(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

130

e High-throughput phenotypic screening of chemical libraries has resulted in the identification of thousands of compounds with potent antimalarial activity, although in most cases, the mechanism(s) of action of these compounds remains unknown. Here we have investigated the mode of action of 90 antimalarial compounds derived from the Malaria Box collection using high-coverage, untargeted metabolomics analysis. Approximately half of the tested compounds induced significant metabolic perturbations in in vitro cultures of Plasmodium falciparum. In most cases, the metabolic profiles were highly correlated with known antimalarials, in particular artemisinin, the 4-aminoquinolines, or atovaquone. Select Malaria Box compounds also induced changes in intermediates in essential metabolic pathways, such as isoprenoid biosynthesis (i.e., 2-C-methyl-D-erythritol 2,4-cyclodiphosphate) and linolenic acid metabolism (i.e., traumatic acid). This study provides a comprehensive database of the metabolic perturbations induced by chemically diverse inhibitors and highlights the utility of metabolomics for triaging new lead compounds and defining specific modes of action, which will assist with the development and optimization of new antimalarial drugs. Malaria remains a major global health problem, and there is a pressing need to discover and develop new antimalarials. Traditional antimalarial drugs have been severely undermined by the emergence of drug-resistant strains and current treatments rely heavily on artemisinin-based combination therapies. Alarmingly, artemisinin resistance has arisen in Southeast Asia during the last decade, highlighting the urgent need for new antimalarials (1). Extensive efforts to produce a malaria vaccine have met limited success and a pipeline of new antimalarial drugs will be required to mitigate extensive morbidity and mortality from malaria for the foreseeable future (2).High-throughput phenotypic screening of chemical libraries against the malaria parasite, Plasmodium falciparum, has provided a major step forward in the discovery of novel antimalarial compounds. Almost 30,000 compounds that selectively inhibit growth of cultured P. falciparum asexual red blood cell (RBC) stages have been identified in these screens, providing excellent starting points for the discovery of new antimalarial drugs (3-5). A prioritized collection of these "hit" compounds, known as the Malaria Box, has been assembled by the Medicines for Malaria Venture (MMV) and provided free to the research community to facilitate this drug development pipeline (6).A key limitation to the further optimization of many of these compounds is the lack of information on their mode of action. While not essential for registration, information on the mode of action of inhibitors discovered in phenotypic screens will significantly accelerate drug development by allowing structure-based drug design, monitoring of activity, toxicity and resistance, and facilitation of rational clinical usage in combination with other medicines. Furthermore, in...

Section: Resultsmentioning

confidence: 94%

Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action

Creek

Chua

Cobbold

et al. 2016

130

“…These tools then are used to provide quantitative efficacy data required to fuel medicinal chemists' efforts to optimize structure-activity properties. Similar strategies and drug discovery pathways are used for novel antimalarial and antileishmanial drugs (40,41).…”

Section: Discussionmentioning

confidence: 99%

“…Advances in drug discovery for emerging infectious and tropical diseases have begun to deliver a pipeline of new chemotypes that are optimized for clinical development by academic and industrial scientists (40,41). In comparison, pathogenic FLA have been ignored and early discovery studies have focused on a small number of drugs already approved for other indications or screens with nonpathogenic species of FLA.…”

Section: Discussionmentioning

confidence: 99%

Bis-Benzimidazole Hits against Naegleria fowleri Discovered with New High-Throughput Screens

Rice

Colon

Alp

et al. 2015

Self Cite

Naegleria fowleri is a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two new in vitro assays suitable for medium-or high-throughput drug discovery and used these for N. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono-and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC 50 ) of 177 nM. Similarly, we identified 10 additional analogues with IC 50 s of <1 M, with many of these having reasonable selectivity indices. The most potent hits were >500 times more potent than pentamidine. In summary, the mono-and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections with N. fowleri. The first report of animal pathogenicity by free-living amoebae (FLA) was by Culbertson et al., who identified Acanthamoeba in necrotic lesions of monkeys and mice (1). He later suggested that FLA causes similar disease in humans. Fowler and Carter reported the first cases of an acute fatal disease caused by Naegleria fowleri in four victims in Australia in 1965 (2). Since the identification of primary amoebic meningoencephalitis (PAM), hundreds of cases have been reported worldwide, including 142 cases in the United States (3-5). Most infections with N. fowleri occur during the summer months, victims usually are symptomatic within 5 days, and the disease is almost always fatal. Infection usually occurs after the victim swam in warm, fresh, or brackish water or from exposure to contaminated tap water associated with the use of Neti pots for nasal irrigation (4-7). Amoebic invasion occurs via disruption of the olfactory mucosa, penetration of organisms into the submucosal nervous plexus, and ultimately passage through the cribriform plate to the frontal lobes of the brain (8, 9). Although most cases of PAM occur in warm climates, such as the southern tier states of the United States, pathogenic amoeba found in thermally polluted water sources, hot springs, and poorly chlorinated pools have been linked to infections (10, 11). Interestingly, most of the PAM cases have been diagnosed in developed countries, including the United States, Australia, and Europe. Although sporadic cases have been d...

