Targeted Structure-Activity Analysis of Endochin-like Quinolones Reveals Potent Qi and Qo Site Inhibitors of Toxoplasma gondii and Plasmodium falciparum Cytochrome bc(1) and Identifies ELQ-400 as a Remarkably Effective Compound against Acute Experimental Toxoplasmosis.

ACS Infect Dis. 2018 Nov 9;4(11):1574-1584. 

McConnell EV, Bruzual I, Pou S, Winter R, Dodean RA, Smilkstein MJ, Krollenbrock A, Nilsen A, Zakharov LN, Riscoe MK, Doggett JS.

​

Cytochrome bc1 inhibitors have been broadly studied as human and veterinary medicines and agricultural fungicides. For the most part, cytochrome bc1 inhibitors compete with ubiquinol at the ubiquinol oxidation (Qo) site or with ubiquinone at the quinone reduction (Qi) site. 4(1 H)-Quinolones with 3-position substituents may inhibit either site based on quinolone ring substituents. 4(1 H)-Quinolones that inhibit the Qi site are highly effective against toxoplasmosis, malaria, and babesiosis and do not inhibit human cytochrome bc1. We tested a series of 4(1 H)-Quinolones against wild-type and drug resistant strains of Toxoplasma gondii and Plasmodium falciparum. These experiments identified very potent compounds that inhibit T. gondii proliferation at picomolar concentrations. The most potent compounds target the Qo site, and for 

these compounds, an alkyl side chain confers potency against T. gondii greater than that of bulkier side chains. Our experiments also show that substituents on the quinolone ring influenced selectivity between T. gondii and P. falciparum 

and between Qo and Qi site-mediated activity. Comparison of the parasite cytochrome b sequences identified amino acids that are associated with drug resistance in P. falciparum that exist naturally in wild-type T. gondii. These underlying differences may influence drug susceptibility. Finally, a Qo site active 4(1 H)-quinolone-3-diarylether tested in a murine model of toxoplasmosis was superior to atovaquone, resulting in survival from Type I strain T. gondii infection. These experiments identify highly effective compounds for toxoplasmosis and provide valuable insight into the structure-activity 

relationship of cytochrome bc1 inhibitors.

DOI: 10.1021/acsinfecdis.8b00133 PMCID: PMC6884402 PMID: 30117728

​

Orally Bioavailable Endochin-Like Quinolone Carbonate Ester Prodrug Reduces Toxoplasma gondii Brain Cysts.

Antimicrob Agents Chemother. 2020 Aug 20;64(9):e00535-20.

Doggett JS, Schultz T, Miller AJ, Bruzual I, Pou S, Winter R, Dodean R, Zakharov LN, Nilsen A, Riscoe MK, Carruthers VB.

​

Toxoplasmosis is a potentially fatal infection for immunocompromised people and the developing fetus. Current medicines for toxoplasmosis have high rates of adverse effects that interfere with therapeutic and prophylactic regimens. Endochin-like quinolones (ELQs) are potent inhibitors of Toxoplasma gondii proliferation in vitro and in animal models of acute and latent infection. ELQ-316, in particular, was found to be effective orally against acute toxoplasmosis in mice and highly selective for T. gondii cytochrome b over human cytochrome b Despite its oral efficacy, the high crystallinity of ELQ-316 limits oral absorption, plasma concentrations, and therapeutic potential. A carbonate ester prodrug of ELQ-316, ELQ-334, was created to decrease crystallinity and increase oral bioavailability, which resulted in a 6-fold increase in both the 

maximum plasma concentration (C max) and the area under the curve (AUC) of ELQ-316. The increased bioavailability of ELQ-316, when administered as ELQ-334, resulted in efficacy against acute toxoplasmosis greater than that of an 

equivalent dose of ELQ-316 and had efficacy against latent toxoplasmosis similar to that of ELQ-316 administered intraperitoneally. Treatment with carbonate ester prodrugs is a successful strategy to overcome the limited oral 

bioavailability of ELQs for the treatment of toxoplasmosis.

DOI: 10.1128/AAC.00535-20 PMCID: PMC7449172 PMID: 32540978

​

Efficacy of Guanabenz Combination Therapy against Chronic Toxoplasmosis across Multiple Mouse Strains.

