Harry P. de Koning

Pentamidine uptake and resistance in pathogenic protozoa: past, present and future

Diamidines, and pentamidine in particular, have a long history as valuable chemotherapeutic agents against infectious disease. Their selectivity is due mostly to selective accumulation by the pathogen, rather than the host cell; and acquired resistance is frequently the result of changes in transmembrane transport of the drug. Here, recent progress in elucidating the mechanisms of diamidine transport in three important protozoan pathogens, Trypanosoma brucei, Leishmania and Plasmodium falciparum, is reviewed, and the implications for drug resistance are discussed.

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei.

ABSTRACT Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.

Drug resistance in African trypanosomiasis: the melarsoprol and pentamidine story

Melarsoprol and pentamidine represent the two main classes of drugs, the arsenicals and diamidines, historically used to treat the diseases caused by African trypanosomes: sleeping sickness in humans and Nagana in livestock. Cross-resistance to these drugs was first observed over 60 years ago and remains the only example of cross-resistance among sleeping sickness therapies. A Trypanosoma brucei adenosine transporter is well known for its role in the uptake of both drugs. More recently, aquaglyceroporin 2 (AQP2) loss of function was linked to melarsoprol-pentamidine cross-resistance. AQP2, a channel that appears to facilitate drug accumulation, may also be linked to clinical cases of resistance. Here, we review these findings and consider some new questions as well as future prospects for tackling the devastating diseases caused by these parasites.

Transporters in African trypanosomes: role in drug action and resistance.

Sleeping sickness is an increasing problem in many parts of sub-Saharan Africa. The problems are compounded by the lack of new medication, and the increasing resistance against traditional drugs such as melarsoprol, berenil and isometamidium. Over the last few years, much progress has been made in understanding how drug action, and the development of resistance, is related to the mechanisms by which the parasite ingests the drugs. In some cases novel transporters have been identified. In other cases, transporters do not appear to be involved in drug uptake, and selectivity must lie with other parasite features, such as a specific target or activation of the drug. Lessons learned from studying the uptake of drugs currently in use may assist the design of a much needed new generation of trypanocides.

The Trypanocide Diminazene Aceturate Is Accumulated Predominantly through the TbAT1 Purine Transporter: Additional Insights on Diamidine Resistance in African Trypanosomes

ABSTRACT Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals. It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite. We describe here the mechanisms by which [3H]diminazene is transported by Trypanosoma brucei brucei bloodstream forms. Diminazene was rapidly accumulated through a single transporter, with a Km of 0.45 ± 0.11 μM, which was dose dependently inhibited by pentamidine and adenosine. The Ki values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter. Yeast expressing TbAT1 acquired the ability to take up [3H]diminazene and [3H]pentamidine. TbAT1-null mutants had lost almost all capacity for [3H]diminazene transport. However, this cell line still displayed a small but detectable rate of [3H]diminazene accumulation, in a nonsaturable manner. We conclude that TbAT1 mediates [3H]diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1-null mutants and field isolates.

Loss of the High-Affinity Pentamidine Transporter Is Responsible for High Levels of Cross-Resistance between Arsenical and Diamidine Drugs in African Trypanosomes

