As with all other anti-infectives (antibiotics, anti-viral drugs, and anthelminthics), the limited arsenal of anti-protozoal drugs is being depleted by a combination of two factors: increasing drug resistance and the failure to replace old and often shamefully inadequate drugs, including those compromised by (cross)-resistance, through the development of new anti-parasitics. Both factors are equally to blame: a leaking bathtub may have plenty of water if the tap is left open; if not, it will soon be empty. Here, I will reflect on the factors that contribute to the drug resistance emergency that is unfolding around us, specifically resistance in protozoan parasites.

Infections with protozoan pathogens will be with us for the foreseeable future. There is still no effective malaria vaccine despite enormous investment in money and effort over many years. Leishmaniasis is still spreading, including in Southern Europe. At least 100 million women worldwide suffer infection with the sexually transmitted Trichomonas vaginalis. Chagas Disease (American trypanosomiasis) affects communities from Texas to Argentina. Sleeping sickness (human African trypanosomiasis) remains a scourge in sub-Saharan Africa. Billions of people are infected with Toxoplasma gondii. Then, there are Cryptosporidium spp., Entamoeba histolytica, and Giardia spp. — but you get the idea.

In almost all cases, we rely on treatment (no vaccines) with a few old drugs that would never pass safety evaluation if entered into trials now. Despite the clear clinical need, there is in fact very little serious new drug development going on for these protozoan infections, and what little there is, is driven by private organisations including the Medicines for Malaria Venture (MMV), the Drugs for Neglected Diseases initiative (DNDi), the Wellcome Trust (in part through its support of the Drug Development Unit at the University of Dundee), the Bill and Melinda Gates Foundation [in part through the Consortium for Parasitic Drug Development (CPDD)], and the Institute for OneWorld Health rather than governments or the private sector. These organisations do phenomenally good work with limited resources and have taken multiple new drugs or formulations into clinical use or trials. However, their budgets clearly fall far short of the level of effort that is needed to tackle all these neglected protozoan diseases. The investment discrepancy with welfare diseases such as obesity, atherosclerosis, diabetes, and some forms of cancer does not need further elaboration.

Moreover, while malaria and the kinetoplastid diseases have a champion in MMV and DNDi, respectively, there is no such champion for trichomoniasis, cryptosporidiosis, giardiasis, toxoplasmosis, etc. And that is not mentioning important protozoan animal infections such as nagana (e.g. cattle; Trypanosoma congolense, T. brucei, and T. vivax), surra (e.g. camels, buffalo; T. evansi), babesiosis (cattle, dogs; Babesia bovis, B. canis), dourine (horses; T. equiperdum), theileriosis (cattle, sheep, goats; Theileria annulata, Theileria parva), and leishmaniosis (canines; Leishmania spp.), for which usually very few treatment options exist. For the veterinary applications, again, little drug development is taking place, as the problems are mostly those of tropical countries and poor farming communities and herdsmen, and this does not fit the necessarily profit-driven drug discovery model, nor does it translate into easy logistics of delivery and administration.

The inevitable consequence is the use, for decades, of the same old treatments, often inexpertly, predictably giving rise to the drug resistance that follows close behind. So far, so obvious; but is resistance so inevitable that we should just shrug and move on? After a century of chemotherapy, are there any lessons learned and steps that can be taken to minimise the impact of (early onset) drug resistance in protozoan disease?