Antimicrob Agents Chemother. 2020 Aug 20;64(9):e00539-20.

Martynowicz J, Doggett JS, Sullivan WJ Jr.

​

Toxoplasma gondii, an obligate intracellular parasite that can cause life-threatening acute disease, differentiates into a quiescent cyst stage to establish lifelong chronic infections in animal hosts, including humans. This tissue cyst reservoir, which can reactivate into an acute infection, is currently refractory to clinically available therapeutics. Recently, we and 

others have discovered drugs capable of significantly reducing the brain cyst burden in latently infected mice, but not to undetectable levels. In this study, we examined the use of novel combination therapies possessing multiple mechanisms of action in mouse models of latent toxoplasmosis. Our drug regimens included combinations of pyrimethamine, clindamycin, guanabenz, and endochin-like quinolones (ELQs) and were administered to two different mouse strains in an attempt to eradicate brain tissue cysts. We observed mouse strain-dependent effects with these drug treatments: pyrimethamine-guanabenz showed synergistic efficacy in C57BL/6 mice yet did not improve upon guanabenz monotherapy in BALB/c mice. Contrary to promising in vitro results demonstrating toxicity to bradyzoites, we observed an antagonistic effect between guanabenz and ELQ-334 in vivo While we were unable to completely eliminate the brain cyst burden, we found that a combination treatment with ELQ-334 and pyrimethamine impressively reduced the brain cyst burden by 95% in C57BL/6 mice, which approached the limit of detection. These analyses highlight the importance of evaluating anti-infective drugs in multiple mouse strains and will help inform further preclinical studies of cocktail therapies designed to treat chronic toxoplasmosis.

DOI: 10.1128/AAC.00539-20 PMCID: PMC7449173 PMID: 32540979

​

Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii.

Antimicrob Agents Chemother. 2017 Jan 24;61(2):e01866-16.

Alday PH, Bruzual I, Nilsen A, Pou S, Winter R, Ben Mamoun C, Riscoe MK, Doggett JS.

 

Toxoplasma gondii is an apicomplexan parasite that causes fatal and debilitating brain and eye disease. Endochin-like quinolones (ELQs) are preclinical compounds that are efficacious against apicomplexan-caused diseases, including 

toxoplasmosis, malaria, and babesiosis. Of the ELQs, ELQ-316 has demonstrated the greatest efficacy against acute and chronic experimental toxoplasmosis. Although genetic analyses in other organisms have highlighted the importance of 

the cytochrome bc1 complex Qi site for ELQ sensitivity, the mechanism of action of ELQs against T. gondii and the specific mechanism of ELQ-316 remain unknown. Here, we describe the selection and genetic characterization of T. gondii clones 

resistant to ELQ-316. A T. gondii strain selected under ELQ-316 drug pressure was found to possess a Thr222-Pro amino acid substitution that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor. These findings provide further evidence for ELQ Qi site inhibition in T. gondii and greater insight into the interactions of Qi site inhibitors with the apicomplexan cytochrome bc1 complex.

DOI: 10.1128/AAC.01866-16 PMCID: PMC5278733 PMID: 27919897 

​

Endochin-like quinolones are highly efficacious against acute and latent experimental toxoplasmosis.

Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):15936-41.

Doggett JS(1), Nilsen A, Forquer I, Wegmann KW, Jones-Brando L, Yolken RH, Bordón C, Charman SA, Katneni K, Schultz T, Burrows JN, Hinrichs DJ, Meunier B, Carruthers VB, Riscoe MK.

 

Toxoplasma gondii is a widely distributed protozoan pathogen that causes devastating ocular and central nervous system disease. We show that the endochin-like quinolone (ELQ) class of compounds contains extremely potent inhibitors of T. gondii growth in vitro and is effective against acute and latent toxoplasmosis in mice. We screened 50 ELQs against T. gondii and selected two lead compounds, ELQ-271 and ELQ-316, for evaluation. ELQ-271 and ELQ-316, have in vitro IC(50) values of 0.1 nM and 0.007 nM, respectively. ELQ-271 and ELQ-316 have ED(50) values of 0.14 mg/kg and 0.08 mg/kg when administered orally to mice with acute toxoplasmosis. Moreover, ELQ-271 and ELQ-316 are highly active against the cyst form of T. gondii in mice at low doses, reducing cyst burden by 76-88% after 16 d of treatment. To investigate the ELQ mechanism of action against T. gondii, we demonstrate that endochin and ELQ-271 inhibit 

cytochrome c reduction by the T. gondii cytochrome bc(1) complex at 8 nM and 31 nM, respectively. We also show that ELQ-271 inhibits the Saccharomyces cerevisiae cytochrome bc(1) complex, and an M221Q amino acid substitution in the 

