Type II NADH dehydrogenase of the respiratory chain of Plasmodium falciparum and its inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.11.071

Plasmodium falciparum NDH2 (pfNDH2) is a non-proton pumping, rotenone-insensitive alternative enzyme to the multi-subunit NADH:ubiquinone oxidoreductases (Complex I) of many other eukaryotes. Recombinantly expressed pfNDH2 prefers coenzyme CoQ₀ as an acceptor substrate, and can also use the artificial electron acceptors, menadione and dichlorophenol-indophenol (DCIP). Previously characterized NDH2 inhibitors, dibenziodolium chloride (DPI), diphenyliodonium chloride (IDP), and 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) do not inhibit pfNDH2 activity. Here, we provide evidence that HDQ likely targets another P. falciparum mitochondrial enzyme, dihydroorotate dehydrogenase (pfDHOD), which is essential for de novo pyrimidine biosynthesis.

Full text of DOI:10.1016/j.bmcl.2008.11.071

Bioorganic & Medicinal Chemistry Letters 19 (2009) 972–975
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Type II NADH dehydrogenase of the respiratory chain of
Plasmodium falciparum and its inhibitors
Carolyn K. Dong a, Vishal Patel a,b, Jimmy C. Yang b,c, Jeffrey D. Dvorin a,d, Manoj T. Duraisingh a,
Jon Clardy b,c, Dyann F. Wirth a,c,
*
a Department of Immunology and Infectious Disease, Harvard School of Public Health, Building 1, Boston, MA 02115, USA
b Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
c The Broad Institute of Harvard and MIT, Infectious Disease Initiative, Cambridge, MA 02142, USA
d Children’s Hospital Boston, Division of Infectious Diseases, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Plasmodium falciparum NDH2 (pfNDH2) is a non-proton pumping, rotenone-insensitive alternative
enzyme to the multi-subunit NADH:ubiquinone oxidoreductases (Complex I) of many other eukaryotes.
Recombinantly expressed pfNDH2 prefers coenzyme CoQ0 as an acceptor substrate, and can also use the
artificial electron acceptors, menadione and dichlorophenol–indophenol (DCIP). Previously characterized
NDH2 inhibitors, dibenziodolium chloride (DPI), diphenyliodonium chloride (IDP), and 1-hydroxy-2-
dodecyl-4(1H)quinolone (HDQ) do not inhibit pfNDH2 activity. Here, we provide evidence that HDQ
likely targets another P. falciparum mitochondrial enzyme, dihydroorotate dehydrogenase (pfDHOD),
which is essential for de novo pyrimidine biosynthesis.
Received 23 October 2008
Revised 17 November 2008
Accepted 19 November 2008
Available online 24 November 2008
Keywords:
Type II NADH dehydrogenase
NDH2
Ó 2008 Elsevier Ltd. All rights reserved.
Dibenziodolium chloride
DPI
Diphenyliodonium chloride
IDP
1-Hydroxy-2-dodecyl-4(1H)quinolone
HDQ
Dihydroorotate dehydrogenase
DHOD
Ubiquinone
CoQ
CoQn
Malaria is
a
vector-born disease caused by
a
protozoan
in a rotenone-sensitive manner. In contrast, P. falciparum
parasite of the genus Plasmodium. It accounts for 300–500 mil-
lion cases each year and over a million deaths, mostly of chil-
dren under the age of five in sub-Saharan Africa.1 Given the
emergence and spread of resistance to current antimalarials such
as chloroquine, pyrimethamine, sulfadoxine, and atovaquone, the
identification of novel drugs and drug targets in the Plasmodium
parasite is critical for effective disease control.2–12 The electron
transport chain (ETC), a component of which is targeted by the
commonly used drug, atovoquone, was examined to expand
the repertoire of exploitable chemotherapeutic targets.13
Although the ETC is generally well conserved across species,
the first component (Complex I) of the Plasmodium falciparum
ETC is DNA sequence divergent and relatively uncharacterized.
