Novel inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with anti-malarial activity in the mouse model

Abstract

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED(50) values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

FIGURE 1.

X-ray structure of Genz-667348 in complex with PfDHODH. A, ribbon diagram of Genz-667348 bound to PfDHODH. α-Helices are displayed in teal, β-strands are displayed in sand, ligands are displayed as space-filling balls. Atoms are colored as follows: carbon atoms are yellow in FMN, pink in G348, and green in orotate; nitrogen is blue; oxygen is red; fluorine is light blue; and sulfur is yellow. B, comparison of PfDHODH inhibitor-binding sites. PfDHODH-G348 (teal) superimposed with PfDHODH-DSM1 (pink) and A77 1726 (orange). Colors refer to carbon atoms, other atoms are colored as in A. Inhibitors are displayed as balls and sticks. A subset of residues within 4 Å of the bound inhibitor is displayed. C, comparison with the human DHODH inhibitor-binding site. PfDHODH-G348 (teal) superimposed with hDHODH-bre (pink). Residue numbers for hDHODH are marked with an apostrophe, while PfDHODH numbers are not. Inhibitors are displayed as balls and sticks.

FIGURE 2.

Oral exposure of DHODH inhibitors in the mouse. Genz-667348 (A), -668857 (B), and -669178 (C) were administered to mice by oral gavage using twice daily dosing for a single day, and plasma samples were collected over a 24-h period for analysis as described under “Experimental Procedures.” Exposure levels are shown relative to the IC₉₀ values derived from in vitro parasite viability studies. Results represent the means for three animals per dose ± S.D.

FIGURE 3.

Compound efficacy in the P. berghei mouse model (N-clone). Animals were dosed once on day 1 and with split twice daily dosing on days 2–4. A, reduction in parasitemia for Genz-667348, -668857, and -669178 as a function of dose. ED₅₀ values: 667348, 16.7 mg/kg/day; 668857, 13.0 mg/kg/day; and 669178, 21.0 mg/kg/day. Results represent the means for five animals per dose ± S.D. B, Kaplan-Meyer plot demonstrating the effect of dose of Genz-667348 on time to recrudescence. The high dose of 200 mg/kg/day delayed the reappearance of parasites until day 15; a sterile cure was not achieved.

FIGURE 4.

Efficacy of Genz-667348 in the P. berghei mouse model (ANKA strain). Animals were dosed twice daily. A, reduction in parasitemia for Genz-667348 as a function of dose. ED₅₀ value: 6.1 mg/kg/day. Results represent the means for five animals per dose ± S.D. B, Kaplan-Meyer plot demonstrating the effect of dose of Genz-667348 on time to recrudescence. A sterile cure was achieved at 4 days of twice daily dosing at 100 mg/kg/day.