AAC Accepted Manuscript Posted Online 8 June 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.04332-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Salinomycin and other ionophores as new class of antimalarial drugs with transmission blocking
2
activity
3 4 5
Sarah D’Alessandroa, Yolanda Corbetta, Denise Ilboudoa, Paola Misianoa, Nisha Dahiyab, Solomon
6
M. Abayb, Annette Habluetzelb, Romualdo Grandec, Maria R. Gismondoc, Koen Decheringd, Karin
7
Koolend, Robert W. Sauerweine, Donatella Taramellia, Nicoletta Basilicof#, Silvia Parapinia
8 9
Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan,
10
Italy a; Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino,
11
Camerino, MC, Italyb; L.Sacco University Hospital, Milan, Italyc; TropIQ Health Sciences,
12
Nijmegen 6525 GA, The Netherlandsd; Department of Medical Microbiology, Radboud University
13
Nijmegen Medical Center, Nijmegen, The Netherlands, 6525 GAe; Dipartimento di Scienze
14
Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Milan, Italyf
15 16
Running Head: Ionophores as antimalarials
17 18
#Address correspondence to Nicoletta Basilico,
[email protected]
19
1
20
Abstract
21
The drug target profile proposed by Medicine for Malaria Venture for the malaria
22
elimination/eradication policy focuses on molecules active on both asexual and sexual stages of
23
Plasmodium, thus with both curative and transmission blocking activity. The aim of the present
24
work was to investigate whether the class of monovalent ionophores, which includes drugs used in
25
veterinary medicine and recently proposed as human anticancer agents, meets these requirements.
26
Here, the activity of salinomycin, monensin and nigericin on Plasmodium falciparum (Pf) asexual
27
and sexual erythrocytic stages and on the development of the P. berghei (Pb) and Pf mosquito
28
stages is reported.
29
Gametocytogenesis of the 3D7 Pf strain was induced in vitro, gametocytes at stage II-III or stage
30
IV-V of development were treated for different lengths of time with the ionophores and their
31
viability measured with the pLDH assay. The monovalent ionophores efficiently killed both asexual
32
parasites and gametocytes with nanomolar IC50. Salinomycin showed a fast speed of kill compared
33
to standard drugs and the potency was higher on stage IV-V than on stage II and III gametocytes.
34
The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito
35
midgut, confirming a transmission blocking activity. Potential toxicity due to hemolysis was
36
excluded since only infected and not normal erythrocytes were damaged by ionophores.
37
Our data strongly support downstream exploration of monovalent ionophores for repositioning as
38
new antimalarial and transmission blocking leads.
39
2
40
Introduction
41
Malaria caused 627.000 deaths and 207 million reported cases in 2012 (1). Among the five species
42
that infect humans, Plasmodium falciparum (Pf) is responsible for the majority of deaths and severe
43
cases. The recommended malaria control measures include drug treatment, in particular with
44
artemisinin-based combination therapy (ACT), and protection from the vectors with insecticide
45
treated bed-nets and indoor residual spraying (1). However, the effectiveness of control tools is
46
seriously threatened by the emergence and spread of drug and insecticide resistance (2). Even for
47
artemisinins, which up to now were safe and effective, resistance is a growing issue in Asia,
48
particularly in the Cambodia-Thailand border that is the cradle of antimalarial resistance (3, 4). New
49
drugs and new lead compounds for antimalarial drug development are highly needed (5).
50
In the past few years, the international strategy against malaria has changed towards malaria
51
elimination, and ultimately eradication. The Medicines for Malaria Venture (MMV) has defined
52
four Target Candidate Profiles (TCPs) that describe the requirements for novel tools for control and
53
elimination of malaria (6). In particular, new multi-stage antimalarial drugs able to kill the liver or
54
sexual stages of the parasite and/or capable of preventing the parasite development in the mosquito
55
are needed (5, 6). Gametocytes are the sexual stage of Plasmodium, which appear in the circulation
56
concomitantly or after the asexual intraerythrocytic stage. They undergo five stages of maturation,
57
from I to V. Stage V gametocytes, when taken up by Anopheles mosquitoes during a blood meal,
58
become gametes and fuse to form a zygote (7). Subsequently, the zygote transforms into a motile
59
ookinete, and becomes an oocyst, which divides to produce sporozoites, ready to restart the cycle.
60
Being the stage responsible for transmission, the gametocytes are an essential target for malaria
61
elimination/eradication and the identification of gametocytocidal compounds has become an
62
absolute priority.
