Moelcular recognition of small molecules by the mitochondrial F1F0-ATPase.

Abstract

The mitochondrial F₁F₀-ATPase, the enzyme responsible for the production of cellular ATP, is one of the most complex and best-studied enzymes in biology. Inhibition of this enzyme has been suggested as a potential therapeutic mode of action to treat a diverse array of diseases including cancer and autoimmunity. Bz-423 is a pro-apoptotic 1,4-benzodiazepine with potent therapeutic activity in animal models of immune and autoimmune diseases. Target identification and validation studies have demonstrated that Bz-423 in an inhibitor of mitochondrial F₁F₀-ATPase and inhibition is mediated through Bz-423 binding to the oligomycin sensitivity conferring protein (OSCP) subunit of the enzyme. While a high resolution crystal structure exists for the F₁ domain of the enzyme, limited structural and functional information is known about the OSCP. This thesis describes the synthesis and analysis of small molecules designed to probe the structural features important for Bz-423&middot;OSCP recognition. One unique feature of Bz-423 is that the <italic>R</italic> and <italic> S</italic> enantiomers exhibit equal potency in enzyme inhibition and cell-based assays. Based on this observation, a series of experiments was designed and executed to elucidate the molecular basis for this finding. Through the design, synthesis, and evaluation of 1,5-benzodiazepines analogues of Bz-423, it was determined that this benzodiazepine possesses pseudo-symmetry elements (i.e., both enantiomers adopt conformations that are roughly superimposable). These data suggest that the stereogenic center in Bz-423 may not be required for binding to the OSCP. To test this hypothesis, a large group of Bz-423 analogues lacking the chiral center were designed and evaluated. These molecules inhibit the F₁F₀-ATPase in a manner consistent with what is predicted from structure-activity studies of Bz-423, suggesting the stereogenic center found in Bz-423 does not contribute to recognition by the OSCP. Combining the enzyme assay results with molecular modeling studies led to a proposed pharmacophore model that describes the three-dimensional arrangement of substituents and their physicochemical properties that correlate to Bz-423 recognition. To test this model, the proposed physicochemical properties and 3-dimensional positioning of the pharmacophore substituents were arranged on a scaffold unrelated to the benzodiazepine. These molecules inhibit the F₁F₀-ATPase in a manner that is generally consistent with the proposed pharmacophore of Bz-423. These compounds ultimately may prove useful in studying how binding to the OSCP mediates inhibition of the F₁F₀-ATPase. In addition, these molecules are starting points for the development of F₁F₀-ATPase inhibitors with therapeutic potential.