Plasmodium falciparumprotein Pfs16 is a target for transmission-blocking antimalarial drug development

Abstract

Phenotypic cell-based screens are critical to the discovery of new antimalarial lead compounds. However, identification and validation of cellular targets of lead compounds is required following discovery in a phenotypic screen. We recently discovered a Plasmodium transmission-blocking N-((4-hydroxychroman-4-yl)methyl)-sulfonamide (N-4HCS) compound, DDD01035881, in a phenotypic screen. DDD01035881 and its potent derivatives have been shown to block Plasmodium male gamete formation (microgametogenesis) with nanomolar activity. Here, we synthesised a photoactivatable N-4HCS derivative, probe 2, to identify the N-4HCS cellular target. Using probe 2 in photo-affinity labelling coupled with mass spectrometry, we identified the 16 kDa Plasmodium falciparum parasitophorous vacuole membrane protein Pfs16 as the likely cellular target of the N-4HCS series. Further validating Pfs16 as the cellular target of the N-4HCS series, the Cellular Thermal Shift Assay (CETSA) confirmed DDD01035881 stabilised Pfs16 in lysate from activated mature gametocytes. Additionally, photo-affinity labelling combined with in-gel fluorescence and immunoblot analysis confirmed the N-4HCS series interacted with Pfs16. High-resolution, widefield fluorescence and electron microscopy of N-4HCS-inhibited parasites was found to result in a cell morphology entirely consistent with targeted gene disruption of Pfs16. Taken together, these data strongly implicate Pfs16 as the target of DDD01035881 and establish the N-4HCS scaffold family as a powerful starting point from which future transmission-blocking antimalarials can be developed.

Adapt or Die: Targeting Unique Transmission-Stage Biology for Malaria Elimination

“…New developments in inducible gene manipulation systems will allow a deeper investigation of the importance of gametocyte-specific regulators ( Tiburcio et al., 2019 ), and this information could be translated to identify druggable candidates for guided, target-based discovery of novel transmission-blocking chemotypes. Additionally, the use of proteomics to identify and validate drug targets in gametocytes have been developed ( Dziekan et al., 2019 ; Yahiya et al., 2021 ), yet, we are far from a streamlined drug target identification and mode-of-action platform for gametocytes similar to the extensive pipeline used to characterize ABS actives ( Yang et al., 2021 ). Indeed, if any of the new transmission-selective hits are to be further developed, this area needs investment and development.…”

“…This extends to CDPK3 and PK7 during fertilization, with other activities such as PDI and PDE also potentially important ( Angrisano et al., 2019 ). Targeting energy metabolism is important in the metabolically active ookinetes and oocysts, as evident by sporonticides atovaquone and ELQ-300 as potent inhibitors of cytochrome bc1 ( Table 3 ) and recent evidence also points to the importance of targeting cytoskeletal proteins such as Pf s16 ( Yahiya et al., 2021 ) that plays a role during the immediate phase of microgametogenesis with DDD01035881 ( Delves et al., 2018b ).…”

Transmission-blocking drugs for malaria elimination

“…More rarely, for example, for ganaplacide (KAF156targeting protein secretion) and the resulting mutations in pfcarl, the selected trait is considered a marker/mechanism of resistance rather than a target for drug activity. The identification of target candidates in gametocytes that are not transferred from ABS is more complicated and relies on alternative, protein-based strategies [28,75] due to the nonproliferative nature of these parasites.…”