A molecular mechanism for eflornithine resistance in African trypanosomes.

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.

A glucose transporter can mediate ribose uptake: definition of residues that confer substrate specificity in a sugar transporter.

Sugars, the major energy source for many organisms, must be transported across biological membranes. Glucose is the most abundant sugar in human plasma and in many other biological systems and has been the primary focus of sugar transporter studies in eukaryotes. We have previously cloned and characterized a family of glucose transporter genes from the protozoan parasite Leishmania. These transporters, called LmGT1, LmGT2, and LmGT3, are homologous to the well characterized glucose transporter (GLUT) family of mammalian glucose transporters. We have demonstrated that LmGT proteins are important for parasite viability. Here we show that one of these transporters, LmGT2, is a more effective carrier of the pentose sugar d-ribose than LmGT3, which has a 6-fold lower relative specificity (V(max)/K(m)) for ribose. A pair of threonine residues, located in the putative extracellular loops joining transmembrane helices 3 to 4 and 7 to 8, define a filter that limits ribose approaching the exofacial substrate binding pocket in LmGT3. When these threonines are substituted by alanine residues, as found in LmGT2, the LmGT3 permease acquires ribose permease activity that is similar to that of LmGT2. The location of these residues in hydrophilic loops supports recent suggestions that substrate recognition is separated from substrate binding and translocation in this important group of transporters.

Trypanocidal furamidine analogues: influence of pyridine nitrogens on trypanocidal activity, transport kinetics, and resistance patterns.

Current therapies for human African trypanosomiasis (HAT) are unsatisfactory and under threat from emerging drug resistance linked to the loss of transporters, e.g., the P2 aminopurine transporter (TbAT1). Here we compare the uptake and trypanocidal properties of furamidine (DB75), recently evaluated in clinical trials against stage 1 (haemolymphatic) HAT, and two aza analogues, DB820 and CPD0801 (DB829), which are candidate compounds for treatment of stage 2 (neurological) disease. Values of 50% inhibitory concentrations (IC50s) determined in vitro against both wild-type and transporter mutant parasites were submicromolar, with DB75 trypanotoxicity shown to be better than and DB820 trypanotoxicity similar to that of the widely used veterinary trypanocide diminazene, while CPD0801 was less active. Activity correlated with uptake and with the minimum drug exposure time necessary to kill trypanosomes: DB75 accumulated at double and 10-fold the rates of DB820 and CPD0801, respectively. All three compounds inhibited P2-mediated adenosine transport with similar Ki values, indicating affinity values for this permease in the low to submicromolar range. Uptake of DB75, DB820, and CPD0801 was significantly reduced in tbat1-/- parasites and was sensitive to inhibition by adenine, showing that all three compounds are substrates for the P2 transporter. Uptake in vitro was significantly less than that seen with parasites freshly isolated from infected rats, correlating with a downregulation of P2 activity in vitro. We conclude that DB75, DB820, and CPD0801 are actively accumulated by Trypanosoma brucei brucei, with P2 as the main transport route. The aza analogues of DB75 accumulate more slowly than furamidine itself and reveal less trypanocidal activity in standard in vitro drug sensitivity assays.

Synthesis and biological evaluation of 1,4-benzodiazepin-2-ones with antitrypanosomal activity.

A library of 1,4-benzodiazepines has been synthesized and evaluated against Trypanosoma brucei, a causative parasite of Human African trypanosomiasis. Benzodiazepines possessing a P2- transporter motif were found to have MIC values as low as 0.78 µM.

Multiple genetic mechanisms lead to loss of functional TbAT1 expression in drug-resistant trypanosomes.

The P2 aminopurine transporter, encoded by TbAT1 in African trypanosomes in the Trypanosoma brucei group, carries melaminophenyl arsenical and diamidine drugs into these parasites. Loss of this transporter contributes to drug resistance. We identified the genomic location of TbAT1 to be in the subtelomeric region of chromosome 5 and determined the status of the TbAT1 gene in two trypanosome lines selected for resistance to the melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), and in a Trypanosoma equiperdum clone selected for resistance to the diamidine, diminazene aceturate. In the Trypanosoma brucei gambiense STIB 386 melarsamine hydrochloride-resistant line, TbAT1 is deleted, while in the Trypanosoma brucei brucei STIB 247 melarsamine hydrochloride-resistant and T. equiperdum diminazene-resistant lines, TbAT1 is present, but expression at the RNA level is no longer detectable. Further characterization of TbAT1 in T. equiperdum revealed that a loss of heterozygosity at the TbAT1 locus accompanied loss of expression and that P2-mediated uptake of [(3)H]diminazene is lost in drug-resistant T. equiperdum. Adenine-inhibitable adenosine uptake is still detectable in a DeltaTbat1 T. b. brucei mutant, although at a greatly reduced capacity compared to that of the wild type, indicating that an additional adenine-inhibitable adenosine permease, distinct from P2, is present in these cells.

Selective delivery of 2-hydroxy APA to Trypanosoma brucei using the melamine motif.