Treatment of many infectious diseases is under threat from drug resistance. Understanding the mechanisms of resistance is as high a priority as the development of new drugs. We have investigated the basis for cross-resistance between the diamidine and melaminophenyl arsenical classes of drugs in African trypanosomes. We induced high levels of pentamidine resistance in a line without the tbat1 gene that encodes the P2 transporter previously implicated in drug uptake. We isolated independent clones that displayed very considerable cross-resistance with melarsen oxide but not phenylarsine oxide and reduced uptake of [3H]pentamidine. In particular, the high-affinity pentamidine transport (HAPT1) activity was absent in the pentamidine-adapted lines, whereas the low affinity pentamidine transport (LAPT1) activity was unchanged. The parental tbat1–/– line was sensitive to lysis by melarsen oxide, and this process was inhibited by low concentrations of pentamidine, indicating the involvement of HAPT1. This pentamidine-inhibitable lysis was absent in the adapted line KO-B48. Likewise, uptake of the fluorescent diamidine 4′,6-diamidino-2-phenylindole dihydrochloride was much delayed in live KO-B48 cells and insensitive to competition with up to 10 μM pentamidine. No overexpression of the Trypanosoma brucei brucei ATP-binding cassette transporter TbMRPA could be detected in KO-B48. We also show that a laboratory line of Trypanosoma brucei gambiense, adapted to high levels of resistance for the melaminophenyl arsenical drug melarsamine hydrochloride (Cymelarsan), had similarly lost TbAT1 and HAPT1 activity while retaining LAPT1 activity. It seems therefore that selection for resistance to either pentamidine or arsenical drugs can result in a similar phenotype of reduced drug accumulation, explaining the occurrence of cross-resistance.

Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes

African trypanosomes cause sleeping sickness in humans, a disease that is typically fatal without chemotherapy. Unfortunately, drug resistance is common and melarsoprol-resistant trypanosomes often display cross-resistance to pentamidine. Although melarsoprol/pentamidine cross-resistance (MPXR) has been an area of intense interest for several decades, our understanding of the underlying mechanisms remains incomplete. Recently, a locus encoding two closely related aquaglyceroporins, AQP2 and AQP3, was linked to MPXR in a high-throughput loss-of-function screen. Here, we show that AQP2 has an unconventional “selectivity filter.” AQP2-specific gene knockout generated MPXR trypanosomes but did not affect resistance to a lipophilic arsenical, whereas recombinant AQP2 reversed MPXR in cells lacking native AQP2 and AQP3. AQP2 was also shown to be disrupted in a laboratory-selected MPXR strain. Both AQP2 and AQP3 gained access to the surface plasma membrane in insect life-cycle–stage trypanosomes but, remarkably, AQP2 was specifically restricted to the flagellar pocket in the bloodstream stage. We conclude that the unconventional aquaglyceroporin, AQP2, renders cells sensitive to both melarsoprol and pentamidine and that loss of AQP2 function could explain cases of innate and acquired MPXR.

Curcuminoid analogs with potent activity against Trypanosoma and Leishmania species.

The natural curcuminoids curcumin (1), demethoxycurcumin (2) and bisdemethoxycurcumin (3) have been chemically modified to give 46 analogs and 8 pairs of 1:1 mixture of curcuminoid analogs and these parent curcuminoids and their analogs were assessed against protozoa of the Trypanosoma and Leishmania species. The parent curcuminoids exhibited low antitrypanosomal activity (EC(50) for our drug-sensitive Trypanosoma brucei brucei line (WT) of compounds 1, 2 and 3 are 2.5, 4.6 and 7.7 microM, respectively). Among 43 curcuminoid analogs and 8 pairs of 1:1 mixture of curcuminoid analogs tested, 8 pure analogs and 5 isomeric mixtures of analogs exhibited high antitrypanosomal activity in submicromolar order of magnitude. Among these highly active analogs, 1,7-bis(4-hydroxy-3-methoxyphenyl)hept-4-en-3-one (40) was the most active compound, with an EC(50) value of 0.053+/-0.007 microM; it was about 2-fold more active than the standard veterinary drug diminazene aceturate (EC(50) 0.12+/-0.01 microM). Using a previously characterized diminazene-resistant T. b. brucei (TbAT1-KO) and a derived multi-drug resistant line (B48), no cross-resistance of curcuminoids was observed to the diamidine and melaminophenyl arsenical drugs that are the current treatments. Indeed, curcuminoids carrying a conjugated keto (enone) motif, including 40, were significantly more active against T. b. brucei B48. This enone motif was found to contribute to particularly high trypanocidal activity against all Trypanosoma species and strains tested. The parent curcuminoids showed low antileishmanial activity (EC(50) values of compounds 1 and 2 for Leishmania mexicana amastigotes are 16+/-3 and 37+/-6 microM, respectively) while the control drug, pentamidine, displayed an EC(50) of 16+/-2 microM. Among the active curcuminoid analogs, four compounds exhibited EC(50) values of less than 5 microM against Leishmania major promastigotes and four against L. mexicana amastigotes. No significant difference in sensitivity to curcuminoids between L. major promastigotes and L. mexicana amastigotes was observed. The parent curcuminoids and most of their analogs were also tested for their toxicity against human embryonic kidney (HEK) cells. All the curcuminoids exhibited lower toxicity to HEK cells than to T. b. brucei bloodstream forms and only one of the tested compounds showed significantly higher activity against HEK cells than curcumin (1). The selectivity index for T. b. brucei ranged from 3-fold to 1500-fold. The selectivity index for the most active analog, the enone 40, was 453-fold.