“…The sensitivity of P. falciparum parasites to increases in intracellular Na ϩ levels has recently been demonstrated to be a common property of several classes of antimalarial drugs believed to act through inhibition of a P-type Na ϩ ATPase, PfATP4 (16)(17)(18)23). The potential for PfATP4 inhibitors to block the parasite's adaptive response to the ion flux induced by maduramicin and augment the effects of increases in intracellular Na ϩ concentration prompted us to test for synergy between these compounds.…”

Section: Resultsmentioning

confidence: 99%

“…An increase in intracellular Na ϩ has also recently been associated with three new classes of antimalarials, spiroindolones (15,16), pyrazoleamides (17), and dihydroisoquinolones (18), as well as 28 of the novel antimalarial compounds included in the Malaria Box (19), a small-molecule library compiled and distributed by Medicines for Malaria Venture. Mutations in PfATP4 (PF3D7_1211900), which has homology to a P-type cation pump that could play a role in actively transporting Na ϩ out of the cell, have been found to confer resistance to the three new antimalarial candidates.…”

mentioning

confidence: 99%

Maduramicin Rapidly Eliminates Malaria Parasites and Potentiates the Gametocytocidal Activity of the Pyrazoleamide PA21A050

Maron

Magle

Czesny

et al. 2016

c New strategies targeting Plasmodium falciparum gametocytes, the sexual-stage parasites that are responsible for malaria transmission, are needed to eradicate this disease. Most commonly used antimalarials are ineffective against P. falciparum gametocytes, allowing patients to continue to be infectious for over a week after asexual parasite clearance. A recent screen for gametocytocidal compounds demonstrated that the carboxylic polyether ionophore maduramicin is active at low nanomolar concentrations against P. falciparum sexual stages. In this study, we showed that maduramicin has an EC 50 (effective concentration that inhibits the signal by 50%) of 14.8 nM against late-stage gametocytes and significantly blocks in vivo transmission in a mouse model of malaria transmission. In contrast to other reported gametocytocidal agents, maduramicin acts rapidly in vitro, eliminating gametocytes and asexual schizonts in less than 12 h without affecting uninfected red blood cells (RBCs). Ring stage parasites are cleared by 24 h. Within an hour of drug treatment, 40% of the normally crescent-shaped gametocytes round up and become spherical. The number of round gametocytes increases to >60% by 2 h, even before a change in membrane potential as monitored by MitoProbe DiIC1 (5) is detectable. Maduramicin is not preferentially taken up by gametocyte-infected RBCs compared to uninfected RBCs, suggesting that gametocytes are more sensitive to alterations in cation concentration than RBCs. Moreover, the addition of 15.6 nM maduramicin enhanced the gametocytocidal activity of the pyrazoleamide PA21A050, which is a promising new antimalarial candidate associated with an increase in intracellular Na ؉ concentration that is proposed to be due to inhibition of PfATP4, a putative Na ؉ pump. These results underscore the importance of cation homeostasis in sexual as well as asexual intraerythrocytic-stage P. falciparum parasites and the potential of targeting this pathway for drug development. R eports of decreasing sensitivity to artemisinin in Southeast Asia provide a sense of urgency to the development of novel antimalarial compounds (1). Although both gametocytes and asexual parasites reside within the red blood cell (RBC), their physiologies differ, resulting in various sensitivities to common antimalarial drugs (2). After RBC invasion, asexual parasites undergo four or five rounds of DNA replication, resulting in 16 to 32 new parasites every 48 h. In contrast, gametocytes do not replicate in the human host. Instead, after RBC invasion, sexual differentiation of Plasmodium falciparum progresses through five stages of development over the course of 10 to 12 days, resulting in a single female or male gametocyte (3). Once taken up in a blood meal by a mosquito, gametocytes are stimulated to emerge from the RBC and develop into female and male gametes (4). After fertilization, the zygote differentiates into an ookinete that migrates out of the mosquito midgut, where it forms an oocyst that produces tens of thousands of sporozoites that...