Sovitj Pou

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

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.

ELQ-300 Prodrugs for Enhanced Delivery and Single-Dose Cure of Malaria

ABSTRACT ELQ-300 is a preclinical candidate that targets the liver and blood stages of Plasmodium falciparum, as well as the forms that are crucial to transmission of disease: gametocytes, zygotes, and ookinetes. A significant obstacle to the clinical development of ELQ-300 is related to its physicochemical properties. Its relatively poor aqueous solubility and high crystallinity limit absorption to the degree that only low blood concentrations can be achieved following oral dosing. While these low blood concentrations are sufficient for therapy, the levels are too low to establish an acceptable safety margin required by regulatory agencies for clinical development. One way to address the challenging physicochemical properties of ELQ-300 is through the development of prodrugs. Here, we profile ELQ-337, a bioreversible O-linked carbonate ester prodrug of the parent molecule. At the molar equivalent dose of 3 mg/kg of body weight, the delivery of ELQ-300 from ELQ-337 is enhanced by 3- to 4-fold, reaching a maximum concentration of drug in serum (Cmax) of 5.9 μM by 6 h after oral administration, and unlike ELQ-300 at any dose, ELQ-337 provides single-dose cures of patent malaria infections in mice at low-single-digit milligram per kilogram doses. Our findings show that the prodrug strategy represents a viable approach to overcome the physicochemical limitations of ELQ-300 to deliver the active drug to the bloodstream at concentrations sufficient for safety and toxicology studies, as well as achieving single-dose cures.

Inhibition of Cytochrome bc1 as a Strategy for Single-Dose, Multi-Stage Antimalarial Therapy

Single-dose therapies for malaria have been proposed as a way to reduce the cost and increase the effectiveness of antimalarial treatment. However, no compound to date has shown single-dose activity against both the blood-stage Plasmodium parasites that cause disease and the liver-stage parasites that initiate malaria infection. Here, we describe a subset of cytochrome bc1 (cyt bc1) inhibitors, including the novel 4(1H)-quinolone ELQ-400, with single-dose activity against liver, blood, and transmission-stage parasites in mouse models of malaria. Although cyt bc1 inhibitors are generally classified as slow-onset antimalarials, we found that a single dose of ELQ-400 rapidly induced stasis in blood-stage parasites, which was associated with a rapid reduction in parasitemia in vivo. ELQ-400 also exhibited a low propensity for drug resistance and was active against atovaquone-resistant P. falciparum strains with point mutations in cyt bc1. Ultimately, ELQ-400 shows that cyt bc1 inhibitors can function as single-dose, blood-stage antimalarials and is the first compound to provide combined treatment, prophylaxis, and transmission blocking activity for malaria after a single oral administration. This remarkable multi-stage efficacy suggests that metabolic therapies, including cyt bc1 inhibitors, may be valuable additions to the collection of single-dose antimalarials in current development.

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

Human babesiosis is a tick-borne multisystem disease, and current treatments have both adverse side effects and a significant rate of drug failure. Lawres et al. report that endochin-like quinolones, in combination with atovaquone, cure experimental babesiosis in immunodeficient mice.

Sontochin as a Guide to the Development of Drugs against Chloroquine-Resistant Malaria

ABSTRACT Sontochin was the original chloroquine replacement drug, arising from research by Hans Andersag 2 years after chloroquine (known as “resochin” at the time) had been shelved due to the mistaken perception that it was too toxic for human use. We were surprised to find that sontochin, i.e., 3-methyl-chloroquine, retains significant activity against chloroquine-resistant strains of Plasmodium falciparum in vitro. We prepared derivatives of sontochin, “pharmachins,” with alkyl or aryl substituents at the 3 position and with alterations to the 4-position side chain to enhance activity against drug-resistant strains. Modified with an aryl substituent in the 3 position of the 7-chloro-quinoline ring, Pharmachin 203 (PH-203) exhibits low-nanomolar 50% inhibitory concentrations (IC50s) against drug-sensitive and multidrug-resistant strains and in vivo efficacy against patent infections of Plasmodium yoelii in mice that is superior to chloroquine. Our findings suggest that novel 3-position aryl pharmachin derivatives have the potential for use in treating drug resistant malaria.