The Complex I of eukaryotes is typically composed of a multi-
subunit NADH:Ubiquinone oxidoreductase that oxidizes NADH
encodes an alternative single polypeptide non-proton pumping
enzyme that is rotenone-insensitive. Alternative NADH dehydro-
genase (NDH2) enzymes are flavoproteins that catalyze the
transfer of electrons from NADH to ubiquinone (CoQn), using a
ping-pong mechanism, in order to maintain a pool of oxidized
NADH for reductive metabolic pathways, such as glycolysis or
the TCA cycle.14 NDH2 enzymes are found throughout the plant
kingdom and in some bacteria, yeast, and protists.15–23 These en-
zymes are characterized by their lack of a transmembrane do-
main, the presence of a conserved GxGxxG motif, and their
insensitivity to Complex I inhibitors such as rotenone and pieri-
cidin A.24 Like other NDH2 proteins, the respiratory chain NDH2
of P. falciparum (pfNDH2) is a single polypeptide, approximately
52 kDa in size.14
In an effort to explore targets along the ETC in P. falciparum,
specific inhibitors for each component of the chain must be
identified and characterized. While there are specific inhibitors
* Corresponding author. Tel.: +1 617 432 4629; fax: +1 617 432 4766.
E-mail address: dfwirth@hsph.harvard.edu (D.F. Wirth).
to Complex III (cytochrome bc1), such as antimycin
A and
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.071

C. K. Dong et al. / Bioorg. Med. Chem. Lett. 19 (2009) 972–975
973
Table 1
CoQD were not efficiently utilized by pfNDH2. It should be noted
that in vivo, pfNDH2 is predicted to be membrane-associated,
however, the in vitro enzyme assay is carried out in the absence
of membrane components. CoQ0 does not have the carbon tail of
the endogenous substrate, but the catalytic rings of the molecule
are intact.29 The observed differences in kcat may be due to differ-
ential binding affinities of CoQn in the absence of mitochondrial
membrane association. The inorganic electron acceptor, DCIP,
functioned with similar efficiency to CoQ0, albeit with increased
substrate specificity and lower catalytic rate. In comparison, the
efficiency of menadione was significantly reduced due to an in-
Kinetic properties of purified pfNDH2 in the presence of electron-accepting substrates
kcat (1/min)a
Km
a,b
Electron acceptor
CoQ0
CoQ1
CoQ4
CoQD
Menadione
DCIP
20.0 0.3
0.9 0.1
n.a.
n.a.
4.6 0.2
1.9 0.0
104.3 6.6
2.2 1.2
n.a.
n.a.
239.3 22.4
4.6 0.4
a
Km and kcat values and standard deviations were calculated from three inde-
pendent experiments. n.a., enzyme activity not observed.
b
For each Km determination, the concentration of either the electron acceptor or
crease in the Km,app
.
NADH was varied while the other substrate was fixed at 500
were determined using best-fit Michaelis–Menton curves.
lM. Km and kcat values
In the absence of a crystal structure for NDH2 enzymes, predic-
tions for the flavin-binding region and binding sites for NADH and
CoQn have been based on sequence and structural similarities to
other redox enzymes.30 The biochemical relevance of these predic-
tions, however, has not yet been demonstrated. An N-terminal
atovaquone, the specificity of inhibitors to alternative Complex I,
pfNDH2, has been debated.25,26 Recombinant pfNDH2 has been
difficult to express and purify, and previous biochemical studies
have been confounded by the use of crude or partially purified
lysate in enzymatic assays.14,26,27 In this study, biochemically
active pfNDH2 was recombinantly expressed in Escherichia coli
in order to thoroughly evaluate substrate and inhibitor specific-
ity. Full-length protein was expressed in-frame with a C-terminal
6xHis tag. The presence of detergent (0.5% Triton X-100) was
critical for purification of active enzyme. The enzymatic activity
of pfNDH2 was measured by chemical quantification of NAD+
using an assay adapted from Putt et al. in which addition of ace-
tophenone base, followed by incubation at 100 °C with formic
truncation product of pfNDH2 (ND214) was expressed and purified
in order to determine if the conserved GxGxxG domains are neces-
sary for catalytic activity. Truncated pfNDH2 showed significantly
less activity compared to full-length product arguing that the
N-terminal region of the enzyme is critical for full catalytic activity
(data not shown).
