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Results indicated a decreased susceptibility to QN, MQ and LMF (i.e. in reduced hemozoin levels and might involve inhibition of host hemoglobin uptake into intra-erythrocytic parasites. Furthermore, parasites resistant to HHQs displayed increased susceptibility to several first-line antimalarial drugs including lumefantrine, confirming that HHQs have a different mode of action than other antimalarials drugs for which PfMDR1 is known to confer resistance. This work evokes therapeutic strategies that combine opposing selective pressures on this parasite transporter as an approach to countering the emergence and transmission of multidrug-resistant malaria. (Pf). Improved malaria treatment and control has reduced morbidity and mortality worldwide by 40C50% since 20001, due largely to highly effective artemisinin-based combination therapies and mosquito vector control measures1,2. An important strategy to further advance the malaria elimination agenda is to include transmission-blocking agents in combination therapies3. Such agents inhibit the development of parasite gametocytes (GAMs) that represent an alternative form of intra-erythrocytic development BMS 299897 to the pathogenic asexual blood stage (ABS) parasites, and that mature over five stages4. Only stage V GAMS, which may survive days to weeks in circulation5, can continue the Pf lifecycle in mosquitoes following ingestion of a parasitized blood meal. In the mosquito midgut, male GAMs exflagellate and produce microgametes that can fuse with female macrogametes to produce zygotes. Following subsequent development as ookinetes and oocysts, sporozoites are formed. Once inoculated in a new host, they will develop in the liver and initiate a new ABS cycle6. Primaquine is currently the only available drug with potent activity against stage V GAMs7,8, but can be toxic for glucose-6-phosphate dehydrogenase-deficient patients8. Hence, there is a compelling need to develop alternative transmission-blocking drugs. Recent advances in GAM-specific high-throughput screen technology9C19, combined with functional validation assays and drug action/resistance studies19C21, provide BMS 299897 a powerful foundation to identify leads with Pf transmission-blocking activity. In this study, we report a screen of the Novartis-GNF Malaria Box of ABS-active compounds22 on Pf GAM stages and describe the identification and antimalarial properties of hexahydroquinolines (HHQs), a chemical class with considerable transmission-blocking potential. Results Identifying HHQs as potent gametocytocidal inhibitors Using a luciferase (LUC)-based assay, we screened 3,825 compounds from the Novartis-GNF Malaria Box for activity against NF54Pfs16 early-stage GAMs. This screen yielded 797 compounds exhibiting 50% inhibition at 2.6 M. Re-screening confirmed activity for 683 compounds, of which 176 showed 50% inhibition at 0.65 M (Figure 1a). Screening the Novartis-GNF Malaria Box against late-stage GAMs using high-content imaging identified 572 hits with 50% inhibition at 2 M in at least two of the three replicates (Figure 1a; Supplementary Figure 1a). Open in a separate window Figure 1 Identifying HHQs as potent Pf ABS and GAM inhibitors(a) Outcomes BMS 299897 of the primary screening of the GNF-Novartis Malaria Box library against early- and late-stage Pf NF54Pfs16 GAMs, using luciferase-based and high-content imaging assays, respectively. (b) Scatterplot of early- and late-stage gametocytocidal potency of 63 compounds with dual activity (expressed as pIC50s which equal the ?log(IC50); a value of 9 corresponds to 10?9 M, i.e. 1 nM). The red stars correspond to the four compounds selected for the study. Data are based on two independent experiments, each probing both early- and late-stage GAMs. (c) Intra-erythrocytic stage-specific activity of the four selected compounds was obtained from two independent experiments for each parasite stage (Supplementary Table 3). (d) Structure of the 3 HHQs confirmed to have transmission-blocking activity. (e) Impact of PfMDR1 mutations and copy number on HHQ potency. The X-axis depicts the Rab21 individual mutant lines and their wild-type parental lines, and indicates the GNF-Pf compound number, the clone number and in parentheses the PfMDR1 mutation and its allele balance. Edited implies that the Dd2-B2 line was edited using a CRISPR/Cas9 system to introduce the designated PfMDR1 mutation. Mean SEM IC50 values were calculated from three or more independent experiments (detailed in Supplementary Table 5) and significance was assessed by a Welchs corrected transmission-blocking activity Using an dual gamete formation assay (DGFA), we investigated whether these four potent GAM-active compounds would BMS 299897 also inhibit the development BMS 299897 of cultured female and male gametes. Results indicated a potent inhibitory effect of the 3 HHQs on exflagellation when GAMs were exposed for 24 h and the compound carried over during the.