Q(i) site of the protein leads to >100-fold resistance. We conclude that ELQ-271 and ELQ-316 are orally bioavailable drugs that are effective against acute and latent toxoplasmosis, likely acting as inhibitors of the Q(i) site of the T. gondii cytochrome bc(1) complex.

DOI: 10.1073/pnas.1208069109 PMCID: PMC3465437 PMID: 23019377

​

A New Scalable Synthesis of ELQ-300, ELQ-316, and other Antiparasitic Quinolones.

Org Process Res Dev. 2021 Aug 20;25(8):1841-1852.

Pou S, Dodean RA, Frueh L, Liebman KM, Gallagher RT, Jin H, Jacobs RT, Nilsen A, Stuart DR, Doggett JS, Riscoe MK, 

Winter RW

 

The Endochin-Like Quinolone (ELQ) compound class may yield effective, safe treatments for a range of important human and animal afflictions. However, to access the public health potential of this compound series, a synthetic route needed to be devised that lowers costs and is amenable to large scale production. In the new synthetic route described here, a substituted β-keto ester, formed by an Ullmann reaction and subsequent acylation, is reacted with an aniline via a Conrad-Limpach reaction to produce 3-substituted 4(1H)-quinolones such as ELQ-300 and ELQ-316. This synthetic route, the first 

described to be truly amenable to industrial scale production, is relatively short (5 reaction steps), does not require palladium, chromatographic separation or protecting group chemistry, and may be performed without high vacuum 

distillation.

DOI: 10.1021/acs.oprd.1c00099 PMCID: PMC8802981 PMID: 35110959

​

​

​

​

Acridones Are Highly Potent Inhibitors of Toxoplasma gondii Tachyzoites.

ACS Infect Dis. 2021 Jul 9;7(7):1877-1884.

Alday PH, McConnell EV, Boitz Zarella JM, Dodean RA, Kancharla P, Kelly JX, Doggett JS

​

Acridone derivatives, which have been shown to have in vitro and in vivo activity against Plasmodium spp, inhibit Toxoplasma gondii proliferation at picomolar concentrations. Using enzymatic assays, we show that acridones inhibit 

both T. gondii cytochrome bc1 and dihydroorotate dehydrogenase and identify acridones that bind preferentially to the Qi site of cytochrome bc1. We identify acridones that have efficacy in a murine model of systemic toxoplasmosis. Acridones have potent activity against T. gondii and represent a promising new class of preclinical compounds.

DOI: 10.1021/acsinfecdis.1c00016 PMCID: PMC8273083 PMID: 33723998 

​

​

​

​

​

Pharmacokinetics and In Vivo Efficacy of Pyrazolopyrimidine, Pyrrolopyrimidine, and 5-Aminopyrazole-4-Carboxamide Bumped Kinase Inhibitors against Toxoplasmosis.

J Infect Dis. 2019 Apr 16;219(9):1464-1473.

Hulverson MA, Bruzual I, McConnell EV, Huang W, Vidadala RSR, Choi R, Arnold SLM, Whitman GR, McCloskey MC, Barrett LK, Rivas KL, Scheele S, DeRocher AE, Parsons M, Ojo KK, Maly DJ, Fan E, Van Voorhis WC, Doggett JS.

