Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 A resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

We report the high-resolution (1.9 Å) crystal structure of oligomycin bound to the subunit c 10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c 10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common "drug-binding site." We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design. (1). Studies in the 1960s from the laboratory of Efraim Racker demonstrated that the mitochondrial ATP synthase can be separated into two parts, coupling factor 1, or F 1 , which contains the catalytic site for ATP synthesis, and coupling factor o, or F o , which is able to confer sensitivity to oligomycin (2-4). Despite more than 50 y of studies on mitochondrial F 1 F o ATP synthase, the binding site of oligomycin on F o has been elusive. Here we report the oligomycin-binding site on subunit-c of the F o portion of the ATP synthase.Subunit-c of the ATP synthase is an integral membrane protein consisting of two helices, 1 and 2, which span the inner mitochondrial membrane (Fig. 1). Subunit-c assembles as a homomeric ring consisting of 10 subunits in the yeast ATP synthase and eight subunits in the bovine ATP synthase (5, 6). The c-ring forms an essential component of the proton turbine of the ATP synthase, which spins coupled to the movement of protons down a potential gradient. The essential carboxylate of Glu59 in helix 2 of the yeast subunit-c is postulated to participate directly in the movement of protons from the cytosol to the mitochondrial matrix during ATP synthesis. The side-chain carboxyl of Glu59 is nearly in the middle of helix 2, positioning it in the lipid bilayer in the protonated, "closed" conformation (7). Subunit-a is postulated to form two aqueous half-channels that allow protons to gain access to the carboxylate of Glu59 in the "open" conformation, allowing protonation and deprotonation reaction (7).Based on the results presented here, we propose that in the intact ATP synthase, oligomycin binds at the c-ring positioned at the proton channel and blocks proton translocation by blocking access to the essential carboxyl. Furthermore, we propose that the binding site framed out by oligomycin is a common drug-binding site for inhibitors that bind to the ba...