aveE mutation

H22O

OR1H
H
CH3OH8aH
8CH325

X mutation

2H5H

YO 1 OOH H
528aX

11OOH

O

23O

8aaveB mutation

2213

1311

H

15

15

H3C

R2
HCH3

aveC mutation

OCH3

H3C

CH322

CH3O

aveF mutation

Characteristics of Mutants

01020

30

40

50

60

70

80kpb

Bam HI map

aveA1

1 kbp

aveA2

aveA3

1 kbp

aveR aveF aveD

aveC aveEaveA41 kbp

orf1 aveBI
aveBIII aveBV aveBVII
aveBII aveBIV aveBVI aveBVIII

Organization of the gene cluster for avermectin biosynthesis.
The direction of transcription and relative sizes of the ORFs deduced
from analysis of the nucleotide sequence are indicated.

L-Isoleucine

L-Valine
Transaminase (IlvE)
orValine dehydrogenase (Vdh)

3-methyl-2-oxovalerate

ATPCoASH

CO2AMP NADH+H+
PPi

2-methylbutyrate

2-oxoisovalerate

AMP
PPi

isobutyryl-CoA

2-methylbutyryl-CoA

Acyl-CoA synthase

CoASH
NAD+

Branched-chain2-oxo acid dehydrogenase
CO2(BkdF, G & H)
NADH+H+methylmalonyl-CoA

AVM-PKS module2 (AVES1)
DHmalonyl-CoA

9 C precursor
to avermectin "1b"

10 C precursor
to avermectin "1a"10 C precursor
to avermectin "2a"CO2malonyl-CoA
AVM-PKS module2 (AVES1)
DH+ (AveC ?)
CO2

CO2

isobutyrate

7 C precursor
to avermectin "b"

8 C precursor
to avermectin "a"
AVM-PKS module2 (AVES1)
DH-

Acyl-CoA synthasemethylmalonyl-CoA

AVM-PKS module1(AVES1)

CO2

ATPCoASH

Polyketide synthesis

CoASH
NAD+

9 C precursor
to avermectin "2b"6 x malonyl-CoA
4 x methylmalonyl-CoA
AVM-PKS module3 - module12(AVES1, 2, 3 & 4)

10 x CO22322

HO

Avermectin B2a and A2aO
13OO

25

Avermectin B1b and A1bO

OH
8aAvermectin B2b and A2bGlucose-1-phosphate

8dTDP-glucose synthetase (AveBIII)
56

dTDP-glucose
dTDP-glucose 4,6-dehydratase (AveBII)

O

6,8a-Seco-6,8a-deoxy-5-oxoavermectin "1a" aglycone

dTDP-4-oxo-6-deoxyglucose

O2

AveBV, BVI, BVIII & BIV

C6-C8a cyclase (AveE)

dTDP-3-demethyloleandrose
dTDP-3-demethyloleandrose
O-methyltransferase (AveBVII)

OH8adTDP-oleandrose

8O

56OCH3OHO

5-Oxoavermectin "1a" aglycone

4"

NADPH + H+O 1"

OCH3O
4'C5 ketoreductase (AveF)

NADP+dTDP-oleandrose

OH8a3"

O 1'OCH3HO
4'8O

63'3'O 1'

523
22OO
13OO

dTDP-oleandrose
O25O

OH
8a138O

OH

65OH

Avermectin B1a aglycone

Avermectin B1a

Avermectin B1a monosaccharide

S-Adenosylmethionine
C13 O-glycosyltransferase (AveBI)

C5 O-methyltransferase (AveD)

C4' O-glycosyltransferase (AveBI)

S-AdenosylhomocysteindTDP-oleandrose

OH8aO

dTDP-oleandrose

Avermectin A1a monosaccharide

86

Avermectin A1a5

OCH3

Avermectin A1a aglycone

Proposed biosynthetic pathway of avermectins in S. avermitilis.

The 12 modules of avermectin PKS (AVES1-4) act sequentially to add 12 acyl units (seven acetate and fivepropionate units). The initial stage of avermectin biosynthesis, designated as “initial aglycon”, is considered
to be up until the formation of 6,8a-seco-6,8a-deoxy-5-oxoavermectin aglycon. The middle stage of biosynthesis is a post-polyketide modification from AveE to AveD and the last stage is glycosylation.

