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.
Highlights
The biguanides are a family of drugs with diverse clinical applications
Experimental strategy To delineate the effects that determine whether biguanides are able to cross the mitochondrial inner membrane we focused on phenformin and proguanil
Improved transmembrane diffusion as a result of their greater hydrophobicity has previously been suggested as a reason why phenformin and buformin are more potent inhibitors of cellular respiration than metformin [30], but our data explain that this correlation is observed because the more hydrophobic compounds are better complex I inhibitors
Summary
The 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. Biguanides are commonly prescribed drugs for the treatment of type II diabetes and to prevent malaria, and are under investigation for their uses in cardiovascular disease and cancer [1,2,3] Their modes of action in all of these applications are still debated, biguanides with known anti-hyperglycemic properties (metformin, phenformin, and buformin) have consistently been observed to inhibit mitochondrial respiratory complex I (NADH:ubiquinone oxidoreductase) at millimolar and high micromolar concentrations [4,5,6]. These inhibitory concentrations appear high, they are physiologically relevant because biguanides are positively charged molecules and are concentrated inside mitochondria by the mitochondrial membrane potential. The antimalarial biguanide cycloguanil and its pro-drug
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