NDH2 has been implicated as an activator of the plant-derived
antimalarial, artemisinin.31 A knockout screen using Saccharamyces
cerevisiae homozygous deletion strains showed that deletion of
two S. cerevisiae NDH2 genes resulted in artemisinin resistance.
Although expression of pfNDH2 in S. cerevisiae NDH2 knockout
strains partially restored artemisinin sensitivity, and over-expres-
sion of the S. cerevisiae NDH2 genes was shown to increase arte-
misinin sensitivity, no biochemical link between pfNDH2 and
artemisinin has yet been established.31 Therefore artemisinin was
tested for either activation or inhibition of pfNDH2 activity. We
found that arteminsin, even at high concentrations, did not perturb
catalysis by pfNDH2 (Table 2). These results suggest that artemis-
inin may act via an indirect mechanism with NDH2 rather than by
direct enzyme binding.
acid, yields
a product with strong fluorescence emission at
444 nm when excited at 372 nm (Supplemental Scheme 1).28
The pH dependence of the pfNDH2 reaction was assessed using
this fluorescence-based assay at fixed concentrations of NADH
and CoQ0 (0.1 mM for both substrates). pfNDH2 was maximally
active at
a pH range between 7.0 and 9.0 (Supplemental
Fig. 1). Given these data, a HEPES buffer equilibrated to pH 8.0
was used in subsequent kinetics assays.
Similar to other CoQn-utilizing enzymes, NDH2 homologs show
differential substrate specificities for various CoQn. To gauge the
substrate specificity of recombinant pfNDH2, the concentration
of NADH was held constant, and in excess at 200
substrates, CoQ0, CoQ1, CoQ4, CoQD, menadione, and DCIP, were
varied from 0.5 M to 2 mM. The Km,app and kcat for the various
Previously described inhibitors of Complex I and NDH2 were
evaluated for inhibition of recombinant pfNDH2 activity. Inhibition
of enzymatic activity was assessed in the presence of excess NADH
lM, while the
(200
(100
l
M) and CoQ0 approximately equivalent to the Km,app
l
lM). As expected, Complex I inhibitors had no effect on
substrates were calculated based on best fit Michaelis–Menten
kinetic curves, and are reported in Table 1 (Supplemental Fig. 2).
Among the CoQn substrates, only CoQ0 and CoQ1 showed detect-
able catalysis with pfNDH2. CoQ0 afforded the maximal catalytic
rate; however, the Km,app was significantly higher for CoQ0 than
CoQ1, reducing the catalytic efficiency (kcat/Km,app) of the substrate.
Electron acceptors with longer carbon chains such as CoQ4, and
pfNDH2 enzyme activity (Table 2). Eschemann et al. identified
1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) as an inhibitor of
yeast (Yarrowia lipolytica) NDH2 by isolating mitochondrial mem-
brane fractions in lieu of purified protein.14 In addition, HDQ was
shown to be a potent inhibitor of P. falciparum parasite prolifera-
tion.14,32 Dibenziodolium chloride (DPI) and diphenyliodonium
chloride (IDP) have been reported to inhibit pfNDH2 activity in
Table 2
Inhibition of in vitro enzyme activity and in vivo parasite proliferation
Compound
IC50
(l
M) of enzyme activitya
pfDHOD
IC50
(
l
M) of P. falciparum proliferationa,b
DD2-scDHOD
pfNDH2
scDHOD
DD2
Atovaquone
Antimycin A
HDQ
DPI
IDP
Rotenone
Flavone
Artemisinin
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.0 0.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.002 0.0002
3.4 1.8 mg/mLc
0.24 0.09
2.7 0.5
12.4 2.0
>10
>10
>10
>10
3.0 0.6
10.6 3.8
>10
>10
0.006 0.0001
>10
0.01 0.0006
a
IC50 values and standard deviations were calculated from three independent experiments.
b
c
IC50 values for DD2 and DD2-scDHOD parasite strains are based upon [3H]-hypoxanthine dose–effect curves.