 

Bumped kinase inhibitors (BKIs) have been shown to be potent inhibitors of Toxoplasma gondii calcium-dependent protein kinase 1. Pyrazolopyrimidine and 5-aminopyrazole-4-carboxamide scaffold-based BKIs are effective in acute and chronic experimental models of toxoplasmosis. Through further exploration of these 2 scaffolds and a new pyrrolopyrimidine scaffold, additional compounds have been identified that are extremely effective against acute experimental toxoplasmosis. The in vivo efficacy of these BKIs demonstrates that the cyclopropyloxynaphthyl, cyclopropyloxyquinoline, and 2-ethoxyquinolin-6-yl substituents are associated with efficacy across scaffolds. In addition, a broad range of plasma concentrations after oral dosing resulted from small structural changes to the BKIs. These select BKIs include anti-Toxoplasma compounds that are effective against acute experimental toxoplasmosis and are not toxic in human cell assays, nor to mice when administered for therapy. The BKIs described here are promising late leads for improving anti-Toxoplasma therapy.

DOI: 10.1093/infdis/jiy664 PMCID: PMC6467197 PMID: 30423128

​

In vitro activity, safety and in vivo efficacy of the novel bumped kinase inhibitor BKI-1748 in non-pregnant and pregnant mice experimentally infected with Neospora caninum tachyzoites and Toxoplasma gondii oocysts.

Int J Parasitol Drugs Drug Resist. 2021 Aug;16:90-101.

Imhof D, Anghel N, Winzer P, Balmer V, Ramseier J, Hänggeli K, Choi R, Hulverson MA, Whitman GR, Arnold SLM, Ojo KK, Van Voorhis WC, Doggett JS, Ortega-Mora LM, Hemphill A

​

Bumped kinase inhibitors (BKIs) target the apicomplexan calcium-dependent protein kinase 1 (CDPK1). BKI-1748, a 5-aminopyrazole-4-carboxamide compound when added to fibroblast cells concomitantly to the time of infection, inhibited 

proliferation of apicomplexan parasites at EC50s of 165 nM (Neospora caninum) and 43 nM (Toxoplasma gondii). Immunofluorescence and electron microscopy showed that addition of 2.5 μM BKI-1748 to infected HFF monolayers transformed parasites into multinucleated schizont-like complexes (MNCs) containing newly formed zoites, which were unable to separate and form infective tachyzoites or undergo egress. In zebrafish (Danio rerio) embryo development assays, no embryonic impairment was detected within 96 h at BKI-1748 concentrations up to 10 μM. In pregnant mice, BKI-1748 applied at days 9-13 of pregnancy at a dose of 20 mg/kg/day was safe and no pregnancy interference was observed. The efficacy of BKI-1748 was assessed in standardized pregnant mouse models infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. In both models, treatments resulted in increased pup survival and profound inhibition of vertical transmission. However, in dams and non-pregnant mice, BKI-1748 treatments resulted in significantly decreased cerebral parasite loads only in T. gondii infected mice. In the T. gondii-model, ocular infection was detected 

in 10 out of 12 adult mice of the control group, but only in 3 out of 12 mice in the BKI-1748-treated group. Thus, TgShSp1 oocyst infection is a suitable model to study both cerebral and ocular infection by T. gondii. BKI-1748 represents an 

interesting candidate for follow-up studies on neosporosis and toxoplasmosis in larger animal models.

DOI: 10.1016/j.ijpddr.2021.05.001 PMCID: PMC8144743 PMID: 34030110

​

Development of an Orally Available and Central Nervous System (CNS) Penetrant Toxoplasma gondii Calcium-Dependent Protein Kinase 1 (TgCDPK1) Inhibitor with Minimal Human Ether-a-go-go-Related Gene (hERG) Activity for the Treatment of Toxoplasmosis.

J Med Chem. 2016 Jul 14;59(13):6531-46.

Vidadala RS, Rivas KL, Ojo KK, Hulverson MA, Zambriski JA, Bruzual I, Schultz TL, Huang W, Zhang Z, Scheele S, DeRocher AE, Choi R, Barrett LK, Siddaramaiah LK, Hol WG, Fan E, Merritt EA, Parsons M, Freiberg G, Marsh K, Kempf DJ, Carruthers VB, Isoherranen N, Doggett JS, Van Voorhis WC, Maly DJ.

