Atpenin ©

1. Discovery, producing organism and structure1-6)
Atpenins were isolated from the culture broth of Penicillium oxalicum FO-125. Atpenins inhibited growth of both fatty acid synthase (FAS) deficient (A-1) and acyl-CoA synthetase I (ACSI)
deficient (L-7) mutants of Candida lipolytica, and also inhibited incorporation of long chain fatty
acids into lipid fractions in Raji cells. The absolute configuration of atpenin A4 was confirmed
by X-ray crystallographic analysis2). The total synthesis of atpenins was reported by Quéguiner’sgroup3) and Ōmura’s group10,11) (See Appendix-I). Recently, atpenins and their structurally related compound, harzianopyridone, have been recognized to have potent mitochondrial complex II(succinate-ubiquinone oxidoreductase) inhibitory activity5). The co-crystallyzation study of atpenin A5 and E. coli complex II provided a new information for the quinone-binding site.
OHO

Cl

H3CO
H3CO

CH3
CH3 CH3OH

NOH

O

N

CH3 CH3OH

H3COCH3H3COAtpenin A4OH

Atpenin BClO

Penicillium sp. FO-125(Penicillium oxalicum FO-125)
Bar: 5 µmO

NCH3OH

H3COH3COH3COOH

N

CH3 CH3 ClOH

Atpenin A5CH3

H3COHerzianopyridone

2. Physical data
White powder. C15H21NO5Cl2; mol wt 366.24. Sol. in DMSO, MeOH, EtOH, CHCl3. Insol. in
H2O.
3. Biological activity1,7-9)1) Antimicrobial activity1)		
Test organisms
Atpenin A4
Staphylococcus aureus FDA 209P
Bacillus subtilis PCI 219
Micrococcus luteus PCI 1001
Mycobacterium smegmatis ATCC 607
Escherichia coli NIHJ JC-2 IFO 12734
Pseudomonas aeruginosa P-3 KB 105
Candida albicans KF 1
C. lipolytica L7 KF 236
Saccharomyces cerevisiae KF 26
Aspergillus niger ATCC 6275
Pyricularia oryzae KF180
Mucor racemosus IFO 4581
Trichophyton mentagrophytes KF 213
T. interdigitale KF 62
Microsporum gypseum KF 64>100>100>100>100>100>100>1006.25>10050
3.12>1006.25
12.5
3.12MIC (µg/ml)
Atpenin A5>100>100>100>100>100>100>1000.4>10012.5
3.12>1000.05
0.050.78

Atpenin B>100>100>100>100>100>100>1001.56>100500.78>1006.25
6.25
6.25

Bacteria: Sensitivity Disc Agar (Nissui); 37°C, 20 hrs. Fungi: Potato-glucose agar; 27°C, 72 hrs.

2) Atpenin B decreased cellular ATP levels in Raji cells with an IC50 value of 20 nM, suggesting
inhibition of an ATP-generating system by the drug7).
3) Atpenins inhibited nematode and mammalian mitochondrial complex II8).			

IC50 (µM)

Species Activities			A. suum
Complex I + II
Adult muscle Complex Ib
Complex II (QFR) c
Complex II (SQR) da								Bovine heart

Rat liverComplex I+ IIIe
Complex IId
Complex I+ IIIe
Complex IId

Atpenin A4

Atpenin A5

Harzianopyridone

1400.011

820.0036

420
0.017

0.11>1000.22
0.220.014>1000.012
0.032

>500
0.024

>500
0.0037

1.6>1000.36
2>5000.2

Measured by NADH-fumarate reductase activity.
Measured by NADH-quinone reductase activity. Ubiquinone 1, ubiquinone 2, or decylrhodoquinone was used as electron acceptor.
c
Measured by rhodoquinol-fumarate reductase activity.
dMeasured by succinate-UQ reductase activity.
e
Measured by NADH-cytochrome c reductase activity.
QFR, quinol-fumarate reductase; SQR, succinate-ubiquinone reductase.
ab4) Atpenin A5 inhibited bovine heart succinate-ubiquinone reductase much more potently than
known complex II inhibitors8).