Actually, the best strategy is to avoid over-reliance on chemotherapy in the first place — anything that reduces transmission rates brings the disease burden down. Good sanitation reduces transmission of waterborne parasites such as Giardia, Entamoeba, and Cryptosporidium, just as condoms prevent trichomoniasis. Clearly, effective vaccines would be our best chance of actually eradicating specific diseases, but none are likely to be approved any time soon. While a commercial vaccine for canine leishmaniasis is now available, it appears to be more effective in preventing disease progression than infection rates [1], and one study concluded that a simple anti-flea collar on the dog prevented infection much more effectively [2]. Similarly, insecticide-treated bed nets have saved many people from malaria [3]; housing improvements, insecticide and the screening of blood donors greatly reduced transmission of Chagas disease [4]; and mathematical modelling shows that spraying cattle with insecticide is much more effective against tsetse-transmitted trypanosomiasis than chemotherapy [5], especially when combined with scent-baited tsetse traps [6] or insecticide-treated tsetse ‘targets’ that can be as small as 0.06 m2 to be effective [7]. One form of tsetse control that has attracted much attention is the use of sterile insect technology (SIT), which eliminated the insects from the island of Zanzibar [8], but this is a very expensive option, and the release of large numbers of (sterile) tsetse risks a dramatic increase in transmission in the short term. Moreover, reinvasion by wild-type flies from adjourning areas would be impossible to prevent on much of the African continent. An example of an alternative to vector control is the use of trypanotolerant indigenous cattle, but these breeds usually have much lower food production value than imported ones, and can still carry and transmit the parasite, and may therefore perpetuate the problem by masking it. Indeed, there is a strong case to be made to also treat asymptomatic infections for two main reasons: continued transmission, and the risk that the infection will not remain asymptomatic. For T. b. gambiense sleeping sickness, asymptomatic carriers present a serious obstacle to eradication [9]; however, even if they could be identified, the treatment of healthy carriers raises ethical issues, especially with the currently available drugs and their well-documented side effects.

Vector control, repellents and barriers such as bed nets are all important in reducing transmission but more so for some protozoan diseases than others; Toxoplasma and Giardia, to name just two human parasites, are not transmitted by insect vectors, nor is dourine, which is caused by T. equiperdum. Moreover, full eradication by vector/transmission control is at best a long-term prospect, and meanwhile infected patients and animals must be effectively treated. Given that no new treatments will be introduced for most of these infections, we must make the best possible use of the current pharmacopoeia and not lose any to drug resistance. One good strategy is to go all-in with a full eradication programme of active case finding, mass treatment, diverse and aggressive vector control measures and the monitoring of treatment outcomes, with a second-line treatment option for the cases of drug failure – and see it through to the end. This does not allow for resistance to take hold and spread, picking off one valuable (near-irreplaceable) drug at the time. Of course, this requires a very high level of funding and organisation, and if not successfully implemented, the mass treatment programme can lead exactly to early-onset resistance as was arguably the case with chloroquine and malaria. Such an approach is likely to work, but this ideal scenario is probably unrealistic given current realities such as the lack of political leadership, prioritisation and funding for the control of the diseases of the poor.

Is drug resistance really inevitable, or is it possible to develop drugs to which the parasites simply cannot develop resistance? It is worth exploring this question, especially at this time, when confidence in antibiotics is fast disappearing because of rapidly adapting pathogens. However, antibiotics are generally products produced by an organism to defend themselves against (other) bacteria, which means that bacteria have already been exposed to them for periods on an evolutionary scale, achieving an ecological balance. In other words, antibiotic resistance adaptations are as much part of the natural world as are the antibiotics themselves. But, is the situation any different for eukaryotic parasites and the anti-parasitic drugs, which are, with a few exceptions such as tetracycline for malaria, not natural compounds? Not fundamentally, no: protozoan parasites still adapt, and have become resistant to many standard treatments. Common adaptations include: target enzyme mutations reducing interactions with the drug (antifolates in malaria [10]); reduced drug uptake (diminazene aceturate in T. b. brucei [11] or antimonials in L. donovani [12]); up-regulation of a metabolic bypass (methotrexate in Leishmania [13]); failing to activate a prodrug (nifurtimox in T. cruzi [14]); increased drug efflux (chloroquine in malaria [15]); and even the failure to produce the target (amphotericin B in Leishmania [16]). The genetic mechanisms of resistance include gene deletions [17], point mutations in targets [10] or transporters (enabling [15] or disabling [18]), copy number variations [19,20], base pair insertions/deletions causing frame shifts in the target gene [12] and the formation of chimeric genes through recombination [21].