Diphenylether-Modified 1,2-Diamines with Improved Drug Properties for Development against Mycobacterium tuberculosis

New treatments for tuberculosis infection are critical to combat the emergence of multidrug- and extensively drug-resistant Mycobacterium tuberculosis (Mtb). We report the characterization of a diphenylether-modified adamantyl 1,2-diamine that we refer to as TBL-140, which has a minimal inhibitory concentration (MIC99) of 1.2 μg/mL. TBL-140 is effective against drug-resistant Mtb and nonreplicating bacteria. In addition, TBL-140 eliminates expansion of Mtb in cell culture infection assays at its MIC. To define the mechanism of action of this compound, we performed a spontaneous mutant screen and biochemical assays. We determined that TBL-140 treatment affects the proton motive force (PMF) by perturbing the transmembrane potential (ΔΨ), consistent with a target in the electron transport chain (ETC). As a result, treated bacteria have reduced intracellular ATP levels. We show that TBL-140 exhibits greater metabolic stability than SQ109, a structurally similar compound in clinical trials for treatment of MDR-TB infections. Combined, these results suggest that TBL-140 should be investigated further to assess its potential as an improved therapeutic lead against Mtb.

Atovaquone and ELQ-300 Combination Therapy as a Novel Dual-Site Cytochrome bc1 Inhibition Strategy for Malaria

ABSTRACT Antimalarial combination therapies play a crucial role in preventing the emergence of drug-resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc1 complex (cyt bc1) are among the few compounds that are effective for both acute antimalarial treatment and prophylaxis. There are two known sites for inhibition within cyt bc1: atovaquone (ATV) blocks the quinol oxidase (Qo) site of cyt bc1, while some members of the endochin-like quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinone reductase (Qi) site and retain full potency against ATV-resistant Plasmodium falciparum strains with Qo site mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and combination therapy consisting of ATV plus ELQ-300 (ATV:ELQ-300), using P. yoelii murine models of malaria. In our monotherapy assessments, we found that ATV functioned as a single-dose curative compound in suppressive tests whereas ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked recrudescence even in a high-parasitemia acute infection model. ATV:ELQ-300 therapy was highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg of body weight. Compared to the ATV:proguanil (Malarone) formulation, ATV:ELQ-300 was more efficacious in multiday, acute infection models and was equally effective at blocking the emergence of ATV-resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt bc1 is a valuable strategy for antimalarial combination therapy and that Qi site inhibitors such as ELQ-300 represent valuable partner drugs for the clinically successful Qo site inhibitor ATV.

Genetic evidence for cytochrome b Qi site inhibition by 4(1H)-quinolone-3-diarylethers and antimycin in Toxoplasma gondii

ABSTRACT Toxoplasma gondii is an apicomplexan parasite that causes fatal and debilitating brain and eye disease. Endochinlike 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.

Alkoxycarbonate Ester Prodrugs of Preclinical Drug Candidate ELQ-300 for Prophylaxis and Treatment of Malaria

ELQ-300 is a preclinical antimalarial drug candidate that is active against liver, blood, and transmission stages of Plasmodium falciparum. While ELQ-300 is highly effective when administered in a low multidose regimen, poor aqueous solubility and high crystallinity have hindered its clinical development. To overcome its challenging physiochemical properties, a number of bioreversible alkoxycarbonate ester prodrugs of ELQ-300 were synthesized. These bioreversible prodrugs are converted to ELQ-300 by host and parasite esterase action in the liver and bloodstream of the host. One such alkoxycarbonate prodrug, ELQ-331, is curative against Plasmodium yoelii with a single low dose of 3 mg/kg in a murine model of patent malaria infection. ELQ-331 is at least as fully protective as ELQ-300 in a murine malaria prophylaxis model when delivered 24 h before sporozoite inoculation at an oral dose of 1 mg/kg. Here, we show that ELQ-331 is a promising prodrug of ELQ-300 with improved physiochemical and metabolic properties and excellent potential for clinical formulation.

Mitochondrial type II NADH dehydrogenase of Plasmodium falciparum is dispensable and not the functional target of putative NDH2 quinolone inhibitors

The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite’s susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results call into question the validity of PfNDH2 as an antimalarial drug target. Importance For a long time, PfNDH2 has been considered an attractive antimalarial drug target. However, the conclusion that PfNDH2 is essential was based on preliminary and incomplete data. Here we generate a PfNDH2 KO (knockout) parasite in the blood stages of Plasmodium falciparum, showing that the gene is not essential. We also show that previously reported PfNDH2-specific inhibitors kill the parasites primarily via targeting the cytochrome bc1 complex, not PfNDH2. Overall, we provide genetic and biochemical data that help to resolve a long-debated issue in the field regarding the potential of PfNDH2 as an antimalarial drug target.