Doxycycline has Distinct Apicoplast-Specific Mechanisms of Antimalarial Activity

Abstract

Doxycycline (DOX) is a key antimalarial drug that is thought to kill Plasmodium falciparum parasites by blocking protein translation in the essential apicoplast organelle. Although parasite resistance to DOX has not emerged, clinical use is primarily limited to prophylaxis due to delayed second-cycle parasite death at 1-3 μM, the DOX concentration readily achieved in human plasma at current dosing. Slow antiparasitic activity is thought to be a fundamental limitation of DOX and other antibiotics that target apicoplast maintenance. We report that slightly higher 8-10 μM DOX concentrations kill parasites with first-cycle activity that blocks apicoplast biogenesis. This faster activity is rescued by exogenous isopentenyl pyrophosphate, an essential apicoplast product, confirming an apicoplast-specific target. Exogenous iron rescues first- but not second-cycle activity by 1 or 10 μM DOX, revealing that first-cycle activity involves a metal-dependent mechanism that is distinct from the canonical delayed-death mechanism. These results provide a new paradigm for understanding fundamental antiparasitic mechanisms of DOX and suggest the possibility of repurposing DOX as a faster-acting antimalarial at higher dosing, which remains well tolerated. Multiple mechanisms of DOX activity would be expected to limit parasite resistance.

Divergent Acyl Carrier Protein Decouples Mitochondrial Fe-S Cluster Biogenesis from Fatty Acid Synthesis in Malaria Parasites

“…Parasite growth assays: All parasite studies were performed with P. falciparum Dd2 parasites cultured in RPMI-1640 medium as previously described (58) For bacteria expression studies, BL21/DE3 E. coli were transformed with pET plasmids encoding cloned P. falciparum mACP-HA2 (full-length or the ∆2-50 truncation, WT or F113A mutant), Isd11-His6 (WT, YR/AA, or LYR/AAA mutants), or full-length Nfs1-HA2. Protein expression was induced by 1 mM IPTG, and bacterial pellets were lysed in PBS by sonication and clarified by centrifugation.…”

“…All parasite studies were performed with P. falciparum Dd2 parasites cultured in RPMI-1640 medium as previously described (64). CRISPR/Cas9 was used to tag the mACP gene in Dd2 parasites to encode a C-terminal HA-FLAG epitope tag and the aptamer/TetR-DOZI system (30), whose integration was validated by PCR.…”

Targeting the Plasmodium falciparum proteome and organelles for potential antimalarial drug candidates

“…The apicoplast is involved in the synthesis of isoprenoids ( Tewari et al, 2021 ) from isopentenyl pyrophosphate (IPP) in the blood stage of the parasite ( Miller et al, 2014 ; Rai et al, 2017 ). Inhibitors of prokaryotic DNA replication (ciprofloxacin), prokaryotic translation (chloramphenicol, doxycycline ( Okada et al, 2020 ), tetracycline, clindamycin, azithromycin, erythromycin and clarithromycin), a tRNA synthase (mupirocin), and IPP synthesis pathway (fosmidomycin and FR900098) are regarded as targets of apicoplasts ( Uddin et al, 2018 ). Kumarihamy et al (2020) showed that the in vitro antimalarial activity of Botryosphaeria dothidea was due to the common metabolic pathway shared by the apicoplast in Plasmodium parasites and plastids in plants.…”