The goal of this project is to understand the molecular structural basis of transport and select inhibition of lipid flippases/multidrug resistance ABC (MDR-ABC) transporters. We will be integrating structure, function, and chemistry to provide a much more unified and comprehensive view of lipid transport coupled to ATP hydrolysis by MsbA. Through this work, we will discover specific and potent inhibitors of MsbA, which has very significant and direct implications for the treatment of cancer and infectious disease. An inhibitor of MsbA will likely disrupt the formation of lipopolysaccharide (IPS) in the outer membrane and kill a broad range of Gram-negative pathogenic bacteria. We have already obtained co-crystals of MsbA with a variety of cancer compounds, which include the MDR reversal compound PSC833. We have recently collected preliminary diffraction data and have initial electron density maps indicating the position of these ligands relative to the structure of MsbA. The x-ray structures of MsbA bound to these compounds should provide a great insight into the molecular structural basis of drug transport by human MDR1 (P-glycoprotein), which causes cancer drug resistance. The co-crystal structures of MsbA with inhibitors could also serve as a close model for other bacterial and mammalian MDR transporters. Finally, we continue to push the frontier of membrane protein structural biology by determining the x-ray structure of a new and distantly related bacterial MDR-ABC transporter from H. influenza. We have already obtained crystals and the x-ray structure determination is well-under way.
Our Specific Aims are: 1. Structural studies of MsbA with anti-cancer compounds and nucleotide analogs. 2. Functional studies of MsbA and the detection of inhibitors. 3. Structural studies of MsbA with compounds that occupy the """"""""ON"""""""" and """"""""OFF"""""""" sites of MsbA (Class I MsbA inhibitors). 4. Structural studies of MsbA with cyclic inhibitors based on cyclosporin A and other MDR cyclopeptides (Class II MsbA inhibitors). 5. Structural studies of a new MDR-ABC transporter from H. Influenza.
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