Conformational Ensembles Explored Dynamically from Disordered Peptides Targeting Chemokine Receptor CXCR4

This work reports on the design and the synthesis of two short linear peptides both containing a few amino acids with disorder propensity and an allylic ester group at the C-terminal end. Their structural properties were firstly analyzed by means of experimental techniques in solution such as CD and NMR methods that highlighted peptide flexibility. These results were further confirmed by MD simulations that demonstrated the ability of the peptides to assume conformational ensembles. They revealed a network of transient and dynamic H-bonds and interactions with water molecules. Binding assays with a well-known drug-target, i.e., the CXCR4 receptor, were also carried out in an attempt to verify their biological function and the possibility to use the assays to develop new specific targets for CXCR4. Moreover, our data indicate that these peptides represent useful tools for molecular recognition processes in which a flexible conformation is required in order to obtain an interaction with a specific target.     

A journey in structure-based drug discovery: from designed peptides to protein surface topomimetics as antibiotic and antiangiogenic agents

Most biological events are mediated through molecular interactions by proteins, and because proteins are composed of structural units like helices, beta-sheets and turns, small peptides and peptidomimetics may be used to mimic their biological effects and even as therapeutic agents in the clinic. Here, we present a structure-based, scaffold-driven approach to design bioactive peptides and peptidomimetics. Initially, we designed a novel series of beta-sheet-forming peptides that mimic the activities of both antibiotic bacterial membrane disrupting peptides and antiangiogenic proteins. We subsequently used structure-activity relationships to reduce the design to partial peptide mimetics and then to fully nonpeptide topomimetics. Some of these agents are currently in extensive preclinical studies for further development as drug candidates against infectious disease and cancer.     

Structural Aspects and Prediction of Calmodulin-Binding Proteins

Calmodulin (CaM) is an important intracellular protein that binds Ca²⁺ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM’s ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca²⁺ concentration through interactions with CaM.     

Peptide-Lipid Interactions: Experiments and Applications

The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated cellular processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Moreover, a major goal of modern biotechnology is obtaining new potent pharmaceutical agents whose biological action is dependent on the binding of peptides to lipid-bilayers. Several issues need to be addressed such as secondary structure, orientation, oligomerization and localization inside the membrane. At the same time, the structural effects which the peptides cause on the lipid bilayer are important for the interactions and need to be elucidated. The structural characterization of membrane active peptides in membranes is a harsh experimental challenge. It is in fact accepted that no single experimental technique can give a complete structural picture of the interaction, but rather a combination of different techniques is necessary.     

Quantitative Use of Paramagnetic Relaxation Enhancements for Determining Orientations and Insertion Depths of Peptides in Micelles

We describe the background and implementation of a method to determine, at atomic resolution, the insertion depths and orientations of peptides embedded in micelles. A nonperturbing paramagnetic agent—Gd(DTPA–BMA)—was used to induce paramagnetic relaxation enhancements (PREs) of peptide atoms inside the micelle. By calibrating these PREs it was possible to translate them into distance restraints that could be used for structure calculation. We demonstrate this here on the antimicrobial peptides novicidin and novispirin. Characterization of the interactions between antimicrobial peptides and membranes is important for understanding of their biological activities and functions, and a further development of tools to study these interactions is described.     

G4 Matters—The Influence of G-Quadruplex Structural Elements on the Antiproliferative Properties of G-Rich Oligonucleotides

G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich sequences of DNA or RNA that have attracted increased attention as anticancer agents. This systematic study aimed to investigate the anticancer potential of five G4-forming, sequence-related DNA molecules in terms of their thermodynamic and structural properties, biostability and cellular uptake. The antiproliferative studies revealed that less thermodynamically stable G4s with three G-tetrads in the core and longer loops are more predisposed to effectively inhibit cancer cell growth. By contrast, highly structured G4s with an extended core containing four G-tetrads and longer loops are characterized by more efficient cellular uptake and improved biostability. Various analyses have indicated that the G4 structural elements are intrinsic to the biological activity of these molecules. Importantly, the structural requirements are different for efficient cancer cell line inhibition and favorable G4 cellular uptake. Thus, the ultimate antiproliferative potential of G4s is a net result of the specific balance among the structural features that are favorable for efficient uptake and those that increase the inhibitory activity of the studied molecules. Understanding the G4 structural features and their role in the biological activity of G-rich molecules might facilitate the development of novel, more potent G4-based therapeutics with unprecedented anticancer properties.     