AveBIIIaveBVII

PstI 9990

XhoI 9801

aveBVIClaI 9761

NcoI 8455
BamHI 8743

XhoI 7350

BamHI 4085
NcoI 4166
EcoRI 4579

aveBV

aveBVIII

CH3

H2O

O
O

O
CH3

O

OH

O dTDP AveBII

OH

OH

OH

aveBIV

OH

O P O

OH

aveBIII

O

OH

OH

aveBIICH2OH

PPidTTPO

aveBI

BamHI 5698
BamHI 5862
EcoRI 6272
SacI 6363

orf-1

XhoI 3748

EcoRI 522
BamHI 1323
BamHI 1348
BstBI 1364
SacI 1468
BamHI 1830
SacI 2744
BglII 2755
SacI 2861
BglII 2902

NcoI 362

BamHI 1

aveA4CH2OH

O dTDP AveBVOH

O dTDP

OHOH

OH

Glucose-1-phosphate

AveBVI

H2O

S-adenosyl-L-homocysteineHO

S-adenosyl-L-methionineO
HOCH3O

CH3AveBVII

O dTDP

NAD(P)+NAD(P)H+H+CH3

O

TDP-L-oleandrose

O

O
CH3O

O dTDP AveBIVO dTDP AveBVIII

OH

OCH3NAD(P)H+H+NAD(P)+O dTDP
O

OH

AveBI

aglycons ormonosaccharides

O

HO

CH3dTDPO R
OCH3Avermectins or monosaccharides

Scheme for the biosynthesis of L-oleandrose.

The putative gene products believed to be associated with various steps
are indicated at respective points in the pathway.
aveA1

aveA2translatetranslate

AVES 1module 2

KR

SO

AT

module 6module 3

ACP KS
SDHKR

AT

ACP
S

23O

2125

OH

23
25

AT ACP KS
S

O

21ATS

O

1725

ACP KS
SO
OH

O

23OH

AT

21OH

1721

25

OH

O

KR

AT

ACP
13KSO

OH
1721OH

DH

KR

DH

KR

AT

AT

ACP KSATACPACP KS
S

O

13OH

17OH

13OH
21OH

S

2521OHACP KS
S

5O

7O

OH

13OH

17OH

OH

17OH

O

O

21OH

AT ACP KSSOH

2321OH

255O

7O

13OH

17OH
OH

O

232521

OH

O
OOO

O

HOO
O

O

OH

AveE, AveF, AveD,AveBI - AveBVIII

13O

1721O

25O

KR

AT

ACP TE
S2OH

O

5O

7O

13OH

17OH
OH

21

O

23
25

23

O

DHO

2325O
HOO

OH

OH

2325

ATO

7OH

O

23

KS

SO

end

KR

1317module 12module 11

DH KR

O

2325module 9

OHOH

O

2325

DHS

OH

O

23

module 10module 7

KR

ACP KS

AVES 4module 8module 5KRKS

translate

AVES 3module 4module 1

aveA4translate

AVES 2load

AT ACP KSaveA3OHAveC ?

OOH 27OOAvermectin A1a5O

6,8a-seco-6,8a-deoxy-5-oxo-avermectin aglycon

Model for 6,8a-seco-6,8a-deoxy-5-oxoavermectin aglycon formation andpredicted domain structure of the avermectin PKS.

Each circle represents an enzymatic domain in the PKS multifunctional polypeptide. Abbreviations: AT, acyltransferase; DH, dehydratase; KR, β-ketoacyl-ACP reductase; KS, β-ketoacyl-ACP synthase; TE, thioesterase. The reaction order from module 7 to 9, and from 10 to 12 in aveA3 and aveA4, respectively, is drawn inthe direction opposite to the gene order on the genome. The crossed-out domain in module 7 is nonfunctional.
The shaded domain in module 10 does not function in polyketide-chain elongation.

6. Mode of action22-24,32-38)
The molecular target of avermectins is a glutamate-gated chloride channel22-24). Cully et al. reported in nematodes and in insects to be a target of the antiparasitic agent avermectins and revealed the molecular basis underlying it36).
Recently, Althoff et al., Ghosh et al., and Hibbs and Gouaux reported an understanding mechanism of avermectins-binding site and atomic interaction of the target molecule by X-ray crystallography analysis32-34).
7. 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 filariasis25,39-44).

