Synthesis of Scyllatoxin-Based BH3 Domain Mimetics with Diverse Patterns of Native Disulfide Bonds.

This article outlines the design and development of scyllatoxin (ScTx)-based BH3 domain mimetics with diverse patterns of native disulfide bonds. More specifically, this method summarizes the total chemical synthesis of ScTx-based peptides that contain zero, one, two, or three disulfide linkages, including techniques to generate variants with any combination of native disulfides. Each peptide reported herein is generated on solid-phase support using microwave-assisted coupling procedures, and all reaction parameters related to the peptide synthesis are described in detail.

Versatile Gold-Coupled Te-Carbon Dots for Quantitative Monitoring and Efficient Scavenging of Superoxide Anions.

The superoxide anion (O) is a reactive oxygen species (ROS) that functions as an important regulator of signal transduction in living systems. However, excess O can cause metabolic imbalances and oxidative damage inside cells. Quantitative detection and efficient scavenging of O are therefore critical for maintaining intracellular redox balance and homeostasis.

A novel peptide antagonist of the human growth hormone receptor.

Excess circulating human growth hormone (hGH) in vivo is linked to metabolic and growth disorders such as cancer, diabetes, and acromegaly. Consequently, there is considerable interest in developing antagonists of hGH action. Here, we present the design, synthesis, and characterization of a 16-residue peptide (site 1-binding helix [S1H]) that inhibits hGH-mediated STAT5 phosphorylation in cultured cells.

Monitoring ligand-mediated helix 12 transitions within the human estrogen receptor α using bipartite tetracysteine display.

Estrogen receptor α ligand-binding domains (ERα-LBD) expressing tetracysteine motifs bind FlAsH-EDT upon transition of helix 12 (H12) to a folded state. Changes in fluorescence intensity allowed surveillance of ligand-mediated H12 transitions and facilitated the determination of FlAsH association rates (k) and apparent equilibrium dissociation constants (K) to ERα-LBDs in the presence of estrogenic ligands.

Synthesis and Biological Activity of Scyllatoxin-Based BH3 Domain Mimetics Containing Two Disulfide Linkages.

The B cell lymphoma 2 (BCL2) proteins are a family of evolutionarily related proteins that act as positive or negative regulators of the intrinsic apoptosis pathway. Overexpression of anti-apoptotic BCL2 proteins in cells is associated with apoptotic resistance, which can result in cancerous phenotypes and pathogenic cell survival. Consequently, anti-apoptotic BCL2 proteins have attracted considerable interest as therapeutic targets.

Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ).

Protein kinase C delta (PKCδ) is a Ser/Thr-specific kinase involved in many fundamental cellular processes including growth, differentiation and apoptosis. PKCδ is expressed ubiquitously in all known cell types, and can be activated by diacylglycerol, phorbol esters and other kinases. Multiple lines of evidence have indicated that the mode of activation greatly influences the role PKCδ plays in cellular function.

Small Scaffolds, Big Potential: Developing Miniature Proteins as Therapeutic Agents.

Preclinical Research Miniature proteins are a class of oligopeptide characterized by their short sequence lengths and ability to adopt well-folded, three-dimensional structures. Because of their biomimetic nature and synthetic tractability, miniature proteins have been used to study a range of biochemical processes including fast protein folding, signal transduction, catalysis and molecular transport. Recently, miniature proteins have been gaining traction as potential therapeutic agents because their small size and ability to fold into defined tertiary structures facilitates their development as protein-based drugs.

Role of single disulfide linkages in the folding and activity of scyllatoxin-based BH3 domain mimetics.

Anti-apoptotic Bcl-2 proteins are implicated in pathogenic cell survival and have attracted considerable interest as therapeutic targets. We recently developed a class of synthetic peptide based on scyllatoxin (ScTx) designed to mimic the helical BH3 interaction domain of the pro-apoptotic Bcl-2 protein Bax. In this communication, the contribution of single disulfides in the folding and function of ScTx-Bax peptides was investigated.

Targeting anti-apoptotic Bcl2 proteins with scyllatoxin-based BH3 domain mimetics.

BH3 domain mimetics based on the small protein scyllatoxin (ScTx) were designed to target the anti-apoptotic protein Bcl2 in vitro. Intrinsically disordered ScTx variants were found to bind Bcl2 with nanomolar affinity, indicating that an induced fit binding mechanism is required for favorable BH3 : Bcl2 interaction.

Improved assays for determining the cytosolic access of peptides, proteins, and their mimetics.

Proteins and other macromolecules that cross biological membranes have great potential as tools for research and next-generation therapeutics. Here, we describe two assays that effectively quantify the cytosolic localization of a number of previously reported peptides and protein domains. One assay, which we call GIGI (glucocorticoid-induced eGFP induction), is an amplified assay that informs on relative cytosolic access without the need for sophisticated imaging equipment or adherent cells.

Modulation of human estrogen receptor α activity by multivalent estradiol-peptidomimetic conjugates.