The Interfacial Interactions of Glycine and Short Glycine Peptides in Model Membrane Systems

The interactions of amino acids and peptides at model membrane interfaces have considerable implications for biological functions, with the ability to act as chemical messengers, hormones, neurotransmitters, and even as antibiotics and anticancer agents. In this study, glycine and the short glycine peptides diglycine, triglycine, and tetraglycine are studied with regards to their interactions at the model membrane interface of Aerosol-OT (AOT) reverse micelles via ¹H NMR spectroscopy, dynamic light scattering (DLS), and Langmuir trough measurements. It was found that with the exception of monomeric glycine, the peptides prefer to associate between the interface and bulk water pool of the reverse micelle. Monomeric glycine, however, resides with the N-terminus in the ordered interstitial water (stern layer) and the C-terminus located in the bulk water pool of the reverse micelle.     

The Dynamic Regulation of G-Quadruplex DNA Structures by Cytosine Methylation

It is well known that certain non B-DNA structures, including G-quadruplexes, are key elements that can regulate gene expression. Here, we explore the theory that DNA modifications, such as methylation of cytosine, could act as a dynamic switch by promoting or alleviating the structural formation of G-quadruplex structures in DNA or RNA. The interaction between epigenetic DNA modifications, G4 formation, and the 3D architecture of the genome is a complex and developing area of research. Although there is growing evidence for such interactions, a great deal still remains to be discovered. In vivo, the potential effect that cytosine methylation may have on the formation of DNA structures has remained largely unresearched, despite this being a potential mechanism through which epigenetic factors could regulate gene activity. Such interactions could represent novel mechanisms for important biological functions, including altering nucleosome positioning or regulation of gene expression. Furthermore, promotion of strand-specific G-quadruplex formation in differentially methylated genes could have a dynamic role in directing X-inactivation or the control of imprinting, and would be a worthwhile focus for future research.     

Small and Simple,yet Sturdy: Conformationally Constrained Peptides with Remarkable Properties

The sheer size and vast chemical space (i.e., diverse repertoire and spatial distribution of functional groups) underlie peptides’ ability to engage in specific interactions with targets of various structures. However, the inherent flexibility of the peptide chain negatively affects binding affinity and metabolic stability, thereby severely limiting the use of peptides as medicines. Imposing conformational constraints to the peptide chain offers to solve these problems but typically requires laborious structure optimization. Alternatively, libraries of constrained peptides with randomized modules can be screened for specific functions. Here, we present the properties of conformationally constrained peptides and review rigidification chemistries/strategies, as well as synthetic and enzymatic methods of producing macrocyclic peptides. Furthermore, we discuss the in vitro molecular evolution methods for the development of constrained peptides with pre-defined functions. Finally, we briefly present applications of selected constrained peptides to illustrate their exceptional properties as drug candidates, molecular recognition probes, and minimalist catalysts.     

Dissipative Self‐Assembly of Peptides

In molecular self‐assembly, molecules interact with each other by non‐covalent interactions to form larger structures. The process occurs in‐equilibrium, which means that molecules can leave the assembly and reassemble elsewhere, but that occurs on average with equal rates. For self‐assembly, peptides have proven to be a particularly useful building block, in part because of their versatility. Biology also uses self‐assembly to create functional materials. For example, components of the cytoskeleton, like actin filament and microtubules, are self‐assembled proteins. In other words, biology uses the same building blocks and design rules as supramolecular chemists to create functional structures. However, biological assemblies are vastly more complex than their synthetic counterparts. The discrepancy is in part because proteins are more complex than the peptides we use as building blocks. Another contributing factor to the difference in complexity is that most assemblies in living systems exist out of equilibrium. To use peptides for complex functions as biology does, we should understand and be able to create peptide assemblies out of equilibrium. In this work, we review recent advances towards the creation of peptide assemblies that exist out of equilibrium driven by an external energy source.     