IC50 values are given in mg/mL as antimycin A was a mixture of the components A1, A2, A3, and A4.

974
C. K. Dong et al. / Bioorg. Med. Chem. Lett. 19 (2009) 972–975
undetermined, therefore additional work is required to identify
specific inhibitors of pfNDH2 for further biochemical
characterization.47
Acknowledgments
The authors thank Ralph Mazitschek for suggestions and advice.
This work was supported by NICHD K12-HD000850 (J.D.D.), and by
the NSF Graduate Research Fellowship Program (V.P.), and Harvard
Malaria Initiative (D.F.W.).
Figure 1. Chemical structures of (A) DPI, (B) IDP, and (C) HDQ.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.11.071.
crude lysate fractions (IC50 = 0.24 0.03 and 5.99 0.36, respec-
tively), and both show efficacy against whole parasite prolifera-
tion.26 It has been suggested that the antimalarial mechanisms of
DPI, IDP, and HDQ may be attributed to the inhibition of pfNDH2
activity, however, dose–effect profiles using purified recombinant
pfNDH2 did not corroborate these findings. In fact, these com-
pounds did not inhibit pfNDH2 activity at concentrations of up to
References and notes
1. World malaria situation in 1994. Part I. Population at risk. Wkly Epidemiol. Rec.
1997, 72(36), 269–274.
2. Cadigan, F. C., Jr.; Sadudee, N.; Bourke, A. T.; Gould, D. J.; Winter, P. E. Trans. Roy.
Soc. Trop. Med. Hyg. 1968, 62, 562.
10 lM (Table 2). Both DPI and IDP are well-known flavoprotein
3. Mahoney, L. E. Lancet 1968, 2, 1139.
oxidoreductase inhibitors, suggesting that previous observations
of reduction in NADH consumption using crude parasite lysate
may have been due to inhibition of a different flavoenzyme-depen-
dent reaction.33–38
The chemical structure of HDQ is similar to that of the CoQn
substrate (Fig. 1) and thus, we speculated that its mode of action
might be related to another CoQn-dependent enzyme, P. falciparum
Type II dihydroorotate dehydrogenase (pfDHOD). pfDHOD is a
mitochondrial flavoenzyme that catalyzes the oxidation of dihy-
4. Peters, W. Lancet 1969, 2, 54.
5. Young, M. D.; Moore, D. V. Am. J. Trop. Med. Hyg. 1961, 10, 317.
6. Charles, L. J.; Van Der Kaay, H. J.; Vincke, I. H.; Brady, J. Bull. World Health Organ.
1962, 26, 103.
7. Clyde, D. F.; Shute, G. T. Trans. Roy. Soc. Trop. Med. Hyg. 1954, 48, 495.
8. Young, M. D.; Contacos, P. G.; Stitcher, J. E.; Millar, J. W. Am. J. Trop. Med. Hyg.
1963, 12, 305.
9. Hess, U.; Timmermans, P. M.; Jones, M. Am. J. Trop. Med. Hyg. 1983, 32, 217.
10. Rumans, L. W.; Dennis, D. T.; Atmosoedjono, S. Lancet 1979, 2, 580.
11. Vleugels, M. P.; Wetsteyn, J. C.; Meuwissen, J. H. Trop. Geogr. Med. 1982, 34, 263.
12. Srivastava, I. K.; Morrisey, J. M.; Darrouzet, E.; Daldal, F.; Vaidya, A. B. Mol.
Microbiol. 1999, 33, 704.
