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. S1H was designed as a direct sequence mimetic of the site 1 mini-helix (residues 36-51) of wild-type hGH and acts by inhibiting the interaction of hGH with the human growth hormone receptor (hGHR). In vitro studies indicated that S1H is stable in human serum and can adopt an α-helix in solution. Our results also show that S1H mitigates phosphorylation of STAT5 in cells co-treated with hGH, reducing intracellular STAT5 phosphorylation levels to those observed in untreated controls. Furthermore, S1H was found to attenuate the activity of the hGHR and the human prolactin receptor, suggesting that this peptide acts as an antagonist of both lactogenic and somatotrophic hGH actions. Finally, we used alanine scanning to determine how discrete amino acids within the S1H sequence contribute to its structural organization and biological activity. We observed a strong correlation between helical propensity and inhibitory effect, indicating that S1H-mediated antagonism of the hGHR is largely dependent on the ability for S1H to adopt an α-helix. Taken together, these results show that S1H not only acts as a novel peptide-based antagonist of the hGHR but can also be applied as a chemical tool to study the molecular nature of hGH-hGHR interactions.

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

The superoxide anion (O2•-) is a reactive oxygen species (ROS) that functions as an important regulator of signal transduction in living systems. However, excess O2•- can cause metabolic imbalances and oxidative damage inside cells. Quantitative detection and efficient scavenging of O2•- are therefore critical for maintaining intracellular redox balance and homeostasis. In this work, a nanomaterial (Au-TeCD) composed of BSA-modified gold nanoparticles (AuNPs) complexed with tellurium-containing carbon dots (TeCDs) was constructed. The introduction of Au-TeCDs to solutions containing superoxide resulted in enhanced elimination of the anion, indicating that Au-TeCDs are able to scavenge O2•- from the surrounding environment. Notably, the respective TeCD and AuNP components of the Au-TeCDs were found to emit fluorescence at 425 and 640 nm upon exposure to superoxide anions. This unique spectroscopic property of Au-TeCDs allowed levels of O2•- in solution to be quantified using dual-fluorescence detection. The Au-TeCDs developed herein also exhibited low-cytotoxicity, versatile capabilities for in situ fluorescence imaging, and effective scavenging of O2•- in living cells. Taken together, these results suggest that Au-TeCDs act as effective tools for monitoring superoxide concentrations in complex mixtures and may be developed as possible therapeutics designed to scavenge excess ROS from diseased 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-EDT2 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 (kon) and apparent equilibrium dissociation constants (Kapp) to ERα-LBDs in the presence of estrogenic ligands.

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. We synthesized five ScTx-Bax variants, each presenting a different combination of native disulfide linkage and evaluated their ability to directly bind Bcl-2 in vitro. It was determined that the position of the disulfide linkage had significant implications on the structure and function of ScTx-Bax peptides. This study underscores the importance of structural dynamics in BH3:Bcl-2 interactions and further validates ScTx-based ligands as potential modulators of anti-apoptotic Bcl-2 function. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

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. This research overview discusses emerging developments involving the use of miniature proteins as scaffolds to design novel therapeutics for the treatment and study of human disease. Specifically, this review will explore strategies to: (i) stabilize miniature protein tertiary structure; (ii) optimize biomolecular recognition by grafting functional epitopes onto miniature protein scaffolds; and (iii) enhance cytosolic delivery of miniature proteins through the use of cationic motifs that facilitate endosomal escape. These objectives are discussed not only to address challenges in developing effective miniature protein-based drugs, but also to highlight the tremendous potential miniature proteins hold for combating and understanding human disease. Drug Dev Res 78 : 268-282, 2017. © 2017 Wiley Periodicals, Inc.

