Cytochrome bc1 complex (ubiquinol oxidation pocket at center P) [1];
Saccharomyces cerevisiae bc1 complex (competitive inhibitor, apparent Ki = 9 nM, IC50 = 50 nM under standard assay conditions with enzyme 2.5 nM and substrate 50 μM) [1];
Bos taurus bc1 complex (Ki = 80 nM, IC50 = 400 nM) [1];
S. cerevisiae L275F mutant bc1 complex (Ki = 100 nM, IC50 = 500 nM) [1];
Rieske iron-sulfur protein (midpoint potential shifts from 285 mV to 385 mV upon atovaquone binding; EPR g value shifts: gz from 2.028 to 2.034, gy from 1.899 to 1.888, gx from 1.75 to 1.76) [1].

Atovaquone (atavaquone) is a chemical compound that belongs to the class of naphthalenes. Atovaquone is a hydroxy-1,4-naphthoquinone, an analog of ubiquinone, with antipneumocystic activity. Atovaquone is an anti-protozoal mitochondrial electron transport inhibitor; Antimalarial; Antipneumocystic, and has also been used to treat toxoplasmosis. It acts by inhibiting the cytochrome bc(1) complex via interactions with the Rieske iron-sulfur protein and cytochrome b in the ubiquinol oxidation pocket.

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Kinase Assay: Atovaquone is a medication used to treat or prevent for pneumocystis pneumonia, toxoplasmosis, malaria, and babesia. Target: Antiparasitic Atovaquone (atavaquone) is a chemical compound that belongs to the class of naphthalenes. Atovaquone is a hydroxy-1,4-naphthoquinone, an analog of ubiquinone, with antipneumocystic activity. Atovaquone is an anti-protozoal mitochondrial electron transport inhibitor; Antimalarial; Antipneumocystic, and has also been used to treat toxoplasmosis. It acts by inhibiting the cytochrome bc(1) complex via interactions with the Rieske iron-sulfur protein and cytochrome b in the ubiquinol oxidation pocket. Atovaquone is a unique naphthoquinone with broad-spectrum antiprotozoal activity. It is effective for the treatment and prevention of Pneumocystis carinii pneumonia (PCP), it is effective in combination with proguanil for the treatment and prevention of malaria, and it is effective in combination with azithromycin for the treatment of babesiosis.

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Atovaquone competitively inhibits the yeast cytochrome bc1 complex with apparent Ki = 9 nM. In the absence of inhibitor, the turnover number (Kcat) of the yeast enzyme is 130 s^-1 (or 170 s^-1 in another measurement) and the Km for decylubiquinol analogue is 11 μM. Under the same assay conditions, the bovine bc1 complex is much less sensitive with IC50 = 400 nM, corresponding to Ki = 80 nM. A L275F mutation introduced into yeast cytochrome b confers atovaquone resistance, raising the IC50 to 500 nM (Ki = 100 nM) without any loss of enzyme activity (Kcat = 190 s^-1) [1].
Atovaquone shifts the EPR spectrum of the Rieske iron-sulfur cluster in the yeast bc1 complex: gz shifts from 2.028 to 2.034, gy from 1.899 to 1.888, and gx from 1.75 to 1.76. These shifts are similar to those caused by stigmatellin but less pronounced [1].
Atovaquone raises the midpoint potential of the Rieske iron-sulfur cluster from 285 mV to 385 mV, indicating that the inhibitor binds approximately 50-fold more tightly when the Rieske center is reduced. For comparison, nonyl-HDBT raises the midpoint potential to 350 ± 12 mV [1].
Molecular modeling shows that Atovaquone binds in the ubiquinol oxidation pocket at center P. The hydroxyl group on the naphthoquinone ring forms a hydrogen bond to His181 of the Rieske iron-sulfur protein. The carbonyl group at position 4 of the quinone ring interacts with Glu272 of cytochrome b via a water-mediated hydrogen bond. Removal of the hydroxyl group increases nonbonding interaction energy by 14-54 kcal/mol, confirming its critical role in binding. The chlorophenyl tail extends into a hydrophobic pocket near residue 275 of cytochrome b. In the L275F mutant, the phenylalanine side chain creates steric hindrance, increasing the energy cost of binding by 16.21 kcal/mol and reducing affinity [1].