Purine nucleobase transport in bloodstream forms of trypanosoma brucei brucei is mediated by two novel transporters

The mechanism and inhibitor sensitivity of hypoxanthine transport by bloodstream forms of Trypanosoma brucei brucei was investigated. The dose response curve for the inhibition of hypoxanthine transport (1 μM) by guanosine was biphasic; ≈90% of transport activity was inhibited with a Ki value of 10.8±1.8 μM, but 10% of the activity remained insensitive to concentrations as high as 2 mM. These two components of hypoxanthine transport are defined as guanosine-sensitive (H2) and guanosine-insensitive (H3). Hypoxanthine influx by both components was saturable, but there was a marked difference in their Km values (123±15 nM and 4.7±0.9 μM for H2 and H3, respectively) although the Vmax values (1.1±0.2 and 1.1±0.1 pmol (107 cells)−1 s−1, n=3) were similar. Hypoxanthine uptake via the H2 carrier was inhibited by purine bases and analogues as well as by some pyrimidine bases and one nucleoside (guanosine), whereas the H3 transporter was sensitive only to inhibition by purine nucleobases. H2-mediated hypoxanthine uptake was inhibited by ionophores, ion exchangers and the potential H+-ATPase inhibitors, N,N′-dicyclohexylcarbodiimide (DCCD) and N-ethylmaleimide (NEM). Measurements of the intracellular pH and membrane potential of bloodstream trypanosomes in the presence and absence of these agents established a linear correlation between protonmotive force and rate of [3H]hypoxanthine (30 nM) uptake. We conclude that hypoxanthine transport in bloodstream forms of T. b. brucei occurs by two transport systems with different affinities and substrate specificities, one of which, H2, appears to function as a H+/hypoxanthine symporter.

Uptake of pentamidine in Trypanosoma brucei brucei is mediated by the P2 adenosine transporter and at least one novel, unrelated transporter

Diamidine drugs such as pentamidine and berenil (diminazene aceturate) are vital drugs for the treatment of early stage human African trypanosomiasis and the corresponding veterinary condition, respectively. The action of diamidines on trypanosomes is critically dependent on their efficient uptake by the parasite. We have therefore investigated the mode of uptake of pentamidine by Trypanosoma brucei brucei, using [(125)I]iodopentamidine as a permeant. [(125)I]Iodopentamidine uptake was linear for up to 15 min and inhibited by adenosine with a K(i) value of 0.64+/-0.03 microM to a maximum of 50-70%. The adenosine-sensitive flux was also inhibited by adenine with a K(i) value of 0.44+/-0.04 microM. Iodopentamidine uptake was saturable, with the adenosine-insensitive flux displaying a K(m) of 22+/-2 microM and a V(max) of 2.2+/-0.9 pmol(10(7) cells)(-1)s(-1), whereas the adenosine-sensitive flux was inhibited by much lower iodopentamidine concentrations. These results clearly demonstrate that iodopentamidine is taken up by at least two different T. b. brucei transporters, an adenosine-sensitive pentamidine transporter (ASPT1) and a low-affinity pentamidine transporter (LAPT1). The identity of these transporters was investigated, and their significance for drug uptake and resistance in African trypanosomes is discussed.