Metal-Promoted Higher-Order Assembly of Disulfide-Stapled Helical Barrels.

Peptide-based helical barrels are a noteworthy building block for hierarchical assembly, with a hydrophobic cavity that can serve as a host for cargo. In this study, disulfide-stapled helical barrels were synthesized containing ligands for metal ions on the hydrophilic face of each amphiphilic peptide helix. The major product of the disulfide-stapling reaction was found to be composed of five amphiphilic peptides, thereby going from a 16-amino-acid peptide to a stapled 80-residue protein in one step. The structure of this pentamer, 5HB1, was optimized in silico, indicating a significant hydrophobic cavity of ~6 Å within a helical barrel. Metal-ion-promoted assembly of the helical barrel building blocks generated higher order assemblies with a three-dimensional (3D) matrix morphology. The matrix was decorated with hydrophobic dyes and His-tagged proteins both before and after assembly, taking advantage of the hydrophobic pocket within the helical barrels and coordination sites within the metal ion-peptide framework. As such, this peptide-based biomaterial has potential for a number of biotechnology applications, including supplying small molecule and protein growth factors during cell and tissue growth within the matrix.

Metal-Assembled Collagen Peptide Microflorettes as Magnetic Resonance Imaging Agents.

Magnetic resonance imaging (MRI) is a medical imaging technique that provides detailed information on tissues and organs. However, the low sensitivity of the technique requires the use of contrast agents, usually ones that are based on the chelates of gadolinium ions. In an effort to improve MRI signal intensity, we developed two strategies whereby the ligand DOTA and Gd(III) ions are contained within Zn(II)-promoted collagen peptide (NCoH) supramolecular assemblies. The DOTA moiety was included in the assembly either via a collagen peptide sidechain (NHdota) or through metal-ligand interactions with a His-tagged DOTA conjugate (DOTA-His6). SEM verified that the morphology of the NCoH assembly was maintained in the presence of the DOTA-containing peptides (microflorettes), and EDX and ICP-MS confirmed that Gd(III) ions were incorporated within the microflorettes. The Gd(III)-loaded DOTA florettes demonstrated higher intensities for the T1-weighted MRI signal and higher longitudinal relaxivity (r1) values, as compared to the clinically used contrast agent Magnevist. Additionally, no appreciable cellular toxicity was observed with the collagen microflorettes loaded with Gd(III). Overall, two peptide-based materials were generated that have potential as MRI contrast agents.

Recent advances in coiled-coil peptide materials and their biomedical applications.

Extensive research has gone into deciphering the sequence requirements for peptides to fold into coiled-coils of varying oligomeric states. More recently, additional signals have been introduced within coiled-coils to promote higher order assembly into biomaterials with a rich distribution of morphologies. Herein we describe these strategies for association of coiled-coil building blocks and biomedical applications. With many of the systems described herein having proven use in protein storage, cargo binding and delivery, three dimensional cell culturing and vaccine development, the future potential of coiled-coil materials to have significant biomedical impact is highly promising.

Co-assembled Coiled-Coil Peptide Nanotubes with Enhanced Stability and Metal-Dependent Cargo Loading.

Peptide nanotube biomaterials are attractive for their range of applications. Herein, we disclose the co-assembly of coiled-coil peptides, one with ligands for metal ions that demonstrate hierarchical assembly into nanotubes, with spatial control of the metal-binding ligands. Enhanced stability of the nanotubes to phosphate-buffered saline was successfully accomplished in a metal-dependent fashion, depending on the levels and placement of the ligand-containing coiled-coil peptide. This spatial control also allowed for site-specific labeling of the nanotubes with His-tagged fluorophores through the length of the tubes or at the termini, in a metal-dependent manner.

Supramolecular Assembly of His-Tagged Fluorescent Protein Guests within Coiled-Coil Peptide Crystal Hosts: Three-Dimensional Ordering and Protein Thermal Stability.

