Structural basis of mammalian respiratory complex I inhibition by medicinal biguanides

Abstract

The molecular mode of action of biguanides, including the drug metformin, which is widely used in the treatment of diabetes, is incompletely characterized. Here, we define the inhibitory drug-target interaction(s) of a model biguanide with mammalian respiratory complex I by combining cryo-electron microscopy and enzyme kinetics. We interpret these data to explain the selectivity of biguanide binding to different enzyme states. The primary inhibitory site is in an amphipathic region of the quinone-binding channel, and an additional binding site is in a pocket on the intermembrane-space side of the enzyme. An independent local chaotropic interaction, not previously described for any drug, displaces a portion of a key helix in the membrane domain. Our data provide a structural basis for biguanide action and enable the rational design of medicinal biguanides.

Fig. 1. Characterization of biguanide effects on catalysis and regions of structural interest.

A) Chemical structures of metformin, phenformin and IM1092 in monoprotonated form. B) Correlation between membrane deactive complex I content and IC₅₀ for metformin (gray), phenformin (teal) and IM1092 (orchid). Error bars represent S.E.M for deactive content and 95% confidence intervals for IC₅₀. Data are fit to an exponential regression for visualization. C) Effect of pH on IC₅₀ in bovine heart membranes for metformin (gray), phenformin (teal) and IM1092 (orchid). 9D: pH 9 deactivated membranes; 9D(i): pH 9 deactivated membranes measured in the presence of antimycin A to inhibit complex III and the alternative oxidase (AOX) to oxidize quinol by a different route, confirming inhibition is on complex I. Error bars represent 95% confidence intervals.

Fig. 2. Binding of IM1092 in the Q-channel.

A) Overview showing the location of the biguanide binding site and inset showing a closer view and bonding interactions of the chloro-iodo-phenyl group for Deactive-1092-i. Orange arrow shows the route for exit from the Q-channel into the lipid bilayer. B) Overlay of models for the Active-1092-i, Deactive-1092-i, ii and iii, and Slack-1092-i and ii states, aligned to subunit ND1, showing the location and variability of biguanide binding orientations, and relative position of NDUFS7-Arg77. NDUFS7-Arg77 and ND1-Phe224 are white in the active models, mint or orchid in the slack models and black in the deactive models. C-H) Cryo-EM difference map densities (composite vs models) for biguanides bound to C) Deactive-1092-i, D) Deactive-1092-ii, E) Deactive-1092-iii, F) Active-1092-i, G) Slack-1092-i and H) Slack-1092-ii. The insets show the difference map density for the biguanide for each model shown. Biguanides are not modelled in F and G due to uncertainties in the ligand identity or orientation. Sidechain and ligand density for panels C-H are shown in Fig. S16.

Fig. 3. Biguanide-induced distortion and disordering of the ND5 lateral helix and NDUFB4 loop.

A) Location of the structure disturbance and two views of the model of Deactive-1092-iv with hydrogen-bonding interactions indicated in black dotted lines and distances indicated. Orchid, NDUFB4; teal, ND5; orange, NDUFA11; dark gray, NDUFB8. Sidechain density for this region of deactive-1092-iv is shown in Fig. S18. B-D) Models and composite cryo-EM maps for B) Active-1092-ii, C) Active -1092-iii, D) Active-1092-iv showing progressive disordering within the series. Details of π-bulge stabilizing interactions and equivalent disordering for the deactive states (Deactive-1092-iv, v, and vi) are shown in Fig. S18.

Fig. 4. Location of the biguanide binding at the ND2/NDUFB5/NDUFA11 interface.

A) Overall location of the binding site. Purple, NDUFC2; teal, ND2; orange, NDUFB5. B) An overlay of all models aligned to ND2. C) Surface representation (Active-1092-ii model) showing subunit NDUFC2 in cartoon with atoms shown for IM1092. D) The same view from the active inhibitor-free model showing the atoms from residues 1-7 of NDUFC2. Local interactions and density for the individual maps and models are shown in Figs. S26-27.