Hundreds of dual-stage antimalarial molecules discovered by a functional gametocyte screen

Abstract

Plasmodium falciparum stage V gametocytes are responsible for parasite transmission, and drugs targeting this stage are needed to support malaria elimination. We here screen the Tres Cantos Antimalarial Set (TCAMS) using the previously developed P. falciparum female gametocyte activation assay (Pf FGAA), which assesses stage V female gametocyte viability and functionality using Pfs25 expression. We identify over 400 compounds with activities <2 μM, chemically classified into 57 clusters and 33 singletons. Up to 68% of the hits are chemotypes described for the first time as late-stage gametocyte-targeting molecules. In addition, the biological profile of 90 compounds representing the chemical diversity is assessed. We confirm in vitro transmission-blocking activity of four of the six selected molecules belonging to three distinct scaffold clusters. Overall, this TCAMS gametocyte screen provides 276 promising antimalarial molecules with dual asexual/sexual activity, representing starting points for target identification and candidate selection.

Figure 1. Progression cascade of the TCAMS screen in the Pf FGAA.

The different steps followed during the TCAMS screen as well as the number of compounds identified in the first phases and those selected for further profiling are shown. The first four steps can be defined as ‘hit identification'. After clustering, compounds are progressed to characterize their biological profiles and finally tested in the gold standard SMFA.

Figure 2. Correlation graph of hits identified in the Pf> FGAA screen compared to their activities in the ATP-depletion assay.

276 compounds (green circles and all stars) out of the 405 hits were exclusively identified in the Pf FGAA while 129 compounds (grey circles) were active in both assays. Stars represent those compounds further profiled: slow-acting (blue stars), fast-acting (yellow starts) and gamete-targeting (magenta stars). Compounds tested in the SMFA are marked as big stars.

Figure 3. TCAMS chemical series representatives of the compound set further progressed.

Scaffolds of 15 chemotypes, belonging to 24 of the 90 compounds profiled, that are within an appropriate physicochemical space (clogP<5, number of aromatic rings <3).

Figure 4. Diversity of biological profiles determined by use of different incubation times in the Pf FGAA.

Stage V gametocytes were exposed to the 90 selected compounds for 24 or 48 h to determine the drug inhibitory speed of action. In addition, compounds were added after triggering gametocyte activation to evaluate their activity in female gametes. The figure shows the number of compounds and chemical diversity classified under each category, that is, slow-acting, fast-acting or gamete-targeting compounds.

Figure 5. Exflagellation inhibition by six TCAMS compounds.

Stage V gametocyte cultures were exposed to 2 μM of TCMDC-141070 and 1 μM of the five remaining compounds for 48 h. The percentage inhibition was determined compared to DMSO-treated controls. Compounds were then progressed into SMFA to further determine the transmission blocking in mosquitoes. Each bar represents mean value of two (TCMDC-125345, TCMDC-141698, TCMDC-123767, TCMDC-141154) or three (TCMDC-141070, TCMDC-124559) independent replicates with s.d.

Figure 6. Effect of selected TCAMS compounds in the SMFA.

The following parameters were measured: (a) Transmission-blocking activity (prevalence reduction) and (b) reduction in mean P. falciparum oocyst intensity. Each bar represents mean value of two (TCMDC-125345, TCMDC-141698, TCMDC-123767, TCMDC-141154) or three (TCMDC-141070, TCMDC-124559) independent replicates with s.d.

Figure 7. Effect of two TCAMS hits in the P. berghei in vivo mouse model.

Each experiment included a control group treated with vehicle (green circle) used as a reference to calculate the percentage of inhibition of parasitaemia in peripheral blood (dotted red line). The response of standard antimalarials in the same assay is also presented (blue, magenta and grey circles). Represented data are the mean±s.e.m. log₁₀ [% asexual blood stages at day 4] of two mice per point.