Surface Modification of Polyhydroxyalkanoates toward Enhancing Cell Compatibility and Antibacterial Activity

Biomaterials for in vivo application should induce positive interaction with various histocytes and inhibit bacteria inflection as well. Cells and/or bacteria response to the extracellular environment is therefore the basic principle to design the biomaterials surface in order to induce the specific biomaterial–biological interaction. Polyhydroxyalkanoate (PHAs) are of growing interests because of their natural origin, biodegradability, biocompatibility, and thermoplasticity; however, quite inert and intrinsic hydrophobic characteristics have hindered their extensive usage in medical applications. Surface modification of PHAs tailors the chemistry, wettability, and topography without altering the bulk properties, and introduces specific proteins/peptides and/or antibacterial agents to mediate cell–matrix interactions. This review describes the recent developments on the surface modification of PHAs to construct cell compatible and antibacterial surfaces.     

Deciphering Specificity Determinants for FR900359‐Derived Gqα Inhibitors Based on Computational and Structure–Activity Studies

Direct targeting of intracellular Gα subunits of G protein‐coupled receptors by chemical tools is a challenging task in current pharmacological studies and in the development of novel therapeutic approaches. In this study we analyzed novel FR900359‐based analogs from natural sources, synthetic cyclic peptides, as well as all so‐far known G_qα inhibitors in a comprehensive study to devise a strategy for the elucidation of characteristics that determine interactions with and inhibition of G_q in the specific FR/YM‐binding pocket. Using 2D NMR spectroscopy and molecular docking we identified unique features in the macrocyclic structures responsible for binding to the target protein correlating with inhibitory activity. While all novel compounds were devoid of effects on G_i and G_s proteins, no inhibitor surpassed the biological activity of FR. This raises the question of whether depsipeptides such as FR already represent valuable chemical tools for specific inhibition of G_q and, at the same time, are suitable natural lead structures for the development of novel compounds to target Gα subunits other than G_q.     

Functional Peptide Monolayers at Interfaces

Amphiphilic peptides can be designed to form ordered supramolecular structures at hydrophilic-hydrophobic interfaces. These systems rely on the ability of peptides to fold into certain secondary structures at interfaces. This review focuses on the design of amphiphilic β-sheet peptide assemblies in monolayers at interfaces, and their relevance to inducing mineralization and interactions with specific ions. In addition, the review discusses recent studies demonstrating the applicability of designed amphiphilic β-sheet peptides to detection of specific small molecules and to elucidating intermolecular interactions relevant to drug delivery and enzyme catalysis systems.     

