Visible light-promoted C(sp3)-H α-carbamoylation of cyclic ethers with isocyanides.

A protocol exploiting isocyanides as carbamoylating agents for the α-C(sp3)-H functionalization of cyclic ethers has been optimized via a combined visible light-driven hydrogen atom transfer/Lewis acid-catalyzed approach. The isocyanide substrate scope revealed an exquisite functional group compatibility (18 examples, with yields up to 99%). Both radical and polar trapping, kinetic isotopic effect and real-time NMR studies support the mechanistic hypothesis and provide insightful details for the design of new chemical processes involving the generation of oxocarbenium ions.

Glucopeptides derived from myelin-relevant proteins and hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin cross-react with multiple sclerosis specific antibodies: A step forward in the identification of native autoantigens in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory and autoimmune disorder, in which an antibody-mediated demyelination mechanism plays a critical role. We prepared two glucosylated peptides derived from the human myelin proteins, that is, oligodendrocyte-myelin glycoprotein (OMGp) and reticulon-4 receptor (RTN4R), selected by a bioinformatic approach for their conformational homology with CSF114(Glc), a designed ß-turn antigenic probe derived from myelin oligodendrocyte glycoprotein (MOG), a glycoprotein present in the CNS. This synthetic antigen is specifically recognized by antibodies in sera of MS patients. We report herein the antigenic properties of these peptides, showing, on the one hand, that MS patient antibodies recognize the two glucosylated peptides and, on the other hand, that these antibodies cross-react with CSF114(Glc) and with the previously described hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin protein HMW1ct(Glc). These observations point to an immunological association between human and bacterial protein antigens, underpinning the hypothesis that molecular mimicry triggers the breakdown of self-tolerance in MS and suggesting that RTN4R and OMGp can be considered as autoantigens.

Introduction of constrained Trp analogs in RW9 modulates structure and partition in membrane models.

Over the past decades, many cell-penetrating peptides (CPP) have been studied for their capacity to cross cellular membranes, mostly in order to improve cellular uptake of therapeutic agents. Even though hydrophobic and anionic CPPs have been described, many of them are polycationic, due to the presence of several arginine (Arg) residues. Noteworthy, however, the presence of aromatic amino acids such as tryptophan (Trp) within CPPs seems to play an important role to reach high membranotropic activity. RW9 (RRWWRRWRR) is a designed CPP derived from the polyarginine R9 presenting both features. In general, when interacting with membranes, CPPs adopt an optimal conformation for membrane interactions - an amphipathic helical secondary structure in the case of RW9. Herein, we assumed that the incorporation of a locally constrained amino acid in the peptide sequence could improve the membranotropic activity of RW9, by facilitating its structuration upon contact with a membrane, while leaving a certain plasticity. Therefore, two cyclized Trp derivatives (Tcc and Aia) were synthesized to be incorporated in RW9 as surrogates of Trp residues. Thus, a series of peptides containing these building blocks has been synthesized by varying the type, position, and number of modifications. The membranotropic activity of the RW9 analogs was studied by spectrofluorescence titration of the peptides in presence of liposomes (DMPG), allowing to calculate partition coefficients (Kp). Our results indicate that the partitioning of the modified peptides depends on the type, the number and the position of the modification, with the best sequence being [Aia4]RW9. Interestingly, both NMR analysis and molecular dynamic (MD) simulations indicate that this analog presents an extended conformation similar to the native RW9, but with a much-reduced structural flexibility. Finally, cell internalization properties were also confirmed by confocal microscopy.

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands.

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.

The Inhibition of DNA Viruses by the Amphibian Antimicrobial Peptide Temporin G: A Virological Study Addressing HSV-1 and JPCyV.

Herpes simplex virus type-1 (HSV-1) and John Cunningham polyomavirus (JCPyV) are widely distributed DNA viruses causing mainly asymptomatic infection, but also mild to very severe diseases, especially when these viruses reach the brain. Some drugs have been developed to inhibit HSV-1 replication in host cells, but their prolonged use may induce resistance phenomena. In contrast, to date, there is no cure for JCPyV. The search for alternative drugs that can reduce viral infections without undermining the host cell is moving toward antimicrobial peptides (AMPs) of natural occurrence. These include amphibian AMPs belonging to the temporin family. Herein, we focus on temporin G (TG), showing that it strongly affects HSV-1 replication by acting either during the earliest stages of its life cycle or directly on the virion. Computational studies have revealed the ability of TG to interact with HSV-1 glycoprotein B. We also found that TG reduced JCPyV infection, probably affecting both the earliest phases of its life cycle and the viral particle, likely through an interaction with the viral capsid protein VP1. Overall, our results are promising for the development of short naturally occurring peptides as antiviral agents used to counteract diseases related to HSV-1 and JCPyV.