Atovaquone is a unique naphthoquinone with broad-spectrum antiprotozoal activity. It is effective for the treatment and prevention of Pneumocystis carinii pneumonia (PCP), it is effective in combination with proguanil for the treatment and prevention of malaria, and it is effective in combination with azithromycin for the treatment of babesiosis.
Oral Atovaquone (750 mg twice daily for 21 days) successfully treated mild-to-moderate PCP in AIDS patients with success rates of 62-79% in clinical trials. Treatment success correlated with plasma levels ≥15 μg/ml (98% success vs. 66% for lower levels) [1].
Compared to trimethoprim-sulfamethoxazole (SXT) for PCP, Atovaquone had slightly lower efficacy (20% failure vs. 7% for SXT) but better tolerability (7% vs. 20% required therapy change). Overall success rates (effective treatment plus no treatment-limiting adverse effects) were 62% for atovaquone and 64% for SXT [1].
For PCP prophylaxis, Atovaquone (1,500 mg once daily) was compared to dapsone (100 mg daily) in 1,057 SXT-intolerant patients; failure rates were 15.7 vs. 18.4 cases per 100 person-years, respectively. Atovaquone had similar efficacy to monthly aerosolized pentamidine (300 mg) with PCP incidences of 22-25% vs. 17% [1].
For malaria treatment, atovaquone-proguanil (A-P) (1,000/400 mg daily for 3 days) showed 100% cure rates in multiple trials, superior to mefloquine, amodiaquine, chloroquine, and equivalent to quinine-tetracycline and halofantrine. A-P was effective against multidrug-resistant P. falciparum [1].
For malaria prophylaxis, A-P (250/100 mg daily) demonstrated >95% protective efficacy in semi-immune adults in Kenya and Zambia, and 100% protective efficacy in nonimmune travelers against P. falciparum. A causal prophylactic effect was shown: a single 250 mg dose given 1 day before challenge protected 100% of subjects [1].
For babesiosis, Atovaquone (750 mg twice daily) plus azithromycin (500 mg then 250 mg daily) for 7-10 days was comparable to clindamycin plus quinine: fever resolved by 8 days, all symptoms resolved by 3 months in 65%, and all patients cleared Babesia DNA by 12 weeks. Side effects were much lower with atovaquone-azithromycin (15% vs. 72%) [1].
For toxoplasmosis salvage therapy in AIDS patients intolerant to standard regimens, Atovaquone (750 mg four times daily for 6 weeks) produced complete or partial response in 37% (radiologic) and 52% (clinical) of patients. Response correlated with higher drug levels [1].

Cytochrome c reductase activity was assayed in 50 mM potassium phosphate pH 7.0, 250 mM sucrose, 0.2 mM EDTA, 1 mM NaN3, 2.5 mM KCN, 0.01% Tween 20, and 40 μM cytochrome c at 23°C. The bc1 complex was diluted to 2.5 nM in assay buffer, and the reaction was started by adding a decylubiquinol analogue. To determine inhibition, atovaquone and substrate were mixed simultaneously into the assay buffer, then enzyme was added. Reduction of cytochrome c was monitored at 550 nm versus 539 nm in dual wavelength mode. Ki values for competitive inhibition were calculated from IC50 using the formula Ki = IC50 / (1 + [S]/Km) [1].
EPR spectroscopy: The bc1 complex was diluted to 24 μM in buffer containing 250 mM sucrose, 50 mM potassium phosphate, 1 mM sodium azide, 0.2 mM EDTA, and 0.01% Tween 20, pH 7.0. Samples were reduced with 5 mM ascorbate. 3 equivalents of atovaquone (72 μM) or stigmatellin were added and incubated for 20 min on ice. EPR spectra were recorded at a temperature of 20 K, microwave frequency of 9.47 GHz, microwave power of 1 mW, and modulation amplitude of 0.64 mT. A sample of oxidized bc1 complex (without addition) was subtracted from the spectra of ascorbate-reduced samples for baseline correction [1].
Potentiometric titrations by CD spectroscopy: The bc1 complex at a concentration of at least 300 μM was placed in an OTTLE cell with a path length of 100 μm. A mixture of redox mediators spanning the potential range from -358 to +644 mV (benzyl viologen, anthraquinone-2-sulfonate, 2-hydroxy-1,4-naphthoquinone, menadione, duroquinone, phenazine ethosulfate, phenazine methosulfate, trimethyl-1,4-benzquinone, 1,2-naphthoquinone, tetramethyl-p-phenylenediamine, potassium hexacyanoferrate, ferrocene-1,1'-dicarboxylic acid) was added at final concentrations of 20-25 μM each. NHDBT or atovaquone was added in 3-fold molar excess. CD signals at 500 nm were monitored, and the degree of reduction of the Rieske protein was calculated from the amplitude change. The potential curves were fitted by Nernstian functions assuming an n = 1 electron transition [1].

Atovaquone is highly lipophilic with very low aqueous solubility. It is marketed as a micronized suspension (750 mg/5 ml). Absorption is doubled when taken with fatty food. Steady-state plasma concentrations of 15-30 μg/ml are achieved with doses of 750-1,500 mg [1].
Plasma protein binding is >99%, with no significant displacement of other highly protein-bound drugs [1].
Concomitant rifampin reduces atovaquone levels by 40-50%; tetracycline reduces levels by 40%; rifabutin has a lesser interaction. Atovaquone increases zidovudine AUC by ~33% via inhibition of hepatic glucuronidation [1].
Cerebrospinal fluid penetration is minimal (<1% of plasma concentration) [1].
No significant hepatic metabolism; <1% renally excreted. Extensive enterohepatic cycling occurs, with >94% of the drug eliminated in feces over 3 weeks [1].
Half-life ranges from 51 to 77 hours [1].
For atovaquone-proguanil combination: proguanil bioavailability ~60%, steady-state levels ~40 ng/ml; cycloguanil (active metabolite) ~10 ng/ml. Proguanil half-life 12-21 hours. No pharmacokinetic interaction between atovaquone and proguanil [1].

