Design, synthesis, and study of a mycobactin-artemisinin conjugate that has selective and potent activity against tuberculosis and malaria

Article abstract of DOI:10.1021/ja109665t

Although the antimalarial agent artemisinin itself is not active against tuberculosis, conjugation to a mycobacterial-specific siderophore (microbial iron chelator) analogue induces significant and selective antituberculosis activity, including activity against multi- and extensively drug-resistant strains of Mycobacterium tuberculosis. The conjugate also retains potent antimalarial activity. Physicochemical and whole-cell studies indicated that ferric-to-ferrous reduction of the iron complex of the conjugate initiates the expected bactericidal Fenton-type radical chemistry on the artemisinin component. Thus, this "Trojan horse" approach demonstrates that new pathogen-selective therapeutic agents in which the iron component of the delivery vehicle also participates in triggering the antibiotic activity can be generated. The result is that one appropriate conjugate has potent and selective activity against two of the most deadly diseases in the world.

Full text of DOI:10.1021/ja109665t

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pubs.acs.org/JACS
Design, Synthesis, and Study of a Mycobactin-Artemisinin Conjugate
That Has Selective and Potent Activity against Tuberculosis and
Malaria
Marvin J. Miller,*,† Andrew J. Walz,† Helen Zhu,† Chunrui Wu,† Garrett Moraski,† Ute M€ollmann,‡
Esther M. Tristani,§ Alvin L. Crumbliss,§ Michael T. Ferdig,|| Lisa Checkley,|| Rachel L. Edwards,^ and
Helena I. Boshoff^
†Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, Indiana 46556,
United States
‡Leibniz Institute for Natural Product Research and Infection Biology-Hans Knoell Institute, Beutenbergstr. 11a, D-07745 Jena,
Germany
§Department of Chemistry, Duke University, Room 2104, French Family Science Center, 124 Science Drive, Box 90346, Durham,
North Carolina 27708, United States
Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States
^Tuberculosis Research Section, LCID, NIAID, NIH, Room 2W20G, Building 33, MSC 3206, 9000 Rockville Pike, Bethesda, Maryland
20892, United States
S Supporting Information
b
treatments of both malaria and tuberculosis are inadequate. New
ABSTRACT: Although the antimalarial agent artemisi-
nin itself is not active against tuberculosis, conjugation to
a mycobacterial-specific siderophore (microbial iron
chelator) analogue induces significant and selective anti-
tuberculosis activity, including activity against multi- and
extensively drug-resistant strains of Mycobacterium tuber-
culosis. The conjugate also retains potent antimalarial
activity. Physicochemical and whole-cell studies indi-
cated that ferric-to-ferrous reduction of the iron com-
plex of the conjugate initiates the expected bactericidal
Fenton-type radical chemistry on the artemisinin com-
ponent. Thus, this “Trojan horse” approach demon-
strates that new pathogen-selective therapeutic agents
in which the iron component of the delivery vehicle also
participates in triggering the antibiotic activity can be
generated. The result is that one appropriate conjugate
has potent and selective activity against two of the most
deadly diseases in the world.
leads for drug development are urgently needed. Herein we
report that an artemisinin conjugate of a mycobactin T analogue
(3; Figure 1) not only retains antimalarial activity similar to that
of artemisinin itself but is exquisitely microbe-selective and has
remarkably potent activity against Mtb.
