Improved efficacy of fosmidomycin against plasmodium and mycobacterium species by combination with the cell-penetrating peptide octaarginine

Article abstract of DOI:10.1128/AAC.00427-13

Cellular drug delivery can improve efficacy and render intracellular pathogens susceptible to compounds that cannot permeate cells. The transport of physiologically active compounds across membranes into target cells can be facilitated by cell-penetrating peptides (CPPs), such as oligoarginines. Here, we investigated whether intracellular delivery of the drug fosmidomycin can be improved by combination with the CPP octaarginine. Fosmidomycin is an antibiotic that inhibits the second reaction in the nonmevalonate pathway of isoprenoid biosynthesis, an essential pathway for many obligate intracellular pathogens, including mycobacteria and apicomplexan parasites. We observed a strict correlation between octaarginine host cell permeability and its ability to improve the efficacy of fosmidomycin. Plasmodium berghei liver-stage parasites were only partially susceptible to an octaarginine-fosmidomycin complex. Similarly, Toxoplasma gondii was only susceptible during the brief extracellular stages. In marked contrast, a salt complex of octaarginine and fosmidomycin greatly enhanced efficacy against blood-stage Plasmodium falciparum. This complex and a covalently linked conjugate of octaarginine and fosmidomycin also reverted resistance of Mycobacteria to fosmidomycin. These findings provide chemical genetic evidence for vital roles of the nonmevalonate pathway of isoprenoid biosynthesis in a number of medically relevant pathogens. Our results warrant further investigation of octaarginine as a delivery vehicle and alternative fosmidomycin formulations for malaria and tuberculosis drug development. Copyright

Full text of DOI:10.1128/AAC.00427-13

Improved Efficacy of Fosmidomycin against Plasmodium and
Mycobacterium Species by Combination with the Cell-Penetrating
Peptide Octaarginine
a,b
a
a,c
d
e
f
Christof Sparr, * Nirupam Purkayastha, Beata Kolesinska, * Martin Gengenbacher, Borko Amulic, Kai Matuschewski,
a
f
Dieter Seebach, Faustin Kamena
a
ETH-Zürich, Laboratorium für Organische Chemie, Departement für Chemie und Angewandte Biowissenschaften, Hönggerberg HCI, Zürich, Switzerland ; Universität
b
c
d
Basel, Departement Chemie, Basel, Switzerland ; Technical University of Lodz, Institute of Organic Chemistry, Lodz, Poland ; Department of Immunology , Department of
e
f
Cellular Microbiology , and Parasitology Unit , Max Planck Institute for Infection Biology, Berlin, Germany
Cellular drug delivery can improve efficacy and render intracellular pathogens susceptible to compounds that cannot per-
meate cells. The transport of physiologically active compounds across membranes into target cells can be facilitated by
cell-penetrating peptides (CPPs), such as oligoarginines. Here, we investigated whether intracellular delivery of the drug
fosmidomycin can be improved by combination with the CPP octaarginine. Fosmidomycin is an antibiotic that inhibits
the second reaction in the nonmevalonate pathway of isoprenoid biosynthesis, an essential pathway for many obligate in-
tracellular pathogens, including mycobacteria and apicomplexan parasites. We observed a strict correlation between octa-
arginine host cell permeability and its ability to improve the efficacy of fosmidomycin. Plasmodium berghei liver-stage
parasites were only partially susceptible to an octaarginine-fosmidomycin complex. Similarly, Toxoplasma gondii was only
susceptible during the brief extracellular stages. In marked contrast, a salt complex of octaarginine and fosmidomycin
greatly enhanced efficacy against blood-stage Plasmodium falciparum. This complex and a covalently linked conjugate of
octaarginine and fosmidomycin also reverted resistance of Mycobacteria to fosmidomycin. These findings provide chemi-
cal genetic evidence for vital roles of the nonmevalonate pathway of isoprenoid biosynthesis in a number of medically rele-
vant pathogens. Our results warrant further investigation of octaarginine as a delivery vehicle and alternative fosmidomy-
cin formulations for malaria and tuberculosis drug development.
alaria and tuberculosis remain two of the most devastat- shapes and sizes, thereby mainly acting as a delivery vehicle of
M
ing diseases of mankind, causing altogether more than drug molecules into cells (7, 8).
1
.5 million fatalities annually (1, 2). Decades of intensive re-
Compared to the naturally existing polycationic cell-penetrat-
search were unable to deliver vaccines that fully protect from ing peptides, such as peptides from the human immunodeficiency
either of the two diseases, although numerous promising can- virus (HIV) trans-activator of transcription (TAT) protein or the
didates entered advanced clinical development (3, 4). There- Antennapedia-homeodomain-derived antennapedia (Antp) pep-
fore, control of malaria and tuberculosis highly relies on access tide from Drosophila melanogaster, synthetic oligoarginines show
to diagnostics and treatment with potent drugs. However, non- excellent cell-penetrating properties across biological membranes
compliance of patients and incomplete treatment schedules, (9). Our previous studies demonstrated that human red blood
among many other factors, accelerate selection for drug-resis- cells are impermeable to 6-carboxyfluorescein (FAM)-octaargin-
tant strains of Plasmodium falciparum and Mycobacterium tu- ine amide (compound 1) (Fig. 1) but become permeable when
berculosis, the intracellular pathogens causing malaria and tu- infected with the malaria parasite Plasmodium falciparum (10),
berculosis, respectively. Drug resistance to antibiotics and thereby offering an opportunity for antimalarial drug delivery.
several antimalarial drugs is now widespread around the globe
2, 5). Hence, there is an urgent and continuous need to design
Here, we investigate the potential of octaarginine derivatives as
drug carriers for the antimalaria drug fosmidomycin (3-[formyl(hy-
(
and develop new drugs and to reduce treatment failures by
improving the efficacy of current anti-infectives.
