Antiparasite and insecticide antibiotics

1. Helminth NADH-fumarate reductase inhibitors

Many adult parasites are living in low oxygen environment. Such organisms generate ATP in different pathway from aerobic mammals. Anaerobic parasites fix CO₂ to phosphoenolpyruvate and produce oxaloacetate. It is converted to malate and transported into mitochondria. Then malate is converted to fumarate, and it is reduced to succinate by complex II using NADH as reducing equivalent. This system is called NADH-fumarate reductase and composed of complexes I and II. Quinone used in this system is not ubiquinone but rhodoquinone. We carried out screening for NADH-fumarate reductase inhibitors of microbial origin to find new anthelmintics and found nafuredin and atpenins.

(1) Nafuredin
Nafuredin was purified from the cultured broth of Aspergillus niger FT-0554 isolated from a marine sponge.^1-3) Nafuredin inhibited NADH-fumarate reductase of Ascaris suum at nM order, while it showed very weak inhibition to the mammalian enzyme.¹⁾ Its target was revealed as complex I, and it also showed anthelmintic activity against Haemonchus contortus (barberpole worm) in in vivo trials using sheep. Thus, nafuredin is a new potential lead compound as a novel anthelmintic. The total synthesis of nafuredin has been already achived.⁴⁾ Nafuredin is easily converted to a gamma-lactone type compound (nafuredin-gamma) by weak alkaline treatment, and it also shows NADH-fumarate reductase inhibitory activity.⁵⁾

(2) Atpenins
Atpenins were originally isolated from the cultured broth of Penicillium sp. FO-125 as inhibitors of lipid metabolism in Kitasato.⁶⁾ Recently, atpenins were rediscovered in the screening for NADH-fumarate reductase inhibitors and revealed to have potent mitochondrial complex II inhibitory activity.⁷⁾ Though atpenins did not show selectivity between mammalian and helminth complex II, their inhibitions are more than 100 times potent compared to known complex II inhibitors. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues.

(3) References

1. S. Omura, H. Miyadera, H. Ui, K. Shiomi, Y. Yamaguchi, R. Masuma, T. Nagamitsu, D. Takano, T. Sunazuka, A. Harder, H. Kolbl, M. Namikoshi, H. Miyoshi, K. Sakamoto, K. Kita: An anthelmintic compound, nafuredin, shows selective inhibition of complex I in helminth mitochondria. Proc. Natl. Acad. Sci. USA 98(1), 60-62 (2001) [PubMed]
   
2. H. Ui, K. Shiomi, Y. Yamaguchi, R. Masuma, T. Nagamitsu, D. Takano, T. Sunazuka, M. Namikoshi, S. Omura: Nafuredin, a novel inhibitor of NADH-fumarate reductase, produced by Aspergillus niger FT-0554. J. Antibiot. 54(3), 234-238 (2001) [PubMed]
   
3. D. Takano, T. Nagamitsu, H. Ui, K. Shiomi, Y. Yamaguchi, R. Masuma, I. Kuwajima, S. Omura: Absolute configuration of nafuredin, a new specific NADH-fumarate reductase inhibitor. Tetrahedron Lett. 42(16), 3017-3020 (2001)
4. D. Takano, T. Nagamitsu, H. Ui, K. Shiomi, Y. Yamaguchi, R. Masuma, I. Kuwajima, S. Omura: Total synthesis of nafuredin, a selective NADH-fumarate reductase inhibitor. Org. Lett. 3(15), 2289-2291 (2001) [PubMed]
   
5. T. Nagamitsu, D. Takano, K. Shiomi, H. Ui, Y. Yamaguchi, R. Masuma, Y. Harigaya, I. Kuwajima, S. Omura: Total synthesis of nafuredin-gamma, a gamma-lactone related to nafuredin with selective inhibitory activity against NADH-fumarate reductase. Tetrahedron Lett. 44(34), 6441-6444 (2003)
6. S. Omura, H. Tomoda, K. Kimura, D. Z. Zhen, H. Kumagai, K. Igarashi, N. Imamura, Y. Takahashi, Y. Tanaka, Y. Iwai: Atpenins, new antifungal antibiotics produced by Penicillium sp. Production, isolation, physico-chemical and biological properties. J. Antibiot. 41(12), 1769-1773 (1988) [PubMed]
   