Estradiol-peptidomimetic conjugates (EPCs) are linear, sequence-specific peptoid oligomers that site-specifically display multiple copies of 17β-estradiol (E2), a ligand for the human estrogen receptor α (hERα). We evaluate the ability of multivalent EPCs to activate hERα-mediated transcription. EPCs activated the hERα in both a length- and valence-dependent manner, with the highest levels of activation generated by divalent peptoid 6-mers, divalent 18-mers, and trivalent 9-mers.

Tricks with clicks: modification of peptidomimetic oligomers via copper-catalyzed azide-alkyne [3 + 2] cycloaddition.

This tutorial review examines recent developments involving use of Copper-catalyzed Azide-Alkyne [3 + 2] Cycloaddition (CuAAC) reactions in the synthesis, modification, and conformational control of peptidomimetic oligomers. CuAAC reactions have been used to address a variety of objectives including: (i) ligation of peptidomimetic oligomers; (ii) synthesis of ordered "foldamer" architectures; (iii) conjugation of ligands to peptidomimetic scaffolds; and (iv) macrocyclization of peptidomimetics using triazole linkages as conformational constraints. Variations in synthesis protocols, such as the use of different solvent systems, temperatures and copper species are evaluated herein to present a range of variables for the optimization of CuAAC reactions.

Peptide cyclization and cyclodimerization by Cu(I)-mediated azide-alkyne cycloaddition.

Head-to-tail cyclodimerization of resin-bound oligopeptides bearing azide and alkyne groups occurs readily by 1,3-dipolar cycloaddition upon treatment with Cu(I). The process was found to be independent of peptide sequence, sensitive to the proximity of the alkyne to the resin, sensitive to solvent composition, facile for alpha- and beta-peptides but not for gamma-peptides, and inhibited by the inclusion of tertiary amide linkages. Peptides shorter than hexamers were predominantly converted to cyclic monomers.

Lipid-lowering effects of ethyl 2-phenacyl-3-aryl-1H-pyrrole- 4-carboxylates in rodents.

A series of substituted 2-phenacyl-3-phenyl-1H-pyrrole-4-carboxylates were prepared from substituted acetophenones in 6 steps. The final condensations between a chloroenal and an aminoketone were carried out under neutral conditions in parallel to yield the series listed below. Selected pyrrole derivatives proved to be potent hypolipidemic agents lowering serum triglyceride concentrations in CF-1 male mice after 14 days of I.

Fit to be tied: conformation-directed macrocyclization of peptoid foldamers.

Covalent macrocyclic constraints can be readily installed on N-substituted glycine "peptoid" oligomer substrates. Cu(I)-catalyzed [3+2] cycloaddition reactions were conducted on solid support to ligate peptoid side chain azide and alkyne functionalities. Intramolecular macrocycle formation is facilitated by preorganizing the reactive groups across one turn of the helical secondary structure.

A conserved RAS/mitogen-activated protein kinase pathway regulates DNA damage-induced cell death postirradiation in Radelegans.

Although the epidermal growth factor receptor (EGFR) signaling pathway is overactive in more than half of human cancers and mediates resistance to cytotoxic therapy, the molecular mechanisms of EGFR pathway-mediated resistance have remained elusive in cancer research. This difficulty partly stems from the lack of tissue models enabling clear separation of the many forms of cell death that the downstream signaling pathways of EGFR affect. We have created a model in Caenorhabditis elegans of radiation-induced reproductive cell death ("Radelegans") in isolation of all other forms of cell death.

Clickity-click: highly functionalized peptoid oligomers generated by sequential conjugation reactions on solid-phase support.

N-Substituted glycine peptoid oligomers were used as substrates for azide-alkyne [3 + 2] cycloaddition conjugation reactions and then elaborated through additional rounds of oligomerization and cycloaddition. This novel sequential conjugation technique allowed for the generation of complex peptidomimetic products in which multiple heterogeneous pendant groups were site-specifically positioned along the oligomer scaffold. Studies of a water-soluble estradiol-ferrocene peptoid conjugate demonstrated a potential application for the modular synthesis of biosensors.

Click to fit: versatile polyvalent display on a peptidomimetic scaffold.

We describe an efficient protocol to effect multisite conjugation reactions to oligomers on solid-phase support. Sequence-specific N-substituted glycine "oligopeptoids" were utilized as substrates for azide-alkyne cycloaddition reactions. Diverse groups, including nucleobases and fluorophores, were conjugated at up to six positions on peptoid side chains with yields ranging from 88 to 96%.

Synthesis and cytotoxicity of substituted ethyl 2-phenacyl-3-phenylpyrrole-4-carboxylates.

The substituted ethyl-2-phenacyl-3-phenylpyrrole-4-carboxylates were synthesized by a condensation of a beta-chloroenal and an alpha-aminoketone under neutral conditions. They proved to be potent cytotoxic agents against the growth of murine L1210 and P388 leukemias and human HL-60 promyelocytic leukemia, HuT-78 lymphoma, and HeLa-S(3) uterine carcinoma. Selective compounds were active against the growth of Tmolt(3) and Tmolt(4) leukemias and THP-1 acute monocytic leukemia, liver Hepe-2, ovary 1-A9, ileum HCT-8 adenocarcinoma, and osteosarcoma HSO.