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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 multi-dose 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 hours 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.

Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and pi-stacking interactions with complementary amino acids of the protein. In this study, analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potential hydrophobic, aromatic active-site residues which could pi-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles. However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significant metabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and (S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a pi-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with pi-stacking to F114 for certain aromatic ligands.

The cytochromes P450 (P450) involved in the epoxidation of the rat carcinogen acrylonitrile (ACN) to the mutagen 2-cyanoethylene oxide (CEO) have been investigated in hepatic microsomes from F-344 rats and humans. Induction of P450 2E1 by acetone treatment increased the Vmax for rat microsomal CEO formation 5-fold, while ACN treatment had little effect. Treatment with beta-naphthoflavone, dexamethasone, and phenobarbital had little effect upon Vmax but increased the KM 3- to 5-fold. The P450 ligand 1-phenylimidazole and substrate ethanol were potent inhibitors of ACN epoxidation after all treatments. 2-(Diethylamino)ethyl 2,2-diphenylvalerate (SKF 525A; 0.1 mM) was not an effective inhibitor with microsomes from untreated or acetone-treated rats, but inhibited approximately 50% following dexamethasone or phenobarbital treatment. Antibodies to P450 2E1 inhibited > 85% of the ACN epoxidation activity in microsomes from untreated or beta-naphthoflavone- or acetone-treated rats, but only produced 40% and 60% inhibition following dexamethasone or phenobarbital treatments, respectively. These results indicate that P450 2E1 is the major catalyst of ACN epoxidation in untreated rats and that other forms of P450 can also epoxidize ACN. Diethyldithiocarbamate (0.1 mM) was a potent irreversible inhibitor of ACN epoxidation after all of the induction treatments, indicating that it is not specific for P450 2E1. Chlorzoxazone (2 mM) produced 75-90% inhibition after all of the induction treatments, indicating that it interacts with several rodent P450 isoforms in addition to 2E1. Human hepatic microsomes (n = 6) epoxidized ACN with Vmax'S ranging from 129 to 315 pmol of CEO formed/(min.mg of protein) and KM's from 12 to 18 microM.(ABSTRACT TRUNCATED AT 250 WORDS)