Despite the plethora of mechanisms by which protozoa can acquire drug resistance, for some drugs this happens much faster than for others. In many cases, a single mutation or gene deletion is sufficient for a loss of sensitivity, sometimes followed by secondary mutations to give higher levels of resistance [10,18]. Whereas a target protein is of course usually essential, active-site mutations that selectively lose affinity for an inhibitor, yet retain sufficient functionality, are certainly possible. In the case of a transporter mutation, the transporter is usually not essential, because of redundancy [22] or its substrate is non-essential to the cell [23], and in such cases the mutation can be disabling to the transport of both the physiological substrate and the drug [17,18,21]. In other cases, however, resistance does not come about so easily. The drug may not have a single protein target that can be mutated but may bind instead to DNA as intercalator or minor groove binder (e.g. phenanthridines, diamidines [2426]), or e.g. to haem (chloroquine [27]), or be more generally cytotoxic, with multiple targets (polypharmacology), as may be the case for heavy metal drugs (arsenicals, antimonials) and suramin [28], in which case selectivity depends mostly on selective entry into the parasite rather than into the host cells, through unique transporters or other uptake mechanisms [20,28,29].

In some such cases, resistance still occurs, through mutation or deletion of these transporters [20,29], but in the case of pentamidine, for instance, clinical resistance for sleeping sickness has not been reported despite being virtually the only drug used against early-stage T. b. gambiense sleeping sickness since the mid-1930s, including population-scale mass treatment campaigns [30], although various levels of resistance can be induced in vitro [31]. The lack (of reports) of clinical resistance, in this case, can be traced to the fact that pentamidine is taken up by three different transport mechanisms, at least two of which, the aminopurine transporter AT1 and the High Affinity Pentamidine Transporter, HAPT1 (now identified as an aquaglyceroporin, TbAQP2 [21]), are highly efficient, allowing a fast accumulation of the drug [32]; moreover, the drug action becomes irreversible after only a brief exposure [33], giving little scope for resistance to develop. Recently, AQP2-mutated T. b. gambiense strains have been isolated from sleeping sickness patients in the Democratic Republic of Congo and from South Sudan, who relapsed after melarsoprol treatment; these isolates were verified to be significantly resistant to both melarsoprol and pentamidine in vitro [34]. However, it is of note that the relapses were after treatment with melarsoprol (which is also a substrate of both AT1 and AQP2 [21]), not pentamidine. As explained by Graf et al. [34], the pharmacokinetics of pentamidine (but not of melarsoprol), especially the high peak plasma concentration and long clearance time, may prevent treatment failure even though the rate by which the drug enters the parasite is reduced dramatically, leading to a significant in vitro resistance phenotype. Similarly, there have been no reports of resistance to suramin, still in use for T. b. rhodesiense sleeping sickness after 100 years, and this is believed to be linked to the drug being internalised by endocytosis, which is uniquely fast in bloodstream trypanosomes, after binding to the invariant surface glycoprotein ISG75, of which there are multiple paralogues [28].

Interestingly, the known protozoan ‘drug transporters’ are mostly members of highly conserved gene families, and often involved in nutrient uptake or other household functions. For example, in Leishmania the miltefosine transporter is a phospholipid translocase [35] and methotrexate is taken up by a folate transporter [13]; the T. brucei carrier for eflornithine is an amino acid transporter [17], while pentamidine and melarsoprol are taken up by an aminopurine transporter and an aquaglyceroporin [18,21]. Thus, it is often hard to predict what might be a (potential) drug transporter in a protozoan: they are hidden in plain sight, masked by their innocuous roles. Conversely, creative use of the uniqueness of protozoan transporters from conserved families can be exploited for the selective targeting of therapeutic agents [36,37].

Thus, we see that polypharmacology, combined with an uptake mechanism that is not easily disabled by mutations in a single Open Reading Frame (ORF), can prolong a drug's lifespan to (most of) a century. This point can be illustrated with our work on curcumin and a series of analogues that display in vitro activity against T. brucei species, with an EC50 value of 53 nM for the most potent analogue, AS-HK014, compared with 2.7 µM for curcumin itself [38]. We were unable to induce any level of resistance against curcumin in vitro, although we easily induced a 50-fold level of resistance to the analogue, which brought it to the exact same level as curcumin, which we could not then surpass [39]. Curcumin is believed to diffuse across the membrane and indeed act on the membrane [40], and we certainly were unable to measure any saturable uptake using 3H-curcumin (unpublished data). The failure to induce curcumin resistance is consistent with the current model that attributes the many biological actions of curcumin to polypharmacology [41] and a capacity to interact with membrane proteins [40]. In contrast, the activity of analogue AS-HK014 was well defined (reaction with cellular thiols leading to depletion of trypanothione and glutathione [39]), and resistance developed rapidly.

