Retargeting azithromycin analogues to have dual-modality antimalarial activity

Background: Resistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium spp. malaria parasites is urgently needed. Azithromycin is a clinically used macrolide antibiotic proposed as a partner drug for combination therapy in malaria, which has also been tested as monotherapy. However, its slow-killing 'delayed-death' activity against the parasite's apicoplast organelle and suboptimal activity as monotherapy limit its application as a potential malaria treatment. Here, we explore a panel of azithromycin analogues and demonstrate that chemical modifications can be used to greatly improve the speed and potency of antimalarial action. Results: Investigation of 84 azithromycin analogues revealed nanomolar quick-killing potency directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin with less than 48 hrs treatment in vitro. Analogues were effective against zoonotic Plasmodium knowlesi malaria parasites and against both multi-drug and artemisinin-resistant Plasmodium falciparum lines. Metabolomic profiles of azithromycin analogue-treated parasites suggested activity in the parasite food vacuole and mitochondria were disrupted. Moreover, unlike the food vacuole-targeting drug chloroquine, azithromycin and analogues were active across blood-stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on 'quick-killing' activity. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity. Conclusion: We show that azithromycin and analogues can rapidly kill malaria parasite asexual blood stages via a fast action mechanism. Development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed-death mechanism of action in a single, multifactorial chemotype.Amy L. Burns, Brad E. Sleebs, Ghizal Siddiqui, Amanda E. De Paoli, Dovile Anderson, Benjamin Liffner, Richard Harvey, James G. Beeson, Darren J. Creek, Christopher D. Goodman, Geoffrey I. McFadden, and Danny W. Wilso

Cell biological analysis reveals an essential role for Pfcerli2 in erythrocyte invasion by malaria parasites

Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum. Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to a proportion of merozoites failing to invade and was associated with elongation of the rhoptry organelle during merozoite development and inhibition of rhoptry antigen processing. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion.Benjamin Liffner, Juan Miguel Balbin, Gerald J. Shami, Ghizal Siddiqui, Jan Strauss, Sonja Frölich, Gary K. Heinemann, Ella May Edwards, Arne Alder, Jan Stephan Wichers, Darren J. Creek, Leann Tilley, Matthew W.A. Dixon, Tim-Wolf Gilberger, Danny W. Wilso

PfCERLI1 is a conserved rhoptry associated protein essential for Plasmodium falciparum merozoite invasion of erythrocytes

The disease-causing blood-stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is a rhoptry associated protein identified to have a direct role in function of this essential merozoite invasion organelle, which has broader implications for understanding apicomplexan invasion biology.Benjamin Liffner, Sonja Frölich, Gary K. Heinemann, Boyin Liu, Stuart A. Ralph, Matthew W.A. Dixon, Tim-Wolf Gilberger, Danny W. Wilso

More Prevalent Prescription of Medicine for Hypertension and Metabolic Syndrome in Males from Couples Undergoing Intracytoplasmic Sperm Injection

Register-based studies have indicated that men with impaired fertility are at higher risk for developing various adult-onset diseases than fertile men. The majority of men undergoing ICSI treatment are sub-fertile and since they are in contact with the health care system, these men are well suited as target for preventive measures. Our study included all men (N = 459 766) who had fathered children in Sweden between 2006 and 2016. Swedish registry data was used for obtaining information regarding conception method and defining three groups of fathers – ICSI -treated, IVF – treated and non IVF/ICSI. By sourcing data from the Swedish Prescribed Drug Register, we specifically searched for information regarding prescription and usage of at least one prescription for diabetes mellitus, hypertension (HT) or dyslipidemia to serve as a proxy for metabolic disease among the study groups. If all three types of medicine were prescribed, the patient was considered as having metabolic syndrome. Our results indicate male partners in couples who became parents using ICSI to be at higher risk for being treated for hypertension (HR = 1.15 95% CI: 1.06–1.24, p = 0.001) and metabolic syndrome (HR = 1.28 95% CI: 1.01–1.58, p = 0.042) when compared to non IVF/ICSI men