13. Vaidya, A. B. Mitochondrial Physiology as a Target for Atovaquone and Other
Antimalarials. In Malaria: Parasite Biology, Pathogenesis, and Protection;
Sherman, I. W., Ed.; American Society for Microbiology: Washington, DC,
1998; pp 355–368.
droorotate (L-DHO) using a FMN cofactor that is re-oxidized by
CoQn.39 The malaria parasite relies upon pfDHOD as it catalyzes
the rate-limiting step for de novo pyrimidine biosynthesis.40 Inhi-
bition of pfDHOD activity by HDQ was assessed in the presence of
14. Eschemann, A.; Galkin, A.; Oettmeier, W.; Brandt, U.; Kerscher, S. HDQ (1-
excess
Km,app (115
(Supplemental Scheme 2). HDQ inhibited pfDHOD activity with
an IC50 of 4.0 0.1 M (Table 2).
L-DHO (500
lM) and CoQ0 approximately equivalent to the
hydroxy-2-dodecyl-4(1H)quinolone),
mitochondrial alternative NADH dehydrogenase: evidence for a ping-pong
mechanism. J. Biol. Chem. 2005, 280, 3138.
a
high
affinity
inhibitor
for
lM), by coupling the assay to the chromogen, DCIP
15. Rasmusson, A. G.; Svensson, A. S.; Knoop, V.; Grohmann, L.; Brennicke, A. Plant
J. 1999, 20(1), 79.
l
16. de Vries, S.; Grivell, L. A. Eur. J. Biochem. 1988, 176, 377.
17. Kerscher, S. J.; Okun, J. G.; Brandt, U. J. Cell Sci. 1999, 112, 2347.
18. Melo, A. M.; Duarte, M.; Moller, I. M.; Prokisch, H.; Dolan, P. L.; Pinto, L.; Nelson,
M. A.; Videira, A. J. Biol. Chem. 2001, 276, 3947.
19. Bjorklof, K.; Zickermann, V.; Finel, M. FEBS Lett. 2000, 467, 105.
20. Matsushita, K.; Otofuji, A.; Iwahashi, M.; Toyama, H.; Adachi, O. FEMS Microbiol.
Lett. 2001, 204, 271.
21. Gomes, C. M.; Bandeiras, T. M.; Teixeira, M. J. Bioenerg. Biomembr. 2001, 33, 1.
22. Bandeiras, T. M.; Salgueiro, C.; Kletzin, A.; Gomes, C. M.; Teixeira, M. FEBS Lett.
2002, 531, 273.
23. Bandeiras, T. M.; Salgueiro, C. A.; Huber, H.; Gomes, C. M.; Teixeira, M. Biochim.
Biophys. Acta 2003, 1557, 13.
24. Melo, A. M.; Bandeiras, T. M.; Teixeira, M. Microbiol. Mol. Biol. Rev. 2004, 68,
603.
25. Vaidya, A. B.; Painter, H. J.; Morrisey, J. M.; Mather, M. W. Trends Parasitol. 2008,
24, 8.
26. Biagini, G. A.; Viriyavejakul, P.; O’Neill P, M.; Bray, P. G.; Ward, S. A. Antimicrob.
Agents Chemother. 2006, 50, 1841.
27. Krungkrai, J.; Kanchanarithisak, R.; Krungkrai, S. R.; Rochanakij, S. Exp. Parasitol.
2002, 100, 54.
A DD2 transgenic P. falciparum strain expressing a Type I cyto-
plasmic DHOD from S. cerevisiae (scDHOD) was used to pinpoint
the antimalarial mechanism of HDQ (Supplemental methods). Pain-
ter et al. had previously shown that the role of the mitochondrial
electron potential in the asexual stage of P. falciparum growth was
to maintain a pool of CoQn in order to sustain pfDHOD activity and
subsequent de novo pyrimidine biosynthesis.40 It was demonstrated
that addition of exogenous scDHOD results in a bypass of the endog-
enous electron transport chain through Complex III.40 ScDHOD uti-
lizes fumarate or NAD+ rather than CoQn to reoxidize the flavin
(FMN) prosthetic group in the second half-reaction of the redox pro-
cess.41–45 Therefore, inhibition of scDHOD activity was assessed in
the presence of excess
L
-DHO (500
lM) and fumarate approximately
equivalent to the Km,app (115
l
M), by coupling the assay to the chro-
mogen, DCIP (Supplemental Scheme 2). None of the compounds
tested inhibited scDHOD enzyme activity. Proliferation of the
scDHOD-expressing transgenic strain was unaffected by HDQ at a
28. Putt, K. S.; Hergenrother, P. J. Anal. Biochem. 2004, 326, 78.
29. de Macedo, C. S.; Uhrig, M. L.; Kimura, E. A.; Katzin, A. M. FEMS Microbiol. Lett.
