A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria

Margaret A. Phillips; Julie Lotharius; Kennan Marsh; John White; Anthony Dayan; Karen L. White; Jacqueline W. Njoroge; Farah El Mazouni; Yanbin Lao; Sreekanth Kokkonda; Diana R. Tomchick; Xiaoyi Deng; Trevor Laird; Sangeeta N. Bhatia; Sandra March; Caroline L. Ng; David A. Fidock; Sergio Wittlin; Maria Lafuente-Monasterio; Francisco Javier Gamo Benito; Laura Maria Sanz Alonso; Maria Santos Martinez; Maria Belen Jimenez-Diaz; Santiago Ferrer Bazaga; Iñigo Angulo-Barturen; John N. Haselden; James Louttit; Yi Cui; Arun Sridhar; Anna-Marie Zeeman; Clemens Kocken; Robert Sauerwein; Koen Dechering; Vicky M. Avery; Sandra Duffy; Michael Delves; Robert Sinden; Andrea Ruecker; Kristina S. Wickham; Rosemary Rochford; Janet Gahagen; Lalitha Iyer; Ed Riccio; Jon Mirsalis; Ian Bathhurst; Thomas Rueckle; Xavier Ding; Brice Campo; Didier Leroy; M. John Rogers; Pradipsinh K. Rathod; Jeremy N. Burrows; Susan A. Charman

