PUBLISHED VERSION Danny W Wilson, Christopher D Goodman, Brad E Sleebs, Greta E Weiss, Nienke WM de Jong, Fiona Angrisano, Christine Langer, Jake Baum, Brendan S Crabb, Paul R Gilson, Geoffrey I McFadden, and James G Beeson Macrolides rapidly inhibit red blood cell invasion by the human malaria parasite, Plasmodium falciparum BMC Biology, 2015; 13(1):52-1-52-19 © 2015 Wilson et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Originally published at: http://doi.org/10.1186/s12915-015-0162-0 PERMISSIONS http://creativecommons.org/licenses/by/4.0/ http://hdl.handle.net/2440/93434 Wilson et al. BMC Biology (2015) 13:52 DOI 10.1186/s12915-015-0162-0 RESEARCH ARTICLE Open Access Macrolides rapidly inhibit red blood cell invasion by the human malaria parasite, Plasmodium falciparum Danny W Wilson1,2,3,4*, Christopher D Goodman5, Brad E Sleebs2,3, Greta E Weiss4, Nienke WM de Jong4, Fiona Angrisano2,3,6, Christine Langer4, Jake Baum2,3,6, Brendan S Crabb3,4,7, Paul R Gilson4,7, Geoffrey I McFadden5 and James G Beeson2,4,8* Abstract Background: Malaria invasion of red blood cells involves multiple parasite-specific targets that are easily accessible to inhibitory compounds, making it an attractive target for antimalarial development. However, no current antimalarial agents act against host cell invasion. Results: Here, we demonstrate that the clinically used macrolide antibiotic azithromycin, which is known to kill human malaria asexual blood-stage parasites by blocking protein synthesis in their apicoplast, is also a rapid inhibitor of red blood cell invasion in human (Plasmodium falciparum) and rodent (P. berghei) malarias. Multiple lines of evidence demonstrate that the action of azithromycin in inhibiting parasite invasion of red blood cells is independent of its inhibition of protein synthesis in the parasite apicoplast, opening up a new strategy to develop a single drug with multiple parasite targets. We identified derivatives of azithromycin and erythromycin that are better invasion inhibitors than parent compounds, offering promise for development of this novel antimalarial strategy. Conclusions: Safe and effective macrolide antibiotics with dual modalities could be developed to combat malaria and reduce the parasite’s options for resistance. Keywords: Plasmodium, Merozoite, Invasion, Macrolide, Dual modality Background is the emerging development of drug resistance to the Malaria is a mosquito-borne parasitic disease, which highly effective artemisinin family of compounds in causes nearly 600,000 deaths every year, mainly in chil- Southeast Asia [7–9]. The prospect of widespread resist- dren under 5 years of age in sub-Saharan Africa [1]. ance to the most effective antimalarials becoming Most deaths are attributed to the most virulent human entrenched has led to the implementation of an emer- malaria parasite, Plasmodium falciparum. Current dis- gency response plan by the World Health Organization ease control measures rely on reducing exposure to the (WHO) to prevent the spread of resistance and the inev- mosquito vector or treatment of clinical malaria cases itable increase in childhood mortality that would result with antimalarial drugs that target the disease causing [10]. This developing emergency highlights the urgent blood-stage trophozoite; most of these act against the need for new antimalarials with novel mechanisms of ac- parasite’s food vacuole or interfere with pyrimidine syn- tion for use in combination with artemisinins, or to re- thesis of maturing trophozoites [2–6]. Of major concern place these drugs in the future. Invasion of blood-stage merozoites into host cells has been proposed as a potential target for antimalarial * Correspondence: [email protected]; [email protected] – 1Research Centre for Infectious Diseases, School of Biological Sciences, The chemotherapy [11 14]. Invasion of the extracellular University of Adelaide, Adelaide, South Australia merozoite into a new host erythrocyte (red blood cell) is 2Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, a complex process requiring the coordinated interaction Australia Full list of author information is available at the end of the article of unique parasite ligands, signaling pathways, and a © 2015 Wilson et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wilson et al. BMC Biology (2015) 13:52 Page 2 of 