Alkyl Galactofuranosides Strongly Interact with Leishmania donovani Membrane and Provide Antileishmanial Activity

Abstract

fWe investigated the in vitro effects of four alkyl-galactofuranoside derivatives, i.e., octyl-␤-D-galactofuranoside (compound 1), 6-amino-␤-D-galactofuranoside (compound 2), 6-N-acetamido-␤-D-galactofuranoside (compound 3), and 6-azido-␤-D-galactofuranoside (compound 4), on Leishmania donovani. Their mechanism of action was explored using electron paramagnetic resonance spectroscopy (EPR) and nuclear magnetic resonance (NMR), and ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Compound 1 showed the most promising effects by inhibiting promastigote growth at a 50% inhibitory concentration (IC 50 ) of 8.96 ؎ 2.5 M. All compounds exhibit low toxicity toward human macrophages. Compound 1 had a higher selectivity index than the molecule used for comparison, i.e., miltefosine (159.7 versus 37.9, respectively). EPR showed that compound 1 significantly reduced membrane fluidity compared to control promastigotes and to compound 3. The furanose ring was shown to support this effect, since the isomer galactopyranose had no effect on parasite membrane fluidity or growth. NMR showed a direct interaction of all compounds (greatest with compound 1, followed by compounds 2, 3, and 4, in descending order) with the promastigote membrane and with octyl-galactopyranose and octanol, providing evidence that the n-octyl chain was primarily involved in anchoring with the parasite membrane, followed by the putative crucial role of the furanose ring in the antileishmanial activity. A morphological analysis of compound 1-treated promastigotes by TEM revealed profound alterations in the parasite membrane and organelles, but this was not the case with compound 3. Quantification of annexin V binding by flow cytometry confirmed that compound 1 induced apoptosis in >90% of promastigotes. The effect of compound 1 was also assessed on intramacrophagic amastigotes and showed a reduction in amastigote growth associated with an increase of reactive oxygen species (ROS) production, thus validating its promising effect.

Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major

“…Differentiation and culture was performed in RPMI-1640 medium (Gibco ® ) supplemented with 10% decomplemented fetal calf serum and antibiotics (100 UI/mL penicillin and 100 μg/mL streptomycin), as previously described. 44 …”

Synthesis and evaluation of 1,2-trans alkyl galactofuranoside mimetics as mycobacteriostatic agents

“…Interestingly, previous screening on eukaryote parasites of the Leishmania genus concluded on the importance of the substituent 15 on the galactofuranoside scaffold to measure some growth inhibition. 25 In this context, and in an attempt to improve the biological activity of the parent furanolipid, we decided to modulate the lipophilicity of the alkyl chain and the polarity of the sugar head. 20 Synthesis 5-Membered ring hexofuranoses generally tend to isomerise into 6-membered one, the most thermodynamically stable form.…”

“…It is thus critical to first lock the ring size and then modulate the functional groups on the scaffold. To access to the A-family, we 25 started from the known octyl β-D-galactofuranoside 1 26, 27 whose C-6 position could be tuned and whose anomeric configuration was already installed, thus avoiding complex purification process.…”

“…1 This deadly infection is difficult to cure and doing so successfully requires long treatment periods with multiple antibiotics. 2 Recent first-line drugs against tuberculosis include 25 ethambutol and isoniazid in association with rifampicin and pyrazinamid. Such Directly Observed Treatment, Short course therapies (DOTS) present multiple shortcomings, like resistance and side-effects.…”

“…The resulting compounds were eventually evaluated against their inhibition of M. smegmatis growth. 25 …”