doi:10.1126/scitranslmed.aaa6645

Figures

Tables
Supplementary Materials
Additional Files

Fig. 1. Chemical and protein-bound inhibitor structures.
(A) Chemical structures of DSM265 (415 daltons), DSM430 (430 daltons), and DSM450 (431 daltons). (B) X-ray structure of the inhibitor binding site of PfDHODH bound to DSM265 [Protein Data Bank (PDB) 5BOO]. DSM265 (pink); protein, orotate (Oro), and flavin mononucleotide (FMN) (carbons in teal); oxygen (red); nitrogen (blue); sulfur (yellow); phosphate (orange); and fluorine (light blue).
Fig. 2. In vitro activity of DSM265 and its analogs on DHODH and P. falciparum parasites.
(A) DHODH inhibition. IC₅₀ values are reported in Table 1. Error bars show SEM for three technical replicates per concentration. Each fitted IC₅₀ was obtained from 30 to 33 data points per fit. (B) In vitro P. falciparum 3D7 growth inhibition. Fitted EC₅₀s were 0.0018 μg/ml (0.0011 to 0.0028), 0.079 μg/ml (0.042 to 0.15), and 0.00020 μg/ml (0.00011 to 0.00056) for DSM265, DSM450, and DSM430, respectively, with the 95% confidence intervals in parenthesis (three technical replicates per concentration and 18 to 24 data points per fit; the plot shows mean ± SEM). (C) Effects on P. falciparum 3D7 blood-stage phenotype and development. Cells were treated with drug at 10 × EC₅₀, and parasites were evaluated by microscopy 48 hours after drug addition (minimally 200 cells were counted per condition). Atovaquone (Ato), artemisinin (Art), and pyrimethamine (Pyr) were used as controls. Images of representative parasites from the counted stages are displayed (R, rings; YT, young trophozoite; MT, mature trophozoite; S, schizont; P, pyknotic). (D) In vitro killing curves for treatment of P. falciparum 3D7 blood-stage parasites. Data for 10 × EC₅₀ and 100 × EC₅₀ (where EC₅₀ = 0.0046 μg/ml) (four technical replicates showing the mean and SD) are displayed for DSM265. Data for artemisinin, atovaquone, pyrimethamine, and chloroquine (Chl) were previously reported (33). (E) Comparative DSM265 kill rates calculated from (D). PRR (parasite reduction ratio), the log number of parasites killed per asexual life cycle (48 hours); Lag phase, time before parasite killing begins; PCT (parasite clearance time), time to achieve 99.9% parasite kill. Primary data are provided in table S19.
Fig. 3. Activity of DSM265 against P. falciparum liver stages.
(A) Inhibitory effect on hepatocyte infection rate. Number of EEF/10,000 hepatocytes quantitated at different concentrations of DSM265 relative to the dimethyl sulfoxide (DMSO) control for three technical replicates using the same human donor; error bars show the SD for the replicates. Primary data are provided in table S20. (B) Dose-response curve showing the effect of DSM265 and primaquine (PQ) on infection rate. Primaquine EC₅₀ = 0.20 μg/ml (0.059 to 0.71) and DSM265 EC₅₀ = 7.0 μg/ml (4.3 to 11) (n = 3). Errors are SEM. (C) Inhibitory effect on EEF diameter for n = 20 to 22 EEFs. (D) Dose-response curve showing the effect on EEF size. Primaquine EC₅₀ = 0.15 μg/ml (0.11 to 0.20) and for DSM265 EC₅₀ = 0.0057 (0.0048 to 0.0069) μg/ml. Error bars show the SD for n = 20 to 22. (E) Representative images of parasites day 3 after infection treated with DMSO or DSM265 (0.42 μg/ml). Statistical significance was determined by analysis of variance [ANOVA; ***P = 0.0002, ****P < 0.0001; for comparison of drug-treated to DMSO controls (A and C)]. Historical data for primaquine (B and D) and atovaquone (A and C) using the same donor are shown. HSP70, heat shock protein 70; DAPI, 4′,6-diamidino-2-phenylindole.
Fig. 4. In vivo efficacy of DSM265 in the P.falciparum–infected SCID mouse.
DSM265 was dosed as the tosylate salt twice daily for 4 days starting on day 3 after infection. (A) Blood parasitemia versus days after infection for administered doses (mg/kg per 12 hours). The ED₉₀ (1.5 mg/kg per 12 hours) and the MPC (dose = 6.4 mg/kg per 12 hours) were determined 24 hours after the last dose. The parasite detection limit was 0.01%. (B) DSM265 blood concentrations (μg/ml) over 10 hours after the first dose for doses as indicated. A single data point was collected per dose with the exception of the no-drug control where n = 3 biological replicates were obtained. Primary data are provided in table S3 (C and D).
Fig. 5. Oral pharmacokinetic profiles for DSM265 in dogs.
Pharmacokinetic behavior of DSM265 was evaluated after a single oral dose (10 mg/kg) of either free base as a suspension in 0.5% carboxymethyl cellulose, 0.4% Tween 80, or SDD formulation administered as a suspension in Methocel A4M, in either the fasted or the fed state. Data represent mean ± SEM for six dogs. Primary data are provided in table S21.

Table 1. Comparative kinetic analysis of DSM265 and derivatives on P. falciparum and mammalian DHODH.

Data represent mean of three technical replicates with 95% confidence intervals for the fit shown in parentheses. Primary data are provided in table S19. ND, not determined.

Compound	DSM265	DSM430	DSM450
DHODH		IC₅₀ (μg/ml)
P. falciparum	0.010 (0.0080–0.013)	0.010 (0.0079–0.014)	0.032 (0.026–0.040)
P. vivax	0.020 (0.017–0.024)	ND	0.11 (0.081–0.15)
P. cynomolgi	0.0050 (0.0042–0.0058)	ND	ND
Human	~41	3.7 (2.6–4.8)	>43
Mouse	1.1 (1.2–1.5)	0.10 (0.082–0.12)	4.5 (3.1–6.9)
Rat	0.82 (0.63–1.1)	0.082 (0.069–0.099)	1.6 (1.2–2.0)
Dog	11 (8.8–12)	0.56 (0.48–0.65)	25 (21–29)
Rabbit	>41	ND	>43
Pig	>41	ND	ND
Monkey	>41	ND	ND

Table 2. DSM265 activity against P. falciparum parasites at different stages in the life cycle.

Data represent EC₅₀ values unless otherwise stated to be the minimum parasiticidal concentration (MPC).

