African Animal Trypanosomiasis (AAT), caused predominantly by Trypanosoma brucei brucei, T. vivax and T. congolense, is a fatal livestock disease throughout Sub-Saharan Africa. Treatment options are very limited and threatened by resistance. Tubercidin (7-deazaadenosine) analogs have shown activity against individual parasites but viable chemotherapy must be active against all three species. Divergence in sensitivity to nucleoside antimetabolites could be caused by differences in nucleoside transporters. Having previously characterized the T. brucei nucleoside carriers, we here report the functional expression and characterization of the main adenosine transporters of T. vivax (TvxNT3) and T. congolense (TcoAT1/NT10), in a Leishmania mexicana cell line (‘SUPKO’) lacking adenosine uptake. Both carriers were similar to the T. brucei P1-type transporters and bind adenosine mostly through interactions with N3, N7 and 3′-OH. Expression of TvxNT3 and TcoAT1 sensitized SUPKO cells to various 7-substituted tubercidins and other nucleoside analogs although tubercidin itself is a poor substrate for P1-type transporters. Individual nucleoside EC50s were similar for T. b. brucei, T. congolense, T. evansi and T. equiperdum but correlated less well with T. vivax. However, multiple nucleosides including 7-halogentubercidines displayed pEC50>7 for all species and, based on transporter and anti-parasite SAR analyses, we conclude that nucleoside chemotherapy for AAT is viable.
The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from Leishmania mexicana (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells’ ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of L. mexicana NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of T. cruzi NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the Aspergillus nidulans uracil transporter FurD.
The trypanosome alternative
oxidase (TAO), a mitochondrial enzyme
involved in the respiration of the bloodstream form trypomastigotes
of
Trypanosoma brucei
, is a validated
drug target against African trypanosomes. Earlier series of TAO inhibitors
having a 2,4-dihydroxy-6-methylbenzoic acid scaffold (“head”)
and a triphenylphosphonium or quinolin-1-ium cation as a mitochondrion-targeting
group (“tail”) were shown to be nanomolar inhibitors
in enzymatic and cellular assays. We investigated here the effect
of different mitochondrion-targeting cations and other scaffold modifications
on the in vitro activity of this class of inhibitors. Low micromolar
range activities were obtained, and the structure–activity
relationship studies showed that modulation of the tail region with
polar substituents is generally detrimental to the enzymatic and cellular
activity of TAO inhibitors.
Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is a serious public health problem. Current treatment is restricted to two drugs, benznidazole and nifurtimox, displaying serious efficacy and safety drawbacks. Nucleoside analogues represent a promising alternative as protozoans do not biosynthesize purines and rely on purine salvage from the hosts. Protozoan transporters often present different substrate specificities from mammalian transporters, justifying the exploration of nucleoside analogues as therapeutic agents. Previous reports identified nucleosides with potent trypanocidal activity; therefore, two 7-derivatized tubercidins (FH11706, FH10714) and a 3′-deoxytubercidin (FH8513) were assayed against T. cruzi. They were highly potent and selective, and the uptake of the tubercidin analogues appeared to be mediated by the nucleoside transporter TcrNT2. At 10 μM, the analogues reduced parasitemia >90% in 2D and 3D cardiac cultures. The washout assays showed that FH10714 sterilized the infected cultures. Given orally, the compounds did not induce noticeable mouse toxicity (50 mg/kg), suppressed the parasitemia of T. cruzi-infected Swiss mice (25 mg/kg, 5 days) and presented DNA amplification below the limit of detection. These findings justify further studies with longer treatment regimens, as well as evaluations in combination with nitro drugs, aiming to identify more effective and safer therapies for Chagas disease.
A selective mono‐N‐arylation strategy of amidines under Chan‐Lam conditions is described. During the reaction optimization phase, the isolation of a mononuclear Cu(II) complex provided unique mechanistic insight into the operation of Chan‐Lam mono‐N‐arylation. The scope of the process is demonstrated, and then applied to access the first mono‐N‐arylated analogues of pentamidine. Sub‐micromolar activity against kinetoplastid parasites was observed for several analogues with no cross‐resistance in pentamidine and diminazene‐resistant trypanosome strains and against Leishmania mexicana. A fluorescent mono‐N‐arylated pentamidine analogue revealed rapid cellular uptake, accumulating in parasite nuclei and the kinetoplasts. The DNA binding capability of the mono‐N‐arylated pentamidine series was confirmed by UV‐melt measurements using AT‐rich DNA. This work highlights the potential to use Chan‐Lam mono‐N‐arylation to develop therapeutic leads against diamidine‐resistant trypanosomiasis and leishmaniasis.
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