Most frequent adverse effects (10-35%): maculopapular rash, nausea, diarrhea, headache. Rash may correlate with higher plasma atovaquone concentrations. Side effects may resolve with continued treatment. Elevation of liver enzymes has been documented. Atovaquone does not cause bone marrow suppression and lacks antibacterial activity. Overdoses in three patients did not result in permanent injury [1].
When combined with proguanil for malaria, gastrointestinal side effects and headache predominate; elevations of liver transaminase and bilirubin occur in ~15% of treated patients (especially in Thai patients with high hepatitis B carriage), resolving over 28 days [1].
Atovaquone suspension compared to SXT: treatment-limiting side effects 7% vs. 20%; compared to pentamidine: 4% vs. 36%; compared to dapsone: gastrointestinal symptoms more common with atovaquone, hypersensitivity and anemia more common with dapsone [1].
Atovaquone plus azithromycin for babesiosis: side effects 15% (diarrhea or rash) vs. 72% for quinine-clindamycin (diarrhea, tinnitus, hearing loss) [1].
No bone marrow suppression. No significant drug interactions beyond those listed [1].

Am J Trop Med Hyg.1996 Jan;54(1):62-6;J Biol Chem.2003 Aug 15;278(33):31312-8;Antimicrob Agents Chemother. 2002 May; 46(5): 1163–1173.

Atovaquone is a naphthoquinone compound with a 4-(4-chlorophenyl)cyclohexyl group at position 2 and a hydroxyl substituent at position 3. It exhibits various activities including antimalarial activity, antifungal activity, inhibition of dihydroorotate dehydrogenase (quinone) activity, inhibition of NADH/ubiquinone reductase (H(+)- transporter) activity, and inhibition of quinone cytochrome c reductase activity. It belongs to the monochlorobenzene class of compounds and is a hydroxy-1,4-naphthoquinone. Atorvaquinone is an antimalarial and antiseptic drug. It is a hydroxynaphthoquinone with antibacterial activity and is currently used in antimalarial treatment regimens. See also: Atorvaquinone (note moved to).

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Atovaquone is a hydroxynaphthoquinone with broad-spectrum activity against apicomplexan parasites including Plasmodium, Toxoplasma, Theileria, and Babesia, as well as the fungus Pneumocystis carinii. Resistance to atovaquone has been associated with mutations in the cytochrome b gene. The yeast Saccharomyces cerevisiae is used as a surrogate model because its cytochrome b shares ~40% identity with Plasmodium and ~60% with P. carinii, while bovine cytochrome b is ~80% identical to human. The L275F mutation in yeast cytochrome b (leucine is present in yeast and P. carinii; phenylalanine in bovine and human) reduces atovaquone sensitivity to a level similar to the bovine enzyme, explaining the differential efficacy between fungal and mammalian bc1 complexes. The computed binding structure of atovaquone differs from two previously postulated structures; in this model the hydroxyl group interacts with the Rieske protein (His181) rather than with Glu272, and a water molecule mediates the interaction with Glu272 [1].

C22H19CLO3

366.84

366.102

C, 72.03; H, 5.22; Cl, 9.66; O, 13.08

95233-18-4

Atovaquone-d4;2070015-14-2;Atovaquone-d5;1329792-63-3;cis-Atovaquone-d4

74989

Light yellow to yellow solid powder

1.4±0.1 g/cm3

542.2±50.0 °C at 760 mmHg

216-2190C

281.7±30.1 °C

0.0±1.5 mmHg at 25°C

1.653

5.86

1

3

2

26

595

0

O=C1C2C=CC=CC=2C(=O)C(O)=C1[C@@H]1CC[C@@H](C2C=CC(Cl)=CC=2)CC1

KUCQYCKVKVOKAY-CTYIDZIISA-N

InChI=1S/C22H19ClO3/c23-16-11-9-14(10-12-16)13-5-7-15(8-6-13)19-20(24)17-3-1-2-4-18(17)21(25)22(19)26/h1-4,9-13,15,26H,5-8H2/t13-,15-

trans-2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthalenedione

566C; 566C80; 566C80 hydroxynaphthoquinone; 566C80, hydroxynaphthoquinone; Atovaquone; atovaquone GlaxoSmithKline brand; compound 566; Glaxo Wellcome brand of atovaquone; GlaxoSmithKline brand of atovaquone; hydroxynaphthoquinone 566C80; hydroxynaphthoquinone, 566C80; Mepron; Wellvone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :8.33 ~ 11 mg/mL ( 22.71 ~29.98 mM )

Solubility in Formulation 1: 0.83 mg/mL (2.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.83 mg/mL (2.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5% DMSO + Corn oil: 0.5mg/ml (1.36mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)