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Schematic of drug treatment regimens outlining the times of treatment and stage/time of parasitaemia measurement for assays used in this study. a Merozoite invasion of RBCs: Merozoites were drug treated prior to addition of RBCs. RBC invasion was measured at early ring stages (< 1 hr rings). b In-cycle: highly synchronous, early ring-stage parasites (0–4 hrs post-invasion) were treated with drug, with the resulting growth inhibition analysed at schizont stage (44 hrs post-invasion for P. falciparum and 26 hrs for P. knowlesi). c One cycle (0–72 hrs): highly synchronous, early ring-stage parasites (0–4 hrs post-invasion) were drug-treated and the resulting growth inhibition was measured after ~ 72 hrs of growth, post one cycle of re-invasion, at schizont stages. d 2 cycle (delayed death); highly synchronous, early ring-stage parasites (0–4 hrs post-invasion) were drug-treated and allowed to grow for 92 hrs before washing drug with fresh media (post second invasion cycle). Growth inhibition was assessed approximately 30 hrs later, at schizont stages (0–120 hrs post-invasion for P. falciparum and 0–92 hrs for P. knowlesi)
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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 als...
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Malaria is caused by infection with protozoan parasites of the Plasmodium genus, which is part of the phylum Apicomplexa. Most organisms in this phylum contain a relic plastid called the apicoplast. The apicoplast genome is replicated by a single DNA polymerase (apPOL), which is an attractive target for anti-malarial drugs. We screened small-molecu...
The emergence and spread of Plasmodium falciparum parasites resistant to front-line antimalarial artemisinin-combination therapies (ACT) threatens to erase the considerable gains against the disease of the last decade. Here, we develop a large-scale phenotypic screening pipeline and use it to carry out a large-scale forward-genetic phenotype screen...
Please cite this article as: Martins-Duarte, E.S., Sheiner, L., Reiff, S.B., de Souza, W., Striepen, B., Replication and partitioning of the apicoplast genome of Toxoplasma gondii is linked to the cell cycle and requires DNA polymerase and gyrase, International Journal for Parasitology (2021), doi: https://doi. Abstract Apicomplexans are the causat...
Plasmodium, the causative agent of malaria, belongs to the phylum Apicomplexa. Most apicomplexans, including Plasmodium, contain an essential nonphotosynthetic plastid called the apicoplast that harbors its own genome that is replicated by a dedicated organellar replisome. This replisome employs a single DNA polymerase (apPol), which is expected to...
Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting Plasmodium-secreted protein functions are limited. We report knockER, a unique DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show...
Citations
... The eradication of Plasmodium falciparum parasites is accomplished through two distinct mechanisms: delayed death by means of inhibiting the apicoplast ribosomes and rapid elimination throughout the blood stage development (112). A trial carried out in Burkina Faso discovered that a solitary oral dose of azithromycin did not result in a reduction in malaria positivity; however, it did alleviate caregiverreported fever as an adverse event (113). Furthermore, an additional study determined that the supplementation of azithromycin to seasonal malaria chemoprevention did not yield improved nutritional outcomes among children (114). ...
Drug repurposing is a strategic endeavor that entails the identification of novel therapeutic applications for pharmaceuticals that are already available in the market. Despite the advantageous nature of implement- ing this particular strategy owing to its cost-effectiveness and efficiency in reducing the time required for the drug discovery process, it is essential to bear in mind that there are various factors that must be meticulously considered and taken into account. Up to this point, there has been a noticeable absence of comprehensive analyses that shed light on the limitations of repurposing drugs. The primary aim of this review is to con- duct a thorough illustration of the various challenges that arise when contemplating drug repurposing from a clinical perspective in three major fields cardiovascular, cancer and diabetes and to further underscore the potential risks associated with the emergence of antimicrobial resistance when employing repurposed antibiotics for the treatment of noninfectious and infectious diseases. The process of developing repurposed medications necessitates the application of creativity and innovation in designing the development program, as the body of evidence may differ for each specific case. In order to effectively repurpose drugs, it is crucial to consider the clinical implications and potential drawbacks that may arise during this process. By com- prehensively analyzing these challenges, we can attain a deeper comprehension of the intricacies involved in drug repurposing, which will ultimately lead to the development of more efficacious and safe therapeutic approaches.
... The eradication of Plasmodium falciparum parasites is accomplished through two distinct mechanisms: delayed death by means of inhibiting the apicoplast ribosomes and rapid elimination throughout the blood stage development (112). A trial carried out in Burkina Faso discovered that a solitary oral dose of azithromycin did not result in a reduction in malaria positivity; however, it did alleviate caregiverreported fever as an adverse event (113). Furthermore, an additional study determined that the supplementation of azithromycin to seasonal malaria chemoprevention did not yield improved nutritional outcomes among children (114). ...