Received 1 March 2013 Returned for modification 22 April 2013
Accepted 5 July 2013
A major limitation for antibiotics against intracellular patho-
gens such as P. falciparum and M. tuberculosis is the need to cross
several biological membranes to access the target site. Further-
more, pathogenic mycobacteria present an intrinsically imperme-
able thick cell wall (6), which serves as a robust barrier to most
antibiotics. Therefore, the development of novel strategies to im-
prove the delivery and the bioavailability of existing or novel drugs
is a promising avenue to significantly increase efficacy and/or re-
duce systemic toxicity through dose reduction. Reports have re-
vealed the potential of short polycationic cell-penetrating peptides
not only to penetrate most biological cell membranes but also to
retain this desirable attribute when attached to cargos of various
Published ahead of print 15 July 2013
Address correspondence to Dieter Seebach, Seebach@org.chem.ethz.ch, or
Faustin Kamena, kamena@mpiib-berlin.mpg.de.
*
Present address: Christof Sparr, Universität Basel, Department Chemie, Basel,
Switzerland; Beata Kolesinska, Institute of Organic Chemistry, Technical University
of Lodz, Lodz, Poland.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/
AAC.00427-13.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.00427-13
October 2013 Volume 57 Number 10
Antimicrobial Agents and Chemotherapy p. 4689–4698
aac.asm.org 4689

Sparr et al.
NH
NH
2
2
O
R
*
N
O
A
C
B
D
linker,
X
cargo
spacer
N
H
readily
cleavable
bond
O
Fl
n
N
H
X
CPP
n
n = 10 - 30
3h - oligoarginines
X = OH, NH2
R: 5 - n Arg or Lys
side chains
n = 6 - 10
X = OH, NH2
*
:
L
- or
D
-configuration
Fl = fluorescent group
H
N
H
N
C
P
P
C
P
P
H
H
O
O
H
O
O
OH
R
HN
P
HN
C
R
N
H
N
H
H
O
2
HO C
O
H
N
NH
2
8
O
P
OH
N
O
O
Fosmidomycin (R = H)
R
HO
HO
FR-900098 (R = CH3)
N
O
OH
H
2
N
NH
2
1
(6-Fam-octaarginine amide)
NH
2
N
NH
2
2
H
3
C
O
O
CH
2
O
O
O
3
O
P
OH
N
P
N
NH
H
EtO
EtO
.
4 HO
N
H
1
HO
O
O
8
2
3
FIG 1 Attachment of drug molecules as cargo to a carrier cell-penetrating peptide (CPP) through a readily cleavable bond for release of the drug in the target cell.
For peptidolytic stability, a D-␣-peptide (A) or a ␤-peptide (B) derivative can be employed. Instead of covalent attachment, counterion activation has been
demonstrated previously for carboxylate salts (C) and was realized for the first time with a phosphonate salt (D) in the present study. Structures for fosmido-
mycin, FR-900098, FAM-octaarginine amide (compound 1), fosmidomycin-octaarginine salt (compound 2), and fosmidomycin-octaarginine covalent conju-
gate (compound 3) are shown.
droxy)amino]-propylphosphonicacid;CID572)anditsmorepotent isms were reported to be resistant to fosmidomycin due to the poor
analogue, FR-900098 (3-[acetyl(hydroxy)amino]propylphosphonic ability of the drug to permeate the membrane (20, 21). Liver-stage
acid; CID 162204) (11) (Fig. 1). In contrast to mammals, most mi- Plasmodium berghei also is known to be resistant to fosmidomycin,
croorganisms and plants synthesize isoprenoid precursors, such as presumably due to its inability to enter hepatocytes (22). Impor-
isopentenyl diphosphate (IPP), by the nonmevalonate pathway, tantly, the choice of fosmidomycin as the cargo allowed us to extend
called the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway (12). theinvestigationtootherpathogenicmicroorganisms, suchasT. gon-
Fosmidomycin is a potent inhibitor of DOXP reductoisomerase dii and Toxoplasma mycobacteria, which also operate the target path-
(DXR), theenzymecatalyzingthesecondreactionintheDOXPpath- way of fosmidomycin.
way (11). Importantly, fosmidomycin has been tested in clinical
phase II as an antimalaria drug either alone or in combination with
other compounds, e.g., clindamycin and artesunate, and it showed
initial promise in fosmidomycin-based combination therapies (13–
MATERIALS AND METHODS
The detailed chemical synthesis of fosmidomycin-octaarginine conju-
gates is described in the supplemental material.
Uptake of FAM-labeled octaarginine. FAM-labeled octaarginine
18). Successful selection of cultured blood-stage P. falciparum para-
(compound 1; 10 ␮g/ml) was added to cultured P. falciparum (strain
sites that lack an apicoplast through medium supplementation with
the product IPP provided genetic proof that nonmevalonate iso-
prenoid biosynthesis is, at least in vitro, the only essential pathway
inside the apicoplast during asexual replication in erythrocytes (19).
3D7)-infected human red blood cells, P. berghei (strain ANKA)-infected
human hepatoma cells (Huh7), T. gondii (strain RH)-infected human
foreskin fibroblasts (HFF), or cultured Mycobacterium smegmatis or
Mycobacterium bovis BCG, followed by further incubation for 12 h.
