Overexpression of ATP-binding cassette (ABC) transporters is often linked to multidrug resistance (MDR) in cancer chemotherapies. P-glycoprotein (P-gp) is one of the best studied drug transporters associated with MDR. There are currently no approved drugs available for clinical use in cancer chemotherapies to reverse MDR by inhibiting P-glycoprotein. Using computational studies, we previously identified several compounds that inhibit P-gp by targeting its nucleotide binding domain and avoiding its drug binding domains. Several of these compounds showed successful MDR reversal when tested on a drug resistant prostate cancer cell line. Using conventional two-dimensional cell culture of MDR ovarian and prostate cancer cells and three dimensional prostate cancer microtumor spheroids, we demonstrated here that co-administration with chemotherapeutics significantly decreased cell viability and survival as well as cell motility. The P-gp inhibitors were not observed to be toxic on their own. The inhibitors increased cellular retention of chemotherapeutics and reporter compounds known to be transport substrates of P-gp. We also showed that these compounds are not transport substrates of P-gp and that two of the three inhibit P-gp, but not the closely related ABC transporter, ABCG2/BCRP. The results presented suggest that these P-gp inhibitors may be promising leads for future drug development.

P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp.

Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATPbinding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotidebinding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp.

Hsp90 is one of the most abundant proteins in the cytosol of eukaryotic cells. Under physiological conditions Hsp90 has been shown to play a major role in several specific signaling pathways, including maturation of various kinases and maintenance of steroid receptors in an activable state. It is well established that the level of Hsp90 increases severalfold under stress conditions, and it has been shown that the chaperone function of Hsp90 is ATP-independent. Although yeast Hsp90 does not bind ATP, as determined by a number of methods monitoring tight binding, ATP-dependent functions of Hsp90 in the presence of co-factors and elevated temperatures are still under discussion.Here, we have reinvestigated ATP-binding properties and ATPase activity of human Hsp90 under various conditions. We show that human Hsp90 does not bind ATP tightly and does not exhibit detectable ATPase activity. However, using electron spin resonance spectroscopy, weak binding of spin-labeled ATP analogues with halfmaximal binding at 400 M ATP was detected. The functional significance of this weak interaction remains enigmatic.Under stress conditions, e.g. high temperatures, cells overexpress a distinct set of proteins, the so-called heat shock or stress proteins. The major classes of heat shock proteins, Hsp90, 1 Hsp70, Hsp60, and small Hsps, are thought to function as molecular chaperones during protein folding (1-3). The mechanisms of these chaperone functions are still under intensive investigation. In the case of Hsp70 and Hsp60, ATP binding and hydrolysis is a major requirement in chaperone-mediated folding (1). In vivo experiments suggest that Hsp90, one of the most abundant and conserved heat shock proteins, is a specific chaperone involved in regulating signal transduction pathways by assisting structural changes of certain kinases and steroid receptors (4 -8). In addition, results from in vitro studies highlight the general chaperone activities of Hsp90 (2, 3). Like other chaperones, Hsp90 performs at least part of its activity in association with specific partner proteins (9 -14), some of which seem to function as molecular chaperones themselves (15,16). In this context, the involvement of ATP in Hsp90 function is still a controversial subject (11)(12)(13)(17)(18)(19), and ATP binding as well as ATPase activity of Hsp90 have been reported previously (18, 20 -23). In contrast to these findings, we have shown that yeast Hsp90 does not bind ATP (17) by means of assays that are reflecting structural changes of the observed protein in the presence of ligand, detecting binding of the protein to immobilized ATP or binding of fluorescencelabeled ATP analogues to Hsp90. These observation enabled only the investigation of tight interactions between ATP and Hsp90. New results concerning p23, a partner protein of Hsp90 that is thought to play an important role in Hsp90/steroid receptor complexes, readdressed the possibility that human Hsp90 is an ATP-binding protein (18). This brought up the question of whether human Hsp90 differs in its ATP...

The importance of smooth muscle cell pHi and pHo for the hypercapnic vasodilation of rat cerebral arteries was evaluated in vitro. Vessel segments were mounted in a myograph for isometric tension recording; pHi was measured by loading the smooth muscle cells with the fluorescent dye BCECF, and pHo was measured with a glass electrode. In all studies, Ca(2+)-dependent basal tension (in the absence of any agonist) and tension in the presence of arginine vasopressin were investigated. Control solution was physiological saline bubbled with 5% CO2 and containing 25 mmol/L HCO3- (pH 7.45 to 7.50). Induction of hypercapnic acidosis (10% CO2) or normocapnic acidosis (15 mmol/L HCO3-) caused significant inhibition of smooth muscle tension, and both conditions reduced pHi as well as pHo. N-Nitro-L-arginine significantly inhibited the relaxation to hypercapnic acidosis but had no significant effect on relaxation to normocapnic acidosis. Predominant extracellular acidosis, induced by reducing [HCO3-] from 25 to 9 mmol/L and CO2 from 5% to 2.5%, also caused inhibition of tension in steady state. By contrast, predominant intracellular acidosis, induced by increasing [HCO3-] from 25 to 65 mmol/L and CO2 from 5% to 15%, induced a small increase of basal tension and a small decrease of tension in the presence of arginine vasopressin. The responses to predominant intracellular or extracellular acidosis were qualitatively similar in the presence and absence of endothelium and in the presence and absence of N-nitro-L-arginine. It is concluded that the extracellular acidosis and not smooth muscle intracellular acidosis is responsible for the relaxation to hypercapnic acidosis.