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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Metal ion‐mediated assembly of peptides is an intriguing method for developing novel biomaterials. In particular, peptide building blocks based on collagen triple helical and coiled‐coil peptides have been functionalized with metal‐binding ligands and assemble into hierarchical structures with diverse potential applications. This review outlines the use of metal‐mediated assembly with these supramolecular structures and highlights how changes in the building blocks lead to significant differences in structural morphologies, with the identity of the metal‐binding ligands and their placement on the building block as crucial aspects in the bottom‐up development of the assemblies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Regenerative medicine makes use of cell-supporting biomaterials to replace lost or damaged tissue. Collagen holds great potential in this regard caused by its biocompatibility and structural versatility. While natural collagen has shown promise for regenerative medicine, collagen mimetic peptides (CMPs) have emerged that allow far higher degrees of customization and ease of preparation. A wide range of two and three-dimensional assemblies have been generated from CMPs, many of which accommodate cellular adhesion and encapsulation, through careful sequence design and the exploitation of electrostatic and hydrophobic forces. But the methodology that has generated the greatest plethora of viable biomaterials is metal-promoted assembly of CMP triple helices—a rapid process that occurs under physiological conditions. Architectures generated in this manner promote cell growth, enable directed attachment of bioactive cargo, and produce living tissue. [ABSTRACT FROM AUTHOR]

Biofunctional scaffolds for the delivery of living cells are of the utmost importance for regenerative medicine. Herein, a novel, robust "spiraled horn" scaffold was elucidated through the Co2+-promoted hierarchical assembly of two collagen mimetic peptides, NCoH and HisCol. Each "horn" displayed a periodic banding pattern with band lengths corresponding to the length of the collagen peptide triple helix. Strand exchange between the two peptide trimers resulted in failure to form this intricate morphology, lending support to a precise metal-ligand-based mechanism of assembly. Little change occurred to the observed morphology when the Co2+ concentration was varied from 0.5 to 4.0 mM, and the scaffold was found to be fully formed within two minutes of exposure to the metal ion. The horned network also displayed biological functionality by binding to a His-tagged fluorophore and associating with cells. [ABSTRACT FROM AUTHOR]

Abstract: Natural and synthetic three-dimensional (3-D) scaffolds that mimic the microenvironment of the extracellular matrix (ECM), with growth factor storage/release and the display of cell adhesion signals, offer numerous advantages for regenerative medicine and in vitro morphogenesis and oncogenesis modeling. Here we report the design of collagen mimetic peptides (CMPs) that assemble into a highly crosslinked 3-D matrix in response to metal ion stimuli, that may be functionalized with His-tagged cargoes, such as green fluorescent protein (GFP-His8) and human epidermal growth factor (hEGF-His6). The bound hEGF-His6 was found to gradually release from the matrix in vitro and induce cell proliferation in the EGF-dependent cell line MCF10A. The additional incorporation of a cell adhesion sequence (RGDS) at the N-terminus of the CMP creates an environment that facilitated the organization of matrix-encapsulated MCF10A cells into spheroid structures, thus mimicking the ECM environment. [Copyright &y& Elsevier]

Abstract: Alternative modes of inhibition for the design of anti-HIV therapies are sought due to the resistance of HIV to a number of the currently approved drugs. A non-active site strategy for generating potent inhibitors of HIV-1 integrase is described based on blocking protein association. Peptides α5 and α6 derived from the HIV-1 integrase dimeric interface have previously demonstrated efficacious dimerization inhibition of HIV-1 integrase. Due to the proximity of the termini of these peptides within the integrase structure, a focused library of tethered agents was designed based on crosslinking the peptides α5 and α6 to mimic a larger interfacial region. The best crosslinked inhibitors are approximately five-fold more potent against HIV-1 integrase than the individual peptides alone or in combination. The most active agents have an inhibitory constant in the mid-nM range and function via a dissociative mechanism of inhibition. [Copyright &y& Elsevier]