Although fosmidomycin strongly inhibits DXR orthologues from Intracellular FAM-labeled octaarginine was visualized microscopically
Toxoplasma gondii and M. tuberculosis in enzyme assays, these organ- either by direct fluorescence or in an immunofluorescence assay to-
4690 aac.asm.org
Antimicrobial Agents and Chemotherapy

Octaarginine Improves Efficacy of Fosmidomycin
gether with pathogen-specific antibodies. For the direct fluorescence measurements. Mean values with standard deviations from three repli-
assay, cells were fixed with 4% paraformaldehyde (PFA) for 20 min at cates were plotted.
room temperature, followed by extensive washing. 4,6-Diamidino-2-
phenylindole (DAPI; 1 ␮g/ml) then was added and incubated for 10 RESULTS
min at room temperature. After extensive washing, cells were mounted
Chemical synthesis of fosmidomycin-octaarginine conjugates.
using fluoromount-G (Southern Biotech). For the immunofluores-
In recent years, cell-penetrating peptides (CPPs) with positively
cence assay, infected Huh7 cells were fixed (4% paraformaldehyde) for
charged side chains, such as the natural Tat[48-60] or penetratin
2
0 min at room temperature and permeabilized (0.2% Triton X-100)
(
AntpHD43-58) and simple oligoarginines (R ), have been devel-
n
for 15 min at room temperature. After 1 h of blocking with 1% fetal calf
serum (FCS) in phosphate-buffered saline (PBS) at room temperature,
cells were incubated overnight with anti-P. berghei HSP70 (PbHSP70)
antibodies in PBS containing 1% FCS at 4°C. Cells were washed three
times with PBS and incubated with the corresponding fluorescently
labeled secondary antibody (Invitrogen) in PBS for 1 h at room tem-
oped for carrying physiologically active compounds into target
cells (23–28). In these studies, the carrier and the cargo were
linked by a readily cleavable bond (23, 28) (Fig. 1A). In some cases,
the carrier peptide was built of D-amino acids instead of the nat-
ural L-amino acids in order to prevent premature degradation
perature. Prior to microscopy, the cells were incubated with DAPI (10 (24, 29, 30). In other cases, proteolytically stable CPPs containing
␮
g/ml) for 10 min and washed three times with PBS.
␤-oligoarginines or lysines were used (31–36) (Fig. 1B).
Estimation of the partial permeability of EEFs by octaarginine. The
Recently, Matile and his group have introduced a conceptually
total number of EEFs was counted in one well of an 8-well Permanox new mode of noncovalent attachment of the carrier CPP to a cargo
Lab-Tek chamber slide system (Nunc). Percentages were obtained as a (37, 38), namely, salt formation of the guanidinium cations of the
ratio of either FAM-octaarginine-positive EEFs to the total number or of
CPP with carboxylate anions of a cargo (Fig. 1C). Anticipating
FAM-octaarginine-negative EEFs to the total number. Mean values from
that a similar, possibly even stronger ionic interaction would oc-
three biological replicates were plotted.
cur with a phosphonate anion, such as that in fosmidomycin and
its analogues (Fig. 1D), we generated the salt (compound 2) of the
Plaque assay. For the plaque assay, HFF cells in 6-well plates were
infected with T. gondii tachyzoites in the presence of the corresponding
FAM-labeled octaarginine amide (compound 1) with four fos-
drug, cultured for 7 days, fixed with Ϫ80°C methanol, stained with crystal
midomycin units by mixing the two compounds in water and
violet, and documented using a Zeiss microscope with a ϫ5 magnification
objective. Experiments were performed in three independent replicates.
For the tachyzoite pretreatment assay, freshly harvested T. gondii
lyophilizing the resulting solution (Fig. 1).
In another approach to carrying fosmidomycin or FR900098
into infected cells, we envisioned that acylation on oxygen of the
tachyzoites were preincubated with the corresponding drug for 1 h in a
hydroxylamine moiety would produce an active ester from which
CO incubator at 37°C prior to infection of HFF cells.
2
Growth inhibition assay of blood-stage P. falciparum. P. falciparum the antibiotic could be released. We expected that the phospho-
ϩ
clone 3D7 was cultured at 5% hematocrit of human O red blood cells nate ester group would also be hydrolyzed, since both the free
in RPMI 1640 supplemented with 0.05 g/liter hypoxanthine (Sigma- phosphonic acid and the free N(OH)COR group are believed to be
Aldrich), 25 mM HEPES, 0.5% Albumax II (Life Technologies), 0.25% essential for binding to the DXR enzyme target. Thus, we also
sodium bicarbonate, and 0.01 mg/ml gentamicin (Sigma-Aldrich). All
generated the octaarginine amide carrying one FR900098 diethyl-
cultures were kept at 37°C under 90% nitrogen, 5% oxygen, and 5%
ester unit (compound 3) (Fig. 1).
carbon dioxide. Cultures were treated with fosmidomycin or fosmido-
Synthesis of the targeted derivative of fosmidomycin com-
mycin-octaarginine conjugates at the concentrations indicated. Ex-
pound 9 (Fig. 2A) was achieved by starting from the commercially
periments were done in triplicate for 72 h. Medium was changed daily,
available 3-bromopropylphosphonic acid diethyl-ester 5. Com-
and blood smears were prepared every 24 h to monitor parasitemia.
pound 5 was first transformed into the corresponding O-benzyl-
Briefly, samples were fixed with methanol and stained with Giemsa
hydroxylamino compound 6 by the nucleophilic substitution
(
Merck). For parasitemia determination, 30 microscopy fields were
2
(
SN ) reaction of the bromide group by O-benzylhydroxylamine
counted. Mean values with standard deviations from three replicates
were plotted.