The use of biomaterials for the inclusion and stabilization of biopolymers is an ongoing challenge. Herein, we disclose three-dimensional (3D) coiled-coil peptide crystals with metal ions that include and overgrow His-tagged fluorescent proteins within the crystal. The protein guests are found within two symmetry-related growth sectors of the crystalline host that are associated with faces of the growing crystal that display ligands for metal ions. The fluorescent proteins are included within this "hourglass" region of the crystals at a notably high level, display order within the crystal hosts, and demonstrate sufficiently tight packing to enable energy transfer between a donor-acceptor pair. His-tagged fluorescent proteins display remarkable thermal stability to denaturation over extended periods of time (days) at high temperatures when within the crystals. Ultimately, this strategy may prove useful for the prolonged storage of thermally sensitive biopolymer guests within a 3D crystalline matrix.

Reversible crosslinked assembly of a trimeric coiled-coil peptide into a three-dimensional matrix for cell encapsulation and release.

Mimicking the extracellular matrix (ECM) continues to be a goal in the field of regenerative medicine. Herein, we report a modified trimeric GCN4 coiled-coil sequence containing three ligands for metal ions specifically positioned for crosslinked assembly (TriCross). In the presence of metal ions, TriCross assembles into a three-dimensional (3D) matrix with significant cavities to accommodate cells. The matrix was found to be stable in media with serum, and mild removal of the metal leads to disassembly. By assembling TriCross with a suspension of cells in media, the matrix encapsulates cells during the assembly process leading to high cell viability. Further disassembly under mild conditions allows for the release of cells from the scaffold. As such, this peptide-based material displays many of the characteristics necessary for successful 3D cell culture.

Formation of Microcages from a Collagen Mimetic Peptide via Metal-Ligand Interactions.

Here, the hierarchical assembly of a collagen mimetic peptide (CMP) displaying four bipyridine moieties is described. The CMP was capable of forming triple helices followed by self-assembly into disks and domes. Treatment of these disks and domes with metal ions such as Fe(II), Cu(II), Zn(II), Co(II), and Ru(III) triggered the formation of microcages, and micron-sized cup-like structures. Mechanistic studies suggest that the formation of the microcages proceeds from the disks and domes in a metal-dependent fashion. Fluorescently-labeled dextrans were encapsulated within the cages and displayed a time-dependent release using thermal conditions.

Antibiotic-cell-penetrating peptide conjugates targeting challenging drug-resistant and intracellular pathogenic bacteria.

The failure to treat everyday bacterial infections is a current threat as pathogens are finding new ways to thwart antibiotics through mechanisms of resistance and intracellular refuge, thus rendering current antibiotic strategies ineffective. Cell-penetrating peptides (CPPs) are providing a means to improve antibiotics that are already approved for use. Through coadministration and conjugation of antibiotics with CPPs, improved accumulation and selectivity with alternative and/or additional modes of action against infections have been observed. Herein, we review the recent progress of this antibiotic-cell-penetrating peptide strategy in combatting sensitive and drug-resistant pathogens. We take a closer look into the specific antibiotics that have been enhanced, and in some cases repurposed as broad-spectrum drugs. Through the addition and conjugation of cell-penetrating peptides to antibiotics, increased permeation across mammalian and/or bacterial membranes and a broader range in bacterial selectivity have been achieved.

The roles of the human ATP-binding cassette transporters P-glycoprotein and ABCG2 in multidrug resistance in cancer and at endogenous sites: future opportunities for structure-based drug design of inhibitors.

The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp) and ABCG2 are multidrug transporters that confer drug resistance to numerous anti-cancer therapeutics in cell culture. These findings initially created great excitement in the medical oncology community, as inhibitors of these transporters held the promise of overcoming clinical multidrug resistance in cancer patients. However, clinical trials of P-gp and ABCG2 inhibitors in combination with cancer chemotherapeutics have not been successful due, in part, to flawed clinical trial designs resulting from an incomplete molecular understanding of the multifactorial basis of multidrug resistance (MDR) in the cancers examined. The field was also stymied by the lack of high-resolution structural information for P-gp and ABCG2 for use in the rational structure-based drug design of inhibitors. Recent advances in structural biology have led to numerous structures of both ABCG2 and P-gp that elucidated more clearly the mechanism of transport and the polyspecific nature of their substrate and inhibitor binding sites. These data should prove useful helpful for developing even more potent and specific inhibitors of both transporters. As such, although possible pharmacokinetic interactions would need to be evaluated, these inhibitors may show greater effectiveness in overcoming ABC-dependent multidrug resistance in combination with chemotherapeutics in carefully selected subsets of cancers. Another perhaps even more compelling use of these inhibitors may be in reversibly inhibiting endogenously expressed P-gp and ABCG2, which serve a protective role at various blood-tissue barriers. Inhibition of these transporters at sanctuary sites such as the brain and gut could lead to increased penetration by chemotherapeutics used to treat brain cancers or other brain disorders and increased oral bioavailability of these agents, respectively.