Purpose: Efficient delivery of therapeutic biopolymers across cell membranes remains a daunting challenge. The development of cell-penetrating peptides (CPPs) has been useful; however, many CPPs are found trapped within endosomes, limiting their use as delivery agents. We optimize a class of CPPs, cationic amphiphilic polyproline helices (CAPHs), for direct transport into cells with mitochondrial localization through dimerization. Methods: The CAPH P11LRR used for this study has been found to enter cells by two distinct pathways: an endocytotic pathway was favored at low concentrations; internalization by direct transport was observed at higher concentrations. CAPH was dimerized to probe if direct transport within cells may be enhanced through increased association of CAPH with the membrane and through the association of individual peptides within the membrane. Results: The dimerization of the CAPH was found to significantly increase cellular uptake over its monomeric counterpart, with a concomitant lowering of the concentration threshold favoring direct transport. Evidence for direct transport within cells and mitochondrial localization was observed. Conclusions: CAPH cellular uptake efficiency can be significantly enhanced through peptide dimerization while favoring cell entry via direct transport at low concentration with low cell toxicity. [ABSTRACT FROM AUTHOR]

The triple-helical Structure of collagen peptides has recently been harnessed as a subunit in the higher-order assembly of unique biomaterials. Specific assembly signals have been designed within collagen peptides, including hydrophobic groups, electrostatic interactions, and metal—ligand binding, to name a few. In this way, a range of novel assemblies have been obtained, including nano- to microscale fibers, gels, spheres, and meshes, each with the potential for novel biological applications in drug delivery, tissue engineering, and regenerative medicine. [ABSTRACT FROM AUTHOR]

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 His2 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 Zn2+, Cu2+, Ni2+, and Co2+. 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 Zn2+-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. [ABSTRACT FROM AUTHOR]

Abstract: There is a great need for alternative modes of inhibition for the design of anti-HIV therapies, due to the increased resistance of HIV to currently approved drugs. A novel strategy for generating potent dimerization inhibitors of HIV-1 protease is described based on sidechain-linked interfacial peptides. In a number of cases the activity of these agents against HIV-1 protease was found to be among the most potent reported, with inhibitory constants in the low nM range. [Copyright &y& Elsevier]

Cell-penetrating peptides present an attractive and efficient tool for the delivery of a variety of cell impermeable cargoes across the cellular membrane. Cell-penetrating peptides usually consist of short basic peptide sequences that are internalized by a variety of cell lines. Most cell-penetrating peptides lack cell specificity, however, which greatly limits their use as efficient therapeutic agents. Herein, we present two cell-penetrating peptides displaying a type II polyproline helical backbone that are functionalized to contain six cationic moieties and two distinctive hydrophobic functionalities, namely isobutyl or benzyl groups. The uptake efficiency of these cationic amphiphilic polyproline helices was studied in seven different cell lines, six cancerous (MCF-7, HOS, HT1080, HeLa, KB-FD, KB3-1) and one non-cancerous (WI 38). The cationic amphiphilic polyproline helix P11LRR at 50 μm showed high specificity toward MCF-7 breast cancer cells. Co-culture experiments with P11LRR demonstrated almost exclusive internalization by MCF-7 cells and not WI38. The replacement of the isobutyl hydrophobic group with a benzyl moiety resulted in a shift in uptake efficiency and specificity across some cell lines. These results demonstrate that the type of hydrophobic residues utilized in the creation of cell-penetrating peptides can strongly influence the extent and specificity of cellular internalization. [ABSTRACT FROM AUTHOR]

Abstract: Cell penetrating peptides (CPP) displaying a type II polyproline helix backbone of different length and amphiphilic character were synthesized and their cellular uptake was compared. The longer CPP sequence, P14LRR, displayed a 7- to 12-fold higher uptake in MCF-7 cells as compared to its shorter counterpart, P11LRR, and a 35-fold higher uptake as compared to Tatp. These results demonstrate that an increased number of cationic and hydrophobic residues can strongly influence the extent of cellular internalization. Mechanistic investigations suggest internalization via a receptor independent endocytotic pathway with these agents. [Copyright &y& Elsevier]