Growth inhibition assay of liver-stage P. berghei. The human liver
cell line Huh7 was seeded into 8-well Lab-Tek chamber slides (Nalge
in refluxing acetonitrile. N-acetylated compound 7 was achieved
by treatment with acetyl chloride (AcCl) in the presence of trieth-
ylamine (Et N). Subsequent hydrogenolysis in the presence of cat-
3
Nunc International Corp.) at a density of 50,000 cells/well. The next day, alytic Pd-C (10%) removed the benzyl protection to give the free
cells were infected with 10,000 freshly dissected P. berghei sporozoites per N-hydroxylamine compound 8.
well. Twenty-four h after infection, either fosmidomycin or fosmidomy-
Treatment of compound 8 with glutaric acid anhydride in the
cin-octaarginine conjugate 2 was added at various concentrations. Mero- presence of Et N and 4-N,N-dimethylaminopyridine (DMAP)
3
somes were isolated from cell culture supernatant 65 h after infection and
yielded compound 9.
counted. Mean numbers of merosomes were generated from three inde-
Coupling of the fosmidomycin derivative 9 to the resin-bound
pendent wells of an 8-well Lab-Tek Permanox chamber slide system
carrier (octaarginine) was performed on a peptide amide linker-
(
Nunc).
polyethylene glycol (PAL-PEG) solid support using O-(7-aza-
H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluo-
Growth inhibition assay of Mycobacterium bovis BCG. Mycobacte-
1
ria were grown in Middlebrook 7H9 broth (Becton, Dickinson) supple-
mented with Middlebrook ADC enrichment (Becton, Dickinson), 0.2%
glycerol, and 0.5% Tween 80. Exponentially growing M. bovis BCG was
diluted in fresh 7H9 complete medium to an optical density at 600 nm
rophosphate (HATU) as the coupling agent and diisopropyl ethyl
amine (DIPEA) as the base in anhydrous dimethylformamide
(
DMF). Subsequent deprotection and cleavage from the resin
with a mixture of trifluoroacetic acid (TFA)-H O-triisopropylsi-
lane (TIPS) provided the fosmidomycin-Arg8 conjugate 3
(
9
OD600) of 0.005, and 200 ␮l per well then was incubated in a translucent
6-well flat-bottom culture plate (Nunc) in the presence of either isonia-
2
zid (INH), fosmidomycin, or fosmidomycin-octaarginine conjugates (Fig. 2B).
salt 2 and conjugate 3) at various concentrations. Plates were incubated
Uptake of FAM-octaarginine by Plasmodium-infected cells.
at 37°C and 80 rpm. Bacterial growth was monitored over time by OD600 Previous work established that fosmidomycin is efficiently trans-
(
October 2013 Volume 57 Number 10
aac.asm.org 4691

Sparr et al.
O
P
O
P
Bn
NH3Cl
H
N
AcCl
Br
O
Bn
EtO
EtO
O
N
K2CO3, KI
CH3CN, Reflux
Et3N
EtO
EtO
5
6
8
CH2Cl2, RT
H3C
O
H3C
O
O
P
Pd-C (10%)
O
P
O
O
O
+
N
Bn
H2
EtO
EtO
EtO
EtO
O
OH
MeOH, RT
7
H3C
O
O
O
O
O
DMAP
Et3N
P
N
EtO
EtO
OH
CH2Cl2, RT
9
H3C
O
O
H3C
O
O
O
O
1. HATU, DIPEA
DMF
O
P
O
O
P
N
N
EtO
EtO
(CH2)3
EtO
(CH2)3
(CH2)3
OH
O
(CH2)3
N
(Arg)8 NH2
2. H2N (Arg(Pbf))8 PEG PAL
. TFA:H2O:TIPS (95:2.5:2.5)
EtO
H
9
3
3
FIG 2 Chemical synthesis of fosmidomycin-octaarginine conjugates. The 3-bromopropylphosphosphonic acid diethyl-ester 5 was transformed into the corre-
2
sponding O-benzylhydroxylamino compound 6 by a nucleophilic substitution (SN ) reaction. Subsequent N-acetylation (compound 7) and hydrogenolysis
yielded the free N-hydroxylamine compound 8. Treatment with glutaric anhydride yielded the targeted derivative of the fosmidomycin compound 9. Coupling
of fosmidomycin derivative 9 with the resin-bound carrier (octaarginine) and subsequent deprotection and cleavage from the resin provided the fosmidomycin-
Arg conjugate 3 (Fig. 1). Et, ethyl; Me, methyl; Bn, benzyl; RT, room temperature; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofurane. See also Results and the
8
supplemental material for more details.
ported into P. falciparum-infected erythrocytes through the para- cin showed clear inhibition of parasite growth. When the nonco-
site-induced new permeability pathways (22). In order to estimate valent complex of fosmidomycin and octaarginine (salt 2) was
the potential of octaarginine to serve as a carrier for drug delivery, used, a striking increase in the activity was observed. Complete
we first evaluated its ability to cross the multiple membrane bar- inhibition of P. falciparum growth already occurred at a concen-
riers separating the parasite from the extracellular compartment. tration of 0.1 ␮M (Fig. 3D). This result indicates that oligoargin-
Plasmodium species are obligate intracellular pathogens that re- ine is an efficient delivery vehicle for fosmidomycin to the parasite
side inside a parasitophorous vacuole (PV) in the cytoplasm of the inside red blood cells.
host cell. The PV membrane (PVM), which is formed upon host
When the covalently attached FR-900098-octaarginine conju-
cell invasion by the parasite, represents the major protective bar- gate 3 was tested, the inhibitory effect on parasite growth was
rier between the parasite and the host cytoplasm. The PVM selec- weaker than that of the salt complex or fosmidomycin (Fig. 3E),
tively controls the exchange of molecules between the host cell and suggesting that FR-900098 was, at least in this experimental setup,
the parasitophorous vacuole. We performed uptake assays with only partially released from the covalent derivative (conjugate 3).