63
The strategy of drug repositioning (the usage of existing drugs for new therapeutic indications)
64
allows to significantly reduce the development costs, the time-to-market and the risks of failure.
65
This is particularly important for diseases of developing countries, for which research funds are 3
66
limited (8). Here, we provide evidence in support of the repurposing of salinomycin and/or other
67
ionophores as antimalarial and transmission blocking agents. Salinomycin is a polyether antibiotic
68
isolated from Streptomyces spp. and a monovalent ionophore for alkali ions with relative K+
69
selectivity, able thus of interfering with mitochondrial functions (9). Salinomycin was patented as
70
an anticoccidial agent in 1974 and has been used in poultry and other livestock since then. More
71
recently, salinomycin and other ionophores such as monensin and nigericin (sodium and potassium
72
antiporters, respectively) were found to inhibit cancer stem cell growth by modulating the Wnt
73
pathway (10-12). This finding has prompted the usage of salinomycin for compassionate use in a
74
few cancer patients with promising results (13).
75
Salinomycin and other ionophores (gramicidin, lasalocid and monensin) have already been reported
76
to be active against Pf parasites (14-16), but the potency of this class of compounds as transmission
77
blocking agents has not been fully investigated, yet. The aim of the present work was to evaluate
78
the antimalarial activity of salinomycin, monensin and nigericin on both asexual and transmission
79
stages of Pf.
80 81
Materials and methods
82
Plasmodium falciparum cultures
83
P. falciparum (Pf) cultures were carried out according to Trager and Jensen (17) with minor
84
modifications. The strains used in this study are either CQ-sensitive (D10, 3D7 and Pf Ghana
85
isolate) or CQ-resistant (W2 and Pf Burkina Faso isolate). The resistance profile of W2 is well
86
documented in the literature (19), whereas the Pf Burkina Faso isolate is considered resistant since
87
its IC50 to chloroquine is higher than 100 nM, as commonly accepted as threshold for resistance
88
(18). All the strains were maintained at 5% hematocrit (human type A-positive red blood cells for
89
D10, W2 and Pf isolates; 0-positive red blood cells for 3D7) in RPMI 1640 medium containing 24
90
mM sodium bicarbonate (EuroClone, Celbio) with the addition of 0.01 % hypoxantine, 20 mM
91
Hepes, 2 mM glutamine. All the parasites were cultured in the presence of 1 % AlbuMaxII (lipid4
92
rich bovine serum albumin), except the 3D7 strain that was cultured in the presence of 10% (v/v)
93
naturally-clotted heat-inactivated 0+ human serum (Interstate Blood Bank, Inc.), which ensures
94
constant and high gametocyte production. All the cultures were maintained at 37 °C in a standard
95
gas mixture consisting of 1 % O2, 5 % CO2, 94 % N2. The Pf Ghana and Burkina Faso isolates
96
derived from two different patients with malaria admitted to the Ospedale Sacco (Milano, Italy)
97
returning to Milan from Ghana or Burkina Faso, respectively. The parasites were adapted to grow in
98
culture and are not clonal strains.
99
Gametocytogenesis was triggered as previously described (20). Briefly, Pf 3D7 asexual parasites
100
cultures were diluted to 0.5% parasitaemia and medium was changed daily without of the addition
101
of fresh red blood cells (RBC). When a parasitaemia higher than 5% was obtained and the parasites
102
were stressed by nutrient deprivation, the cultures were treated for 48-72h with N-
103
acetylglucosamine (NAG) (Sigma-Aldrich) to clear residual asexual parasites. Stage II-III
104
gametocytes were obtained and used for the experiments after 4 days from the addition of NAG to
105
the culture, while stage IV-V after 7-8 days. Gametocytes stages were routinely checked in Giemsa
106
stained smears.
107 108
In vitro Plasmodium falciparum drug susceptibility assay
109
Salinomycin, monensin and nigericin sodium salts (Sigma Aldrich, Figure 1) were dissolved in
110
either DMSO (salinomycin and nigericin) or ethanol (monensin) and then diluted with medium to
111
achieve the required concentrations (final DMSO or ethanol concentration 500
14.3 ± 8.7
IV-V
>500
156.3 ± 78.3
II-III
>100
12.1 ± 4.5
IV-V
>100
6.7 ± 1.9
SI* 72h# 72+72h##
11.4
25.0
27.8
29.8
9.3
96.0
NA
19.8
NA
1.7
HMEC-1 IC50 (nM) 157.5 ± 45.0
169.6 ± 48.0
86.4 ± 31.0
3094.8 ± 316.5
11.7 ± 3.1
656
§
657
$
p