Summary: A library of dimerization inhibitors of HIV-1 protease is described based on crosslinked interfacial peptides. The 54 component library was designed to contain two modifications to the starting structure, one each in the Northern and Southern fragments. A rapid synthesis and in situ screening method in microtiter plates was developed to facilitate the generation and evaluation of the library members. More than 90% of the doubly modified agents were more potent than their respective singly mutated parent compounds, and five of the most potent dimerization inhibitors of HIV-1 protease described to date were identified. The free energy of binding for the combined two modifications was generally found to be additive, demonstrating the predictive value of earlier libraries. [Copyright &y& Elsevier]

Somatostatin owes its biological activity to the presence of a well-defined β-turn centered around the tetrapeptide Phe-Trp-Lys-Thr. We have developed a light-activated β-turn scaffold, 1, with the ability to template a β-turn conformation within the somatostatin tetrapeptide only upon photolysis. The three-dimensional structure of the trans cyclic peptide I obtained by NMR revealed no β-turn conformation; however, when isomerized to the cis form II with light, the solution structure of the resulting cyclic peptide was found to contain a type II′ β-turn within the Phe-Trp-Lys-Thr sequence. Binding assays with the SRIF receptor demonstrated that the cis peptide displayed enhanced affinity for the receptor over the trans form. [ABSTRACT FROM AUTHOR]

Continuing investigations into protein–protein interactions have revealed their key role in regulating a wide range of cellular processes. Although efforts to modulate these interactions are more challenging and much less mature than work on conventional drug discovery pathways, significant progress has been made on several fronts. Highlights of recent advances involve peptide-based inhibitors, including sidechain and backbone cross-linked agents, and peptide scaffolds, as well as small-molecule inhibitors of protein–protein interactions, such as those containing terephthalate or bis-imidazole scaffolds. [Copyright &y& Elsevier]

Excellent catalytic efficiency has been obtained within a series of self replicating peptides, and nucleobase inclusion into a salt-switchable self replicating peptide is found to override the switch. Interestingly, cross-catalytic formation of an RNA aptamer is reported with a cationic peptide, and novel, amide-based biopolymers have been designed to self assemble. [Copyright &y& Elsevier]

A novel strategy to identify potent HIV-1 protease dimerization inhibitors was developed using 12-aminododecanoic acid as a tether to crosslink interfacial peptides. The directionality of the southern peptide was changed from N → C to C → N as compared to previously reported inhibitors. The terminal amine of the southern peptide and side chains were further diversified to find essential functional groups for dimerization inhibition of HIV-1 protease. [Copyright &y& Elsevier]

An inhibitor based on crosslinked peptides from the interfacial region of HIV-1 protease, previously shown to act by dimerization inhibition, was modified by N-methylation to ascertain the importance of the amide hydrogens on inhibition. The effects of N-methylation on HIV-1 protease inhibition, as well as the effects on degradation by proteases are described. [Copyright &y& Elsevier]

The article presents a study which focused on the assembly of metal-triggered collagen peptide through the use of staggered bipyridine ligands as demonstrated by the synthesis of H-(byp)2. The study utilized solid-phase methods for H-(byp)2 synthesis, and the helix and metal ion binding properties of the resulting material were examined. The study showed that radial assembly of collagen peptide triple helices with metal ions led into the formation of collagen peptide disks.

A small-molecule inhibitor of the obligate dimeric protease of human Kaposi's sarcoma-associated herpesvirus was identified. The agent functions by a "monomer trap" mechanism in which the compound binds to a partially unfolded monomer and disrupts the formation of the enzymatically active protease dimer. [ABSTRACT FROM AUTHOR]

The article discusses the concept of collagen fiber formation through the radial growth of a collagen mimetic peptide, H-byp, via a metal assisted self-assembling trigger. It says that the fiber formation was triggered through the addition of metal iron Fe(II) and the assembly consisted of branched fibers at concentrations below the ligand. Furthermore, future studies are to investigate how the ligand placement and coordination chemistry can control the structures of the collagen fibers.