FAM-octaarginine (compound 1) in P. falciparum-infected hu- The observed significant activity at lower concentrations (0.1 ␮M
man red blood cells and in Plasmodium berghei-infected human and 0.5 ␮M) indicated that the active antibiotic must have become
liver cells (Huh7) representing the exoerythrocytic form (EEF) of available. In order to quantify how octaarginine improves the ef-
the parasite (Fig. 3A and 4A). In line with our previous findings ficacy of fosmidomycin, we calculated the 50% inhibitory concen-
(
10), FAM-octaarginine (compound 1) efficiently penetrated in- trations (IC s) of salt complex 2 and compared them to those of
50
fected red blood cells. After 12 h of incubation, most of the fluo- fosmidomycin (Fig. 3F). Growth inhibition experiments with ei-
rescent signal was located within the PV (Fig. 3A). In contrast to ther fosmidomycin or salt conjugate 2 were performed over a
P. falciparum-infected erythrocytes, where virtually every in- concentration range of 1 nM to 5 ␮M. The IC50 of salt complex 2
fected cell incorporated the peptide, in P. berghei-infected hepa- was 4.4 nM, whereas it was 181 nM for free fosmidomycin (Fig.
toma cells peptide uptake was not complete, and a subset of in- 3F). Hence, the ϳ40-fold increase in activity could be attributed
fected cells (ϳ30%) did not incorporate the peptide (Fig. 4A and directly to the addition of octaarginine.
B). Together, the findings show that FAM-octaarginine (com-
pound 1) is able to cross various biological membranes.
A previous report described the broad-spectrum activity of
TP10, a fragment of the cell-penetrating peptide transportan
Conjugation of octaarginine to fosmidomycin improves ef- against P. falciparum and the kinetoplastid protozoan parasite
ficacy against asexual blood-stage P. falciparum. We next eval- Trypanosoma brucei (39). To test whether the observed improved
uated the potential of octaarginine to deliver fosmidomycin into efficacy of fosmidomycin in combination with octaarginine was a
target cells. Growth inhibition assays were performed in the pres- result of a potential synergistic growth-inhibitory property of oc-
ence of fosmidomycin or its octaarginine conjugate (salt 2 or con- taarginine, we included control experiments with FAM-octaargi-
jugate 3). We first tested whether combining fosmidomycin with nine alone (Fig. 3C). We found that the use of up to 100 ␮M
octaarginine could enhance the inhibitory effect of fosmidomycin FAM-octaarginine has no effect on the growth of P. falciparum.
on blood-stage P. falciparum (Fig. 3B). As expected, fosmidomy- This result supports the notion that octaarginine acts as a delivery
4692 aac.asm.org
Antimicrobial Agents and Chemotherapy

Octaarginine Improves Efficacy of Fosmidomycin
A
B
C
Fosmidomycin
6-Fam-octaarginine
4
3
2
1
0
8
6
4
2
0
untreated
untreated
0
0
1
2
5
.1 µM
.5 µM
µM
.5 µM
µM
10 µM
1
00 µM
h
h
h
0
0h
24h
48h
72
6
-Fam-octaarginine
24
48h
72h
D 4
E 4
Salt 2
Conjugate 3
untreated
3
2
1
0
3
2
1
0
0
0
1
2
5
.1 µM
.5 µM
µM
.5 µM
µM
untreated
0.1 µM
0.5 µM
1 µM
2.5 µM
5
µM
DAPI
h
h
0
h
h
h
h
h
2
4
48
72h
0
2
4
48
72
F 1
5
0
5
0
Fosmidomycin
IC = 181.4 nM
1
50
salt 2
5
µM
DIC
IC = 4.4 nM
50
-2
-1
100
101
1
0-4 10-3 10
10
Concentration (µM)
FIG 3 Octaarginine improves efficacy of fosmidomycin against Plasmodium falciparum asexual blood-stage parasites. (A) FAM-octaarginine efficiently penetrates the
parasitophorous vacuole of blood-stage P. falciparum. Shown are fluorescence microscopy images of FAM-octaarginine (green) in P. falciparum-infected human red
blood cells. Parasite DNA was stained with DAPI (blue). DIC, differential interference contrast. (B) Time course of cultured P. falciparum parasites in the presence of
fosmidomycin at the indicated concentrations (0.1 to 5 ␮M). (C) Control experiment with 10 or 100 ␮M FAM-octaarginine. (D) Inhibition of P. falciparum asexual
blood-stage growth by fosmidomycin-octaarginine noncovalent salt 2. Note that inhibition is evident already at 0.1 ␮M. (E) Inferior inhibition by covalent conjugate 3.
(
F) IC s for growth inhibition for fosmidomycin alone (red) and the fosmidomycin-octaarginine salt complex 2 (orange).
50
vehicle only. Our results also indicate that the inhibitory activity of stages in the presence of either fosmidomycin or the salt complex
TP10 on parasite growth is not a general property of cell-penetrat- (data not shown). Enumeration of merosomes 65 h after infection
ing peptides but rather is a distinct characteristic of transportan.
revealed a moderate but significant reduction when either 10 or
Collectively, our results show that the efficacy of the antibiotic 100 ␮M fosmidomycin-octaarginine salt complex 2 was applied
fosmidomycin against blood-stage P. falciparum can be substan- (Fig. 4C). In marked contrast, addition of fosmidomycin alone
tially increased when complexed with cell-penetrating peptides.
had no effect (Fig. 4C). Of note, the inhibitory activity of the
Fosmidomycin-octaarginine complex displays activity fosmidomycin-octaarginine salt complex 2 on liver-stage para-
against P. berghei preerythrocytic stages. We next investigated sites remained incomplete, and we failed to achieve full inhibition
the effect of fosmidomycin-octaarginine salt complex 2 on liver- of merosome release even at higher concentrations (data not
stage parasites. There have been contradictory reports on the abil- shown). This incapacity to fully inhibit Plasmodium liver-stage
ity of fosmidomycin to inhibit the growth of liver-stage P. berghei. growth might be at least partially explained by the observed in-
While two studies claimed a complete lack of activity on liver- complete uptake of FAM-octaarginine into Plasmodium-infected
stage parasites, presumably due to poor drug uptake (22, 40), an- hepatoma cells (Fig. 4B). In conclusion, these findings provide
other study reported significant inhibition of apicoplast biogene- chemical genetic evidence for an important function of the non-
sis in liver-stage parasites; as a consequence, merosome formation mevalonate isoprenoid biosynthesis pathway in preerythrocytic
occurred at 10 mM fosmidomycin (21).
development of the malaria parasite.
