The self-assembly of synthetic biomaterials, such as collagen peptides, can be harnessed for a range of biomedical applications. In an effort to obtain collagen-based macromolecular assemblies with temporal control, we designed a system that assembled only in the presence of external stimuli. We report a collagen triple helical peptide that is modified with a His(2) moiety on its C-terminus and a nitrilotriacetic acid unit on its N-terminus that rapidly and reversibly assembles in the presence of metal ions. Dynamic light scattering and turbidity experiments confirmed the presence of higher order aggregates in solution upon the introduction of Zn(2+), Cu(2+), Ni(2+), and Co(2+). This assembly process was found to be fully reversible using EDTA as a metal ion chelator. Control peptides that contain only a single ligand-modified terminus were not responsive to the same metal ions, thus demonstrating the requirement of both ligand modifications for peptide assembly. Scanning electron microscopy imaging of the peptide-metal assemblies revealed micrometer-sized florettes in addition to curved, stacked sheets. More detailed analysis of the Zn(2+)-generated microflorettes showed that the surface of these particles contains ruffled structures with a highly dense surface area. Potential folding intermediates in the formation of the microflorettes were observed at lower temperatures and at early time points in the assembly that are composed of curved layered sheets. Significantly, the assembly process proceeded under mild conditions using neutrally buffered aqueous solution at room temperature. These microscopic structures offer opportunities in many areas, including drug delivery, tissue engineering, and regenerative medicine.

P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, has been implicated in multidrug resistance of several cancers as a result of its overexpression. In this work, rationally designed second-generation P-gp inhibitors are disclosed, based on dimerized versions of the substrates quinine and quinidine. These dimeric agents include reversible tethers with a built-in clearance mechanism. The designed agents were potent inhibitors of rhodamine 123 efflux in cultured cancer cell lines that display high levels of P-gp expression at the cell surface and in transfected cells expressing P-gp. The quinine homodimer Q2, which was tethered by reversible ester bonds, was particularly potent (IC(50) approximately 1.7 microM). Further studies revealed that Q2 inhibited the efflux of a range of fluorescent substrates (rhodamine 123, doxorubicin, mitoxantrone, and BODIPY-FL-prazosin) from MCF-7/DX1 cells. The reversibility of the tether was confirmed in experiments showing that Q2 was readily hydrolyzed by esterases in vitro (t(1/2) approximately 20 h) while demonstrating high resistance to nonenzymatic hydrolysis in cell culture media (t(1/2) approximately 21 days). Specific inhibition of [(125)I]iodoarylazidoprazosin binding to P-gp by Q2 verified that the bivalent agent interacted specifically with the drug binding site(s) of P-gp. Q2 was also an inhibitor of verapamil-stimulated ATPase activity. In addition, low concentrations of Q2 stimulated basal P-gp ATPase levels. Finally, Q2 was shown to inhibit the transport of radiolabeled paclitaxel (Taxol) in MCF-7/DX1 cells, and it completely reversed the P-gp-mediated paclitaxel resistance phenotype.

Glutathione is a crucial component of the redox homeostasis of cells, and altered levels have been linked to human pathologies. We constructed a latent fluorophore (RhoSS) that responds to cellular thiols in vitro and in cyto following intracellular reduction by glutathione to yield rhodamine 110. Importantly, RhoSS was demonstrated to respond to changing levels of glutathione in cells. This compound represents a class of rationally designed latent fluorophores with exciting potential for monitoring cellular thiols.

An investigation of gas-phase methanol clusters (CH3OH)n, where n = 2-12, 16, and 20, was completed with a range of computational methods:  PM3, Hartree-Fock, B3LYP, MP2, and their combination using an ONIOM (our own n-layered integrated molecular orbital and molecular mechanics) method. Geometries, binding energies, and vibrational frequencies are reported. For all ab initio optimized structures, the cyclic isomer was found to be the most stable structure of all isomers investigated. The scaled OH frequency shift for n = 1-4 is found to be in good agreement with experimentally measured shifts. An ONIOM method, with the methyl group calculated at the low level and the hydroxyl group at the high level, proved to be an excellent way of reducing computational expense. The calculated enthalpies, geometries, and infrared spectra using an ONIOM method were comparable to that of a high-level calculation. Clusters were solvated using the integral equation formalism for the polarized continuum model method to compare with the microsolvation studies.

A small library of bivalent agents was designed to probe the substrate binding sites of the human multidrug transporter P-glycoprotein (P-gp). The bivalent agents were composed of two copies of the P-gp substrate emetine, linked by tethers of varied composition. An optimum distance between the emetine molecules of approximately 10 A was found to be necessary for blocking transport of the known fluorescent substrate rhodamine 123. Additionally, it was determined that hydrophobic tethers were optimal for bridging the bivalent compounds; hydrophilic or cationic moieties within the tether had a detrimental effect on inhibition of transport. In addition to acting as probes of P-gp's drug binding sites, these agents were also potent inhibitors of P-gp. One agent, EmeC5, had IC50 values of 2.9 microM for inhibiting transport of rhodamine 123 and approximately 5 nM for inhibiting the binding of a known P-gp substrate, [125I]iodoarylazidoprazosin. Although EmeC5 is an inhibitor of P-gp and was shown to interact directly with P-gp in one or more of the substrate binding sites, our data suggest that it is either not a P-gp transport substrate itself or a poor one. Most significantly, EmeC5 was shown to reverse the MDR phenotype of MCF-7/DX1 cells when co-administered with a cytotoxic agent, such as doxorubicin.