A class of tricyclic compounds blocking malaria parasite oocyst development and transmission

Abstract

Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.

Fig 1

Comparison of growth and drug response phenotypes between Plasmodium falciparum 3D7 and the two pfabcg2 null lines (B2 and C11 were obtained from a single transformation experiment and may be derived from a common progenitor). (A) Parasitemia of asexual stages. (B) Gametocytemia of all gametocyte stages from day 1 (setup day) to day 17. (C) Exflagellation center counts from days 15 to 17. (D) Inhibition curves in response to CQ. (E) Inhibition curves in response to DHA. (F) Inhibition curves in response to ketotifen (KET). The results are consistent with those from the initial high-throughput screening; the ketotifen IC₅₀ for 3D7 was 6.3 μM (log IC₅₀, 5.2), and it was >30 μM for both B2 and C11 (or log IC₅₀ ≥4.5). For the experiments shown in panels A to C, two independent repeats, designated 1 and 2, in duplicates were plotted separately due to a large variation in 3D7 gametocytemia; for panels D to F, the results of four independent experiments were averaged and plotted. Standard errors of means were estimated from the repeats/duplicates accordingly.

Fig 2

Complementation of the pfabcg2 KO C11 parasite with a pfabcg2 transgene partially restored parasite sensitivity to ketotifen. (A) PCR products with expected sizes amplified from DNA samples extracted from parasites with or without the plasmids to introduce the genes encoding PfABCG2 or luciferase. Resc-1 and Resc-2 were derived from C11 lines transfected with the pfabcg2 expression plasmid shown in Fig. S1E (top) in the supplemental material. Luc-1 and Luc-2 were derived from C11 lines transfected with the control luciferase expression plasmid (see Fig. S1E, bottom). (B) Immunoblot showing expression of the PfABCG2-GFP fusion protein in the transfected parasites, as detected by antibodies against GFP. (C) Changes in parasite response to ketotifen in the presence or absence of the pfabcg2 transgene. The parasites were the same as those shown in panel A. Standard errors of means were estimated from 3 to 4 repeat experiments. The IC₅₀s of Resc-1 and Resc-2 were not significantly different from that of 3D7 (unpaired t tests of IC₅₀s; P = 0.62 for 3D7 versus Resc-1 and P = 0.64 for 3D7 versus Resc-2), the IC₅₀s of controls (Luc-1 and Luc-2) remained significantly different from that of 3D7 or close to significance (unpaired t tests of IC₅₀s; P = 0.03 for 3D7 versus Luc-1 and P = 0.08 for 3D7 versus Luc-2), and the differences between the IC₅₀s of the complemented lines and the parental knockout clone C11 were also significant (unpaired t test, P = 0.04 for C11 versus Resc-1and P = 0.03 for C11 versus Resc-2).

Fig 3

Inhibition of oocyst development by ketotifen (KET), PQ, ART, and CQ. Mice (three per group) infected with Plasmodium yoelii nigeriensis N67 were treated with KET at different concentrations for various time periods and were exposed to mosquitoes. Oocysts were counted from dissected midguts 9 days after feeding. (A) Numbers of oocysts from mosquitoes fed on mice treated with different concentrations of KET for 1 h before feeding were compared with those treated with PQ (5 and 10 mg/kg), ART (5 mg/kg), or CQ (10 mg/kg). (B) Numbers of oocysts from mosquitoes fed on mice treated with a single dose of 0.1 mg/kg (filled dots) or 0.5 mg/kg (open circles) of KET for 1, 6, or 24 h before feeding to mosquitoes. (C) Number of oocysts from mosquitoes fed on mice treated with two doses (4-hour interval) of 0.1 mg/kg (filled dots) or 0.5 mg/kg KET for 6 or 24 h. Con, no-drug control. The short horizontal bars are mean oocyst counts.

Fig 4

Relapse patterns in Plasmodium cynomolgi-infected rhesus monkeys after 5 days of quinine treatment to clear the initial parasitemia. (A) Control monkeys, treated with quinine only (32 mg/kg twice daily for 5 consecutive days); (B) monkeys treated with 8 mg/kg artesunate for 4 days after quinine treatment; (C) monkeys treated with 15 mg/kg ketotifen for 4 days after quinine treatment; (D) no-drug controls, treated as for panel A (repeat); (E) monkeys treated as for panel C, except ketotifen treatment was for 8 days. EZD, DB36, DB9H, C67, DA4AB, CE49, DB1X, DB4W, DB31, DBXW, BLX, DC77, and G31 are designations for individual monkeys.