Rolf Walter Winter

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.

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.

No evidence for mevalonate shunting in moderately affected children with Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is caused by a genetic deficiency in 7-dehydrocholesterol (7-DHC) reductase (EC 1.3.1.21), the last enzyme of the cholesterol synthetic pathway. In SLOS, plasma cholesterol concentration is reduced and immediate precursor concentration (7-DHC) is elevated. Surprisingly, total sterol synthesis is reduced but HMG-CoA reductase activity, a rate-limiting enzyme in cholesterol synthesis is unaltered as judged by normal urinary excretion of mevalonic acid (MVA) (Pappu et al. J Lipid Res 43:1661–1669, 2002). These findings raise the possibility of increased diversion of MVA into the MVA shunt pathway away from sterol synthesis, by activation of the shunt pathway enzymes. To test this hypothesis, we measured the urinary excretion of 3-methylglutaconic acid (U-3MGC), a by-product of the shunt pathway, in 19 mildly to moderately severely affected SLOS subjects (ten males, nine females) receiving either a cholesterol-free or a high cholesterol diet, and in 20 age- and sex-matched controls. U-3MGC was similar in SLOS and controls, and was unaffected by dietary cholesterol intake. Further, no change in U-3MGC was observed in a subset of SLOS subjects (n = 9) receiving simvastatin. In contrast, U-MVA was reduced by cholesterol supplementation (~54%, p < 0.05) and by simvastatin (~50%, p < 0.04). There was no correlation between U-3MGC and either plasma sterol concentrations, urinary isoprenoids, or the subjects’ clinical severity score. However U-3MGC was inversely correlated with age (p < 0.04) and body weight (p < 0.02), and higher in females than in males (~65%, p < 0.025). The data show that DHCR7 deficiency does not result in 3MGC accumulation in SLOS and suggest that the MVA shunt pathway is not activated in patients with the condition.

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.