A more important example, of course, is the essential antimalarial drug artemisinin; it is estimated that widespread resistance to this drug, as is now spreading in South-East Asia [42], will result in well over 100 000 additional malaria deaths and US$385M/year in additional loss of economic activity [43]. As artemisinin appears to act, when activated by haem, through highly promiscuous covalent binding to many cellular targets in the parasite [44], resistance through target mutations/gene deletion would appear impossible, and no transporter mutations have been found. Indeed, as noted by Wang et al. [45] no Plasmodium falciparum strain has so far been found to be insensitive to artemisinin in a standard in vitro protocol. Yet treatment failure with artemisinin combination therapy (ACT) is undoubtedly real, and associated with mutations in a single gene, kelch13 (K13) [42,46], which is neither an artemisinin target nor a transporter [45]. However, drug resistance is not an all-or-nothing proposition, and the answer to the Sphinx's riddle is that the parasites remain sensitive but that the parasite clearance time is significantly increased in the adapted strains [42]. The K13 mutations have the effect of shortening the duration of the trophozoite stage, during which the parasite is particularly susceptible to artemisinin, and lengthening the duration of the ring stage that is relatively insensitive due its low haem content [47]. This adaptation of intra-erythrocyte development is yet another unimagined way by which parasites can develop clinical resistance, but the clinical resistance would probably not have taken hold if the ACT partner drugs had not suffered from resistance first. It is the function of these partner drugs to ‘mop up’ the parasites that survive the relatively short exposure to artemisinin, caused by the rapid clearance of the drug.

We thus see that combination chemotherapy as in ACT, or nifurtimox/eflornithine in late-stage sleeping sickness, can keep resistance effectively at bay, unless the parasite can either develop transmissible resistance to one of the component drugs separately (which is not true combination therapy) or if resistance to one of the drugs in the combination already exists in the pathogen population. In other words, it may be a really counterproductive idea to introduce a combination in order to try to ‘salvage’ a drug after resistance has been reported, because that may lead to the functional loss of both components in the combined formulation. This is not a trivial concern, as we truly cannot lose any of the main anti-protozoal drugs, and should feature in current discussions about the potential introduction of combinations against visceral leishmaniasis. Resistance against all the major anti-leishmanial drugs has been reported [16,48,49] and, alarmingly, resistance against several combinations can be induced readily in vitro [50].

In conclusion, we see that it is extremely difficult to prevent the onset, or spread of drug resistance and that, therefore, the reality is that we are and will continue to lose life-saving treatments. While more can be done on the side of prevention, vector control, sanitation, etc., the only way to prevent a catastrophic inability to treat protozoan disease is by investing in new treatments. For that, genuine, long-term partners with very deep pockets are eagerly sought.

Abbreviations

     
  • ACT

    artemisinin combination therapy

  •  
  • DNDi

    Drugs for Neglected Diseases initiative

  •  
  • MMV

    Medicines for Malaria Venture

Competing Interests

The Author declares that there are no competing interests associated with this manuscript.