2002, 207, 13.
30. Fisher, N.; Bray, P. G.; Ward, S. A.; Biagini, G. A. Trends Parasitol. 2007, 23, 305.
31. Li, W.; Mo, W.; Shen, D.; Sun, L.; Wang, J.; Lu, S.; Gitschier, J. M.; Zhou, B. PLoS
Genet. 2005, 1, e36.
drug concentration of up to 10
half-maximal growth inhibition at 0.24 0.09
l
M, while the parental strain showed
M (Table 2) (Supple-
l
mental Fig. 3). This rescued growth phenotype associated with the
addition of exogenous yeast DHOD argues that pfDHOD is the likely
in vivo target of HDQ rather than pfNDH2 as formerly described.
The results of this study have shown that previously
characterized NDH2 inhibitors are not effective inhibitors of
pfNDH2. Such an observation is not unexpected since inhibitors
for alternative rotenone-insensitive NADH dehydrogenases are
rare and non-specific.46 The essentiality of pfNDH2 remains
32. Saleh, A.; Friesen, J.; Baumeister, S.; Gross, U.; Bohne, W. Antimicrob. Agents
Chemother. 2007, 51, 1217.
33. Cross, A. R.; Jones, O. T. Biochem. J. 1986, 237, 111.
34. Stuehr, D. J.; Fasehun, O. A.; Kwon, N. S.; Gross, S. S.; Gonzalez, J. A.; Levi, R.;
Nathan, C. F. FASEB J. 1991, 5, 98.
35. Sanders, S. A.; Eisenthal, R.; Harrison, R. Eur. J. Biochem. 1997, 245, 541.
36. Tew, D. G. Biochemistry 1993, 32, 10209.
37. Luetjens, C. M.; Bui, N. T.; Sengpiel, B.; Munstermann, G.; Poppe, M.; Krohn, A.
J.; Bauerbach, E.; Krieglstein, J.; Prehn, J. H. J. Neurosci. 2000, 20, 5715.

C. K. Dong et al. / Bioorg. Med. Chem. Lett. 19 (2009) 972–975
975
38. Majander, A.; Finel, M.; Wikstrom, M. J. Biol. Chem. 1994, 269, 21037.
39. Malmquist, N. A.; Gujjar, R.; Rathod, P. K.; Phillips, M. A. Biochemistry 2008, 47, 2466.
40. Painter, H. J.; Morrisey, J. M.; Mather, M. W.; Vaidya, A. B. Nature 2007, 446, 88.
41. Taylor, M. L.; Taylor, W. H.; Eames, D. F.; Taylor, C. D. J. Bacteriol. 1971, 105,
1015.
43. Skophammer, R. G.; Servin, J. A.; Herbold, C. W.; Lake, J. A. Mol. Biol. Evol. 2007,
24, 1761.
44. Denis-Duphil, M. Biochem. Cell. Biol. 1989, 67, 612.
45. Jordan, D. B.; Bisaha, J. J.; Picollelli, M. A. Arch. Biochem. Biophys. 2000, 378, 84.
46. Fang, J.; Beattie, D. S. Biochemistry 2002, 41, 3065.
47. Kawahara, K.; Mogi, T.; Tanaka, T. Q.; Hata, M.; Miyoshi, H.; Kita, K. J. Biochem.
2008 [Epub ahead of print].
42. van der Plas, J.; Hellingwerf, K. J.; Seijen, H. G.; Guest, J. R.; Weiner, J. H.;
Konings, W. N. J. Bacteriol. 1983, 153, 1027.