19 form of gliding motility powered by an actomyosin of azithromycin and clindamycin for the treatment of motor. The multiple accessible targets make this essen- clinical disease as stand-alone drugs, and successful com- tial stage of the lifecycle an attractive drug target. In binations with other antimalarials remain elusive [34]. addition, inhibition of merozoite invasion into the In this study, we aimed to identify existing, and novel, erythrocyte would lead to instant disruption of the para- compounds with invasion inhibitory activity that have site lifecycle and prevent sequestration, dormancy and the potential for development as effective antimalarials. commitment to the mosquito-transmissible gametocyte We demonstrate that the antibiotic azithromycin can stage, which have been reported to occur after drug specifically inhibit merozoite invasion. Related macrolide treatment with current first-line antimalarials [15–17]. antibiotics were also found to inhibit invasion, and the Application of antimalarial drugs that target invasion IC50 of invasion inhibitory activity could be lowered has the potential to result in quicker resolution of clin- through modification. These results provide the basis for ical disease if used in combination with antimalarials a novel strategy of antimalarial drug development by that act at other developmental stages. advancing compounds that have dual mechanisms of Few compounds have been directly tested for ability to action. Given the established safety and low cost of inhibit P. falciparum merozoite invasion, partly through macrolides in widespread clinical use, development of lack of tractable invasion inhibition assays. The ability to macrolides as antimalarials with dual modalities against assess the invasion inhibitory potential of compounds merozoite invasion and protein translation in the apico- and their biological activity in vitro has been greatly en- plast is a promising strategy to counter the rapid emer- hanced with the development of a robust and efficient gence of drug resistance. merozoite purification protocol and techniques to dir- ectly measure invasion inhibition [13, 18–20], allowing Results the accurate and rapid assessment of a compounds inva- Azithromycin inhibits merozoite invasion sion inhibitory potential. While some invasion inhibitory Application of the merozoite purification method of compounds (e.g. protease inhibitors [18, 21, 22], heparin Boyle et al. [18] identified the macrolide antibiotic [23] and calcium signaling modulators [23, 24]) have azithromycin as a candidate inhibitor of P. falciparum been reported, to date no drug used clinically to treat asexual blood-stage merozoite invasion of the host malaria has been shown to have inhibitory activity erythrocyte (see Fig. 1a for representation of assay setup, against merozoite invasion [13]. In this study, we have Fig. 2 for structure of azithromycin and other drugs used identified clinically relevant macrolide antibiotics and re- in this study). Initial screens indicated that the invasion lated compounds, exemplified by azithromycin, as rapid inhibitory IC50 differed between azithromycin prepared inhibitors of P. falciparum and P. berghei merozoite in- in ethanol (10 μM) or DMSO (38 μM), suggesting that vasion in vitro. This represents the first time that a drug choice of vehicle can impact azithromycin potency or drug class that has clinical applications against mal- in vitro. Comparative screens of azithromycin potency aria has been demonstrated to inhibit parasite invasion over different treatment times demonstrated that the of the red blood cell. IC50 of merozoite invasion assays (1 hour exposure to Antibiotics such as azithromycin and clindamycin drug) were surprisingly similar to the in cycle (40 hour (lincosamide antibiotic) have been proposed as potential incubation, no invasion step, Fig. 1b) and 1 cycle (90 hour, partner drugs for artemisinin combination therapies one invasion step, Fig. 1c) assays (IC50 –(drug pre- owing to their extremely long half-life and good safety pared in ethanol) invasion 10 μm; in cycle 6 μM; 1 profile in vivo [25–29]. Both azithromycin and clindamy- cycle 7 μM, −(drug prepared in DMSO) invasion 38 μM cin inhibit apicoplast ribosomal protein synthesis of (DMSO); in cycle 12 μM; 1 cycle assay 7 μM, Fig. 3a, asexual blood-stage parasites