Conservation of Cdc14 phosphatase specificity in plant fungal pathogens: implications for antifungal development.

Cdc14 protein phosphatases play an important role in plant infection by several fungal pathogens. This and other properties of Cdc14 enzymes make them an intriguing target for development of new antifungal crop treatments. Active site architecture and substrate specificity of Cdc14 from the model fungus Saccharomyces cerevisiae (ScCdc14) are well-defined and unique among characterized phosphatases. Cdc14 appears absent from some model plants. However, the extent of conservation of Cdc14 sequence, structure, and specificity in fungal plant pathogens is unknown. We addressed this by performing a comprehensive phylogenetic analysis of the Cdc14 family and comparing the conservation of active site structure and specificity among a sampling of plant pathogen Cdc14 homologs. We show that Cdc14 was lost in the common ancestor of angiosperm plants but is ubiquitous in ascomycete and basidiomycete fungi. The unique substrate specificity of ScCdc14 was invariant in homologs from eight diverse species of dikarya, suggesting it is conserved across the lineage. A synthetic substrate mimetic inhibited diverse fungal Cdc14 homologs with similar low µM Ki values, but had little effect on related phosphatases. Our results justify future exploration of Cdc14 as a broad spectrum antifungal target for plant protection.

Targeting Intracellular Pathogenic Bacteria Through N-Terminal Modification of Cationic Amphiphilic Polyproline Helices.

Intracellular pathogens can thrive within mammalian cells and are inaccessible to many antimicrobial agents. Herein, we present a facile method of enhancing the cell penetrating and antibacterial properties of cationic amphiphilic polyproline helices (CAPHs) with modifications to the hydrophobic moiety at the N-terminus. These altered CAPHs display superior cell penetration within macrophage cells, and in some cases, minimal cytotoxicity. Furthermore, one CAPH, Pentyl-P14 exhibited excellent antibacterial activity against multiple strains of pathogenic bacteria and promoted the clearance of intracellular Shigella within macrophages.

Fluorescent Probes for Monitoring Serine Ubiquitination.

In a radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD+ to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD+ analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.

Potential Tools for Eradicating HIV Reservoirs in the Brain: Development of Trojan Horse Prodrugs for the Inhibition of P-Glycoprotein with Anti-HIV-1 Activity.

Combination antiretroviral therapy is the mainstay of HIV treatment, lowering plasma viral levels below detection. However, eradication of HIV is a major challenge due to cellular and anatomical viral reservoirs that are often protected from treatment by efflux transporters, such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Herein we described a Trojan horse approach to therapeutic evasion of P-gp based on a reversibly linked combination of HIV reverse transcriptase and protease inhibitors. Potent inhibition of P-gp efflux in cells, including human brain endothelial cells, was observed with the linked heterodimeric compounds. In vitro regeneration of active monomeric drugs was observed in a reducing environment with these dimeric prodrugs, with the superior leaving group promoting more facile release from the tether. These release trends were mirrored in the efficacy of the in cyto anti-HIV-1 activity of the Trojan horse heterodimers.

Self-Assembling Coiled-Coil Peptide Nanotubes with Biomolecular Cargo Encapsulation.

We report a coiled-coil trimeric peptide based on the known GCN4 zipper motif that self-assembles into nanotubes with diameters on the order of a few hundred nanometers to a micron. The dimensional morphology of these tubular structures was observed to be tunable by altering the properties of the buffer during assembly. Structural evidence from X-ray scattering and electron microscopy suggest that tube assembly takes place in structural tiers leading to a hexagonal close-packed arrangement of the coiled-coils. The hollow tubes were observed to selectively encapsulate fluorescein-labeled anionic dextran, as a result of electrostatic interactions between the inner tube surface and negatively charged cargo. The ability of these nanotubes to house biomolecular cargo endows them with potential for molecular storage and drug delivery applications.