A novel ß-hairpin peptide derived from the ARC repressor selectively interacts with the major groove of B-DNA.

Transcription factors (TFs) have a remarkable role in the homeostasis of the organisms and there is a growing interest in how they recognize and interact with specific DNA sequences. TFs recognize DNA using a variety of structural motifs. Among those, the ribbon-helix-helix (RHH) proteins, exemplified by the MetJ and ARC repressors, form dimers that insert antiparallel ß-sheets into the major groove of DNA. A great chemical challenge consists of using the principles of DNA recognition by TFs to design minimized peptides that maintain the DNA affinity and specificity characteristics of the natural counterparts. In this context, a peptide mimic of an antiparallel ß-sheet is very attractive since it can be obtained by a single peptide chain folding in a ß-hairpin structure and can be as short as 14 amino acids or less. Herein, we designed eight linear and two cyclic dodeca-peptides endowed with ß-hairpins. Their DNA binding properties have been investigated using fluorescence spectroscopy together with the conformational analysis through circular dichroism and solution NMR. We found that one of our peptides, peptide 6, is able to bind DNA, albeit without sequence selectivity. Notably, it shows a topological selectivity for the major groove of the DNA which is the interaction site of ARC and many other DNA-binding proteins. Moreover, we found that a type I' ß-hairpin folding pattern is a favorite peptide structure for interaction with the B-DNA major groove. Peptide 6 is a valuable lead compound for the development of novel analogs with sequence selectivity.

Synthesis and Characterization of Bis-Triazolyl-Pyridine Derivatives as Noncanonical DNA-Interacting Compounds.

Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.

Disulfide Bond Replacement with 1,4- and 1,5-Disubstituted [1,2,3]-Triazole on C-X-C Chemokine Receptor Type 4 (CXCR4) Peptide Ligands: Small Changes that Make Big Differences.

Here we investigated the structural and biological effects ensuing from the disulfide bond replacement of a potent and selective C-X-C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4- and 1,5- disubstituted 1,2,3-triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12-mediated cell migration, the 1,4-triazole variant conserved the antagonistic effect in the low-mid nanomolar range, while the 1,5-triazole one displayed the ability to activate the migration, becoming the first in class low-molecular-weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR-interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors.

Constrained Peptides with Fine-Tuned Flexibility Inhibit NF-Y Transcription Factor Assembly.

Protein complex formation depends on the interplay between preorganization and flexibility of the binding epitopes involved. The design of epitope mimetics typically focuses on stabilizing a particular bioactive conformation, often without considering conformational dynamics, which limits the potential of peptidomimetics against challenging targets such as transcription factors. We developed a peptide-derived inhibitor of the NF-Y transcription factor by first constraining the conformation of an epitope through hydrocarbon stapling and then fine-tuning its flexibility. In the initial set of constrained peptides, a single non-interacting α-methyl group was observed to have a detrimental effect on complex stability. Biophysical characterization revealed how this methyl group affects the conformation of the peptide in its bound state. Adaption of the methylation pattern resulted in a peptide that inhibits transcription factor assembly and subsequent recruitment to the target DNA.

Investigation on side-product formation during the synthesis of a lactoferrin-derived lactam-bridged cyclic peptide.

Bovine lactoferrin C-lobe is able to prevent both influenza virus hemagglutination and cell infection. In particular, it was demonstrated that the fragment 418SKHSSLDCVLRP429 is a potent antiviral peptide. Therefore, we tried to increase the stability of this fragment through side-chain lactam cyclization of the peptide, S[KHSSLD]CVLRP (1). However, classic strategy involving solid-supported cyclization of the linear precursor, containing orthogonal allyl/alloc-based protection for the key amino and carboxyl residues, did not provide the desired cyclic peptide. Here, we report the identification of problematic stretches during the sequence assembly process and the optimization of the different parameters involved in the construction of 1. Results indicated a significant influence of ß-protecting group of both aspartic acid and adjacent cysteine residues on the formation of side products. Therefore, the identification of suitable ß-protecting groups of these residues allowed us to optimize the synthesis of designed lactam-bridged cyclic peptide.