8. References
1. [155] R. W. Burg et al., Antimicrob. Agents Chemother. 15, 361-367 (1979)
2. [821] Y. Takahashi et al., Int. J. Syst. Evol. Microbiol. 52, 2163-2168 (2002)
3.		
S. J. Danishefsky et al., J. Am. Chem. Soc. 111, 2967-2980 (1989)
4.		
K. S. Todd et al., Am. J. Vet. Res. 45, 976-977 (1984)
5. [564] C.H. Pang et al., J. Antibiot. 48, 59-66 (1995)
6. [566] C.H. Pang et al., J. Antibiot. 48, 92-94 (1995)
7. [567] H. Ikeda et al., J. Antibiot. 48, 95-97 (1995)
8. [381] H. Ikeda et al., J. Bacteriol. 169, 5615-5621 (1987)
9. [388] H. Ikeda et al., Antimicrob., Agents Chemother. 32, 282-284 (1988)
10. [462] S. Ōmura et al., J. Antibiot. 44, 560-563 (1991)
11. [466] H. Ikeda & S. Ōmura, Kitasato Arch. Exp. Med. 63, 143-155 (1990)
12. [476] H. Ikeda & S. Ōmura, Actinomycetol. 5, 86-99 (1991)
13. [504] H. Ikeda et al., J. Bacteriol. 175, 2077-2082 (1993)
14. [526] H. Ikeda & S. Ōmura, Actinomycetol. 7, 133-144 (1993)
15. [575] H. Ikeda et al., J. Antibiot. 48, 532-534 (1995)
16. [577] H. Ikeda & S. Ōmura, J. Antibiot. 48, 549-562 (1995)
17. [685] H. Ikeda & S. Ōmura, Chem. Rev. 97, 2591-2609 (1997)
18. [693] H. Ikeda et al., Gene 206,175-180 (1998)
19. [722] H. Ikeda et al., Proc. Natl. Acad. Sci. USA 96, 9509-9514 (1999)
20.		
H. Ikeda et al., Actinomycetol. 13, 94-112 (1999)
21. [790] H. Ikeda et al., J. Ind. Microbiol. Biotechnol. 27, 170-176 (2001)
22.		
A. Pomes et al., Biochim. Biophys. Acta 1339, 233-238 (1996)
23.		
D. F. Cully et al., J. Biol. Chem. 271, 20187-20191 (1997)
24. [811] S. Ōmura, In “Macrolide Antibiotics (2nd Edition)” pp. 571-576, Academic Press(2002)
25			
A. Crump & K. Otoguro, Trends Parasitol. 21, 126-132 (2005)
26. [1161] H. Ikeda et al., J. Ind. Microbiol. Biotechnol. 41, 233-250 (2014)
27. [1151] M. Komatsu et al., ACS Synth. Biol. 2, 384-396 (2013)
28. [791] S. Ōmura et al., Proc. Natl. Acad. Sci. USA 98, 12215-12220 (2001)
29. [1098] S. Kitani et al., Proc. Natl. Acad. Sci. USA 108, 16410-16415 (2011)30.		
K. T. Miyamoto et al., Microbiology 157, 2266-2275 (2011)
31.		
S. Kitani et al., Appl. Microbiol. Biotechnol. 82, 1089-1096 (2009)32.		
T. Althoff et al., Nature 512, 333-337 (2014)
33.		
R. Ghosh et al., Science 335, 574-578 (2012)
34.		
R. E. Hibbs & E. Gouaux, Nature 474, 54-60 (2011)35.		
D. K. Vassilatis et al., J. Biol. Chem. 272, 33167-33174 (1997)
36.		
D. F. Cully et al., Nature 371, 707-711 (1994)
37.		
S. P. Rohrer et al., Proc. Natl. Acad. Sci. USA 89, 4168-4172 (1992)
38.		
J. P. Arena et al., Mol. Pharmacol. 40, 368-374 (1991)
39.		
A. Crump, Trends Parasitol. 30, 423-425 (2014)
40. [1172] S. Ōmura & A. Crump, Trends Parasitol. 30, 445-455 (2014)
41. [1124] A. Crump, C. M. Morel & S. Ōmura, Trends Parasitol. 28, 280-288 (2012)
42. [1089] A. Crump & S. Ōmura, Proc. Jpn. Acad. Ser. B 87, 13-28 (2011)
43. [979] S. Ōmura, Int. J. Antimicrob. Agents 31, 91-98 (2008)
44. [876] S. Ōmura & A. Crump, Nat. Rev. Microbiol. 2, 984-989 (2004)