 

New therapies are needed for the treatment of toxoplasmosis, which is a disease caused by the protozoan parasite Toxoplasma gondii. To this end, we previously developed a potent and selective inhibitor (compound 1) of Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) that possesses antitoxoplasmosis activity in vitro and in vivo. Unfortunately, 1 has potent human ether-a-go-go-related gene (hERG) inhibitory activity, associated with long Q-T 

syndrome, and consequently presents a cardiotoxicity risk. Here, we describe the identification of an optimized TgCDPK1 inhibitor 32, which does not have a hERG liability and possesses a favorable pharmacokinetic profile in small and large 

animals. 32 is CNS-penetrant and highly effective in acute and latent mouse models of T. gondii infection, significantly reducing the amount of parasite in the brain, spleen, and peritoneal fluid and reducing brain cysts by >85%. These 

properties make 32 a promising lead for the development of a new antitoxoplasmosis therapy.

DOI: 10.1021/acs.jmedchem.6b00760 PMCID: PMC5100899 PMID: 27309760

​

SAR Studies of 5-Aminopyrazole-4-carboxamide Analogues as Potent and Selective Inhibitors of Toxoplasma gondii CDPK1.

ACS Med Chem Lett. 2015 Oct 22;6(12):1184-1189.

Huang W, Ojo KK, Zhang Z, Rivas K, Vidadala RS, Scheele S, DeRocher AE, Choi R, Hulverson MA, Barrett LK, Bruzual I, Siddaramaiah LK, Kerchner KM, Kurnick MD, Freiberg GM, Kempf D, Hol WG, Merritt EA, Neckermann G, de Hostos EL, Isoherranen N, Maly DJ, Parsons M, Doggett JS, Van Voorhis WC, Fan E.

​

We previously discovered compounds based on a 5-aminopyrazole-4-carboxamide scaffold to be potent and selective inhibitors of CDPK1 from T. gondii. The current work, through structure-activity relationship studies, led to the discovery of compounds (34 and 35) with improved characteristics over the starting inhibitor 1 in terms of solubility, plasma exposure after oral administration in mice, or efficacy on parasite growth inhibition. Compounds 34 and 35 were further demonstrated to be more effective than 1 in a mouse infection model and markedly reduced the amount of T. gondii in the brain, spleen, and peritoneal fluid, and 35 given at 20 mg/kg eliminated T. gondii from the peritoneal fluid.

DOI: 10.1021/acsmedchemlett.5b00319 PMCID: PMC4677665 PMID: 26693272

J Med Chem. 2014 May 8;57(9):3818-34. doi: 10.1021/jm500147k. Epub 2014 Apr 18.

 

Bumped kinase inhibitor 1294 treats established Toxoplasma gondii infection.

Antimicrob Agents Chemother. 2014 Jun;58(6):3547-9.

Doggett JS, Ojo KK, Fan E, Maly DJ, Van Voorhis WC.

​

Toxoplasma gondii is a unicellular parasite that causes severe brain and eye disease. Current drugs for T. gondii are limited by toxicity. Bumped kinase inhibitors (BKIs) selectively inhibit calcium-dependent protein kinases of the apicomplexan pathogens T. gondii, cryptosporidia, and plasmodia. A lead anti-Toxoplasma BKI, 1294, has been developed to be metabolically stable and orally bioavailable. Herein, we demonstrate the oral efficacy of 1294 against toxoplasmosis in vivo.

DOI: 10.1128/AAC.01823-13 PMCID: PMC4068437 PMID: 24687502

​

​

​

​

​

Drugs in development for toxoplasmosis: advances, challenges, and current status.

Drug Des Devel Ther. 2017 Jan 25;11:273-293.

Alday PH, Doggett JS.

 

Toxoplasma gondii causes fatal and debilitating brain and eye diseases. Medicines that are currently used to treat toxoplasmosis commonly have toxic side effects and require prolonged courses that range from weeks to more than a 

year. The need for long treatment durations and the risk of relapsing disease are in part due to the lack of efficacy against T. gondii tissue cysts. The challenges for developing a more effective treatment for toxoplasmosis include decreasing toxicity, achieving therapeutic concentrations in the brain and eye, shortening duration, eliminating tissue cysts from the host, safety in pregnancy, and creating a formulation that is inexpensive and practical for use in resource-poor areas of the world. Over the last decade, significant progress has been made in identifying and developing new compounds for the treatment of toxoplasmosis. Unlike clinically used medicines that were repurposed for toxoplasmosis, these compounds have been optimized for efficacy against toxoplasmosis during preclinical development. Medicines with enhanced efficacy as well as features that address the unique aspects of toxoplasmosis have the potential to greatly improve toxoplasmosis therapy. This review discusses the facets of toxoplasmosis that are pertinent to drug design and the advances, challenges, and current status of preclinical drug research for toxoplasmosis.

