Mark Alan Winter

Structural characteristics of compounds that modulate P-glycoprotein-associated multidrug resistance

Multidrug resistance is mediated by a membrane-bound protein, P-gp, that functions as an energy dependent efflux system to reduce the intracellular concentration of anticancer drugs by binding to these drugs and actively exporting them from the cell. Compounds that interact with P-gp and compete with anticancer drug binding modulate the degree of drug resistance and therefore enhance the cytotoxicity of anticancer drugs against the resistant cell. Effective modulators share certain physical and chemical properties including octanol/water partitioning and molecular size, but the physical properties of size and shape seem to correlate best with modulator effectiveness. Using a photoactivatable analog of vinblastine as a probe, together with a semi-synthetic series of structurally homologous reserpine and yohimbine analogs, the need for two planar aromatic domains and a basic nitrogen atom was established within the structural context of these compounds. The use of three-dimensional comparisons was extended to examine important structural features in other modulator types such as the condensed-ring aromatics. This approach indicates that structural similarities between different classes of compounds are present in compounds recognized by the MDR phenotype. These studies emphasize the importance of a ligand-receptor relationship for modulators of MDR, and begin to define the P-gp-binding pharmacophore. It is likely that this approach will be useful in directing the de novo synthesis of compounds that modulate MDR and help to further define the requirements for molecular recognition by this system.

Effect of modulators on the ATPase activity and vanadate nucleotide trapping of human P-glycoprotein.

P-Glycoprotein (Pgp) is responsible for the energy-dependent efflux of many natural product oncolytics. Overexpression of Pgp may result in multidrug resistance (MDR). Modulators can block Pgp efflux and sensitize multidrug resistant cells to these oncolytics. To study the interaction of modulators with Pgp, Pgp-ATPase activity was examined, using plasma membranes isolated from the multidrug-resistant cell line CEM/VLB100. A survey of modulators indicated that verapamil, trifluoperazine, and nicardipine stimulated ATPase activity by 1.3- to 1.8-fold, whereas two others, trimethoxybenzoylyohimbine (TMBY) and vindoline, had no effect. Further evaluation showed that TMBY completely blocked the stimulation by verapamil of ATPase activity by competitive inhibition, with a Ki of 2.1 microM. When the effects of these two modulators on the formation of the enzyme-nucleotide complex important in the catalytic cycle were examined, verapamil increased the amount of vanadate-trapped 8-azido-[alpha-32P]ATP bound to Pgp by two-fold, whereas TMBY had no effect. Moreover, TMBY blocked the verapamil stimulation of vanadate-8-azido-[alpha-32P]ATP. Together, these data indicate that verapamil and TMBY bind to Pgp at a common site or overlapping sites, but only verapamil results in enhanced Pgp-ATP hydrolysis and formation of the vanadate-nucleotide-enzyme complex.

Reversal of resistance in multidrug resistance protein (MRP1)-overexpressing cells by LY329146.

The benzothiophene LY329146 reverses the drug resistance phenotype in multidrug resistance protein (MRP1)-overexpressing cells when dosed in combination with MRP1-associated oncolytics doxorubicin and vincristine. Additionally, LY329146 inhibited MRP1-mediated uptake of the MRP1 substrate LTC4 into membrane vesicles prepared from MRP1-overexpressing cells.

GluK1 antagonists from 6-(tetrazolyl)phenyl decahydroisoquinoline derivatives: in vitro profile and in vivo analgesic efficacy.

We have explored the decahydroisoquinoline scaffold, bearing a phenyl tetrazole, as GluK1 antagonists with potential as oral analgesics. We have established the optimal linker atom between decahydroisoquinoline and phenyl rings and demonstrated an improvement of both the affinity for the GluK1 receptor and the selectivity against the related GluA2 receptor with proper phenyl substitution. In this Letter, we also disclose in vivo data that led to the discovery of LY545694·HCl, a compound with oral efficacy in two persistent pain models.

GluK1 antagonists from 6-(carboxy)phenyl decahydroisoquinoline derivatives. SAR and evaluation of a prodrug strategy for oral efficacy in pain models

The synthesis and structure-activity relationship of decahydroisoquinoline derivatives with various benzoic acid substitutions as GluK1 antagonists are described. Potent and selective antagonists were selected for a tailored prodrug approach in order to facilitate the evaluation of the new compounds in pain models after oral administration. Several diester prodrugs allowed for acceptable amino acid exposure and moderate efficacy in vivo.