Structure of the NDH-2 – HQNO inhibited complex provides molecular insight into quinone-binding site inhibitors

Abstract

Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ1) interactions in the yeast Ndi1–UQ1 complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors.

Synthesis and Investigation of Phthalazinones as Antitubercular Agents

“…To determine whether the phthalazinones could be accommodated in the NDH‐II Q‐site, molecular docking of 2‐methylphthalazin‐1(2 H )‐one ( 10 ) was undertaken using USCF Chimera/DOCK6 (Figures ) . Different molecular orientations with similar binding scores were observed (Figure B), however, in all instances, phthalazinone 10 was wedged between the hydrophobic residues Q317 and I379 with a similar binding orientation to that reported for MK and HQNO . Hydrogen bonding was also evident between phthalazinone 10 and guanidine residue R382 (Figure B).…”

“…Further analysis of the NDH‐II Q‐site also indicated that the pocket should be able to accommodate substituents at the 2‐ and 7‐positions, thus providing scope to potentially improve the anti‐bacterial activity of this class of compounds. As quinolinyl‐pyrimidines have been found to interact with NDH‐II, [31 ] we also reasoned that the addition of a pyrimidine to the phthalazinone scaffold might assist with NDH‐II binding by way of a hydrogen bond between the pyrimidine amine and a glutamic acid residue (E324) in the Q‐site …”

“… Docking studies of phthalazinone 10 in C. thermarum NDH‐II (PDB: 6BDO), calculated using USCF Chimera/DOCK6. A) Structure of 2‐methylphthalazin‐1(2 H )‐one ( 10 ).…”

“…NDH‐II is absent from mammalian mitochondria yet it is essential for growth in mycobacteria as it catalyses the transfer of electrons from NADH to MK via the aid of a flavin co‐factor (FAD) . There are three distinct binding sites in NDH‐II: the first and second domains, which are situated in the aqueous environment of the cytoplasm and which bind NADH and FAD, respectively; and a hydrophobic membrane‐anchoring domain, which contains the quinone binding site (Q‐site) . To date, the antimycobacterial drug clofazimine ( 8 ) and our recently identified quinolinequinones, such as QQ8c ( 9 ), have been shown to target NDH‐II thereby leading to increased NADH oxidation activity and bactericidal levels of ROS …”

“…Moreover, a hydrogen bond between one of the carbonyl oxygen atoms of the quinones and an amine proton on the FAD isoalloxazine ring, as well as a distant edge‐to‐face π‐π interaction between MK and the FAD isoalloxazine ring, were observed. Co‐crystallisation of Caldalkalibacillus thermarum NDH‐II with a known inhibitor, 2‐heptyl‐4‐hydroxyquinoline‐ N ‐oxide (HQNO), revealed similar binding interactions . Accordingly, we proposed that the ketone‐containing bicyclic planar phthalazinone scaffold could be accommodated between the hydrophobic residues Q317 and I379 in the Q‐site, with the ketone oxygen participating in hydrogen bonding interactions with the ring nitrogen of the FAD isoalloxazine ring.…”