DOI: 10.2147/DDDT.S60973 PMCID: PMC5279849 PMID: 28182168

​

​

​

​

​

Discovery, synthesis, and optimization of antimalarial 4(1H)-quinolone-3-diarylethers.

Nilsen A(1), Miley GP, Forquer IP, Mather MW, Katneni K, Li Y, Pou S, Pershing AM, Stickles AM, Ryan E, Kelly JX, Doggett JS, White KL, Hinrichs DJ, Winter RW, Charman SA, Zakharov LN, Bathurst I, Burrows JN, Vaidya AB, Riscoe MK.

​

The historical antimalarial compound endochin served as a structural lead for optimization. Endochin-like quinolones (ELQ) were prepared by a novel chemical route and assessed for in vitro activity against multidrug resistant strains of Plasmodium falciparum and against malaria infections in mice. Here we describe the pathway to discovery of a potent class of orally active antimalarial 4(1H)-quinolone-3-diarylethers. The initial prototype, ELQ-233, exhibited low nanomolar IC50 values against all tested strains including clinical isolates harboring resistance to atovaquone. ELQ-271 represented the next critical step in the iterative optimization process, as it was stable to metabolism and highly effective in vivo. Continued analoging revealed that the substitution pattern on the benzenoid ring of the quinolone core significantly influenced reactivity with the host enzyme. This finding led to the rational design of highly selective ELQs with outstanding oral efficacy against murine malaria that is superior to established antimalarials chloroquine and atovaquone.

DOI: 10.1021/jm500147k PMCID: PMC4018401 PMID: 24720377 

​

​

​

​

​

Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone.

J Exp Med. 2016 Jun 27;213(7):1307-18.

Lawres LA, Garg A, Kumar V, Bruzual I, Forquer IP, Renard I, Virji AZ, Boulard P, Rodriguez EX, Allen AJ, Pou S, Wegmann KW, Winter RW, Nilsen A, Mao J, Preston DA, Belperron AA, Bockenstedt LK, Hinrichs DJ, Riscoe MK, Doggett JS, Ben Mamoun C.

 

Human babesiosis is a tick-borne multisystem disease caused by Babesia species of the apicomplexan phylum. Most clinical cases and fatalities of babesiosis are caused by Babesia microti Current treatment for human babesiosis consists of two drug combinations, atovaquone + azithromycin or quinine + clindamycin. These treatments are associated with adverse side effects and a significant rate of drug failure. Here, we provide evidence for radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone (ELQ) prodrug and atovaquone. In vivo efficacy studies in mice using ELQ-271, ELQ-316, and the ELQ-316 prodrug, ELQ-334, demonstrated excellent growth inhibitory activity against the parasite, with potency equal to that of orally administered atovaquone at 10 mg/kg. Analysis of recrudescent parasites after ELQ or atovaquone monotherapy identified genetic substitutions in the Qi or Qo sites, respectively, of the cytochrome bc1 complex. Impressively, a combination of ELQ-334 and atovaquone, at doses as low as 5.0 mg/kg each, resulted in complete clearance of the parasite with no recrudescence up to 122 d after discontinuation of therapy. These results will set the stage for future clinical evaluation of ELQ and atovaquone combination therapy for treatment of human babesiosis.

DOI: 10.1084/jem.20151519 PMCID: PMC4925016 PMID: 27270894

​

Cytochrome b Drug Resistance Mutation Decreases Babesia Fitness in the Tick Stages But Not the Mammalian Erythrocytic Cycle.

J Infect Dis. 2022 Jan 5;225(1):135-145. 

Chiu JE, Renard I, George S, Pal AC, Alday PH, Narasimhan S, Riscoe MK, Doggett JS, Ben Mamoun C

​

Human babesiosis is an emerging tick-borne malaria-like illness caused by Babesia parasites following their development in erythrocytes. Here, we show  that a mutation in the Babesia microti mitochondrial cytochrome b (Cytb) that  confers resistance to the antibabesial drug ELQ-502 decreases parasite fitness in the arthropod vector. Interestingly, whereas the mutant allele does not affect B. microti fitness during the mammalian blood phase of the parasite life cycle and is genetically stable as parasite burden increases, ELQ-502-resistant mutant parasites developing in the tick vector are genetically unstable with a high rate of the wild-type allele emerging during the nymphal stage. 