Screening Platform toward New Anti-HIV Aptamers Set on Molecular Docking and Fluorescence Quenching Techniques.

By using a new rapid screening platform set on molecular docking simulations and fluorescence quenching techniques, three new anti-HIV aptamers targeting the viral surface glycoprotein 120 (gp120) were selected, synthesized, and assayed. The use of the short synthetic fluorescent peptide V35-Fluo mimicking the V3 loop of gp120, as the molecular target for fluorescence-quenching binding affinity studies, allowed one to measure the binding affinities of the new aptamers for the HIV-1 gp120 without the need to obtain and purify the full recombinant gp120 protein. The almost perfect correspondence between the calculated Kd and the experimental EC50 on HIV-infected cells confirmed the reliability of the platform as an alternative to the existing methods for aptamer selection and measuring of aptamer-protein equilibria.

Native PAGE to study the interaction between the oncosuppressor p53 and its protein ligands.

In the present study, we investigated a new approach for studying the interaction between p53 and MDM2/X (where MDM is murine double minute protein). The method is based on the different mobility between the interacting domains of the oncosuppressor p53 and its protein ligands MDM2/X on polyacrylamide gels under native conditions. While the two proteins MDM2/X alone were able to enter the gel, the formation of a binary complex between p53 and MDM2/X prevented the gel entry. The novel technique is reliable for determining the different affinity elicited by MDM2 or MDMX toward p53, and can be useful for analyzing the dissociation power exerted by other molecules on the p53-MDM2/X complex.

Nondenaturing polyacrylamide gel electrophoresis to study the dissociation of the p53·MDM2/X complex by potentially anticancer compounds.

A new analytical method to study the dissociation of the complexes between the oncosuppressor p53 and its negative modulators murine double-minute protein 2 (MDM2) or MDMX, is proposed. This technique is reliable to determine the dissociative power exerted by small molecules on the complex taking advantage of the appearance of migrating MDM2 or MDMX in a native polyacrylamide gel, when inhibitors are added to the complex mixture. Therefore, we propose this new approach to easily screen library of compounds, with potential pharmacological anticancer activity.

An investigation into the origin of the biased agonism associated with the urotensin II receptor activation.

The urotensin II receptor (UTR) has long been studied mainly for its involvement in the cardiovascular homeostasis both in health and disease state. Two endogenous ligands activate UTR, i.e. urotensin II (U-II) and urotensin II-related peptide (URP). Extensive expression of the two ligands uncovers the diversified pathophysiological effects mediated by the urotensinergic system such as cardiovascular disorders, smooth muscle cell proliferation, renal disease, diabetes, and tumour growth. As newly reported, U-II and URP have distinct effects on transcriptional activity, cell proliferation, and myocardial contractile activities supporting the idea that U-II and URP interact with UTR in a distinct manner (biased agonism). To shed light on the origin of the divergent activities of the two endogenous ligands, we performed a conformational study on URP by solution NMR in sodium dodecyl sulfate micelle solution and compared the obtained NMR structure of URP with that of hU-II previously determined. Finally, we undertook docking studies between URP, hU-II, and an UT receptor model.

The effect of d-amino acid substitution on the selectivity of temporin L towards target cells: identification of a potent anti-Candida peptide.

The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro(3)]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6-13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro(3)]TL with the corresponding d isomers, known as helix breakers. Structure-activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro(3)]TL, without giving a toxic effect towards human cells.

SAR study and conformational analysis of a series of novel peptide G protein-coupled receptor kinase 2 inhibitors.

G protein-coupled receptor kinase 2 (GRK2) plays a central role in the cellular transduction network. In particular, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. Thereby, its inhibition offers a potential therapeutic solution to several pathological conditions. In the present study, we performed a SAR study and a NMR conformational analysis of peptides derived from HJ loop of GRK2 and able to selectively inhibit GRK2. From Ala-scan and D-Ala point replacement, we found that Arg residues don't affect the inhibitory properties, while a D-amino acid at position 5 is key to the activity. Conformational analysis identified two ß-turns that involve N-terminal residues, followed by a short extended region. These information can help the design of peptides and peptido-mimetics with enhanced GRK2 inhibition properties.

Urantide conformation and interaction with the urotensin-II receptor.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have previously identified the compound termed urantide (H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor (UTR) antagonist described to date. Urantide may have potential clinical value in the treatment of atherosclerosis. In the present study, we studied the conformational preferences of urantide in DPC micelles and developed a urantide/UTR interaction model. This model can help the design of novel peptides and small molecules as UTR antagonists.

