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In addition to playing a central role in the mitochondria as the main producer of ATP, FOF1-ATP synthase performs diverse key regulatory functions in the cell membrane. Its malfunction has been linked to a growing number of human diseases, including hypertension, atherosclerosis, cancer, and some neurodegenerative, autoimmune, and aging diseases. Furthermore, inhibition of this enzyme jeopardizes the survival of several bacterial pathogens of public health concern. Therefore, FOF1-ATP synthase has emerged as a novel drug target both to treat human diseases and to combat antibiotic resistance. In this work, we carried out a computational characterization of the binding sites of the fungal antibiotic aurovertin in the bovine F1 subcomplex, which shares a large identity with the human enzyme. Molecular dynamics simulations showed that although the binding sites can be described as preformed, the inhibitor hinders inter-subunit communications and exerts long-range effects on the dynamics of the catalytic site residues. End-point binding free energy calculations revealed hot spot residues for aurovertin recognition. These residues were also relevant to stabilize solvent sites determined from mixed-solvent molecular dynamics, which mimic the interaction between aurovertin and the enzyme, and could be used as pharmacophore constraints in virtual screening campaigns. To explore the possibility of finding species-specific inhibitors targeting the aurovertin binding site, we performed free energy calculations for two bacterial enzymes with experimentally solved 3D structures. Finally, an analysis of bacterial sequences was carried out to determine conservation of the aurovertin binding site. Taken together, our results constitute a first step in paving the way for structure-based development of new allosteric drugs targeting FOF1-ATP synthase sites of exogenous inhibitors.

With the uncontrolled growth of multidrug-resistant bacteria, there is an urgent need to search for new therapeutic targets, to develop drugs with novel modes of bactericidal action. FoF1-ATP synthase plays a crucial role in bacterial bioenergetic processes, and it has emerged as an attractive antimicrobial target, validated by the pharmaceutical approval of an inhibitor to treat multidrug-resistant tuberculosis. In this work, we aimed to design, through two types of in silico strategies, new allosteric inhibitors of the ATP synthase, by targeting the catalytic β subunit, a centerpiece in communication between rotor subunits and catalytic sites, to drive the rotary mechanism. As a model system, we used the F1 sector of Escherichia coli, a bacterium included in the priority list of multidrug-resistant pathogens. Drug-like molecules and an IF1-derived peptide, designed through molecular dynamics simulations and sequence mining approaches, respectively, exhibited in vitro micromolar inhibitor potency against F1. An analysis of bacterial and Mammalia sequences of the key structural helix-turn-turn motif of the C-terminal domain of the β subunit revealed highly and moderately conserved positions that could be exploited for the development of new species-specific allosteric inhibitors. To our knowledge, these inhibitors are the first binders computationally designed against the catalytic subunit of FOF1-ATP synthase.