Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP-kinase

Abstract

BackgroundThe biguanides are a family of drugs with diverse clinical applications. Metformin, a widely used anti-hyperglycemic biguanide, suppresses mitochondrial respiration by inhibiting respiratory complex I. Phenformin, a related anti-hyperglycemic biguanide, also inhibits respiration, but proguanil, which is widely used for the prevention of malaria, does not. The molecular structures of phenformin and proguanil are closely related and both inhibit isolated complex I. Proguanil does not inhibit respiration in cells and mitochondria because it is unable to access complex I. The molecular features that determine which biguanides accumulate in mitochondria, enabling them to inhibit complex I in vivo, are not known.ResultsHere, a family of seven biguanides are used to reveal the molecular features that determine why phenformin enters mitochondria and inhibits respiration whereas proguanil does not. All seven biguanides inhibit isolated complex I, but only four of them inhibit respiration in cells and mitochondria. Direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. This high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only those biguanides that enter mitochondria and inhibit complex I activate AMP kinase, strengthening links between complex I and the downstream effects of biguanide treatments.ConclusionsBiguanides inhibit mitochondrial complex I, but specific molecular features control the uptake of substituted biguanides into mitochondria, so only some biguanides inhibit mitochondrial respiration in vivo. Biguanides with restricted intracellular access may be used to determine physiologically relevant targets of biguanide action, and for the rational design of substituted biguanides for diverse clinical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0287-9) contains supplementary material, which is available to authorized users.

Synthesis and In Vitro AMPK Activation of Cycloalkyl/Alkarylbiguanides with Robust In Vivo Antihyperglycemic Action

“…On the other hand, aromatic biguanides -were unable to activate the AMPK. These results are in accordance with those reported in a parallel work performed with closely related biguanides [15].…”

“…With these results, it can be concluded that the mechanism of action of the analogues -that confer their antidiabetic activity is similar to metformin, through the activation of AMPK and of some of the pathways that are regulated by this enzyme. Previously in vitro reports agree with the AMPK results obtained with compound [15]. However, in our current study, we have demonstrated the robust in vivo effect produced by this compound after an oral administration.…”

Metformin directly targets the H3K27me3 demethylase KDM6A/UTX

“…Despite its small size, however, metformin was capable of simultaneously interacting, in the crystal structure of KDM6A/UTX (3AVS), with the residues required not only for iron binding in the demethylase reaction but also with the methyl groups of the H3K27me3 side chain as well as NOG—an analog of the cofactor AKG (H1146, E1148, Y1135; Sengoku & Yokoyama, 2011; Shpargel, Sengoku, Yokoyama & Magnuson, 2012). Moreover, metformin‐induced inhibition of catalysis by isolated mCI, which is believed to be the primary target of metformin, requires even higher concentrations of metformin (i.e., 20–100 mmol/L; Bridges et al., 2014, 2016) than those required to inhibit the demethylation activity of the purified KDM6A/UTX enzyme. We experimentally confirmed that neither mCI nor AMPK seem to be required to elicit specific augmentation of the global levels of the KDM6A/UTX substrate H3K27me3 in mouse and human cells growing in the presence of millimolar concentrations of metformin.…”

“…In this regard, norMitoMet, a novel metformin derivative tagged with the mitochondrial vector triphenylphosphonium (TPP + ), which has been shown to notably increase the antineoplastic efficacy of parental metformin (Boukalova et al., 2016), behaved as the most potent KDM6A/UTX inhibitor—in the low micromolar range—likely because it was the sole biguanide that shared with metformin the ability to coordinate with catalytic metal ions of KDM6A/UTX. Thus, the unique side chains that are known to determine the pharmacological differences of biguanides including mitochondrial accumulation and mCI inhibition (Boukalova et al., 2016; Bridges, Sirviö, Agip & Hirst, 2016; Bridges et al., 2014) might also influence the ability of the biguanides to interfere with KDM6A/UTX activity. Our discovery that biguanides do not exhibit either 2D structural‐topological analogies or 3D nonanalogous bioisosteric physicochemical relationships with KDM‐targeted metabolites (Mishur et al., 2016; Tarhonskaya et al., 2017) strongly suggest that different biguanides might be viewed as a new family of pharmacologically active KDM6A/UTX regulators.…”

The effects of metformin on gut microbiota and the immune system as research frontiers

“…At the molecular level, many targets have been studied (for examples, [6,7]) but most studies have focused on respiratory complex I of the electron transport chain [8,9]. The precise interaction between the tiny biguanide molecule and the huge multi-protein complex has not been established, although ongoing research is addressing that question.…”