7. H. Miyadera, K. Shiomi, H. Ui, Y. Yamaguchi, R. Masuma, H. Tomoda, H. Miyoshi, A. Osanai, K. Kita, S. Omura: Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase). Proc. Natl. Acad. Sci. USA 100(2), 473-477 (2003) [PubMed]
   

2. Insect chitinase inhibitors

Chitin occurs in fungi, some algae and many invertebrates including insects, but is not found in vertebrates. Thus chitin synthesis and degradation processes might be expected to be specific targets for fungicides and insecticides. Though chitin synthesis inhibitors, such as fungicidal polyoxins and insecticidal benzoylphenylureas, are commercially available, chitin degradation inhibitors have not yet been used. Chitinase (EC 3.2.1.14) is a chitin degradation enzyme and hydrolyzes chitin into oligomer of N-acetylglucosamine. An inhibitor of chitinase would be expected to inhibit apolysis during the insect molting and prevent maturation to the adult reproductive stage.
In the course of screening for new chitinase inhibitors of microbial origin, we found new cyclic pentapeptides, argadin and argifin, which were the first inhibitor produced by fungi and showed inhibition in a submicromolar range.

(1) Chitinase inhibition

	Lucilia cuprina (sheepblowfly) chitinase (IC50)	Bacillus subtilis chitinase (IC50)	Serratia marcescens chitinase B (Ki)
Argadin	0.15 microM	1.9 microM	0.0192 microM
Argifin	3.7	19.0	32.5

(2) Argadin-chitinase complex and argifin-chitinase complex
The structures of argifin and argadin in complex with Serratia marcescens chitinase B were resolved (2.0 angstrom resolution). These structures give an unprecedented view of how peptide based inhibitors inactivate a carbohydrate-processing enzyme. For example, the carbonyl oxygen of the histidine in argadin occupies almost the same position as the scissile oxygen in the substrate chitooligosaccharide, and hydrogen bonds to the catalytic acid Glu144, while the guanidinium group of the arginine side chain in argifin makes hydrogen bonds to Glu144.

(3) Insecticidal activity
Argifin (0.02 mg) was injected into cockroach larvae and compared to mock injected controls. Three separate trials repeatedly showed the efficacy of argifin against larval stages of Periplaneta americana (american cockroach) and Blattella germanica (german cockroach). Argifin showed 73% mortality of cockroaches. Most of the dead cockroach larvae showed no signs of molting.

(4) References

1. K. Shiomi, N. Arai, Y. Iwai, A. Turberg, H. Kolbl, S. Omura: Structure of argifin, a new chitinase inhibitor produced by Gliocladium sp. FTD-0668. Tetrahedron Lett. 41(13), 2141-2143 (2000)
2. S. Omura, N. Arai, Y. Yamaguchi, R. Masuma, Y. Iwai, M. Namikoshi, A. Turberg, H. Kolbl, K. Shiomi: Argifin, a new chitinase inhibitor, produced by Gliocladium sp. FTD-0668. I. Taxonomy, fermentation, and biological activities. J. Antibiot. 53(6), 603-608 (2000) [PubMed]
3. N. Arai, K. Shiomi, Y. Iwai, S. Omura: Argifin, a new chitinase inhibitor, produced by Gliocladium sp.FTD-0668. II. Isolation, physico-chemical properties, and structure elucidation. J. Antibiot. 53(6), 609-614 (2000) [PubMed]
4. N. Arai, K. Shiomi, Y. Yamaguchi, R. Masuma, Y. Iwai, A. Turberg, H. Kolbl, S. Omura: Argadin, a new chitinase inhibitor, produced by Clonostachys sp. FO-7314. Chem. Pharm. Bull. 48(10), 1442-1446 (2000) [PubMed]
5. D. R. Houston, K. Shiomi, N. Arai, S. Omura, M. G. Peter, A. Turberg, B. Synstad, V. G. H. Eijsink, D. M. F. van Aalten: High resolution structures of chitinase complexed with natural product cyclopentapeptide inhibitors: Mimicry of carbohydrate substrate. Proc. Natl. Acad. Sci. USA 99(14), 9127-9132 (2002) [PubMed]