PROJECT SUMMARY/ABSTRACT
PROJECT TITLE: Studies of P-glycoprotein drug interactions
P-glycoprotein (Pgp) is a molecular pump that detoxifies cells by transporting hundreds of structurally
unrelated toxins out of the cell. Pgp limits uptake in the intestines, and enhances excretion of drugs in
the liver, kidney and blood-brain barrier, of many drugs that are used for treatment of cancers, HIV/AIDS,
psychiatric illnesses, and cardiovascular conditions. It is among the seven most important transporters
responsible for regulating drug absorption and disposition that now require documentation of drug
interactions for approval of any new drugs by the US Food and Drug Administration (FDA). Pgp is an
ATP-binding cassette transporter with two transmembrane domains (TMDs) and two nucleotide-binding
domains (NBDs). It uses ATP hydrolysis to pump substrates across the cell membranes. Our recent X-
ray structures of Pgp identified hydrophobic and aromatic amino acids that contribute to binding of
different inhibitors to the drug-binding site. In this proposal, we will test the hypothesis that therapeutic
drugs bind to different subsets of residues within defined subpockets in the TMDs of the protein. Our
general approach is to introduce tryptophans (Trps) at strategic positions in order to monitor drug
binding. The Trps will be introduced on the background of a new fully functional Trp-less Pgp, or a low-
Trp Pgp that retains three native conformationally sensitive Trps in the cytoplasmic domains. Using
fluorescence changes, such as quenching, and resonance energy transfer (FRET), we will map out
sites of interaction of the purified protein with prototypical substrates that occupy biochemically defined
and distinct binding sites, as well as those of common therapeutic drugs and newly identified inhibitors.
We will further insert a fluorescent Trp analog (L-Anap) into wild-type Pgp using the amber stop codon
suppression strategy to explore monitoring drug binding in biological cell membranes. By determining
how drugs and inhibitors modulate the cooperativity and conformational dynamics of this multidomain
transporter, we will gain unique insight into the mechanisms of drug binding and their effects on Pgp
function. With these new approaches, we will address the molecular mechanism and kinetics of
drug/inhibitor binding, determine synergistic effects, and refine the mechanisms of drug-drug
interactions in Pgp. The information will pave the way to new analytical approaches to refine Pgp drug
interaction studies of old and new drugs, and will be invaluable to redesign drugs with clinically
favorable pharmacokinetics and accelerate pharmacotherapeutic developments.