Abstract
BackgroundGiven the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated.MethodsSix diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (HssLDH) or plasmodial lactate dehydrogenase (PfLDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro.ResultsAll tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of PfLDH; specificity between the residues involved in H-bonds of the PfLDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of PfLDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive.ConclusionsThe presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than PfLDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.
Highlights
Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed
The results showed differences in the binding pockets: inside the PfLDH pocket, the electrostatic potential was totally positive, whereas inside HssLDH, it was mostly negative, as illustrated by CQAn33 docked inside both enzymes (Figures 2 and 3)
The results showed that CQAn33 inhibited β-haematin formation with an IC50 similar to that of CQ, whereas CQAn37 exhibited an IC50 more than 2-fold higher than CQ, which is in accordance with this molecule being less active than CQAn33 against P. falciparum
Summary
Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chemotherapy has been hampered by the low sensitivity of the parasite to most available anti-malarial drugs [3], including artemisinin derivatives [4,5,6]. Resistance to chloroquine, in the case of P. falciparum, is linked to mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene, which alters the transport and accumulation of the drug in the digestive vacuole (DV) of the parasite [7,8]. Neither CQ-R nor mutation markers were detected among P. vivax in recent studies of 47 isolates from the West Amazon, all from patients with non-severe malaria [12]
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