Drug repurposing is a strategic endeavor that entails the identification of novel therapeutic applications for pharmaceuticals that are already available in the market. Despite the advantageous nature of implementing this particular strategy owing to its cost-effectiveness and efficiency in reducing the time required for the drug discovery process, it is essential to bear in mind that there are various factors that must be meticulously considered and taken into account. Up to this point, there has been a noticeable absence of comprehensive analyses that shed light on the limitations of repurposing drugs. The primary aim of this review is to conduct a thorough illustration of the various challenges that arise when contemplating drug repurposing from a clinical perspective in three major fields-cardiovascular, cancer, and diabetes-and to further underscore the potential risks associated with the emergence of antimicrobial resistance (AMR) when employing repurposed antibiotics for the treatment of noninfectious and infectious diseases. The process of developing repurposed medications necessitates the application of creativity and innovation in designing the development program, as the body of evidence may differ for each specific case. In order to effectively repurpose drugs, it is crucial to consider the clinical implications and potential drawbacks that may arise during this process. By comprehensively analyzing these challenges, we can attain a deeper comprehension of the intricacies involved in drug repurposing, which will ultimately lead to the development of more efficacious and safe therapeutic approaches.
... Therefore, combination treatment with hydroxynaphthoquinone atovaquone and the antibiotic azithromycin is usually used (143). Azithromycin and other antibiotics with antiprotozoal properties mainly target the apicoplast, a residual plastid found in protozoa, and eventually cause parasite death (144). Moreover, tafenoquine may be a highly useful drug to treat B. microti infection (145). ...
Protozoan diseases cause great harm in animal husbandry and require human-provided medical treatment. Protozoan infection can induce changes in cyclooxygenase-2 (COX-2) expression. The role played by COX-2 in the response to protozoan infection is complex. COX-2 induces and regulates inflammation by promoting the synthesis of different prostaglandins (PGs), which exhibit a variety of biological activities and participate in pathophysiological processes in the body in a variety of ways. This review explains the roles played by COX-2 in protozoan infection and analyzes the effects of COX-2-related drugs in protozoan diseases.
... We previously investigated a secondary, quick-killing, mechanism of action for azithromycin and select analogues Wilson et al., 2015;Burns et al., 2020). Azithromycin and analogues were demonstrated to rapidly inhibit P. falciparum merozoite invasion of RBCs and effectively kill asexual stages throughout one full blood stage lifecycle (rings to schizonts, in cycle,~48 hrs). ...
... Azithromycin and analogues were demonstrated to rapidly inhibit P. falciparum merozoite invasion of RBCs and effectively kill asexual stages throughout one full blood stage lifecycle (rings to schizonts, in cycle,~48 hrs). Azithromycin was equipotent throughout the entire blood stage lifecycle, and the most potent analogues were active against ring-stage parasites (<6 hrs treatments) at nanomolar potencies (Wilson et al., 2015;Burns et al., 2020), a desirable property for an antimalarial targeting asexual stages which most clinically used drugs have failed to achieve. This 'quick-killing' activity was active against parasites selected for resistance to the delayed death activity of azithromycin and against parasites that had their apicoplast chemically removed, confirming the quick killing mechanism to be independent of apicoplast targeted delayed death (Wilson et al., 2015;Burns et al., 2020). ...
... Azithromycin was equipotent throughout the entire blood stage lifecycle, and the most potent analogues were active against ring-stage parasites (<6 hrs treatments) at nanomolar potencies (Wilson et al., 2015;Burns et al., 2020), a desirable property for an antimalarial targeting asexual stages which most clinically used drugs have failed to achieve. This 'quick-killing' activity was active against parasites selected for resistance to the delayed death activity of azithromycin and against parasites that had their apicoplast chemically removed, confirming the quick killing mechanism to be independent of apicoplast targeted delayed death (Wilson et al., 2015;Burns et al., 2020). Given concerns that repurposing an antibiotic into an antimalarial could potentially select for azithromycin resistance in pathogenic bacteria (Lee et al., 2010) and cause dysbiosis of the human microbiome (Wei et al., 2018), medicinal chemistry synthesis efforts have been directed in making non-antibiotic azithromycin analogues (Pesic et al., 2012;Starcevic et al., 2012). ...
Introduction
The spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: ‘delayed death’ by inhibiting the bacterium-like ribosomes of the apicoplast, and ‘quick-killing’ that kills rapidly across the entire blood stage development.
Methods
Here, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans).
Results
Seventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi, respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (<12 hrs treatment) and were >5-fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment.
Discussion
The azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.
... Interventions such as seasonal malaria chemoprevention (SMC) have reduced deaths from severe malaria, but growing resistance to first line antimalarial drugs threatens to impede progress [3,4]. Azithromycin is a macrolide with modest anti-malarial properties that has been shown to have a negative impact on the asexual stages of the Plasmodium falciparum parasite [5]. While azithromycin is not widely used for malaria control, mass biannual azithromycin distribution has been shown to reduce all-cause child mortality and malaria parasitemia among preschool aged children in some settings [6][7][8]. ...