Assay	MPC or EC₅₀ (μg/ml)	Fraction unbound in assay media	Unbound MPC or EC₅₀ (μg/ml)
Blood-stage: P. falciparum 3D7 in vitro MPC (kill assay)	0.014*	0.17^†	0.0020
SCID mouse in vivo MPC (plasma)	1.1^‡	0.001^§	0.0011
Blood-stage: P. falciparum 3D7 in vitro growth assay	0.0046*	0.17^†	0.00078
Liver-stage: P. falciparum to schizont	0.0057	0.215^¶	0.0012
P. falciparum early-stage gametocytes stages III to IV; strain NF54	>0.42	0.004^∥	>0.0017
Gametocyte assay P. falciparum stages IV to V	Inactive >16	ND	ND
P. falciparum dual gamete formation assay	Inactive >42	ND	ND
P. falciparum stage V oocyst	38% at 1.0	0.001^§	38% at 0.001

*Data taken from Fig. 2D.

†AlbuMAX II (0.5%) in RPMI 1640.

‡Blood to plasma ratio in the humanized SCID mouse of 0.5 was used to convert blood concentrations to plasma concentrations.

§Human plasma.

¶Fetal calf serum (10%) in Dulbecco’s modified Eagle’s medium or similar medium.

∥Human serum (10%) in RPMI 1640.

Table 3. Evaluation of in vitro generated DSM265 Dd2–resistant parasites.

Fold change in parentheses. PfDHODH data are IC₅₀ values determined on the recombinant wild-type (wt) or G181C mutant P. falciparum enzymes. All data were collected with a minimum of three technical replicates and then used for a global fit. Error analysis is provided in table S5. N/A, not applicable.

Clone number	Inoculum	DSM265 selection concentration (μg/ml)	DSM265 Dd2 EC₅₀ (μg/ml)	Gene copy number	DHODH mutation	PfDHODH IC₅₀ (μg/ml)
Dd2/wt	N/A	N/A	0.0022	1.0	N/A	0.0096
PR.Cl1	2 × 10⁷	0.0083	0.0083 (3.8)	2.7	None	N/A
PR.Cl2	2 × 10⁷	0.0083	0.0099 (4.5)	2.3	None	N/A
DF.R10 Clb	2 × 10⁶	0.0095	0.058 (26)	1.1	G181C	0.120 (12)