References

References
1
Oliva
,
G.
,
Nieto
,
J.
,
Foglia Manzillo
,
V.
,
Cappiello
,
S.
,
Fiorentino
,
E.
,
Di Muccio
,
T.
et al. 
(
2014
)
A randomised, double-blind, controlled efficacy trial of the LiESP/QA-21 vaccine in naïve dogs exposed to two Leishmania infantum transmission seasons
.
PLoS Negl. Trop. Dis.
8
,
e3213
2
Brianti
,
E.
,
Napoli
,
E.
,
Gaglio
,
G.
,
Falsone
,
L.
,
Giannetto
,
S.
,
Solari Basano
,
F.
et al. 
(
2016
)
Field evaluation of two different treatment approaches and their ability to control fleas and prevent canine leishmaniosis in a highly endemic area
.
PLoS Negl. Trop. Dis.
10
,
e0004987
3
Eisele
,
T.P.
,
Keating
,
J.
,
Littrell
,
M.
,
Larsen
,
D.
and
Macintyre
,
K.
(
2009
)
Assessment of insecticide-treated bednet use among children and pregnant women across 15 countries using standardized national surveys
.
Am. J. Trop. Med. Hyg.
80
,
209
214
PMID:
[PubMed]
4
Salvatella
,
R.
,
Irabedra
,
P.
and
Castellanos
,
L.G
. (
2014
)
Interruption of vector transmission by native vectors and the ‘art of the possible’
.
Mem. Inst. Oswaldo Cruz
109
,
122
130
5
Hargrove
,
J.W.
,
Ouifki
,
R.
,
Kajunguri
,
D.
,
Vale
,
G.A.
and
Torr
,
S.J.
(
2012
)
Modeling the control of trypanosomiasis using trypanocides or insecticide-treated livestock
.
PLoS Negl. Trop. Dis.
6
,
e1615
6
Muzari
,
M.O.
(
1999
)
Odour-baited targets as invasion barriers for tsetse flies (Diptera: Glossinidae): a field trial in Zimbabwe
.
Bull. Entomol. Res.
89
,
73
77
7
Lindh
,
J.M.
,
Torr
,
S.J.
,
Vale
,
G.A.
and
Lehane
,
M.J.
(
2009
)
Improving the cost-effectiveness of artificial visual baits for controlling the tsetse fly Glossina fuscipes fuscipes
.
PLoS Negl. Trop. Dis.
3
,
e474
8
Vreysen
,
M.J.B.
,
Saleh
,
K.M.
,
Ali
,
M.Y.
,
Abdulla
,
A.M.
,
Zhu
,
Z.-R.
,
Juma
,
K.G.
et al. 
(
2000
)
Glossina austeni (Diptera: Glossinidae) eradicated on the island of Unguja, Zanzibar, using the sterile insect technique
.
J. Econ. Entomol.
93
,
123
135
9
Jamonneau
,
V.
,
Ilboudo
,
H.
,
Kaboré
,
J.
,
Kaba
,
D.
,
Koffi
,
M.
,
Solano
,
P.
et al. 
(
2012
)
Untreated human infections by Trypanosoma brucei gambiense are not 100% fatal
.
PLoS Negl. Trop. Dis.
6
,
e1691
10
Hyde
,
J.E.
(
2002
)
Mechanisms of resistance of Plasmodium falciparum to antimalarial drugs
.
Microbes Infect.
4
,
165
174
11
De Koning
,
H.P.
,
Anderson
,
L.F.
,
Stewart
,
M.
,
Burchmore
,
R.J.S.
,
Wallace
,
L.J.M.
and
Barrett
,
M.P.
(
2004
)
The trypanocide diminazene aceturate is accumulated predominantly through the TbAT1 purine transporter: additional insights on diamidine resistance in African trypanosomes
.
Antimicrob. Agents Chemother.
48
,
1515
1519
12
Imamura
,
H.
,
Downing
,
T.
,
Van den Broeck
,
F.
,
Sanders
,
M.J.
,
Rijal
,
S.
,
Sundar
,
S.
et al. 
(
2016
)
Evolutionary genomics of epidemic visceral leishmaniasis in the Indian subcontinent
.
eLife
5
,
e12613
13
Ouellette
