Probing binding determinants in center P of the cytochrome bc(1) complex using novel hydroxy-naphthoquinones

Abstract

Atovaquone is a substituted 2-hydroxy-naphthoquinone used therapeutically against Plasmodium falciparum (malaria) and Pneumocystis pathogens. 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. As the targeted pathogens have developed resistance to this drug there is an urgent need for new alternatives. To better understand the determinants of inhibitor binding in the ubiquinol oxidation pocket of the bc(1) complex we synthesized a series of hydroxy-naphthoquinones bearing a methyl group on the benzene ring that is predicted to interact with the nuclear encoded Rieske iron-sulfur protein. We have also attempted to overcome the metabolic instability of a potent cytochrome bc(1) complex inhibitor, a 2-hydroxy-naphthoquinone with a branched side chain, by fluorinating the terminal methyl group. We have tested these new 2-hydroxy-naphthoquinones against yeast and bovine cytochrome bc(1) complexes to model the interaction with pathogen and human enzymes and determine parameters that affect efficacy of binding of these inhibitors. We identified a hydroxy-naphthoquinone with a trifluoromethyl function that has potential for development as an anti-fungal and anti-parasitic therapeutic.

Figure 1

(A) Atovaquone (ATV) (B) S-10576 (C) Novel 2-hydroxy-naphthoquinones: 2-OH-3-(2-Me-trifluorooctyl)-naphthoquinone (NQ1), 2-OH-3-(2-Me-octyl)-8-Me-naphthoquinone (NQ2), 2-OH-3-(2-Me-trifluorooctyl)-8-Me-naphthoquinone (NQ3).

Figure 2

Structure of 2-OH-3-(2-Me-octyl)-8-Me-naphthoquinone (NQ2) docked in the ubiquinol oxidation pocket at center P. Portions of the Rieske protein and cytochrome b are shown as gold and cyan ribbons, respectively. Van der Waals radii depicting the non-covalent interaction between the 8-methyl group of the naphthoquinone ring and Cys-180 (C180) of the Rieske protein are shown. The hydrogen bond between the 2-hydroxy group of NQ2 and His-181 of the Rieske protein and the water-mediated hydrogen bond between the 4-carbonyl oxygen of NQ2 and Glu-272 of cytochrome b are indicated by dashed white lines. Carbon atoms are shown in green, oxygens are red, hydrogens are white, nitrogens are blue, sulfurs are yellow, and iron are purple. The structure was created by substitution of NQ2 into the stigmatellin liganded crystal structure of the yeast bc₁ complex [23] and energy minimization as described in the Experimental Procedures.

Figure 3

Effect of 2-hydroxy-naphthoquinones on bc₁ activity of (A) yeast and (B) bovine bc₁ complexes. The cytochrome c reductase activity of purified enzyme was measured as described in Materials and Methods in the presence of increasing concentrations of inhibitor. The assays were performed in duplicate and the results are shown as averages. Activities are expressed as a percentage of the turnover number of each strain in the absence of inhibitor.

Figure 4

Effect of chain length on inhibition of yeast cytochrome bc₁ complexes by 2-hydroxy-naphthoquinones without and with a methyl group on carbon 8 of the naphthoquinone ring. The IC₅₀ values for inhibition of the bc₁ complex activity are shown with light gray bars for the 2-hydroxy-3-alkyl-naphthoquinones and dark gray bars for the 2-hydroxy-3-alkyl-8-methylnaphthoquinones.

Figure 5

Selectivity of 2-hydroxy-naphthoquinone inhibitors of the bovine and yeast cytochrome bc₁ complexes. The bar graphs show the Selectivity Coefficients for inhibition of the yeast versus bovine bc₁ complex by atovaquone (ATV), S-10576 and its derivatives 2-OH-3-(2-Me-trifluorooctyl)-naphthoquinone (NQ1), 2-OH-3-(2-Me-octyl)-8-Me- naphthoquinone (NQ2), and 2-OH-3-(2-Me-trifluorooctyl)-8-Me-naphthoquinone (NQ3).

Figure 6

Experimentally measured IC₅₀ values compared to calculated binding energies of S-10576, 2-OH-3-(2-Me-trifluorooctyl)-naphthoquinone (NQ1), and 2-OH-3-(2-Metrifluorooctyl)-8-Me-naphthoquinone (NQ3) in yeast cytochrome bc₁ complex. The IC₅₀ values for the inhibition of the yeast bc₁ complex activity are shown in light gray bars. The dark gray bars show the calculated binding energies.

Figure 7

Specific binding of 2-hydroxy-naphthoquinone inhibitors to center P of the yeast cytochrome bc₁ complex. Upon rapid mixing of cytochrome c with bc₁ complex that had been partially reduced by incubation with quinol before the addition of antimycin and NQs (yielding the initial absorbance shown at t = 0), the additional oxidant-induced reduction of cytochrome b through center P (A) was greatly inhibited by atovaquone (middle trace), and completely by 2-OH-3-(2-Me-trifluorooctyl)-naphthoquinone (NQ1, lower trace). In contrast, cytochrome b reduction through center N (B) obtained by rapid-mixing of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol with oxidized bc₁ complex in the presence of stigmatellin yielded identical, superimposed kinetic traces in the presence and absence of 2-OH-3-(2-Me-trifluorooctyl)-naphthoquinone (NQ1).