In order to test enhanced causal prophylactic efficacy of fos-
Inhibition of extracellular Toxoplasma gondii tachyzoites by
midomycin conjugation to octaarginine on liver-stage parasites, the fosmidomycin-octaarginine complex. To independently
we monitored P. berghei liver-stage development in the presence confirm the carrier function of octaarginine in antiparasitic drug
of fosmidomycin or its octaarginine salt complex 2 (Fig. 4). We delivery, we decided to extend our investigation to Toxoplasma
assessed liver-stage size and morphology and the number of mero- gondii, another apicomplexan parasite and the causative agent of
somes for the final stage of preerythrocytic development. We did toxoplasmosis. Recent data demonstrated that T. gondii DXR is
not observe any apparent changes in the morphology of liver required for parasite growth (21). We first tested whether FAM-
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Sparr et al.
A
B
1
00
8
6
4
2
0
0
0
0
0
6
-Fam-octaarginine
6
-Fam-octa-
positive
negative
Salt 2
arginine
C
Fosmidomycin
3%
5
00
00
00
00
00
0
4
%
4
3
2
1
PbHSP70
39%
6
5%
5
µM
Merged
FIG 4 Conjugation of fosmidomycin to octaarginine inhibits formation of Plasmodium berghei merosomes, the final stage of preerythrocytic develop-
ment. (A) Fluorescence microscopy images of P. berghei-infected hepatoma cells. Incorporated FAM-octaarginine (green) and PbHSP70 (red) are shown.
The exoerythrocytic form (EEF) on the left did not incorporate the FAM-octaarginine (star), whereas the EEF on the right did. (B) Quantification of P.
berghei-infected hepatoma cells that incorporated FAM-octaarginine. Shown are mean values and standard deviations from three biological replicates.
(
C) Quantification of merosomes at 65 h after infection in the presence of either fosmidomycin or the fosmidomycin-octaarginine salt complex 2. Mean
values with standard deviations from three independent replicates are shown. Numbers represent the percentage of inhibition of merosome formation
compared to that of untreated samples.
octaarginine can enter T. gondii-infected HFFs (Fig. 5A). When T. constitutes the major barrier to the fosmidomycin-octaargin-
gondii-infected HFFs were grown in the presence of FAM-octa- ine complex in intracellular T. gondii.
arginine, the fluorescently labeled peptide was excluded from the
Octaarginine derivatives render fosmidomycin effective
parasitophorous vacuole, although it was clearly visible in the cy- against mycobacteria. Reversal of refractoriness to fosmidomy-
tosol of the host cell. These data indicate that only the first mem- cin of T. gondii tachyzoites and P. berghei liver-stage parasites by
brane barrier, i.e., the host plasma membrane, can be overcome by the octaarginine salt complex 2 prompted us to extend our study
the octaarginine carrier. We next investigated the ability of fos- to a different domain of organisms, bacteria. The genus Mycobac-
midomycin-octaarginine complex 2 to inhibit the intracellular terium contains the causative agents of tuberculosis and leprosy.
growth of T. gondii (Fig. 5B and C). Neither fosmidomycin nor the The respective pathogens, M. tuberculosis and M. leprae, are
salt conjugate had a significant effect on parasite growth. This protected by a cell wall of highly complex structure and excep-
result is probably a direct consequence of the inability of FAM- tional thickness that ranks as one of the most robust among bac-
octaarginine to penetrate the PV of T. gondii (Fig. 5A).
teria. The presence of complex glycoconjugates, such as arabi-
In order to test this hypothesis, we bypassed the PV and nogalactan and lipids of long-chain mycolic acids, contributes to
preincubated free T. gondii tachyzoites in the presence of fos- the substantial rigidity and reduced permeability of the mycobac-
midomycin or fosmidomycin-octaarginine salt complex 2 for 1 terial cell wall to lipophilic and hydrophilic molecules. The high
h prior to infection of fresh HFFs (Fig. 5D and E). Pretreatment intrinsic resistance of mycobacteria to most antibiotics is partly
of tachyzoites with up to 100 ␮M fosmidomycin had no effect attributed to their unique cell envelope.