,
M.
,
Drummelsmith
,
J.
,
El-Fadili
,
A.
,
Kündig
,
C.
,
Richard
,
D.
and
Roy
,
G
. (
2002
)
Pterin transport and metabolism in Leishmania and related trypanosomatid parasites
.
Int. J. Parasitol.
32
,
385
398
14
Wilkinson
,
S.R.
,
Taylor
,
M.C.
,
Horn
,
D.
,
Kelly
,
J.M.
and
Cheeseman
,
I.
(
2008
)
A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes
.
Proc. Natl Acad. Sci. U.S.A.
105
,
5022
5027
15
Ecker
,
A.
,
Lehane
,
A.M.
,
Clain
,
J.
and
Fidock
,
D.A.
(
2012
)
PfCRT and its role in antimalarial drug resistance
.
Trends Parasitol.
28
,
504
514
16
Purkait
,
B.
,
Kumar
,
A.
,
Nandi
,
N.
,
Sardar
,
A.H.
,
Das
,
S.
,
Kumar
,
S.
et al. 
(
2012
)
Mechanism of Amphotericin B resistance in clinical isolates of Leishmania donovani
.
Antimicrob. Agents Chemother.
56
,
1031
1041
17
Vincent
,
I.M.
,
Creek
,
D.
,
Watson
,
D.G.
,
Kamleh
,
M.A.
,
Woods
,
D.J.
,
Wong
,
P.E.
et al. 
(
2010
)
A molecular mechanism for eflornithine resistance in African trypanosomes
.
PLoS Pathog.
6
,
e1001204
18
Munday
,
J.C.
,
Tagoe
,
D.N.A.
,
Eze
,
A.A.
,
Krezdorn
,
J.A.M.
,
Rojas López
,
K.E.
,
Alkhaldi
,
A.A.M.
et al. 
(
2015
)
Functional analysis of drug resistance-associated mutations in the Trypanosoma brucei Adenosine Transporter 1 (TbAT1) and the proposal of a structural model for the protein
.
Mol. Microbiol.
96
,
887
900
19
Papadopoulou
,
B.
,
Kündig
,
C.
,
Singh
,
A.
and
Ouellette
,
M.
(
1998
)
Drug resistance in Leishmania: similarities and differences to other organisms
.
Drug Resist. Updat.
1
,
266
278
20
Monte-Neto
,
R.
,
Laffitte
,
M.-C.N.
,
Leprohon
,
P.
,
Reis
,
P.
,
Frézard
,
F.
and
Ouellette
,
M.
(
2015
)
Intrachromosomal amplification, locus deletion and point mutation in the aquaglyceroporin AQP1 gene in antimony resistant Leishmania (Viannia) guyanensis
.
PLoS Negl. Trop. Dis.
9
,
e0003476
21
Munday
,
J.C.
,
Eze
,
A.A.
,
Baker
,
N.
,
Glover
,
L.
,
Clucas
,
C.
,
Aguinaga Andrés
,
D.
et al. 
(
2014
)
Trypanosoma brucei Aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs
.
J. Antimicrob. Chemother.
69
,
651
663
22
De Koning
,
H.P.
,
Bridges
,
D.J.
and
Burchmore
,
R.J.S.
(
2005
)
Purine and pyrimidine transport in pathogenic protozoa: from biology to therapy
.
FEMS Microbiol. Rev.
29
,
987
1020
23
Ali
,
J.A.M.
,
Creek
,
D.J.
,
Burgess
,
K.
,
Allison
,
H.C.
,
Field
,
M.C.
,
Mäser
,
P.
et al. 
(
2013
)
Pyrimidine salvage in Trypanosoma brucei bloodstream forms and the trypanocidal action of halogenated pyrimidines
.
Mol. Pharmacol.
83
,
439
453
24
Roy Chowdhury
,
A.
,
Bakshi
,
R.
,
Wang
,
J.
,
Yildirir
,
G.
,
Liu
,
B.
,
Pappas-Brown
,
V.
et al. 
(
2010
)
The killing of African trypanosomes by ethidium bromide
.
PLoS Pathog.
6
,
e1001226
25
Wilson
,
W.D.
,
Nguyen
,
B.
,
Tanious
,
F.A.
,
Mathis
,
A.
,
Hall
,
J.E.
,
Stephens
,
C.E.
et al. 
(
2005
)
Dications that target the DNA minor groove: compound design and preparation, DNA interactions, cellular distribution and biological activity