Artemisinin (1), also called qinghaosu, is a natural peroxide-
containing sesquiterpene based on 1,2,4-trioxane, which is a
highly active and relatively nontoxic antimalarial agent.9 The
trioxane derivatives dihydroartemisinin, artemether, and water-
soluble sodium artesunate have been studied extensively and are
widely used, and efforts toward the development of efficient
methods for their preparation continue.10 Mode-of-action stud-
ies indicate that ferrous iron [in the form of heme or iron(II)
salts] triggers reductive cleavage of the peroxide bond in
artemisinin to form oxygen-centered radicals. The oxy radicals
then form carbon-centered radicals that ultimately lead to death
of the malaria parasites. Related mechanistic work has been used
to rationally design new antimalarial peroxides,11 including
dimeric versions.12 Similarly, in cancer-related therapeutic appli-
cations, iron released from transferrin can react with artemisinin
bound to a transferrin-receptor-targeting peptide to generate free
radicals that can kill cells.13 Since cancer cells overexpress trans-
ferrin receptors for iron uptake, the artemisinin-peptide con-
jugates and transferrin-artemisinin conjugates are selective and
potent anticancer agents.14
uberculosis (TB) and malaria are the two most widespread
T
and lethal infectious diseases in the world.1 Approximately
one-third of the world’s population is presently infected with
Mycobacterium tuberculosis (Mtb), the causative pathogen of TB,
and 40% is affected by malaria. Each of these diseases causes
about 2 million deaths worldwide every year.2 The development
and spread of multi-drug-resistant (MDR) and extensively drug-
resistant (XDR) strains of Mtb have stimulated research efforts
globally.3 As a consequence, the pipeline of potential new drugs
has expanded,4-6 but no effective new anti-TB drug has been
marketed in decades.7 Plasmodium falciparum parasites that cause
malaria also have become resistant to standard antimalarial drugs
such as quinine and chloroquine.8 Current chemotherapeutic
The virulence of Mtb depends on its ability to assimilate iron,
and like most microbes, it synthesizes and utilizes siderophores
(low-molecular-weight iron chelators) to sequester iron.15 On
the basis of natural and synthetic examples of siderophore-
antibiotic conjugates that enable active transport of drugs
into specifically targeted bacteria,16 we anticipated that while
Received: November 8, 2010
Published: January 28, 2011
r
2011 American Chemical Society
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Table 1. Activities of Conjugate 3 against P. falciparum
Strains (IC50 in μg/mL)a
HB3
7G8
3D7
Dd2
conjugate 3
0.0047
0.0013
0.0051
0.0004
0.0040
0.0036
0.0041
0.0022
artemisinin
a Abbreviations: HB3, Honduras (chloroquine-sensitive, low-level pyr-
imethamine-resistant); 7G8, Brazil (low-level chloroquine-resistant);
3D7, Airport (sensitive to sulfadoxine); Dd2, Indochina (multi-drug-
resistant).
complex of 3 does initiate the expected lethal radical-producing
Fenton chemistry on the artemisinin component.
The synthesis of mycobactin-artemisinin conjugate 3 is
summarized in Figure 1. Since mycobactin T has no functional
group for use in linking to the artemisinin component, we
resynthesized our previously reported mycobactin analogue
5,20 which incorporates a diaminopropionic acid as a re-
placement for the natural β-hydroxybutyrate. Artemisinin is
remarkably amenable to chemical modification despite contain-
ing inherently sensitive functionality. Thus, a sequence analo-
gous to previous reports21 gave N-hydroxysuccinimide (NHS)-
active ester 4. Treatment of mycobactin analogue 5 with trifluoro-
acetic acid to remove the Boc group followed by neutraliza-
tion and direct treatment with 4 gave the desired conjugate 3
(see the Supporting Information).
As indicated, conjugate 3 not only retained efficacy against P.
falciparum (Table 1) but also was remarkably potent against Mtb
H37Rv [MIC (0.338 μM) = 0.39 μg/mL], while artemisinin (1)
itself and all synthetic intermediates to active ester 4 (see the
Supporting Information) had no activity against Mtb. Further
studies revealed that conjugate 3 was similarly potent against
MDR and XDR strains of Mtb (Tables 2 and 3). Thus, by using a
mycobacterial siderophore to deliver an artemisinin derivative
and provide a source of reactive iron to Mtb, we retained its
antimalarial activity and enabled it to be a potent antitubercu-
larculosis agent.
Conjugate 3 was not active against a broad set of bacteria at the
highest levels tested (2 mM), including Gram-positive bacteria
(Bacillus subtilis ATCC 6633 and Staphylococcus aureus SG 511)
and Gram-negative bacteria [Serratia marcescens SG 621, Kleb-
siella pneumoniae ATCC 10031, Escherichia coli ATCC 25922, E.
coli DC0, E. coli DC2 (a permeability mutant), Pseudomonas
aeruginosa KW 799/WT, P. aeruginosa KW 799/61 (a perme-
ability mutant), P. aeruginosa SG 137, P. aeruginosa ATCC 27853,
and Stenotrophomonas maltophilia GN 12873] (see the Support-
ing Information). As a further indication of the unique anti-
malarial and anti-TB selectivity of 3, it was tested and found to
have negligible activity against a number of fast-growing strains of
mycobacteria [Mycobacterium vaccae IMET 10670, MIC = 50 μg/mL
(43 μM); Mycobacterium smegmatis SG987, MIC = 200 μg/mL
(173 μM); Mycobacterium aurum SB66, MIC = 12.5 μg/mL
(43.3 μM); Mycobacterium fortuitum B, MIC = 100 μg/mL
(87 μM); see the Supporting Information]. Thus, the antibiotic
activity of conjugate 3 is microbe-selective, as anticipated.