Trypanosoma brucei, the parasite that causes human African trypanosomiasis, is auxotrophic for purines and has specialist nucleoside transporters to import these metabolites. In particular, the P2 aminopurine transporter can also selectively accumulate melamine derivatives. In this Letter, we report the coupling of the melamine moiety to 2-hydroxy APA, a potent ornithine decarboxylase inhibitor, with the aim of selectively delivering this compound to the parasite. The best compound described here shows an increased in vitro trypanocidal activity compared with the parent.

Targeted delivery of compounds to Trypanosoma brucei using the melamine motif.

There is an urgent need for the development of new drugs for the treatment of human African trypanosomiasis. The causative organism, Trypanosoma brucei, has been shown to have some unusual plasma membrane transporters, in particular the P2 aminopurine transporter and related permeases, which have been used for the selective targeting of trypanocidal compounds to the organism. In this paper, we report the addition of melamine-based P2-targeting motifs to three different classes of compound in order to try and improve activity through increased selective uptake. The classes reported here are fluoroquinolones, difluoromethylornithine and artesunate derivatives.

An evaluation of Minor Groove Binders as anti-Trypanosoma brucei brucei therapeutics.

A series of 32 structurally diverse MGBs, derived from the natural product distamycin, was evaluated for activity against Trypanosoma brucei brucei. Four compounds have been found to possess significant activity, in the nanomolar range, and represent hits for further optimisation towards novel treatments for Human and Animal African Trypanosomiases. Moreover, SAR indicates that the head group linking moiety is a significant modulator of biological activity.

Structure-based design and synthesis of antiparasitic pyrrolopyrimidines targeting pteridine reductase 1.

The treatment of Human African trypanosomiasis remains a major unmet health need in sub-Saharan Africa. Approaches involving new molecular targets are important; pteridine reductase 1 (PTR1), an enzyme that reduces dihydrobiopterin in Trypanosoma spp., has been identified as a candidate target, and it has been shown previously that substituted pyrrolo[2,3-d]pyrimidines are inhibitors of PTR1 from Trypanosoma brucei (J. Med. Chem. 2010, 53, 221-229). In this study, 61 new pyrrolo[2,3-d]pyrimidines have been prepared, designed with input from new crystal structures of 23 of these compounds complexed with PTR1, and evaluated in screens for enzyme inhibitory activity against PTR1 and in vitro antitrypanosomal activity. Eight compounds were sufficiently active in both screens to take forward to in vivo evaluation. Thus, although evidence for trypanocidal activity in a stage I disease model in mice was obtained, the compounds were too toxic to mice for further development.

Aryl phosphoramidates of 5-phospho erythronohydroxamic acid, a new class of potent trypanocidal compounds.

RNAi and enzymatic studies have shown the importance of 6-phosphogluconate dehydrogenase (6-PGDH) in Trypanosoma brucei for the parasite survival and make it an attractive drug target for the development of new treatments against human African trypanosomiasis. 2,3-O-Isopropylidene-4-erythrono hydroxamate is a potent inhibitor of parasite Trypanosoma brucei 6-phosphogluconate dehydrogenase (6-PGDH), the third enzyme of the pentose phosphate pathway. However, this compound does not have trypanocidal activity due to its poor membrane permeability. Consequently, we have previously reported a prodrug approach to improve the antiparasitic activity of this inhibitor by converting the phosphate group into a less charged phosphate prodrug. The activity of prodrugs appeared to be dependent on their stability in phosphate buffer. Here we have successfully further extended the development of the aryl phosphoramidate prodrugs of 2,3-O-isopropylidene-4-erythrono hydroxamate by synthesizing a small library of phosphoramidates and evaluating their biological activity and stability in a variety of assays. Some of the compounds showed high trypanocidal activity and good correlation of activity with their stability in fresh mouse blood.

Polyamine aza-cyclic compounds demonstrate anti-proliferative activity in vitro but fail to control tumour growth in vivo.

Cationic polyamines such as the poly(propylenimine) dendrimers (DAB16) are anti-tumour agents (Dufes et al., 2005, Cancer Res 65:8079-8084). Their mechanism of action is poorly understood, but the lack of in vivo toxicity suggests cancer specificity. To explore this polyamine pharmacophore we cross-linked the aza-cyclic compound, hexacyclen, with 1,4-dibromobutane or 1,8-dibromooctane to yield the polyamines [poly(butylhexacyclen)--CL4] or [poly(octylhexacyclen)--CL8] respectively, both free of primary amines. We characterised the compounds and their respective nanoparticles and examined their interaction with the putative targets of the cationic polyamines: the cell membrane and DNA. Like DAB 16, CL4 and CL8 bind plasmid DNA and all three compounds interrupted the cell cycle of A431 epidermoid carcinoma cells in the S-phase. Additionally all three compounds disrupted erythrocyte membranes, with CL8 and DAB 16 being more active, in this respect, than CL4. CL4 (IC(50) =775.1 µg mL(-1)) and CL8 (IC(50) =8.4 µg mL(-1)), in a similar manner to DAB 16, were anti-proliferative against A431 cells; although unlike DAB 16, CL4 and CL8 were not tumouricidal against A431 xenografts in mice, indicating that primary amines may play an important role in the in vivo activity of DAB 16.