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. The second, GIGT (glucocorticoid-induced eGFP translocation), is a nonamplified assay that informs on relative cytosolic access and exploits sophisticated imaging equipment to facilitate high-content screens in live cells. Each assay was employed to quantify the cytosolic delivery of several canonical "cell permeable peptides," as well as more recently reported minimally cationic miniature proteins and zinc finger nuclease domains. Our results show definitively that both overall charge as well as charge distribution influence cytosolic access and that small protein domains containing a discrete, helical, penta-Arg motif can dramatically improve the cytosolic delivery of small folded proteins such as zinc finger domains. We anticipate that the assays described herein will prove useful to explore and discover the fundamental physicochemical and genetic properties that influence both the uptake and endosomal release of peptidic molecules and their mimetics.

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. The various disulfide patterns of the ScTx-based constructs are established during peptide synthesis and are ultimately verified by mass analysis of trypsin-digested fragments. The BH3 domain mimetics developed herein were generated by transposing residues from the helical BH3 domain of the pro-apoptotic BCL2 protein Bax to the α-helix of wild-type ScTx. Interestingly, we found that the relative binding affinities of ScTx-Bax peptides for the anti-apoptotic BCL2 protein Bcl-2 (proper) were heavily influenced by the number and position of disulfide linkages within the ScTx-Bax sequence. As a consequence, we were able to utilize ScTx-Bax BH3 domain mimetics with varied patterns of disulfide bonds to survey how structural rigidity within the helical Bax BH3 domain affects binding to promiscuous anti-apoptotic BCL2 proteins. More broadly, the ability to generate ScTx-based molecules that contain any combination of native disulfide bonds expands the utility of such constructs as tools to study the molecular nature of protein-protein interactions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of ScTx-based Bax BH3 domain mimetics Basic Protocol 2: Oxidation of ScTx-Bax BH3 domain mimetics containing one, two, or three disulfide linkages Support Protocol: Mapping of disulfide linkages in oxidized ScTx-Bax BH3 domain mimetics.

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. The overall objectives of these studies are assessed to highlight the widespread applications of the products, which range from bioactive ligands to new materials.

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. Oligoglycine and oligo(beta-alanine) chains underwent oligomerization by 1,3-dipolar cycloaddition in the absence of a copper catalyst. These results suggest that cyclodimerization depends on the ability of the azido-alkyne peptide to form in-frame hydrogen bonds between chains in order to place the reacting groups in close proximity and lower the entropic penalty for dimerization. The properties of the resin and solvent are crucial, giving rise to a productive balance between swelling and interstrand H-bonding. These findings allow for the design of optimal substrates for triazole-forming ring closure and for the course of the reaction to be controlled by the choice of conditions.

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. We have employed Radelegans to genetically define the role of the EGFR signaling pathway in protection from reproductive cell death, the primary form of tumor stem or clonogen cell death postirradiation. We have found that the RAS/mitogen-activated protein kinase (MAPK) downstream signal transduction pathway of EGFR is critical for protection from reproductive cell death in Radelegans. In addition, we have shown that RAS/MAPK pathway signaling is genetically linear with the DNA damage response pathway and acts downstream of the DNA damage checkpoint in the radioresponse, implicating this pathway in DNA repair post-cytotoxic therapy. These findings support the hypothesis that enhanced repair is a mechanism of RAS/MAPK pathway-mediated resistance to cytotoxic therapy through its interaction with the DNA damage response pathway postirradiation. We postulate that these findings also help explain why current treatment strategies, based on the presumption that tumors have ineffective repair compared with normal tissues, are ineffective in EGFR/RAS/MAPK pathway-mediated tumors. Radelegans is a platform to further define the genetic basis of the radiation response in tissues.