on parasite growth, confirming that fosmidomycin does not
We first assessed whether FAM-octaarginine (compound 1) is
cross the parasite plasma membrane. In marked contrast, pre- able to enter M. smegmatis and M. bovis BCG, two common non-
treatment with fosmidomycin-octaarginine salt complex 2 led pathogenic experimental surrogates of pathogenic mycobacteria
to a concentration-dependent inhibition of parasite growth (Fig. 6A and B). Exponentially growing bacilli of both strains were
with a pronounced inhibition already at 1 ␮M. This result incubated with 10 ␮g/ml of FAM-octaarginine (compound 1) for
shows that the PV membrane, but not the parasite membrane, 12 h at 37°C. Microscopic analysis demonstrated that FAM-octa-
4694 aac.asm.org
Antimicrobial Agents and Chemotherapy

Octaarginine Improves Efficacy of Fosmidomycin
A
6
-Fam-octaarginine
anti-T. gondii
DAPI
Merged
5
µM
C 2
50
00
50
00
B
Untreated
Fosmidomycin
Salt 2
Conjugate 3
2
1
1
5
0
0
2
1
00 µM
Fos
Salt
Untreated
D
1
µM
10 µM
100 µM
Conjugate 3
1
00 µM
Untreated
Fosmidomycin
Salt 2
E
2
50
Salt 2
Fos
Untreated
1 uM
2
1
00
50
1
0 uM
100 uM
1
1
1
uM
0 uM
00 uM
100
5
0
0
FIG 5 Fosmidomycin-octaarginine salt complex inhibits extracellular T. gondii tachyzoites but does not affect intracellular replication. (A) Fluorescent
micrographs of T. gondii-infected human foreskin fibroblasts (HFFs) showing incorporated FAM-octaarginine (green), anti-T. gondii antibody (red), and
DAPI-stained DNA (blue). Plaque assay (B) and corresponding quantification (C) after treatment of T. gondii-infected HFFs with either 100 ␮M fosmidomycin,
salt 2, or conjugate 3 are shown. Also shown are plaque assay (D) and corresponding quantification (E) after pretreatment of T. gondii tachyzoites with either
fosmidomycin or fosmidomycin-octaarginine salt complex 2 prior to infecting new HFFs.
arginine efficiently penetrates bacteria of both strains. We next DISCUSSION
sought to investigate whether the combination of fosmidomycin
and octaarginine (salt 2 and conjugate 3) could inhibit mycobac-
terial growth in vitro. The gene dxr, which encodes the enzyme
DXR, is essential for M. tuberculosis growth (20). In addition, re-
combinant DXR is clearly sensitive to fosmidomycin in enzyme
assays (41). However, growth of the pathogen is not affected by
fosmidomycin, presumably due to the poor uptake of the drug by
bacteria (20).
Based on our observation that mycobacteria are highly perme-
able to FAM-octaarginine (Fig. 6A and B), we hypothesized that
combination of fosmidomycin and octaarginine would improve
drug uptake and thereby increase susceptibility of mycobacteria.
We monitored the growth of nonpathogenic M. bovis BCG in the
presence of fosmidomycin alone or in combination with octaargi-
nine (salt 2 and conjugate 3) and used isoniazid as a positive con-
trol (Fig. 6). Mycobacterial growth was reduced when fosmido-
mycin was combined with octaarginine in either noncovalent
Plasmodium berghei liver-stage parasites, Toxoplasma gondii, and
mycobacteria all are known to be resistant to the antibiotic fos-
midomycin, despite an essential role of DRX, the molecular target
of fosmidomycin, in these pathogens (20, 21, 22, 24). Accord-
ingly, the failure of fosmidomycin to inhibit growth of these
pathogens was attributed to poor drug uptake. In the present
study, we have overcome the restriction of fosmidomycin by
carrier-mediated delivery to intracellular pathogens. We eval-
uated the potential of octaarginine, a polycationic cell-pene-
trating peptide, as a delivery vehicle for fosmidomycin to im-
prove efficacy. We hypothesized that octaarginine would
improve the delivery of fosmidomycin across the multiple bi-
ological membranes separating the target compartment of in-
tracellular pathogens from the extracellular space. Our inves-
tigations show that the combination of octaarginine and
fosmidomycin dramatically increases its efficacy toward blood-
stage P. falciparum. In addition, the fosmidomycin-octaargin-
ine combination has a significant inhibitory effect on matura-
(Fig. 6E) or covalent form (Fig. 6F). When octaarginine alone or
unmodified fosmidomycin was used, mycobacterial growth re-
mained unaffected (Fig. 6C and D). In contrast to our findings in tion of liver-stage P. berghei, whereas up to 100 ␮M
apicomplexan parasites, the covalently linked fosmidomycin-oc- fosmidomycin alone had no effect. We note that these results
taarginine conjugate 3 showed significant inhibition in mycobac- are in opposition to a previous report by Nair et al., which
teria (Fig. 6F).
claimed a significant effect on merosome formation of P. ber-
October 2013 Volume 57 Number 10
aac.asm.org 4695

Sparr et al.
A
B
C
E
0
0
0
0
0
0
D
M. smegmatis
M. bovis BCG
Fosmidomycin
6
-Fam-octaarginine
0
0
0
0
0
0
0
.6
.5
.4
.3
.2
.1
.0
0.6
untreated
1 µM
untreated
1 µM
5 µM
0.5
0.4
0.3
0.2
0.1
0.0
5
µM
0
1
2
3
4
5
0
1
2
3
4
5
days
days
F
0
.6
.5
.4
.3
.2
.1
.0
Salt 2
0.6
.5
0.4
Conjugate 3
untreated
1 µM
untreated
1 µM
0
5
µM
5
µM
0
0
0
0
.3
.2
.1
.0
0
1
2
3
4
5
0
1
2
3
4
5
days
days
G
0.6
Isoniazid
untreated
1 µM
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
5
µM
0
1
2
3
4
5
days
FIG 6 Efficacy of fosmidomycin either complexed with or conjugated to octaarginine against Mycobacterium species. (A and B) Fluorescent micrographs of
Mycobacterium smegmatis (A) and Mycobacteria bovis BCG (B) showing incorporation of FAM-octaarginine (green). DAPI (blue) is shown as a reference for
bacterial DNA. (C) Growth curves of cultured M. bovis BCG in the presence of FAM-octaarginine (C), fosmidomycin (D), the fosmidomycin-octaarginine salt
complex 2 (E), the covalent conjugate 3 (F), and isoniazid, a first-line drug for tuberculosis therapy (G).