Discloses the effect of a proline kink on coiled-coil, self-replicating peptides. Design of two proline-containing, coiled-coiled peptides based on the self-replicating peptide E1E2; Promise for developing replicators.

Examines the self-replicating properties of peptide. Catalytic efficiency of the peptide; Destabilization of the coiled coil structure of the protein; Thermal denaturation of the peptide.

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. [ABSTRACT FROM AUTHOR]

The chemistry curriculum for undergraduate life science majors at Purdue University has been transformed to better meet the needs of this student population and prepare them for future success. The curriculum, called the 1-2-1 curriculum, includes four consecutive and integrated semesters of instruction in general chemistry, organic chemistry, and biochemistry, taken by students in their first two years. As part of this curriculum, a one-semester general chemistry course, General Chemistry with a Biological Focus (GCBF), was created and evaluated. The course covers all topics typically taught in general chemistry, but with a shift in emphasis toward topics relevant to chemistry in biological systems. Students who took this new course performed better in subsequent organic chemistry courses compared with their peers who took a traditional two-semester general chemistry course. [ABSTRACT FROM AUTHOR]

Reports on the use of subtilisin in a mixed aqueous-organic medium to catalyze a remarkably facile and high-yielding resynthesis of triose phosphate isomerase from a mixture of its subtilisin-proteolyzed fragments.

Describes a novel method using disulfide bonds to trap the assembly formed by a 16 amino acid amphiphilic peptide. Potential of the peptide to self-assemble into a four-helix bundle; Stability of the four-helix bundle; Circular dichroism experiments.

The ability to reversibly modulate peptide secondary structures, such as the β‐turn, allows for precise control of biological function, including protein interactions. Herein, we describe the design of two scaffolds containing an azobenzene moiety with flanking alanine or β‐alanine residues to probe essential features for photo‐control of a β‐turn within a cyclic peptide. To efficiently cyclize the designed linear peptides, prior isomerization of the azobenzene‐containing amino acid from the trans to the cis form was necessary. The two cyclic peptides (TAp and TApβ) were found to undergo rapid photochemical conversion to the cis isomer of the azobenzene, with a more gradual thermal reversion to the trans isomer over the course of a week at 37 °C. Spectroscopic analysis and restrained molecular dynamics simulation of the cis form of TAp and TApβ revealed type II and type II' β‐turns within the cyclic peptides, respectively. The trans isomer of the TAp cyclic peptide was found to have a kink within the peptide structure, whereas the longer trans‐TApβ contained a more extended conformation. TApβ, therefore, demonstrates a clearer difference in the cyclic peptide conformations when in the cis versus trans form, a feature that may prove beneficial for use with biologically active β‐turn sequences. [ABSTRACT FROM AUTHOR]

Abstract: We report a dimerization strategy to enhance the antibacterial potency of an otherwise weak cationic amphiphilic polyproline helical (CAPH) peptide. Overall, the dimeric CAPHs were more active against Escherichia coli and Staphylococcus aureus than the monomeric counterpart, reaching up to a 60-fold increase in potency. At their minimum inhibitory concentration (MIC), the dimeric peptides demonstrated no hemolytic activity or bacterial membrane disruption as monitored by β-galactosidase release in E. coli. At higher concentrations the dimeric agents were found to induce β-galactosidase release, but maintained negligible hemolytic activity, pointing to a potential shift in the mechanism of action at higher concentrations. Thus, discontinuous dimerization of an unnatural proline-rich peptide was a successful strategy to create potent de novo antibacterial peptides without membrane lysis. [Copyright &y& Elsevier]

