A number of multidrug-resistant (MDR) cancer cells have been shown to have acquired an increased capacity to sequester weakly basic anticancer drugs in their lysosomes relative to drug-sensitive counterparts. In this report we have comparatively evaluated the concentrations of the anticancer agent daunorubicin (DNR) in intracellular compartments of drug-sensitive and MDR HL-60 cell lines, both of which do not express common efflux transporters such as P-glycoprotein at the plasma membrane. Our results suggest that lysosomal sequestration plays a significant role in the emergence of MDR since it effectively limits the drug's ability to interact with target molecules located in the nucleus. Using a series of weakly basic structural isomers with variable basicity, we illustrate that the magnitude of the pKa value correlates with the degree of lysosomal sequestration. Accordingly, a series of structurally modified forms of DNR with reduced basicity were synthesized, and their intracellular distribution was evaluated. Consistent with model compounds, derivatives of DNR with lowered pKa values showed visibly reduced lysosomal sequestration in two separate MDR cell lines. Collectively, this work highlights the importance of understanding the intracellular localization of drugs and proposes a rational strategy to manipulate it.

The degree to which anticancer agents selectively target cancer cells is a key determinant in successful therapeutic outcomes. Inhibitors of the Hsp90 molecular chaperone represent an important new class of anticancer agents. We propose here a novel mechanism by which physiochemical properties of Hsp90 inhibitors can be optimized to increase selectivity towards cancer cells. The basis for this approach relies on differential intracellular pH gradients that have been shown to exist between normal and transformed cells. Five Hsp90 inhibitors containing basic or neutral properties were evaluated in antiproliferation assays using cells with variable lysosomal pH. Inhibitors with basic functionalities had reduced activity in cells with normal (low) lysosomal pH but showed significantly greater activity in cells with abnormally elevated lysosomal pH (similar to what has been recorded in many types of cancer cells). Conversely, such selectivity enhancement was not observed for neutral inhibitors. The mechanistic basis for the observed selectivity was demonstrated quantitatively by determining the concentration of inhibitors within relevant intracellular compartments. Collectively, these findings suggest that Hsp90 inhibitors with optimal basicity and physicochemical properties have enhanced selectivity toward cancer cells than their neutral counterparts. It is anticipated that these findings may be applicable to other classes of anticancer agents for improvement of differential selectivity.

[a] Mammalian fertilization depends upon successful binding and fusion of the membranes of the spermatozoon and the oocyte. These processes are thought to be mediated by a series of protein ± protein interactions in which sperm proteins known as ADAMs (A disintegrin and metalloproteinases) play a key role. [1,2] Fertilinb and cyritestin are members of the ADAM family and their disintegrin domains mediate sperm ± egg binding.[3±6] Two different integrins have been implicated as fertilinb receptors: a 6 b 1 and a 9 b 1 [5, 7±11] The proposed identity of the receptor depends on the experimental approach utilized. However, integrin knockout experiments in the mouse egg call into question whether either a 6 b 1 or a 9 b 1 integrin is the only fertilinb receptor.[12] These genetic experiments suggest that a member of a nonintegrin receptor family may be sufficient for sperm ± egg binding and fusion. The cyritestin receptor has not yet been identified. Thus, there is much uncertainty about the identity and number of sperm receptors on the egg.Peptides derived from the fertilinb and cyritestin disintegrin binding loops inhibit sperm ± egg binding and subsequent fusion in vitro. Mimics of these peptides may be used to probe biological function, for example, to establish the identity of the receptor(s) and to elucidate the role of individual ligand proteins. However, for the probes to be useful, they must have high affinity for their target. Both linear peptides [13±16] and existing peptide mimics [17] of fertilinb and cyritestin are modest inhibitors of sperm ± egg binding, with IC 50 values of around 500 mM. We report herein that norbornyl homopolymers containing 10 pendent fertilinb-derived oligopeptides are significantly improved inhibitors of adhesion and fertilization in vitro and work in a multivalent fashion.We hypothesized that the modest inhibition observed with linear peptides might be due to a monovalent binding interaction that is of low affinity. We previously determined that liposomes bearing 80 copies of fertilinb peptide per outer membrane are 100-fold improved inhibitors of fertilization compared to linear peptides, [18] which demonstrates that polyvalency does effect adhesion in this system. In this work, we further investigated the role of polyvalency in the sperm ± egg adhesion process by synthesizing multivalent peptide mimics that not only allow control of the effective concentration of ligands presented, but are also different macromolecular sizes.The approach adopted was to synthesize peptide polymers by ring-opening metathesis polymerization (ROMP) of norbornyl oligopeptides with Grubbs' ruthenium carbene catalyst, Cl 2 (PCy 3 ) 2 RuCHPh (5). In addition to its functional group tolerance, 5 catalyzes living polymerizations that allow the preparation of polymers of defined molecular weight and of block copolymers.[19] Several strategies have been employed to prepare polynorbornenes substituted with bioactive, watersoluble molecules by using Grubbs' catalyst. We used homogenous polymerization ...

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