ghei at 10 ␮M fosmidomycin (21), but are in full support of permeable to FAM-octaarginine hints at a PVM-resident trans-
studies that show no efficacy against P. berghei liver stages (22, port protein operating in Plasmodium parasites. Patch clamp ex-
4
0). periments identified a solute pore in blood-stage parasites that is
We observed a strict correlation between the permeability of a permeable to molecules up to a size of 1.4 kDa, which is less than
given cell type to octaarginine and its ability to improve the effi- the size of FAM-octaarginine (43, 44). The apparent different per-
cacy of fosmidomycin in these cells. When octaarginine was un- meability of the Plasmodium PVM compared to that of the T. gondii
able to cross a biological membrane, the pathogen beyond this PVM is best explained by a distinct carrier in the Plasmodium PVM,
barrier remained unaffected by fosmidomycin-octaarginine con- which is known to be particularly rich in species-specific proteins
jugates. This was the case for intracellular T. gondii, for example. (45). Although a previous study has reported a successful application
In these cells, FAM-octaarginine did not penetrate the PV, which of oligoarginine for the delivery of triclosan against both extracellular
correlates with resistance to the fosmidomycin-octaarginine con- and intracellular forms of T. gondii (24), we did not observe perme-
jugate. This view is further supported by the observation that free ability of the T. gondii PVM for octaarginine.
T. gondii tachyzoites, which are completely permeable to FAM-
The application of octaarginine as a versatile delivery vehicle for
octaarginine, show a concentration-dependent sensitivity to the fosmidomycin was further broadened by our finding that mycobac-
fosmidomycin-octaarginine complex. We speculate that the lim- terial fosmidomycin resistance could be reverted by combining the
ited activity of the fosmidomycin-octaarginine complex against drug with octaarginine. Interestingly, both the noncovalent salt com-
liver-stage P. berghei is also a consequence of the incomplete up- plex (salt 2) and the covalently linked fosmidomycin-octaarginine
take of the peptide. These results suggest that both the PVM and conjugate (conjugate 3) showed promising activity against M. bovis
the parasite plasma membrane are critical barriers to fosmidomy- BCG. These results open an attractive opportunity for the use of fos-
cin in liver-stage P. berghei parasites and, most importantly, in T. midomycin in combination with octaarginine as a candidate antitu-
gondii-infected host cells. The carrier peptide FAM-octaarginine berculosis agent. Further investigations, including efficacy testing of
used in this study has a molecular weight of 1.6 kDa, which is well fosmidomycin derivatives in animal models of tuberculosis, are be-
above the proposed cut off 1.2 kDa for the PVM of T. gondii- yond the scope of the current study but will be part of our future
infected cells (42), probably explaining the inability of this mole- work. Very recent data demonstrated that M. tuberculosis changes its
cule to penetrate the PV. Strikingly, the observation that the PVM cell wall/membrane permeability to antibiotics depending on the
of both blood-stage and liver-stage Plasmodium organisms are physiological state (46). Dormant bacilli represent a phenotypic
4696 aac.asm.org
Antimicrobial Agents and Chemotherapy

Octaarginine Improves Efficacy of Fosmidomycin
drug-resistant subpopulation that contributes to the prolonged che-
motherapy required to cure tuberculosis. Whether octaarginine-an-
tibiotic conjugates have the potential to shorten tuberculosis treat-
ment by improved eradication of the difficult-to-treat dormant M.
tuberculosis population remains to be determined.
Altogether, our results provide a valuable basis for alternative
formulation of antimicrobial agents using cell-penetrating pep-
tides. Efficacy was observed with the noncovalent drug conjuga-
tion to octaarginine that was previously reported by others (33, 34,
artesunate-fosmidomycin in treatment of uncomplicated Plasmodium falcip-
arum malaria. Antimicrob. Agents Chemother. 49:3749–3754.
1
8. Borrmann S, Lundgren I, Oyakhirome S, Impouma B, Matsiegui PB,
Adegnika AA, Issifou S, Kun JF, Hutchinson D, Wiesner J, Jomaa H,
Kremsner PG. 2006. Fosmidomycin plus clindamycin for treatment of
pediatric patients aged 1 to 14 years with Plasmodium falciparum malaria.
Antimicrob. Agents Chemother. 50:2713–2718.
9. Yeh E, Derisi JL. 2011. Chemical rescue of malaria parasites lacking an
apicoplast defines organelle function in blood-stage Plasmodium falcipa-
rum. PLoS Biol. 9:e1001138. doi:10.1371/journal.pbio.1001138.
0. Brown AC, Parish T. 2008. DRX is essential in Mycobacterium tuberculosis
and fosmidomycin resistance is due to lack of uptake. BMC Microbiol.
8:78. doi:10.1186/1471-2180-8-78.
1
2
2
4
7), and this was confirmed in this study for the antibiotic fos-
midomycin against diverse pathogens, warranting further inves-
tigation of octaarginine as a drug delivery vehicle.
1. Nair SC, Brooks CF, Goodman CD, Sturm A, McFadden GI, Sundriyal
S, Anglin JL, Song Y, Moreno SN, Striepen B. 2011. Apicoplast iso-
prenoid precursor synthesis and the molecular basis of fosmidomycin
resistance in Toxoplasma gondii. J. Exp. Med. 208:1547–1549.
ACKNOWLEDGMENTS
We are grateful to Steffen Bormann and Felista Tansi for critical reading of 22. Baumeister S, Wiesner J, Reichenberg A, Hintz M, Bietz S, Harb OS,
Roos DS, Kordes M, Friesen J, Matuschewski K, Lingelbach K,
Jomaa H, Seeber F. 2011. Fosmidomycin uptake into Plasmodium-
the manuscript.
and Babesia-infected erythrocytes is facilitated by parasite-induced
new permeability pathways. PLoS One 6:e19334. doi:10.1371/journal
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