Figure 1. (A) Conjugate design. (B) Synthesis, anti-Mtb activity, and
antimalaria activity of mycobactin-artemisinin conjugate 3.
artemisinin itself has no antituberculosis activity, attachment of
an artemisinin derivative to an analogue of mycobactin T (2),17
an Mtb siderophore critical for growth under iron restriction,
might allow the peroxide drug to be actively assimilated by the
pathogen. Subsequent reduction of the bound ferric ion to its
lower-affinity ferrous form might then induce Fenton-like chem-
istry from the interaction of the ferrous iron with the nearby
peroxide. The consequent generation of free radicals would then
be expected to cause intracellular damage selectively to the
assimilating bacteria.18 To test this hypothesis, we synthesized 3,
an artemisinin conjugate of a mycobactin T analogue (Figure 1, and
found that it not only retains antimalarial activity similar to that of
artemisinin itself (Table 1) but is exquisitely microbe-selective and has
remarkably potent activity against Mtb (Table 2). The mycobactin-
arteminisin conjugate is very active against not only H37Rv Mtb
[minimum inhibitory concentration (MIC) = 0.39 μg/mL] but
also both MDR Mtb (MIC = 0.16-1.25 μg/mL) and XDR Mtb
(MIC = 0.078-0.625 μg/mL) (Tables 2 and 3).19 Moreover, as
described below, it is selectively active against Mtb relative to fast-
growing species of mycobacteria and a broad set of other Gram-
positive and Gram-negative bacteria. Physicochemical and whole-
cell studies indicated that ferric-to-ferrous reduction of the iron
In order to determine whether the remarkable activity of 3
depends on its ability to bind iron, we synthesized and tested the
activity of derivative 6, in which the iron-binding hydroxamates
were protected (Figure 2). This modification reduced the anti-
malarial activity by >20- to 100-fold. The activity against all
strains of Mtb was completely lost (>12.5 μM). To determine the
effect of conjugation to a different siderophore, we prepared 7, an
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Table 2. Activities of Conjugate 3 against Eight Different MDR Mtb Clinical Strains19 (MIC in μg/mL)a
strain 1 HRESP
strain 2 HREZSP
strain 3 HCPTTh
strain 4 HREKP
strain 5 HRERb
strain 6 HREZSKPTh
strain 7 HREZRbTh
0.3125
1.25
0.625
0.625
0.625
0.3125, 0.156,b 1.25b
0.625
a Abbreviations denote resistance to the following: H, isoniazid; R, rifampicin; E, ethambutol; Z, pyrazinamide; S, streptomycin; C, cycloserine; T,
ethionamide; K, kanamycin; P, p-aminosalicylic acid; Rb, rifabutin; Th, thioacetazone; O, ofloxacin. b The Mtb strains are subsets of those reported
previously.19
Table 3. Activities of Conjugate 3 against XDR Mtb Strains19
and M. smegmatis (MIC in μg/mL)a
strain 8
strain 9
strain 10
strain 11
M.
HRESPOCTh HREPKOTh
HRESPO
HRESPOCTh smegmatis
0.31
0.078
0.625
0.31
>5
a Abbreviations are the same as in Table 2.
Figure 3. Cyclic voltammograms of Fe-loaded mycobactin J, artemisinin,
and the Fe-loaded mycobactin-artemisinin conjugate. Conditions:
glassy carbon working electrode; Pt wire auxiliary electrode; Ag/AgCl
reference electrode; scan rate =10 mV/s; [NaClO4] = 100 mM as the
background electrolyte in 95% EtOH. [Fe-mycobactin J] = 3 mM, Ecp
=
-856 mV vs NHE (7 μA/mM); [artemisinin] = 3 mM, Ecp = -1063
mV vs NHE (9 μA/mM); [Fe-mycobactin-artemisinin conjugate] =
1.625 mM, Ecp = -955 mV vs NHE (18 μA/mM), Ecp = -1090 mV vs
NHE (19 μA/mM).