IC50 (µM) against
bovine heart complex IIAtpenin A5Carboxin

TTFAHQNO

0.0036

1.1

5.8

> 100

Double-reciprocal plots showed mixed inhibition of atpenins with ubiquinone 2. This indicatesthat atpenins may block electron transfer between the enzyme and ubiquinone by binding to a region that partially overlaps with the physiological ubiquinone binding site. The kinetic analyses
showed that Ki values for atpenin A4, atpenin A5, and harzianopyridone were 6.9, 1.0, and 7.0
nM, respectively.
5) Studies of carboxin resistant fungi indicate that amino acid residues close to the [3Fe-4S] center in Ip, as well as a residue in CybS, located in the cytoplasmic loop between transmembranesegments, are important for carboxin binding and possibly for ubiquinone recognition. Thus,
ubiquinone is thought to bind to complex II at the interface between Ip and the membrane-anchor domain. This explains the observation that atpenins affected not only succinate-ubiquinone
reductase (SQR) activity but also succinate dehydrogenase (SDH) activity of the bovine heartcomplex II. Inhibition of SDH by atpenins was less potent than that of SQR, and complete SDH
inhibition was not achieved even at high concentrations. These discoverys of mixed type inhibition of SQR activity are very similar to those reported for carboxin.
6) Atpenin A5-SQR complex9)
The structure of atpenin A5 in complex with E. coli SQR was resolved. Atpenin A5 was locatedwithin the same hydrophobic pocket as ubiquinone at, however, a different position within thepocket. Atpenin A5 was bound deeper into the site prompting further assessment using protein-ligand docking experiments in silico. The initial interpretation of the quinone-binding site(Q-site) was re-evaluated in the light of this complex structure and protein-ligand docking data.
Two binding positions, the Q1-site (formerly presumed Q-site) and Q2-site (atpenin A5-bindingsite), are proposed for the E. coli SQR quinone-binding pocket to explain these data. At the Q2site, the side chains of a Ser and His are suitably positioned to provide hydrogen bonding partners to ubiquinone. This allows to propose a mechanism for the reduction of ubiquinone during
the catalytic turnover of the enzyme.
Succinate

Atpenins
MatrixFumarate

FpFAD[2Fe-2S]

Ip [4Fe-4S]

SDH
Succinate dehydrogenase
activity
PMS

MTT

Ubiquinone

DCIP[3Fe-4S]
CybL CybS(Heme)

Succinate-ubiquinone
reductase activity
SQR
Subunit structure and enzyme activities of complex II

Intermembrane space

5. References
1. [405] S. Ōmura et al., J. Antibiot. 41, 1769-1773 (1988)
2. [454] H. Kumagai et al., J. Antibiot. 43, 1553-1558 (1990)
3.		
F. Trécourt et al., J. Org. Chem. 59, 6173-6178 (1994)
4. [868] K. Shiomi & S. Ōmura, Proc. Jpn. Acad., Ser. B 80, 245-258 (2004)
5. [946] K. Kita et al., Trends Parasitol. 23, 223-229 (2007)
6. [947] S. Ōmura & K. Shiomi, Pure Appl. Chem. 79, 581-591 (2007)
7. [446] K. Oshino et al., J. Antibiot. 43, 1064-1068 (1990)
8. [823] H. Miyadera et al., Proc. Natl. Acad. Sci. USA 100, 473-477 (2003)
9. [910] R. Horsefield et al., J. Biol. Chem. 281, 7309-7316 (2006)
10. [1121] M. Ohtawa et al., Chem. Pharm. Bull. 60, 898-906 (2012)
11. [1032] M. Ohtawa et al., J. Antibiot. 62, 289-294 (2009)