Marcos M. Pires

Fluorescence Imaging of Cellular Glutathione Using a Latent Rhodamine

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.

Self-assembly of Collagen Peptides into Microflorettes via Metal Coordination

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.

Inhibition of P-Glycoprotein-Mediated Paclitaxel Resistance by Reversibly Linked Quinine Homodimers

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 (IC50 ≈ 1.7 μM). 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 (t½ ≈ 20 h) while demonstrating high resistance to nonenzymatic hydrolysis in cell culture media (t½ ≈ 21 days). Specific inhibition of [125I]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.

Metal-mediated tandem coassembly of collagen peptides into banded microstructures.

The ability to recapitulate the features of natural collagen at the micro- and nanoscale with novel biopolymers has the potential to lead to improved biomaterials. Herein we describe stimuli-responsive collagen-based peptides (IdaCol and HisCol) that together form higher order assemblies in the presence of added metal ions. SEM and TEM imaging of these assemblies revealed microscale petal-like and intertwined fiber morphologies, each with periodic banding on the nanometer scale. The observed banding is consistent with tandem coassembly of alternating IdaCol and HisCol triple helical blocks that may laterally associate either in or out of register to form higher order structures, and mimics the banding found in natural collagen fibers.

Structural, Energetic, and Infrared Spectra Insights into Methanol Clusters (CH3OH)n, for n = 2−12, 16, 20. ONIOM as an Efficient Method of Modeling Large Methanol Clusters

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.

Controlling the Morphology of Metal-Promoted Higher Ordered Assemblies of Collagen Peptides with Varied Core Lengths

Self-assembling peptides have become an important subclass of next-generation biomaterials. In particular, materials that mimic the properties of collagen have received considerable attention due to the unique properties of natural collagen. Previous peptide-based designs have been successful in generating structures with morphological properties that were primarily determined by the type of self-assembling mechanism. Herein we demonstrate the metal ion-promoted, supramolecular assembly of collagen-based peptide triple helices into distinct morphologies that are controlled by defining the number of Pro-Hyp-Gly repeating units. We synthesized and characterized collagen-based peptides that incorporated either 5, 7, 9, or 11 Pro-Hyp-Gly repeating units. We found that the number of repeating units, and the resulting stability of the collagen triple helix, is intimately linked with the types of assemblies formed. For instance, collagen peptides that did not form a stable triple helix, such as NCoH5, did not participate in supramolecular assembly with added metal ions. Collagen peptides that formed stable triple helices, such as NCoH11, resulted in microsaddle structures with metal-promoted assembly, whereas a highly cross-linked, three-dimensional mesh formed with NCoH7, albeit at a higher metal ion concentration. These data provide evidence that triple helix formation is required for efficient metal-triggered assembly to the observed microstructures.

D-amino acid mediated recruitment of endogenous antibodies to bacterial surfaces.

The number of antibiotic resistant bacterial strains has been continuously increasing over the last few decades. Nontraditional routes to combat bacteria may offer an attractive alternative to the ongoing problem of drug discovery in this field. Herein, we describe the initial framework toward the development of bacterial d-amino acid antibody recruitment therapy (DART). DART represents a promising antibiotic strategy by exploiting the promiscuity of bacteria to incorporate unnatural d-amino acids and subsequently recruit antibodies to the bacterial surface. The conjugation of 2,4-dinitrophenyl (DNP) to various d-amino acids led to the discovery of a d-amino acid that specifically tags the surface of Bacillus subtilis and Staphylococcus aureus for the recruitment of anti-DNP antibodies (a highly abundant antibody in human serum). This system represents a novel strategy as an antibacterial therapy that targets planktonic Gram-positive bacteria.

Inhibition of human P-glycoprotein transport and substrate binding using a galantamine dimer

The human multidrug resistance transporter P-glycoprotein (P-gp) prevents the entry of compounds into the brain by an active efflux mechanism at the blood-brain barrier (BBB). Treatment of neurodegenerative diseases, therefore, has become a challenge and the development of new reversible inhibitors of P-gp is pertinent to overcome this problem. We report the design and synthesis of a crosslinked agent based on the Alzheimers disease treatment galantamine (Gal-2) that inhibits P-gp-mediated efflux from cultured cells. Gal-2 was found to inhibit the efflux of the fluorescent P-gp substrate rhodamine 123 in cancer cells that over-express P-gp with an IC(50) value of approximately 0.6 microM. In addition, Gal-2 was found to inhibit the efflux of therapeutic substrates of P-gp, such as doxorubicin, daunomycin and verapamil with IC(50) values ranging from 0.3 to 1.6 microM. Through competition experiments, it was determined that Gal-2 modulates P-gp mediated efflux by competing for the substrate binding sites. These findings support a potential role of agents, such as Gal-2, as inhibitors of P-gp at the BBB to augment treatment of neurodegenerative diseases.

d-Amino Acids Do Not Inhibit Biofilm Formation in Staphylococcus aureus

Bacteria can either exist in the planktonic (free floating) state or in the biofilm (encased within an organic framework) state. Bacteria biofilms cause industrial concerns and medical complications and there has been a great deal of interest in the discovery of small molecule agents that can inhibit the formation of biofilms or disperse existing structures. Herein we show that, contrary to previously published reports, d-amino acids do not inhibit biofilm formation of Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), and Staphylococcus epidermis (S. epidermis) at millimolar concentrations. We evaluated a diverse set of natural and unnatural d-amino acids and observed no activity from these compounds in inhibiting biofilm formation.

Metabolic Profiling of Bacteria by Unnatural C‐terminated D‐Amino Acids

Bacterial peptidoglycan is a mesh-like network comprised of sugars and oligopeptides. Transpeptidases cross-link peptidoglycan oligopeptides to provide vital cell wall rigidity and structural support. It was recently discovered that the same transpeptidases catalyze the metabolic incorporation of exogenous D-amino acids onto bacterial cell surfaces with vast promiscuity for the side-chain identity. It is now shown that this enzymatic promiscuity is not exclusive to side chains, but that C-terminus variations can also be accommodated across a diverse range of bacteria. Atomic force microscopy analysis revealed that the incorporation of C-terminus amidated D-amino acids onto bacterial surfaces substantially reduced the cell wall stiffness. We exploited the promiscuity of bacterial transpeptidases to develop a novel assay for profiling different bacterial species.