Figure 2. Protected mycobactin conjugate 6, DFO conjugate 7, and
triacetyl-protected DFO conjugate 8.
artemisinin conjugate of desferrioxiamine (DFO), a natural
siderophore derived from Streptomyces pilosus that is therapeuti-
cally used for treatment of iron overload and also has efficacy for
revival of patients in malaria-induced comas.22 We also synthe-
sized DFO derivative 8 in which all three of the hydroxamates
were acetylated to negate iron binding. With half-maximal
inhibitory concentration (IC50) values ranging from 0.16 to
>2.2 μM, both of the DFO-derived conjugates were more active
against P. falciparum than DFO itself but much less active than
conjugate 3 or artemisinin, and they were completely inactive
against Mtb, since Mtb probably has no uptake route for the
siderophore (DFO) on the basis of studies with M. smegmatis.23
Since our intent was to use the mycobactin to deliver iron and
the redox-sensitive agent artemisinin to Mtb and induce damag-
ing Fenton chemistry during the attempt of the pathogen to
reductively assimilate the iron, we studied the related physico-
chemical properties of conjugate 3. A cyclic voltammogram for
commercially available iron-loaded mycobactin J (used as a
control) is shown in Figure 3. The strongly negative cathodic
peak current is consistent with that observed for other high-iron-
affinity siderophore complexes.24 The absence of a return oxida-
tion wave is indicative of an irreversible Fe3þ/Fe2þ couple, with
dissociation of the labile ferrous ion subsequent to reduction.
A cyclic voltammogram for iron-loaded mycobactin-artemisinin
conjugate 3 is also shown in Figure 3, along with that of
artemisinin alone. Both voltammograms show irreversible behav-
ior, consistent with the literature report for artemisinin.25 The
iron-loaded conjugate shows a shoulder and a main peak, which
we assign to the Fe-mycobactin and artemisinin components,
respectively. The Fe-mycobactin component in the conjugate is
shifted negatively by ∼100 mV relative to the control Fe-
mycobactin J, and the peak current is increased from 7 to 18
μA/mM. The main peak attributable to the artemisinin component
of the conjugate is shifted to a slightly more negative value, and
the peak current is increased from 9 to 19 μA/mM. These results
suggest that the combination of Fe-loaded mycobactin and
artemisinin within the conjugate results in a chemical interaction
between the two redox-active components, apparently subsequent
to the reduction of Fe3þ to Fe2þ in the mycobactin binding site.
This is consistent with the increased currents, suggesting a
chemical event subsequent to the electrochemical (EC) reduc-
tion event (i.e., a modified EC process). We hypothesize that
reduction of the bound ferric iron to its ferrous form induces
Fenton-like chemistry from the interaction of the ferrous iron
with the peroxide linkage of the artemisinin, leading to the
reduction and cleavage of the peroxo bridge. This correlates
with the biological activity observed for this complex, since this
latter reduction would generate free radicals that would be
expected to cause intracellular damage. We propose the coupled
electrochemical/chemical processes shown in Scheme 1 (where
artO and artR represent the artemisinin component of the
conjugate in the oxidized and reduced forms, respectively),
which are consistent with our observations. This scheme illus-
trates an initial reduction of conjugate-bound Fe3þ followed by
dissociation of the labile Fe2þ and/or intramolecular redox
reaction with the artemisinin component and regeneration of
Fe3þ, with subsequent electrochemical reduction and dissocia-
tion of Fe2þ. The free Fe2þ(aq) can generate toxic reactive
oxygen species by Fenton-type reactions, as discussed earlier.
To verify that conjugate 3 fueled the formation of hydroxyl
radicals in Mtb, we exposed washed drug-treated cells to the dye
hydroxyphenylfluorescein, which is oxidized by hydroxyl radicals
to yield a fluorescent derivative.26 Conjugate 3 resulted in
hydroxyl radical formation within 3 h of treatment at 1ꢀ and
10ꢀ MIC (Figure 4). Rifampicin, a transcriptional inhibitor and
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Scheme 1. Proposed Reaction for Mycobactin-Artemisinin
Conjugate 3
’ ACKNOWLEDGMENT
M.J.M. and M.T.F. gratefully acknowledge support from the
U.S. National Institutes of Health (Grants R01 AI054193 and
AI055035). A.L.C. thanks the NSF (CHE 0809466) for financial
support. This research was supported in part by the Intramural
Research Program of the NIH, NIAID.
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Figure 4. Treatment of M. tuberculosis with mycobactin-artemisinin
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures, syn-
b
theses of conjugate 3 and control compounds, details of all
microbiological and biological studies, and complete refs 6 and 19.
This material is available free of charge via the Internet at http://
pubs.acs.org.
809–825.
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’ AUTHOR INFORMATION
Corresponding Author
mmiller1@nd.edu
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