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. We recently reported the development of a novel class of synthetic protein based on scyllatoxin (ScTx) designed to mimic the helical BH3 interaction domain of the pro-apoptotic BCL2 protein Bax. These studies showed that the number and position of native disulfide linkages contained within the ScTx-Bax structure significantly influences the ability for these constructs to target anti-apoptotic BCL2 proteins in vitro. The goal of the present study is to investigate the contribution of two disulfide linkages in the folding and biological activity of ScTx-Bax proteins. Here, we report the full chemical synthesis of three ScTx-Bax sequence variants, each presenting two native disulfide linkages at different positions within the folded structure. It was observed that two disulfide linkages were sufficient to fold ScTx-Bax proteins into native-like architectures reminiscent of wild-type ScTx. Furthermore, we show that select (bis)disulfide ScTx-Bax variants can target Bcl-2 (proper) in vitro and that the position of the disulfide bonds significantly influences binding affinity. Despite exhibiting only modest binding to Bcl-2, the successful synthesis of ScTx-Bax proteins containing two disulfide linkages represents a viable route to ScTx-based BH3 domain mimetics that preserve native-like conformations. Finally, structural models of ScTx-Bax proteins in complex with Bcl-2 indicate that these helical mimetics bind in similar configurations as wild-type Bax BH3 domains. Taken together, these results suggest that ScTx-Bax proteins may serve as potent lead compounds that expand the repertoire of "druggable" protein-protein interactions.

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. Divalent metal ions, such as zinc are released as a response to cellular stress and injury, often resulting in oxidative damage and cell death. In this study, we evaluate the effect increased concentrations of intracellular zinc has on the phosphorylation state and subcellular localization of PKCδ. More specifically, we demonstrate that intracellular zinc inhibits the phosphorylation of PKCδ at Thr505 in a concentration-dependent manner and facilitates the translocation of PKCδ from the cytosol to the Golgi complex. Analysis of a PKCδ structural model revealed a potential His-Cys3 zinc-binding domain adjacent to residue Thr505 and suggests that interaction with a Zn2+ ion may preclude phosphorylation at this site. This study establishes zinc as a potent modulator of PKCδ function and suggests a novel mechanism by which PKCδ is able to "sense" changes in the concentration of intracellular zinc. These findings illuminate a new paradigm of metal ion-protein interaction that may have significant implications on a broad spectrum of cellular processes.

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. These results confirm that conformational ordering can be exploited to assist the macrocyclization of folded oligomers.

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%. This strategy will be broadly applicable for generating polyvalent displays on peptides and other scaffolds, allowing precise control of spacing between the displayed groups.

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.P. administration. One agent orally lowered serum cholesterol in Sprague-Dawley male rats at 2mg/kg/day after 14 days. The agents demonstrated a lowering of mouse serum LDL- cholesterol levels and selected compounds showed an elevation of serum HDL-cholesterol levels. The cholesterol concentrations in the liver were raised while the cholesterol and triglyceride contents of the aorta were significantly lowered by the selected trisubstituted pyrrole.

Arginine topology controls escape of minimally cationic proteins from early endosomes to the cytoplasm.

Proteins represent an expanding class of therapeutics, but their actions are limited primarily to extracellular targets because most peptidic molecules fail to enter cells. Here we identified two small proteins, miniature protein 5.3 and zinc finger module ZF5.3, that enter cells to reach the cytosol through rapid internalization and escape from Rab5+ endosomes. The trafficking pathway mapped for these molecules differs from that of Tat and Arg(8), which require transport beyond Rab5+ endosomes to gain cytosolic access. Our results suggest that the ability of 5.3 and ZF5.3 to escape from early endosomes is a unique feature and imply the existence of distinct signals, encodable within short sequences, that favor early versus late endosomal release. Identifying these signals and understanding their mechanistic basis will illustrate how cells control the movement of endocytic cargo and may allow researchers to engineer molecules to follow a desired delivery pathway for rapid cytosolic access.

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 17ß-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. Hexavalent EPCs did not activate hERα, but instead blocked E2-mediated hERα activation. The physicochemical features of EPCs can be precisely tuned, which may allow the generation of a library of chemical tools for modulating specific effects of estrogens.

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.

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. A mode of action study in HL-60 cells demonstrated that DNA and protein syntheses were inhibited after 60 min at 100 microM. DNA and RNA polymerases, PRPP-amido transferase, dihydrofolate reductase, thymidylate synthase, and TMP kinase activities were interfered with by the agent with reduction of d[NTP] pools. Nonspecific interaction with the bases of DNA and cross-linking of the DNA may play a role in the mode of action of these carboxylates.