Supplementary Materials

Figures
Tables
Additional Files

www.sciencetranslationalmedicine.org/cgi/content/full/7/296/296ra111/DC1
Synthesis
Materials and Methods
Fig. S1. (Fo-Fc) map for DSM265:PfDHODH binding site.
Fig. S2. DHODH sequence alignment.
Fig. S3. In vitro parasite killing curves.
Fig. S4. Activity of DSM265 against P. cynomolgi large (liver schizonts) and small (hypnozoite) forms.
Fig. S5. The effect of DSM265 treatment on P. falciparum Pf3D70087/N9 in vivo.
Fig. S6A. Proposed biotransformation pathways of DSM265 in plasma of mice, rabbits, monkeys, and dogs.
Fig. S6B. Plasma concentrations of DSM265 and DSM450 (hydroxy metabolite).
Fig. S7. Simulated human plasma profiles using a PBPK model (GastroPlus).
Fig. S8. Effect on ECG in the rabbit cardiac ventricular wedge assay.
Fig. S9. Evaluation of the effects of DSM265 on G6PD-deficient human RBCs engrafted into a NOD-SCID mouse.
Table S1. PfDHODH-DSM265 x-ray diffraction data and refinement statistics.
Table S2. In vitro antimalarial activity of DSM265.
Table S3A. Blood pharmacokinetic data for DSM265 in SCID mice.
Table S3B. SCID mouse in vivo antimalarial activity.
Table S3C. SCID mouse parasitemia.
Table S3D. SCID mouse pharmacokinetic individual time point data.
Table S4A. Selection for DSM265-resistant parasites in P. falciparum Dd2: Rathod laboratory.
Table S4B. Selection for atovaquone-resistant parasites in P. falciparum Dd2: Rathod laboratory.
Table S4C. Selection for DSM265-resistant parasites in P. falciparum Dd2: Fidock laboratory.
Table S4D. Selection for atovaquone-resistant parasites in P. falciparum Dd2: Fidock laboratory.
Table S4E. Selection for DSM265-resistant parasites in P. falciparum K1: Fidock laboratory.
Table S4F. Selection for DSM265 and atovaquone P. falciparum HB3: Rathod laboratory.
Table S5. Summary of DSM265-resistant clones: Analysis of parasites in whole-cell assays.
Table S6. Kinetic analysis of PfDHODH mutants.
Table S7. Drug combination analysis.
Table S8A. Stability data for DSM265 free base and tosylate salt.
Table S8B. Solubility data for DSM265 free base.
Table S9. In vitro ADME data for DSM265.
Table S10. In vivo metabolite identification.
Table S11. Relative plasma exposures of DSM265 metabolites in mice, rabbits, monkeys, and dogs.
Table S12. DSM265 plasma pharmacokinetics after a single intravenous dose in mice, rats, dogs, and monkey.
Table S13. DSM265 plasma pharmacokinetics after a single oral dose of DSM265 in mice, rats, dogs, and monkeys.
Table S14. Effect of salt form, formulation, and food on the DSM265 plasma pharmacokinetics after oral dosing in beagle dogs.
Table S15. Safety pharmacology.
Table S16. Exploratory toxicology studies (non-GLP) in rodents and dogs.
Table S17A. Toxicokinetic parameters on days 1 and 7 in a mouse 7-day toxicology study.
Table S17B. Individual mouse plasma concentrations 7-day toxicology study 25 mg/kg.
Table S17C. Individual mouse plasma concentrations 7-day toxicology study 75 mg/kg.
Table S17D. Individual mouse plasma concentrations 7-day toxicology study 200 mg/kg.
Table S18A. Toxicokinetic data from a 10-day toxicology study in male beagle dogs.
Table S18B. Toxicokinetic parameters day 1 of toxicology study in male beagle dogs.
Table. S19. Primary data supporting Fig. 2.
Table. S20. Primary data supporting Fig. 3 (A and B).
Table. S21. Primary data supporting Fig. 5.
References (38–66)

Additional Files

Figures
Tables
Supplementary Materials

Supplementary Material for:
A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria

Margaret A. Phillips,* Julie Lotharius, Kennan Marsh, John White, Anthony Dayan, Karen L. White, Jacqueline W. Njoroge, Farah El Mazouni, Yanbin Lao, Sreekanth Kokkonda, Diana R. Tomchick, Xiaoyi Deng, Trevor Laird, Sangeeta N. Bhatia, Sandra March, Caroline L. Ng, David A. Fidock, Sergio Wittlin, Maria Lafuente-Monasterio, Francisco Javier Gamo Benito, Laura Maria Sanz Alonso, Maria Santos Martinez, Maria Belen Jimenez-Diaz, Santiago Ferrer Bazaga, I�igo Angulo-Barturen, John N. Haselden, James Louttit, Yi Cui, Arun Sridhar, Anna-Marie Zeeman, Clemens Kocken, Robert Sauerwein, Koen Dechering, Vicky M. Avery, Sandra Duffy, Michael Delves, Robert Sinden, Andrea Ruecker, Kristina S. Wickham, Rosemary Rochford, Janet Gahagen, Lalitha Iyer, Ed Riccio, Jon Mirsalis, Ian Bathhurst, Thomas Rueckle, Xavier Ding, Brice Campo, Didier Leroy, M. John Rogers, Pradipsinh K. Rathod, Jeremy N. Burrows, Susan A. Charman*

*Corresponding author. E-mail: margaret.phillips{at}utsouthwestern.edu (M.A.P.); susan.charman{at}monash.edu