Strategic Single-Residue Substitution in the Antimicrobial Peptide Esc(1-21) Confers Activity against Staphylococcus aureus, Including Drug-Resistant and Biofilm Phenotype.

Staphylococcus aureus, a bacterium resistant to multiple drugs, is a significant cause of illness and death worldwide. Antimicrobial peptides (AMPs) provide an excellent potential strategy to cope with this threat. Recently, we characterized a derivative of the frog-skin AMP esculentin-1a, Esc(1-21) (1) that is endowed with potent activity against Gram-negative bacteria but poor efficacy against Gram-positive strains. In this study, three analogues of peptide 1 were designed by replacing Gly8 with α-aminoisobutyric acid (Aib), Pro, and dPro (2-4, respectively). The single substitution Gly8 → Aib8 in peptide 2 makes it active against the planktonic form of Gram-positive bacterial strains, especially Staphylococcus aureus, including multidrug-resistant clinical isolates, with an improved biostability without resulting in cytotoxicity to mammalian cells. Moreover, peptide 2 showed a higher antibiofilm activity than peptide 1 against both reference and clinical isolates of S. aureus. Peptide 2 was also able to induce rapid bacterial killing, suggesting a membrane-perturbing mechanism of action. Structural analysis of the most active peptide 2 evidenced that the improved biological activity of peptide 2 is the consequence of a combination of higher biostability, higher α helical content, and ability to reduce membrane fluidity and to adopt a distorted helix, bent in correspondence of Aib8. Overall, this study has shown how a strategic single amino acid substitution is sufficient to enlarge the spectrum of activity of the original peptide 1, and improve its biological properties for therapeutic purposes, thus paving the way to optimize AMPs for the development of new broad-spectrum anti-infective agents.

Discovery of 2,3-Diaminoindole Derivatives as a Novel Class of NOD Antagonists.

NOD1 and NOD2 are members of the pattern recognition receptors involved in the innate immune response. Overactivation of NOD1 is implicated in inflammatory disorders, multiple sclerosis, and cancer cell metastases. NOD1 antagonists would represent valuable pharmacological tools to gain further insight into protein roles, potentially leading to new therapeutic strategies. We herein report the expansion of the chemical space of NOD1 antagonists via a multicomponent synthetic approach affording a novel chemotype, namely, 2,3-diaminoindoles. These efforts resulted in compound 37, endowed with low micromolar affinity toward NOD1. Importantly, a proof-of-evidence of direct binding to NOD1 of Noditinib-1 and derivative 37 is provided here for the first time. Additionally, the combination of computational studies and NMR-based displacement assays enabled the characterization of the binding modality of 37 to NOD1, thus providing key unprecedented knowledge for the design of potent and selective NOD1 antagonists.

Opposite modulation of cell migration by distinct subregions of urokinase connecting peptide.

Functional analysis of isolated protein domains may uncover cryptic activities otherwise missed. The serine protease urokinase (uPA) has a clear-cut motogen activity that is catalytically independent and resides in its amino-terminal growth factor domain (GFD, residues 1-49) and connecting peptide region (CP, residues 132-158). To functionally dissect the CP region, we analysed the biological activity of two synthetic peptides corresponding to the N-terminal [uPA-(135-143), residues 135-143] and C-terminal [uPA-(144-158), residues 144-158] CP subregions. Most of the chemotactic activity of connecting peptide-derived peptide (CPp, [uPA-(135-158)]) for embryonic kidney HEK293/uPAR-25 cells is retained by uPA-(144-158) at nanomolar concentrations. In contrast, uPA-(135-143) inhibits basal, CPp -, vitronectin- and fibronectin-induced cell migration. Radioreceptor binding assays on intact HEK293 cells revealed that uPA-(135-143) and uPA-(144-158) are both able to compete with [(125)I]-CPp, albeit with different binding affinities. The consequences of phospho-mimicking, S138E substitution, were studied using [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. Unlike CPp, [138E]uPA-(135-158) and [138E]uPA-(135-143) exhibit remarkable inhibitory properties. Finally, analysis of the conformational preferences of the peptides allowed to identify secondary structure elements exclusively characterising the stimulatory CPp and uPA-(144-158) versus the inhibitory uPA-(135-143), [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. In conclusion, these data shed light on the cryptic activities of uPA connecting peptide, revealing the occurrence of two adjacent regions, both competing for binding to cell surface but conveying opposite signalling on cell migration.