Background
Azithromycin is a broad-spectrum antibiotic that has moderate antimalarial activity and has been shown to reduce all-cause mortality when biannually administered to children under five in high mortality settings in sub-Saharan Africa. One potential mechanism for this observed reduction in mortality is via a reduction in malaria transmission.
Methods
We evaluated whether a single oral dose of azithromycin reduces malaria positivity by rapid diagnostic test (RDT). We conducted an individually randomized placebo-controlled trial in Burkina Faso during the high malaria transmission season in August 2020. Children aged 8 days to 59 months old were randomized to a single oral dose of azithromycin (20 mg/kg) or matching placebo. At baseline and 14 days following treatment, we administered a rapid diagnostic test (RDT) to detect Plasmodium falciparum and measured tympanic temperature for all children. Caregiver-reported adverse events and clinic visits were recorded at the day 14 visit.
Results
We enrolled 449 children with 221 randomized to azithromycin and 228 to placebo. The median age was 32 months and 48% were female. A total of 8% of children had a positive RDT for malaria at baseline and 11% had a fever (tympanic temperature ≥ 37.5 °C). In the azithromycin arm, 8% of children had a positive RDT for malaria at 14 days compared to 7% in the placebo arm ( P = 0.65). Fifteen percent of children in the azithromycin arm had a fever ≥ 37.5 °C compared to 21% in the placebo arm ( P = 0.12). Caregivers of children in the azithromycin group had lower odds of reporting fever as an adverse event compared to children in the placebo group (OR 0.41, 95% CI 0.18–0.96, P = 0.04). Caregiver-reported clinic visits were uncommon, and there were no observed differences between arms ( P = 0.32).
Conclusions
We did not find evidence that a single oral dose of azithromycin reduced malaria positivity during the high transmission season. Caregiver-reported fever occurred less often in children receiving azithromycin compared to placebo, indicating that azithromycin may have some effect on non-malarial infections.
Trial registration Clinicaltrials.gov NCT04315272, registered 19/03/2020
... Using nuclear magnetic resonance or mass spectrometry to analyze metabolites present in P. falciparum parasites after drug treatments presents an indirect way to examine their effects on processes within the parasite, including Hb digestion. A build-up or loss of peptides found in Hb can represent downstream or upstream blocking of this digestion pathway, respectively and to date has provided useful information into the effect of novel or potential repurposed compounds on Hb digestion (Birrell et al., 2020;Burns et al., 2020;Giannangelo et al., 2020;Edgar et al., 2021). Metabolomic analysis after DHA treatment has been shown to result in a decrease in Hb-derived peptides, in accordance with resistance to this drug being attributed to a decrease in Hb uptake (Mok et al., 2021). ...
Plasmodium falciparum malaria remains a global health problem as parasites continue to develop resistance to all antimalarials in use. Infection causes clinical symptoms during the intra-erythrocytic stage of the lifecycle where the parasite infects and replicates within red blood cells (RBC). During this stage, P. falciparum digests the main constituent of the RBC, hemoglobin, in a specialized acidic compartment termed the digestive vacuole (DV), a process essential for survival. Many therapeutics in use target one or multiple aspects of the DV, with chloroquine and its derivatives, as well as artemisinin, having mechanisms of action within this organelle. In order to better understand how current therapeutics and those under development target DV processes, techniques used to investigate the DV are paramount. This review outlines the involvement of the DV in therapeutics currently in use and focuses on the range of techniques that are currently utilized to study this organelle including microscopy, biochemical analysis, genetic approaches and metabolomic studies. Importantly, continued development and application of these techniques will aid in our understanding of the DV and in the development of new therapeutics or therapeutic partners for the future.
... Unless it overcomes some of the highlighted challenges and finds a specific niche that is complementary to the current scala of more efficacious antimalarial combinations, its future as an antimalarial does not look promising" [221]. Recently, members of a series of 84 azithromycin analogs were identified with nanomolar quick-killing potency against invading merozoites and the ring stages of parasite development within red blood cells [222]. The feature of targeting early stages of the intraerythrocytic life cycle is rare among existing antimalarials, so this discovery has brought new life for azithromycin (50). ...