Beta-hairpin peptidomimetics: design, structures and biological activities

The folded 3D structures of peptides and proteins provide excellent starting points for the design of synthetic molecules that mimic key epitopes (or surface patches) involved in protein-protein and protein-nucleic acid interactions. Protein epitope mimetics (PEMs) may recapitulate not only the structural and conformational properties of the target epitope but also their biological activities. By transferring the epitope from a recombinant to a synthetic scaffold that can be produced by parallel combinatorial methods, it is possible to optimize properties through iterative cycles of library synthesis and screening, and even to evolve new biological activities. One very interesting scaffold is found in beta-hairpin motifs, which are used by many proteins to mediate molecular recognition events. This motif is readily amenable to PEM design, for example, by transplanting hairpin loop sequences from folded proteins onto hairpin-stabilizing templates, such as the dipeptide D-Pro-L-Pro. In addition, beta-hairpin peptidomimetics can also be exploited to mimic other types of epitopes, such as those based on alpha-helical secondary structures. The size and shape of beta-hairpin PEMs appear well suited for the design of inhibitors of both protein-protein and protein-nucleic acid interactions, endeavors that have so far proven difficult using small "drug-like" molecules. In recent work, it was shown that beta-hairpin PEMs can be designed that mimic the canonical conformations of antibody hypervariable loops, suggesting that novel small-molecule antibody mimics may be feasible. Using naturally occurring peptides as starting points, beta-hairpin mimetics have been discovered that possess antimicrobial activity, while others are potent inhibitors of the chemokine receptor CXCR4. Beta-hairpin PEMs have also been designed and optimized that mimic an alpha-helical epitope in p53 and so block its interaction with HDM2. A crystal structure of one HDM2-mimetic complex revealed how the surface of the protein had adapted to the shape of the hairpin, thereby enhancing inhibitor affinity. Small folded RNA motifs also make interesting targets for inhibitor design. For example, beta-hairpin mimetics have been designed and optimized that bind with high affinity and good selectivity to the TAR and RRE RNA motifs from HIV-1. Solution structures of the mimetics both free and bound to the RNA target provided some surprises, as well as an improved understanding of the mechanisms of binding. These mimetics represent still a relatively new family of RNA-binding molecules, but clearly one with potential for development into novel antiviral agents.     

Synthetic diacylglycerols (DAG) and DAG-lactones as activators of protein kinase C (PK-C)

The central role of protein kinase C (PK-C) in cellular signal transduction has established it as an important therapeutic target for cancer and other diseases. We have developed a series of 4,4-disubstituted-gamma-butyrolactones, which contain a constrained glycerol backbone (DAG-lactones) and behave as potent and selective activating ligands of PK-C with affinities that approach those of the structurally complex natural product agonists, such as the phorbol esters. This Account traces the design and construction of these molecules. Initially, we examined the consequences of reducing the entropic penalty associated with the transformation of a DAG into a DAG-lactone. Then, using molecular modeling to extend insights arising from the newly solved crystal structure of a C1 domain complexed with phorbol ester, we incorporated amino acid-specific branched hydrophobic chains to provide a new generation of DAG-lactones that have the capacity to bind to PK-C with low nanomolar affinity. Depending on the specific pattern of hydrophobic substitution, some DAG-lactones are able to induce selective translocation of individual PK-C isozymes to different cellular compartments, and since the specific nature of these hydrophobic interactions influences biological outcome, some of these compounds exhibit cell-specific antitumor activity. The ability to direct specific PK-C isozyme translocation with sets of structurally simple, yet highly potent molecules provides a powerful tool for engineering a plethora of molecules with novel biological functions.     

Evaluating Molecular Docking Software for Small Molecule Binding to G-Quadruplex DNA

G-quadruplexes are four-stranded nucleic acid secondary structures of biological significance and have emerged as an attractive drug target. The G4 formed in the MYC promoter (MycG4) is one of the most studied small-molecule targets, and a model system for parallel structures that are prevalent in promoter DNA G4s and RNA G4s. Molecular docking has become an essential tool in structure-based drug discovery for protein targets, and is also increasingly applied to G4 DNA. However, DNA, and in particular G4, binding sites differ significantly from protein targets. Here we perform the first systematic evaluation of four commonly used docking programs (AutoDock Vina, DOCK 6, Glide, and RxDock) for G4 DNA-ligand binding pose prediction using four small molecules whose complex structures with the MycG4 have been experimentally determined in solution. The results indicate that there are considerable differences in the performance of the docking programs and that DOCK 6 with GB/SA rescoring performs better than the other programs. We found that docking accuracy is mainly limited by the scoring functions. The study shows that current docking programs should be used with caution to predict G4 DNA-small molecule binding modes.     

