Targeting the Ubiquinol-Reduction (Qi) Site of the Mitochondrial Cytochrome bc1 Complex for the Development of Next Generation Quinolone Antimalarials

Abstract

Antimalarials targeting the ubiquinol-oxidation (Q_o) site of the Plasmodium falciparum bc₁ complex, such as atovaquone, have become less effective due to the rapid emergence of resistance linked to point mutations in the Q_o site. Recent findings showed a series of 2-aryl quinolones mediate inhibitions of this complex by binding to the ubiquinone-reduction (Qi) site, which offers a potential advantage in circumventing drug resistance. Since it is essential to understand how 2-aryl quinolone lead compounds bind within the Qi site, here we describe the co-crystallization and structure elucidation of the bovine cytochrome bc₁ complex with three different antimalarial 4(1H)-quinolone sub-types, including two 2-aryl quinolone derivatives and a 3-aryl quinolone analogue for comparison. Currently, no structural information is available for Plasmodial cytochrome bc₁. Our crystallographic studies have enabled comparison of an in-silico homology docking model of P. falciparum with the mammalian's equivalent, enabling an examination of how binding compares for the 2- versus 3-aryl analogues. Based on crystallographic and computational modeling, key differences in human and P. falciparum Q_i sites have been mapped that provide new insights that can be exploited for the development of next-generation antimalarials with greater selective inhibitory activity against the parasite bc₁ with improved antimalarial properties.

Keywords: Plasmodium falciparum; antimalarial; atovaquone; bc1 inhibitor; crystallography; drug resistance; homology modeling; mitochondria; molecular modeling; quinolone.

Figure 1

Chemical structures of cytochrome bc₁ ligands. (A) Ubiquinone, a native substrate that binds to both Q_o and Q_i sites of cytochrome bc₁; atovaquone, a hydroxy-naphthoquinone targeting the Q_o site; GSK932121 and GW844520, 4(1H)-pyridone-based compounds developed by GlaxoSmithKline; quinolone-based compounds ELQ300 and SCR0911, lead antimalarial compounds; LSPN 178 and LSPN 182, modified 2-quinolone that inhibits both stages of the parasite; (B) CK-2–67, RKA066, and WDH-1U-4 were the 4(1H)-quinolone-based compounds that were co-crystallized with bovine cytochrome bc₁ in this study.

Figure 2

The cytochrome bc₁ Q_i site with bound inhibitors (a) CK-2-67, (b) RKA066, and (c) WDH-1U-4 molecules are shown as green, orange, and magenta sticks, respectively. Upper insert: the omit F_o-F_c map is contoured at 3σ level and shown as green mesh. The Q_i and Q_o sites are marked by black circles. Lower insert: the molecules are shown as sticks surrounded by 2F_o-F_c electron density contoured at 1σ level. Q_i site residues are shown as blue sticks, hydrogen bonds as black dash lines. The cartoon representation of bovine cytochrome b subunit is shown in blue.

Figure 3

Binding modes of selected antimalarial compounds within the bovine Q_i site. (a) CK-2-67 (green sticks). (b) RKA066 (orange sticks). (c) WDH-1U-4 (magenta sticks). (d) SCR0911 (yellow sticks) from the 5OKD structure. (e) GSK932121 (teal sticks) from the 4D6U structure. (f) Superimposed crystal structures with 2-aryl 4(1H)-quinolones: SCR0911, CK-2-67, and RKA066. (g) Superimposed crystal structures of WDH-1U-4 and GSK932121. Black, cyan, green, and red dashes represent hydrogen bonds, electrostatic, aromatic-aromatic, and hydrophobic interactions between ligands and protein residues, respectively. The blue cartoon represents the bovine cytochrome b subunit.

Figure 4

Sequence alignment between bovine, human, and P. falciparum cytochrome b showing conservation in the Q_i site. Fully and partially conserved residues are colored in deep and light blue, respectively. The numbering shown at the top and bottom corresponds to bovine (human) and P. falciparum sequences, respectively. The polar and non-polar residues interacting with 4(1H)-quinolones in crystal structures and docking models are highlighted in red and yellow boxes, respectively. Sequence identity between human and bovine cytochrome b is 93%.

Figure 5

Molecular docking within the Q_i site of P. falciparum cytochrome bc₁. The predicted binding mode of each compound. (a) RKA066 (orange sticks). (b) CK-2-67 (green sticks). (c) GSK932121 (teal sticks). (d) WDH-1U-4 (magenta sticks). (e) ELQ300 (pink sticks). (f) SCR0911 (yellow sticks). Black, cyan, green, and red dashes represent hydrogen bonds, electrostatic, π-π stacking, and hydrophobic interactions between ligands and protein residues, respectively. The grey cartoon represents bovine cytochrome b subunit.

Figure 6

Schemes for the future development of aryl-4(1H)-quinolones targeting the Q_i site binding. (a) 2-Aryl-4(1H)-quinolones in P. falciparum bc₁. (b) 3-Aryl-4(1H)-quinolone in P. falciparum bc₁. (c) 2-Aryl-4(1H)-quinolones in human bc₁. (d) 3-Aryl-4(1H)-quinolone in human bc₁ (based on PDB: 5XTE). Colored dashed bars represent the interactions that could be considered for lead compound design: green—the interactions observed only in P. falciparum; orange—the interactions observed in both species; red—the interactions observed only in humans.