Natural products have made a crucial and unique contribution to human health, and this is especially true in the case of malaria, where the natural products quinine and artemisinin and their derivatives and analogues, have saved millions of lives. The need for new drugs to treat malaria is still urgent, since the most dangerous malaria parasite, Plasmodium falciparum, has become resistant to quinine and most of its derivatives and is becoming resistant to artemisinin and its derivatives. This volume begins with a short history of malaria and follows this with a summary of its biology. It then traces the fascinating history of the discovery of quinine for malaria treatment and then describes quinine’s biosynthesis, its mechanism of action, and its clinical use, concluding with a discussion of synthetic antimalarial agents based on quinine’s structure. The volume then covers the discovery of artemisinin and its development as the source of the most effective current antimalarial drug, including summaries of its synthesis and biosynthesis, its mechanism of action, and its clinical use and resistance. A short discussion of other clinically used antimalarial natural products leads to a detailed treatment of other natural products with significant antiplasmodial activity, classified by compound type. Although the search for new antimalarial natural products from Nature’s combinatorial library is challenging, it is very likely to yield new antimalarial drugs. The chapter thus ends by identifying over ten natural products with development potential as clinical antimalarial agents.
... The delayed effect of azithromycin was explained by the effect of specific toxicity of the drug on apicoplast and death of the parasite in the second cycle due to lack of the apicoplast genome replication in the first cycle (Dahl and Rosenthal, 2007b). In addition to this delayed action, azithromycin demonstrated fast-killing activity which was independent of apicoplast-mediating delayed killing and augmented in azithromycin analogs with side-chain modifications, although the mechanism of this fast-killing mode of the drug was not understood (Burns et al., 2020). Similar to other drugs like tetracycline and fosmidomycin, azithromycin targets an enzymatic pathway in apicoplast, which makes the drug selective to Plasmodium parasites (Sidhu et al., 2007b) but makes the action of the drug slow for its disadvantage also (Dahl and Rosenthal, 2007b). ...
Malaria, a disease caused by the protozoan parasites Plasmodium spp., is still causing serious problems in endemic regions in the world. Although the WHO recommends artemisinin combination therapies for the treatment of malaria patients, the emergence of artemisinin-resistant parasites has become a serious issue and underscores the need for the development of new antimalarial drugs. On the other hand, new and re-emergences of infectious diseases, such as the influenza pandemic, Ebola virus disease, and COVID-19, are urging the world to develop effective chemotherapeutic agents against the causative viruses, which are not achieved to the desired level yet. In this review article, we describe existing drugs which are active against both Plasmodium spp. and microorganisms including viruses, bacteria, and fungi. We also focus on the current knowledge about the mechanism of actions of these drugs. Our major aims of this article are to describe examples of drugs that kill both Plasmodium parasites and other microbes and to provide valuable information to help find new ideas for developing novel drugs, rather than merely augmenting already existing drug repurposing efforts.
... Drugs targeting the apicoplast typically kill the parasites in the second cycle following treatment, a phenomenon referred to as 'delayed death' (reviewed in Ref. [16]). While the slow killing mode is an undesirable feature and does not meet the criteria for new antimalarials, recent studies have highlighted that known apicoplast drugs can exhibit fast-killing modes, presumably due to off-target effects at higher concentrations or following chemical modification [22,23]. Furthermore, inhibition of the apicoplastresident non-mevalonate methylerythritol phosphate (MEP) pathway through fosmidomycin [17], and inhibition of protein import cause rapid death of the parasites [24], rekindling interest in the apicoplast as a drug target. ...
The apicoplast is the relict of a plastid organelle found in several disease-causing apicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii. In these organisms, the organelle has lost its photosynthetic capability but harbours several fitness-conferring or essential metabolic pathways. Although maintaining the apicoplast and fuelling the metabolic pathways within requires the challenging constant import and export of numerous metabolites across its four membranes, only few apicoplast transporters have been identified to date, most of which are orphan transporters. Here we review the roles of metabolic pathways within the apicoplast and what is currently known about the few identified apicoplast metabolite transporters. We discuss what metabolites must get in and out of the apicoplast, the many transporters that are yet to be discovered, and what role these might play in parasite metabolism and as putative drug targets.
... nitazoxanide are examples of antimicrobials that have done both [91][92][93][94] ; azithromycin is clinically used against malarial parasites and nitazoxanide treats bacterial infections such as H. pylori 95,96 . ...
Drug repositioning studies in recent decades have revealed a growing number of antimicrobials effective at treating infection types tangential to their original antimicrobial classification. Such ‘pan-pathogen antimicrobials’ (or ‘broad-spectrum anti-infectives’) have not yet been formally characterised. This review examines historical limitations of the canonical antimicrobial lexicon in light of the contemporary model for infectious disease and propounds a taxonomy that defines antimicrobials according to the host-pathogen interactome, not the pathogen. By doing so, antimicrobials that are effective at treating multiple infection types are highlighted, namely azithromycin, ivermectin, niclosamide, and nitazoxanide. Recognition of the pan-pathogen nature of these antimicrobials can stimulate a more unified approach to antimicrobial development cognisant of generalised anti-infective mechanisms within the host-pathogen interactome and anticipatory of future pandemics and bioterrorist attacks.