.
Curr. Med. Chem. Anticancer Agents
5
,
389
408
26
Millán
,
C.R.
,
Acosta-Reyes
,
F.J.
,
Lagartera
,
L.
,
Ebiloma
,
G.U.
,
Lemgruber
,
L.
,
Nué Martinez
,
J.J.
et al. 
(
2017
)
Functional and structural analysis of AT-specific minor groove binders that disrupt DNA–protein interactions and cause disintegration of the Trypanosoma brucei kinetoplast
.
Nucl. Acid Res.
45
,
8378
8391
27
Roepe
,
P.D.
(
2009
)
Molecular and physiologic basis of quinoline drug resistance in Plasmodium falciparum
.
Future Microbiol.
4
,
441
455
28
Zoltner
,
M.
,
Horn
,
D.
,
De Koning
,
H.P.
and
Field
,
M.C.
(
2016
)
Exploiting the Achilles’ heel of membrane trafficking in trypanosomes
.
Curr. Opin. Microbiol.
34
,
97
103
29
Munday
,
J.C.
,
Settimo
,
L.
and
De Koning
,
H.P.
(
2015
)
Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei
.
Front. Pharmacol.
6
,
32
30
Bray
,
P.G.
,
Barrett
,
M.P.
,
Ward
,
S.A.
and
De Koning
,
H.P.
(
2003
)
Pentamidine uptake and resistance in pathogenic protozoa
.
Trends Parasitol.
19
,
232
239
31
Bridges
,
D.
,
Gould
,
M.K.
,
Nerima
,
B.
,
Mäser
,
P.
,
Burchmore
,
R.J.S.
and
De Koning
,
H.P.
(
2007
)
Loss of the High-Affinity Pentamidine Transporter is responsible for high levels of cross-resistance between arsenical and diamidine drugs in African trypanosomes
.
Mol. Pharmacol.
71
,
1098
1108
32
De Koning
,
H.P.
(
2001
)
Uptake of pentamidine in Trypanosoma brucei brucei is mediated by three distinct transporters. implications for cross-resistance with arsenicals
.
Mol. Pharmacol.
59
,
586
592
PMID:
[PubMed]
33
Ward
,
C.P.
,
Wong
,
P.E.
,
Burchmore
,
R.J.
,
De Koning
,
H.P.
and
Barrett
,
M.P.
(
2011
)
Trypanocidal furamidine analogues: influence of pyridine nitrogens on trypanocidal activity, transport kinetics, and resistance patterns
.
Antimicrob. Agents Chemother.
55
,
2352
2361
34
Graf
,
F.E.
,
Ludin
,
P.
,
Wenzler
,
T.
,
Kaiser
,
M.
,
Brun
,
R.
,
Pyana
,
P.P.
et al. 
(
2013
)
Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol
.
PLoS Negl. Trop. Dis.
7
,
e2475
35
García-Sánchez
,
S.
,
Sánchez-Cañete
,
M.P.
,
Gamarro
,
F.
and
Castanys
,
S.
(
2014
)
Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit
.
Biochem. J.
459
,
83
94
36
Rodenko
,
B.
,
Van der Burg
,
A.M.
,
Wanner
,
M.J.
,
Kaiser
,
M.
,
Brun
,
R.
,
Gould
,
M.
et al. 
(
2007
)
2,N6-Disubstituted adenosine analogs with antitrypanosomal and antimalarial activities
.
Antimicrob. Agents Chemother.
51
,
3796
3802
37
Barrett
,
M.P.
and
Gilbert
,
I.H.
(
2006
)
Targeting of toxic compounds to the trypanosome's interior
.
Adv. Parasitol.
63
,
125
183
38
Changtam
,
C.
,
De Koning
,
H.P.
,
Ibrahim
,
H.
,
Sajid
,
S.
,
Gould
,
M.K.
and
Suksamrarn
,
A.
(
2010
)
Curcuminoid analogs with potent activity against Trypanosoma and Leishmania species
.
Eur. J. Med. Chem.
45
,
941
956
39
Alkhaldi
,