Two molecular scaffolds were designed using the CAVEAT molecular design package to inhibit the oligomerization of protective antigen (PA63), a key protein component of anthrax toxin. The inhibitors were designed to prevent heptamerization of PA63 by mimicking key residues of PA63 needed for the intermolecular interactions that stabilize the heptamer. Using the scaffolds identified by CAVEAT, seven candidate inhibitors were synthesized and tested for their ability to inhibit anthrax toxin-induced cytotoxicity, with three of the agents demonstrating modest inhibition in murine J774A.1 macrophage cells. [ABSTRACT FROM AUTHOR]

Abstract: We demonstrate that P11LRR, a recently developed amphiphilic polyproline, cell penetrating agent, is able to locate inside the mitochondria of various cell lines when administrated at high concentrations. Mitochondrial targeting was verified by confocal fluorescence co-localization of P11LRR-fluorescein with Mitotracker Red. Elimination of mitochondrial membrane potential dramatically inhibits the localization of P11LRR to mitochondria. Concentration-dependency experiments suggest that cellular internalization of P11LRR occurs via two different pathways: endocytosis and direct transport. Results indicate that the latter pathway predominates at high concentrations of P11LRR, resulting in localization of the agent to the mitochondria. The membrane translocation pathway was further confirmed by two endocytosis inhibitors, cytochalasin D and phenylarsine oxide, and by modulation of plasma membrane potential. The potential of using P11LRR as a mitochondrial drug delivery vector was demonstrated through the delivery of a covalently linked small antioxidant, dimethyltyrosine (Dmt), which allowed for the reduction of chemically induced reactive oxygen species within the mitochondria. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: The structural features of an anthrax lethal factor inhibitor, N-oleoyldopamine (OLDA, 1) have been probed. The oleic acid moiety is critical, but, more interestingly, the presence of the double bond and its geometry were found to play an essential role. One compound, 5, was found to be an uncompetitive inhibitor of lethal factor (LF) with a K i value of 2.2μM and a cell-based IC50 value of 4.3μM. [Copyright &y& Elsevier]

Abstract: An inhibitor of anthrax lethal toxin mediated cell death (1) was identified by a medium throughput cell-based screen. This compound was determined to specifically inhibit anthrax lethal factor (LF), and subsequent SAR studies produced an even more potent inhibitor (4). Mechanistic studies identified these agents as uncompetitive inhibitors of LF with K i values of 3.0 and 1.7μM, respectively, with good cell potency and low cytotoxicity. [Copyright &y& Elsevier]

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 Å 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 μM 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. [ABSTRACT FROM AUTHOR]

Cell penetrating agents were designed and synthesized that introduce cationic and hydrophobic moieties along the backbone of a polyproline helix (PPII) in an amphiphilic manner. The CD profile has the features that are expected for a PPII helix, demonstrating that the addition of these groups had little effect on the backbone structure. Dramatic increases in uptake were found with MCF-7 cells when up to six guanidinium groups were positioned on the polyproline helix, whereas only modest increases in cellular uptake were observed with the amine-containing polyproline compounds as compared to their flexible counterparts. Amphiphilicity played a key role in the enhanced cell translocation, as scrambled versions of the designed agents, with hydrophobic and cationic groups on all faces of the helix, were only as effective as their flexible peptide counterparts. Interestingly, the most potent agent, P11 LRR, demonstrated almost an order of magnitude more efficient cellular uptake as compared to that of the well-studied Tat peptide, with minimal cytotoxicity. [ABSTRACT FROM AUTHOR]

Summary: Refining the functional groups on a phenethylamine moiety within an inhibitor of HIV-1 protease led to a switch in the mechanism of inhibition from competitive and allosteric to dimerization inhibition. Phenylether extensions to the phenethylamine group led to agents that target the dimerization interface of HIV-1 protease with high potency. [Copyright &y& Elsevier]