Published 15 July 2015, Sci. Transl. Med. 7, 296ra111 (2015)
DOI: 10.1126/scitranslmed.aaa6645

This PDF file includes:

Synthesis
Materials and Methods
Fig. S1. (Fo-Fc) map for DSM265:PfDHODH binding site.
Fig. S2. DHODH sequence alignment.
Fig. S3. In vitro parasite killing curves.
Fig. S4. Activity of DSM265 against P. cynomolgi large (liver schizonts) and small (hypnozoite) forms.
Fig. S5. The effect of DSM265 treatment on P. falciparum Pf3D70087/N9 in vivo.
Fig. S6A. Proposed biotransformation pathways of DSM265 in plasma of mice, rabbits, monkeys, and dogs.
Fig. S6B. Plasma concentrations of DSM265 and DSM450 (hydroxy metabolite).
Fig. S7. Simulated human plasma profiles using a PBPK model (GastroPlus).
Fig. S8. Effect on ECG in the rabbit cardiac ventricular wedge assay.
Fig. S9. Evaluation of the effects of DSM265 on G6PD-deficient human RBCs engrafted into a NOD-SCID mouse.
Table S1. PfDHODH-DSM265 x-ray diffraction data and refinement statistics.
Table S2. In vitro antimalarial activity of DSM265.
Table S3A. Blood pharmacokinetic data for DSM265 in SCID mice.
Table S3B. SCID mouse in vivo antimalarial activity.
Table S3C. SCID mouse parasitemia.
Table S3D. SCID mouse pharmacokinetic individual time point data.
Table S4A. Selection for DSM265-resistant parasites in P. falciparum Dd2: Rathod laboratory.
Table S4B. Selection for atovaquone-resistant parasites in P. falciparum Dd2: Rathod laboratory.
Table S4C. Selection for DSM265-resistant parasites in P. falciparum Dd2: Fidock laboratory.
Table S4D. Selection for atovaquone-resistant parasites in P. falciparum Dd2: Fidock laboratory.
Table S4E. Selection for DSM265-resistant parasites in P. falciparum K1: Fidock laboratory.
Table S4F. Selection for DSM265 and atovaquone P. falciparum HB3: Rathod laboratory.
Table S5. Summary of DSM265-resistant clones: Analysis of parasites in whole-cell assays.
Table S6. Kinetic analysis of PfDHODH mutants.
Table S7. Drug combination analysis.
Table S8A. Stability data for DSM265 free base and tosylate salt.
Table S8B. Solubility data for DSM265 free base.
Table S9. In vitro ADME data for DSM265.
Table S10. In vivo metabolite identification.
Table S11. Relative plasma exposures of DSM265 metabolites in mice, rabbits, monkeys, and dogs.
Table S12. DSM265 plasma pharmacokinetics after a single intravenous dose in mice, rats, dogs, and monkey.
Table S13. DSM265 plasma pharmacokinetics after a single oral dose of DSM265 in mice, rats, dogs, and monkeys.
Table S14. Effect of salt form, formulation, and food on the DSM265 plasma pharmacokinetics after oral dosing in beagle dogs.
Table S15. Safety pharmacology.
Table S16. Exploratory toxicology studies (non-GLP) in rodents and dogs.
Table S17A. Toxicokinetic parameters on days 1 and 7 in a mouse 7-day toxicology study.
Table S17B. Individual mouse plasma concentrations 7-day toxicology study 25 mg/kg.
Table S17C. Individual mouse plasma concentrations 7-day toxicology study 75 mg/kg.
Table S17D. Individual mouse plasma concentrations 7-day toxicology study 200 mg/kg.
Table S18A. Toxicokinetic data from a 10-day toxicology study in male beagle dogs.
Table S18B. Toxicokinetic parameters day 1 of toxicology study in male beagle dogs.
Table. S19. Primary data supporting Fig. 2.
Table. S20. Primary data supporting Fig. 3 (A and B).
Table. S21. Primary data supporting Fig. 5.
References (38–66)

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A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria

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A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria

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