A.A.M.
,
Creek
,
D.J.
,
Ibrahim
,
H.
,
Kim
,
D.-H.
,
Quashie
,
N.B.
,
Burgess
,
K.E.
et al. 
(
2015
)
Potent trypanocidal curcumin analogs bearing a monoenone linker motif act on Trypanosoma brucei by forming an adduct with trypanothione
.
Mol. Pharmacol.
87
,
451
464
40
Ingólfsson
,
H.I.
,
Thakur
,
P.
,
Herold
,
K.F.
,
Hobart
,
E.A.
,
Ramsey
,
N.B.
,
Periole
,
X.
et al. 
(
2014
)
Phytochemicals perturb membranes and promiscuously alter protein function
.
ACS Chem. Biol.
9
,
1788
1798
41
Nantasenamat
,
C.
,
Simeon
,
S.
,
Hafeez
,
A.
,
Prachayasittikul
,
V.
,
Worachartcheewan
,
A.
,
Songtawee
,
N.
et al. 
(
2014
) Elucidating the structure-activity relationships of curcumin and its biological activities. In
Curcumin: Synthesis, Emerging Role in Pain Management and Health Implications
(
D.L.
Pouliquen
, ed.).
Nova Science Publishers
,
New York, U.S.A.
, pp.
49
86
42
Ashley
,
E.A.
,
Dhorda
,
M.
,
Fairhurst
,
R.M.
,
Amaratunga
,
C.
,
Lim
,
P.
,
Suon
,
S.
et al. 
(
2014
)
Spread of artemisinin resistance in Plasmodium falciparum malaria
.
N. Engl. J. Med.
371
,
411
423
43
Lubell
,
Y.
,
Dondorp
,
A.
,
Guérin
,
P.J.
,
Drake
,
T.
,
Meek
,
S.
,
Ashley
,
E.
et al. 
(
2014
)
Artemisinin resistance–modelling the potential human and economic costs
.
Malar. J.
13
,
452
44
Wang
,
J.
,
Zhang
,
C.-J.
,
Chia
,
W.N.
,
Loh
,
C.C.Y.
,
Li
,
Z.
,
Lee
,
Y.M.
et al. 
(
2015
)
Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum
.
Nat. Commun.
6
,
10111
45
Wang
,
J.
,
Xu
,
C.
,
Lun
,
Z.-R.
and
Meshnick
,
S.R.
(
2017
)
Unpacking ‘artemisinin resistance’
.
Trends Pharmacol. Sci.
38
,
506
511
46
Ariey
,
F.
,
Witkowski
,
B.
,
Amaratunga
,
C.
,
Beghain
,
J.
,
Langlois
,
A.-C.
,
Khim
,
N.
et al. 
(
2014
)
A molecular marker of artemisinin-resistant Plasmodium falciparum malaria
.
Nature
505
,
50
55
47
Hott
,
A.
,
Casandra
,
D.
,
Sparks
,
K.N.
,
Morton
,
L.C.
,
Castanares
,
G.-G.
,
Rutter
,
A.
et al. 
(
2015
)
Artemisinin-resistant Plasmodium falciparum parasites exhibit altered patterns of development in infected erythrocytes
.
Antimicrob. Agents Chemother.
59
,
3156
3167
48
Sundar
,
S.
,
Singh
,
A.
,
Rai
,
M.
,
Prajapati
,
V.K.
,
Singh
,
A.K.
,
Ostyn
,
B.
et al. 
(
2012
)
Efficacy of miltefosine in the treatment of visceral leishmaniasis in India after a decade of use
.
Clin. Infect. Dis.
55
,
543
550
49
Mittal
,
M.K.
,
Rai
,
S.
,
Ashutosh
,
.
,
Ravinder
,
.
,
Gupta
,
S.
,
Sundar
,
S.
et al. 
(
2007
)
Characterization of natural antimony resistance in Leishmania donovani isolates
.
Am. J. Trop. Med. Hyg.
76
,
681
688
PMID:
[PubMed]
50
García-Hernández
,
R.
,
Manzano
,
J.I.
,
Castanys
,
S.
and
Gamarro
,
F.
(
2012
)
Leishmania donovani develops resistance to drug combinations
.
PLoS Negl. Trop. Dis.
6
,
e1974
This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology and distributed under the Creative Commons Attribution License 4.0 (CC BY-NC-ND).