Furthermore, we show that B. microti parasites with this mutation are transmitted from the tick to the host, raising the possibility that the frequency of Cytb resistance mutations may be decreased by passage through the tick vector, but could persist in the environment if present when ticks feed.

DOI: 10.1093/infdis/jiab321 PMCID: PMC8730496 PMID: 34139755 

​

Effective Therapy Targeting Cytochrome bc(1) Prevents Babesia Erythrocytic Development and Protects from Lethal Infection.

Antimicrob Agents Chemother. 2021 Aug 17;65(9):e0066221.

Chiu JE, Renard I, Pal AC, Singh P, Vydyam P, Thekkiniath J, Kumar M, Gihaz S, Pou S, Winter RW, Dodean R, Frueh L, Nilsen AC, Riscoe MK, Doggett JS, Mamoun CB.

 

An effective strategy to control blood-borne diseases and prevent outbreak recrudescence involves targeting conserved metabolic processes that are essential for pathogen viability. One such target for Plasmodium and Babesia,  the infectious agents of malaria and babesiosis, respectively, is the mitochondrial cytochrome bc1 protein complex, which can be inhibited by endochin-like quinolones (ELQ) and atovaquone. We used the tick-transmitted and 

culturable blood-borne pathogen Babesia duncani to evaluate the structure-activity relationship, safety, efficacy, and mode of action of ELQs. We identified a potent and highly selective ELQ prodrug (ELQ-502), which, alone or in combination with atovaquone, eliminates B. microti and B. duncani infections in vitro and in mouse models of parasitemia and lethal infection. The strong efficacy at low dose, excellent safety, bioavailability, and long half-life of this experimental therapy make it an ideal clinical candidate for the treatment of human infections caused by Babesia and its closely related apicomplexan parasites.

DOI: 10.1128/AAC.00662-21 PMCID: PMC8370247 PMID: 34152821

​

​

​

​

​

​

Activities of Endochin-Like Quinolones Against in vitro Cultured Besnoitia besnoiti Tachyzoites.

Front Vet Sci. 2020 Feb 26;7:96. 

Eberhard N, Balmer V, Müller J, Müller N, Winter R, Pou S, Nilsen A, Riscoe M, Francisco S, Leitao A, Doggett JS, 

Hemphill A.

​

Endochin-like quinolones (ELQs) potently inhibit the proliferation of Plasmodium, Toxoplasma, Neospora, and Babesia by targeting the cytochrome b Qo and Qi sites and interfering with oxidative phosphorylation and pyrimidine biosynthesis. The activities of 14 different ELQs were assessed against B. besnoiti tachyzoites grown in human foreskin fibroblasts (HFF) by quantitative real time PCR. The values for 50% proliferation inhibition (IC50) of five ELQs were determined in a 3-days growth assay after an initial screen of 12 ELQs at 0.01, 0.1, and 1 μM. The IC50s of ELQ-121, -136, and -316 were 0.49, 2.36, and 7.97 nM, respectively. The IC50s of ELQs tested against B. besnoiti were higher than IC50s previously observed for P. falciparum and T. gondii. However, the B. besnoiti cytochrome b sequence and the predicted Qo and Qi ELQ binding sites in the Toxoplasma, Neospora, and Besnoitia cytochrome b are virtually identical, suggesting that the differences in ELQ susceptibility are not due to variations in the substrate binding sites. TEM of ELQ-treated parasites primarily 

demonstrated alterations within the parasite mitochondrion, profound thickening of the nuclear membrane, as well as increased vacuolization within the tachyzoite cytoplasm. Long-term treatment assays of intracellular B. besnoiti with ELQs for up to 20 days followed by the release of drug pressure caused a substantial delay in parasite growth and proliferation while ELQs were present, but parasite proliferation resumed days after ELQs were removed. Interestingly, structural alterations persisted after ELQ removal and parasite proliferation was slowed. These findings provide a basis for further in vivo studies of ELQs as therapeutic options against B. besnoiti infection.