Reversible Hierarchical Assembly of Trimeric Coiled-Coil Peptides into Banded Nano- and Microstructures.

We report a set of coiled-coil peptides, radially functionalized with bipyridines, that demonstrate hierarchical assembly into banded rectangular nano- and microstructures, the dimensions of which vary with the strategic placement and number of aromatic groups on the monomer backbone. Finer structural aspects of the hexagonal packing of the individual trimers were determined by X-ray scattering, including intertrimer aromatic interactions between bipyridine moieties. The ease of formation of these biomaterials under physiological conditions and the use of pH to reversibly modulate assembly demonstrate future potential for a range of biological applications, such as drug delivery in a pH-controlled manner.

A Library Approach to Cationic Amphiphilic Polyproline Helices that Target Intracellular Pathogenic Bacteria.

A number of pathogenic bacteria reproduce inside mammalian cells and are thus inaccessible to many antimicrobial drugs. Herein, we present a facile method to a focused library of antibacterial agents known as cationic amphiphilic polyproline helices (CAPHs). We identified three CAPHs from the library with superior cell penetration within macrophages and excellent antibacterial action against both Gram-positive and Gram-negative bacteria. These cell-penetrating antibacterial CAPHs have specific subcellular localizations that allow for targeting of pathogenic bacteria at their intracellular niches, a unique feature that promotes the successful clearance of intracellular pathogens ( Salmonella, Shigella, and Listeria) residing within macrophages. Furthermore, the selected CAPHs also significantly reduced bacterial infections in an in vivo model of Caenorhabditis elegans, with minimal in vivo toxicity.

A short D-enantiomeric antimicrobial peptide with potent immunomodulatory and antibiofilm activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii.

Antimicrobial peptides (AMPs) represent a promising therapeutic alternative for the treatment of antibiotic-resistant bacterial infections. The present study investigates the antimicrobial activity of new, rationally-designed derivatives of a short α-helical peptide, RR. From the peptides designed, RR4 and its D-enantiomer, D-RR4, emerged as the most potent analogues with a more than 32-fold improvement in antimicrobial activity observed against multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii. Remarkably, D-RR4 demonstrated potent activity against colistin-resistant strains of P. aeruginosa (isolated from cystic fibrosis patients) indicating a potential therapeutic advantage of this peptide over several AMPs. In contrast to many natural AMPs, D-RR4 retained its activity under challenging physiological conditions (high salts, serum, and acidic pH). Furthermore, D-RR4 was more capable of disrupting P. aeruginosa and A. baumannii biofilms when compared to conventional antibiotics. Of note, D-RR4 was able to bind to lipopolysaccharide to reduce the endotoxin-induced proinflammatory cytokine response in macrophages. Finally, D-RR4 protected Caenorhabditis elegans from lethal infections of P. aeruginosa and A. baumannii and enhanced the activity of colistin in vivo against colistin-resistant P. aeruginosa.

Dual inhibitors of the human blood-brain barrier drug efflux transporters P-glycoprotein and ABCG2 based on the antiviral azidothymidine.

The brain provides a sanctuary site for HIV due, in part, to poor penetration of antiretroviral agents at the blood-brain barrier. This lack of penetration is partially attributed to drug efflux transporters such as P-glycoprotein (P-gp) and ABCG2. Inhibition of both ABCG2 and P-gp is critical for enhancing drug accumulation into the brain. In this work, we have developed a class of homodimers based on the HIV reverse transcriptase inhibitor azidothymidine (AZT) that effectively inhibits P-gp and ABCG2. These agents block transporter mediated efflux of the P-gp substrate calcein-AM and the ABCG2 substrate mitoxantrone. The homodimers function by interacting with the transporter drug binding sites as demonstrated by competition studies with the photo-affinity agent and P-gp/ABCG2 substrate [125I]iodoarylazidoprazosin. As such, these dual inhibitors of both efflux transporters provide a model for the future development of delivery vehicles for antiretroviral agents to the brain.