We describe the discovery of a novel indazole-based scaffold that represents the "first-in-class" dual Hsp90/tubulin binding compound. Individual known ligands for both targets shared similar 3',4',5'-trimethoxyphenyl cores, and from this it was hypothesized that application of an integrated ligand and structure-based virtual screening (VS) workflow could yield a single scaffold with dual binding affinity. Following validation of the VS protocol, we successfully identified a novel dual inhibitor, sourced from a commercial screening collection of 160 000 compounds.

Multidrug resistance is a complex pleiotropic phenotype of cross-resistance and collateral sensitivity to unrelated compounds observed in many mammalian cell mutants selected for resistance to single agents. In Chinese hamster ovary cells, colchicine-resistant mutants expressing this phenotype have been characterized extensively. Such mutants arise apparently from a single genetic event, and the basis of this phenotype appears to be localized at the membrane level, resulting in altered drug permeability. Expression of a 170,000-dalton surface glycoprotein (P-glycoprotein) has been identified to correlate with the multidrug-resistance phenotype. Selection of a second mutation in colchicine-resistant mutants, for resistance to phytohemagglutinin, results in an alteration of the carbohydrate moiety in P-glycoprotein and other surface components. This mutation does not noticeably affect the multi-drug-resistance phenotype. The altered permeability of mutant cells to drugs, however, can be modulated by nonionic detergents or metabolic inhibitors. These findings are consistent with a molecular mechanism of multidrug resistance whereby the pleiotropic response of the cell is mediated by an overexpression of a cell-surface protein, the P-glycoprotein.

The microtubule cytoskeleton is a highly regulated system. At different times in the cell cycle and positions within the organism, microtubules can be very stable or highly dynamic. Stability and dynamics are regulated by interaction with a large number of proteins that themselves may change at specific points in the cell cycle. Exogenous ligands can disrupt the normal processes by either increasing or decreasing microtubule stability and inhibiting their dynamic behavior. The recent determination of the structure of tubulin, the main component of microtubules, makes it possible now to begin to understand the details of these interactions. We review here the structure of the tubulin dimer, with particular regard to how proteins and drugs may bind and modulate microtubule dynamics.

Considerable uncertainty surrounds the stoichiometry of coupling of ATP hydrolysis to drug pumping by P-glycoprotein, the multidrug transporter. To estimate relative turnovers for pumping of the drug vinblastine and ATP hydrolysis, we began by measuring the number of P-glycoprotein molecules on the surface of murine NIH3T3 cells expressing the human MDR1 gene. Fluorescence of cells treated with monoclonal antibody UIC2 was determined as a function of (i) amount of antibody at a fixed number of cells and (ii) increasing cell number at constant antibody. The two together gives 1.95 x 10(6) P-glycoprotein molecules/cell. Initial uptake rates of vinblastine +/- verapamil measure the ability of P-glycoprotein to extract vinblastine from the plasma membrane before it enters the cell. As a function of [vinblastine] at 37 degrees C, they give the maximum rate of this component of outward pumping as 2.1 x 10(6) molecules s-1 cell-1 or a turnover number of 1.1 s-1. Initial rates of one-way efflux as a function of [vinblastine] at 25 degrees C +/- glucose give the maximum rate of this component of pumping as 0.59 x 10(6) molecules s-1 cell-1. The ratio of ATPase activity of P-glycoprotein at 37 and 25 degrees C is 4.6. Appropriating this ratio for pumping, maximum one-way efflux at 37 degrees C is 4.6 x 0.59 = 2.7 x 10(6) molecules s-1 cell-1, a turnover number of 1.4 s-1. The vinblastine-stimulated ATPase activity of P-glycoprotein has a turnover number of 3.5 s-1 at 37 degrees C, giving 2.8 molecules of ATP hydrolyzed for every vinblastine molecule transported in a particular direction. These calculations involve several approximations, but turnover numbers for pumping of vinblastine and for vinblastine-stimulated ATP hydrolysis are comparable. Thus, ATP hydrolysis is probably directly linked to drug transport by P-glycoprotein.

The PC12 line of nerve growth factor (NGF)-responsive rat pheochromocytoma cells was used as a model system to determine whether properties of microtubules change during neurite growth and maturation. In the absence of NGF, PC12 cells lack processes. After several days with NGF, PC12 cells begin extending neurites and, by 2-3 wk with NGF, PC12 cells have long (approximately 1 mm), highly branched neurites. We examined the effect of colchicine on microtubules of PC12 cells grown without NGF or with NGF for 1 or 21 d. PC12 cells grown under the various conditions were exposed to 50 microM colchicine for 1 or 6 h, and were then assayed for their content of polymerized tubulin using a biochemical assay. Microtubule levels in drug-treated cultures were compared to those in non-drug-treated control sister cultures. PC12 cells grown without NGF or with NGF for 1 d were depleted of MT by 1 h with colchicine. In contrast, microtubule levels in long-term NGF-treated cells exposed to colchicine for 6 h were reduced to only approximately 57% of those in control cells. Control experiments indicated that the observed differential susceptibility to colchicine was not due to differences in colchicine uptake or to the effects of colchicine on cell viability. These observations suggest that microtubules of PC12 cells grown without NGF or with NGF for 21 d differ in their properties. Such differences may be related to one or more of the changes in structure and/or motility that result from treatment with NGF.

We identified a thiol-reactive compound, dibromobimane (dBBn), that was a potent stimulator (8.2-fold) of the ATPase activity of Cys-less P-glycoprotein. We then used this compound together with cysteine-scanning mutagenesis to identify residues in transmembrane segment (TM) 6 and TM12 that are important for function. TM6 and TM12 lie close to each other in the tertiary structure and are postulated to be important for drug-protein interactions. The majority of P-glycoprotein mutants containing a single cysteine residue retained substantial amounts of drug-stimulated ATPase activity and were not inhibited by dBBn. The ATPase activities of mutants L339C, A342C, L975C, V982C, and A985C, however, were markedly inhibited (>60%) by dBBn. The drug substrates verapamil, vinblastine, and colchicine protected these mutants against inhibition by dBBn, suggesting that these residues are important for interaction of substrates with P-glycoprotein. We previously showed that residues Leu339, Ala342, Leu975, Val982, and Ala985 lie along the point of contact between helices TM6 and TM12, when both are aligned in a left-handed coiled coil (Loo, T. W., and Clarke, D. M.(1997) J. Biol. Chem. 272, 20986-20989). Taken together, these results suggest that the interface between TM6 and TM12 likely forms part of the potential drug-binding pocket in P-glycoprotein.