The cellular delivery of therapeutic agents and their localization within cells is currently a great challenge in medicinal chemistry. A few cationic peptides have shown a strong propensity to cross the cytoplasmic membrane and enter cells. Nuclear localization signal (NLS) sequences are a class of highly cationic peptides that may be exploited for cellular import of linked cargo. A series of NLS sequence peptides were investigated for entry into different cancer cell lines by flow cytometry and confocal microscopy. All NLS peptides demonstrated rapid accumulation within cells when added to the cellular media. Covalent adducts of proteins and oligonucleotides with NLS peptides were also effectively imported within cells. An understanding of the structural and mechanistic properties of these sequences will provide great potential for the rational design of efficient and selective peptidic delivery systems. [Copyright &y& Elsevier]

Although liposomes have proven useful for the delivery of drugs and gene therapy vectors, their potencies are often compromised by poor unloading following uptake into their target cells. We have consequently explored the properties of a novel 29-residue amphipathic peptide that was designed by arrangement of hydrophobic and hydrophilic residues to disrupt liposomes at lower peptide concentrations than previously tested peptides. The peptide was indeed found to promote pH-dependent liposome unloading with improved efficiency. A peptide of the same sequence, but half the length, however, promoted pH-dependent permeabilization only at much higher concentrations. Further characterization of the longer peptide revealed that release of liposome contents (i) occurred at a pH of ∼6, (ii) became less efficient as the size of the encapsulated cargo increased, and (iii) was moderately suppressed in cholesterol-containing liposomes. Use of this peptide to enhance the cytotoxicity of cytosine arabinoside encapsulated in folate-targeted liposomes demonstrated an increase in drug potency of ∼30-fold. Gene expression by a serum-stable folate-targeted liposomal vector was also measurably enhanced by inclusion of the peptide. We conclude that intracellular unloading of liposomal contents can be significantly improved by co-encapsulation of an optimally designed, pH-sensitive peptide. [Copyright &y& Elsevier]

A hybrid hydrogel based on collagen-mimetic peptides has been designed with tunable thermosensitive properties. By changing the number of POG repeats within the collagen peptide sequence, the thermal stability of the triple helical physical crosslinks of a peptidepolymer conjugate can be altered, thus tuning the stiffness of the hydrogel as a function of temperature. This report focuses on three different thermally responsive collagen peptide, PEG-based hydrogels and validates their use as stimuli-responsive materials [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A secondary structural model for the neuroprotective octapeptide NAPVSIPQ in DMSO was developed using a combination of NMR spectroscopy and molecular dynamics simulations. On the basis of this model, four cyclized analogues of NAPVSIPQ were designed to improve its cell permeability with a long‐term view to oral delivery and increasing central nervous system (CNS) penetration. Testing of cell permeability through a monolayer of Caco‐2 cells demonstrated that three of the four cyclized analogues did exhibit up to 5‐fold improved permeability through the Caco‐2 monolayer. Additionally, none of the peptides appears to be P‐glycoprotein substrates subject to cellular efflux. [ABSTRACT FROM AUTHOR]

Abstract: P-glycoprotein (P-gp) effluxes a diverse set of drug substrates out of cells in an ATP dependent manner, thereby limiting the effective accumulation of therapeutic agents. Herein we demonstrate the use of click chemistry to rapidly generate bivalent quinine dimers, containing an intervening triazole ring, as potential inhibitors of P-gp mediated efflux. Calcein-AM substrate accumulation assays were performed in an MCF7/DX1 cell line that overexpresses P-gp to monitor the inhibitory activity of the clicked quinine dimers. A small library of potent P-gp inhibitors with varying tether lengths is reported, with the best dimer demonstrating low micromolar efficacy. [Copyright &y& Elsevier]

Abstract: The development of cell penetrating peptides (CPPs) for the cellular delivery of attached cargo is an area of growing interest. Many CPPs, however, are found trapped within endosomes, thereby limiting their use as drug delivery agents with sub-cellular applications. Herein, we detail the properties of a highly efficient class of CPPs, cationic amphiphilic polyproline helices (CAPHs), that are found localized to the mitochondria by direct transport into cells. [Copyright &y& Elsevier]

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 [ 125 I]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. [ABSTRACT FROM AUTHOR]