DOI: 10.3389/fvets.2020.00096 PMCID: PMC7054222 PMID: 32161765

​

​

Endochin-Like Quinolones Exhibit Promising Efficacy Against Neospora Caninum in vitro and in Experimentally Infected Pregnant Mice.

Front Vet Sci. 2018 Nov 19;5:285.

Anghel N, Balmer V, Müller J, Winzer P, Aguado-Martinez A, Roozbehani M, Pou S, Nilsen A, Riscoe M, Doggett JS, Hemphill A

 

We report on the efficacy of selected endochin-like quinolones (ELQs) against N. caninum tachyzoites grown in human foreskin fibroblasts (HFF), and in a pregnant BALB/c mouse model. Fourteen ELQs were screened against transgenic N. caninum tachyzoites expressing β-galactosidase (Nc-βgal). Drugs were added concomitantly to infection and the values for 50% proliferation inhibition (IC50) were determined after 3 days. Three compounds exhibited IC50 values below 0.1 nM, 3 

ELQs had IC50s between 0.1 and 1 nM, for 7 compounds values between 1 and 10 nM were noted, and one compound had an IC50 of 22.4 nM. Two compounds, namely ELQ-316 and its prodrug ELQ-334 with IC50s of 0.66 and 3.33 nM, respectively, were previously shown to display promising activities against experimental toxoplasmosis and babesiosis caused by Babesia microti in mice, and were thus further studied. They were assessed in long-term treatment assays by exposure of infected HFF to ELQs at 0.5 μM concentration, starting 3 h after infection and lasting for up to 17 days followed by release of drug pressure. Results showed that the compounds substantially delayed parasite proliferation, but did not 

exert parasiticidal activities. TEM of drug treated parasites detected distinct alterations within the parasite mitochondria, but not in other parasite organelles. Assessment of safety of ELQ-334 in the pregnant mouse model showed that the compound did not interfere in fertility or pregnancy outcome. In N. caninum infected pregnant mice treated with ELQ-334 at 10 mg/kg/day for 5 days, neonatal mortality (within 2 days post partum) was found in 7 of 44 pups (15.9%), but no postnatal mortality was noted, and vertical transmission was reduced by 49% compared to the placebo group, which exhibited 100% vertical transmission, neonatal mortality in 15 of 34 pups (44%), and postnatal mortality for 18 of the residual 19 pups during the 4 weeks follow-up. These findings encourage more research on the use of ELQs for therapeutic options against N. caninum infection.

DOI: 10.3389/fvets.2018.00285 PMCID: PMC6252379 PMID: 30510935

​

​

​

The Need for Antiviral Drugs for Pandemic Coronaviruses From a Global Health Perspective.

Front Med. 2020 Dec 22;7:596587.

Villamagna AH, Gore SJ, Lewis JS, Doggett JS.

 

Respiratory failure due to SARS-CoV-2 has caused widespread mortality, creating an urgent need for effective treatments and a long-term need for antivirals for future emergent coronaviruses. Pharmacotherapy for respiratory viruses has 

largely been unsuccessful with the exception of early treatment of influenza viruses, which shortens symptom duration and prevents infection in close contacts. Under the rapidly evolving circumstances of the COVID-19 pandemic, most clinical trials of experimental treatments in the United States have focused on later stages of the disease process. Worldwide, the clinical studies of the most impactful drugs, remdesivir and dexamethasone in ACTT-1, RECOVERY, and Solidarity, have studied hospitalized patients. Less than half of clinical trials in the U.S. have investigated oral agents, and the majority have taken place in hospitals at a disease stage where the viral load is already decreasing. The limited success of treatments for respiratory viruses and the viral dynamics of COVID-19 suggest that an antiviral therapy with the greatest impact against pandemic coronaviruses would be orally administered, well-tolerated, target a highly conserved viral protein or host-coronavirus interaction and could be used effectively throughout the world, including resource-poor settings. We examine the treatment of respiratory viral infections and current clinical trials for COVID-19 to provide a framework for effective 

antiviral therapy and prevention of future emergent coronaviruses and call attention to the need for continued preclinical drug discovery.

DOI: 10.3389/fmed.2020.596587 PMCID: PMC7783399 PMID: 33415116

​