Endogenous Opioid Peptides and Alternatively Spliced Mu Opioid Receptor Seven Transmembrane Carboxyl-Terminal Variants

There exist three main types of endogenous opioid peptides, enkephalins, dynorphins and β-endorphin, all of which are derived from their precursors. These endogenous opioid peptides act through opioid receptors, including mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opioid receptor (KOR), and play important roles not only in analgesia, but also many other biological processes such as reward, stress response, feeding and emotion. The MOR gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms. One type of these splice variants, the full-length 7 transmembrane (TM) Carboxyl (C)-terminal variants, has the same receptor structures but contains different intracellular C-terminal tails. The pharmacological functions of several endogenous opioid peptides through the mouse, rat and human OPRM1 7TM C-terminal variants have been considerably investigated together with various mu opioid ligands. The current review focuses on the studies of these endogenous opioid peptides and summarizes the results from early pharmacological studies, including receptor binding affinity and G protein activation, and recent studies of β-arrestin2 recruitment and biased signaling, aiming to provide new insights into the mechanisms and functions of endogenous opioid peptides, which are mediated through the OPRM1 7TM C-terminal splice variants.     

Titin N2A Domain and Its Interactions at the Sarcomere

Titin is a giant protein in the sarcomere that plays an essential role in muscle contraction with actin and myosin filaments. However, its utility goes beyond mechanical functions, extending to versatile and complex roles in sarcomere organization and maintenance, passive force, mechanosensing, and signaling. Titin’s multiple functions are in part attributed to its large size and modular structures that interact with a myriad of protein partners. Among titin’s domains, the N2A element is one of titin’s unique segments that contributes to titin’s functions in compliance, contraction, structural stability, and signaling via protein–protein interactions with actin filament, chaperones, stress-sensing proteins, and proteases. Considering the significance of N2A, this review highlights structural conformations of N2A, its predisposition for protein–protein interactions, and its multiple interacting protein partners that allow the modulation of titin’s biological effects. Lastly, the nature of N2A for interactions with chaperones and proteases is included, presenting it as an important node that impacts titin’s structural and functional integrity.     

Iterative Nonproteinogenic Residue Incorporation Yields α/β‐Peptides with a Helix–Loop–Helix Tertiary Structure and High Affinity for VEGF

Inhibition of specific protein–protein interactions is attractive for a range of therapeutic applications, but the large and irregularly shaped contact surfaces involved in many such interactions make it challenging to design synthetic antagonists. Here, we describe the development of backbone‐modified peptides containing both α‐ and β‐amino acid residues (α/β‐peptides) that target the receptor‐binding surface of vascular endothelial growth factor (VEGF). Our approach is based on the Z‐domain, which adopts a three‐helix bundle tertiary structure. We show how a two‐helix “mini‐Z‐domain” can be modified to contain β and other nonproteinogenic residues while retaining the target‐binding epitope by using iterative unnatural residue incorporation. The resulting α/β‐peptides are less susceptible to proteolysis than is their parent α‐peptide, and some of these α/β‐peptides match the full‐length Z‐domain in terms of affinity for receptor‐recognition surfaces on the VEGF homodimer.     

Self-Assembling Peptides: From Design to Biomedical Applications

Self-assembling peptides could be considered a novel class of agents able to harvest an array of micro/nanostructures that are highly attractive in the biomedical field. By modifying their amino acid composition, it is possible to mime several biological functions; when assembled in micro/nanostructures, they can be used for a variety of purposes such as tissue regeneration and engineering or drug delivery to improve drug release and/or stability and to reduce side effects. Other significant advantages of self-assembled peptides involve their biocompatibility and their ability to efficiently target molecular recognition sites. Due to their intrinsic characteristics, self-assembled peptide micro/nanostructures are capable to load both hydrophobic and hydrophilic drugs, and they are suitable to achieve a triggered drug delivery at disease sites by inserting in their structure’s stimuli-responsive moieties. The focus of this review was to summarize the most recent and significant studies on self-assembled peptides with an emphasis on their application in the biomedical field.