Neuroprotective and Anti-inflammatory Effects of Rubiscolin-6 Analogs with Proline Surrogates in Position 2.

Naturally occurring peptides, such as rubiscolins derived from spinach leaves, have been shown to possess some interesting activities. They exerted central effects, such as antinociception, memory consolidation and anxiolytic-like activity. The fact that rubiscolins are potent even when given orally makes them very promising drug candidates. The present work tested whether rubiscolin-6 (R-6, Tyr-Pro-Leu-Asp-Leu-Phe) analogs have neuroprotective and anti-inflammatory effects. These hypotheses were tested in the 6-hydroxydopamine (6-OHDA) injury model of human neuroblastoma SH-SY5Y and lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The determination of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), Caspase-3 activity, lipid peroxidation and nitric oxide (NO) production allowed us to determine the effects of peptides on hallmarks related to Parkinson's Disease (PD) and inflammation. Additionally, we investigated the impact of R-6 analogs on serine-threonine kinase (also known as protein kinase B, AKT) and mammalian target of rapamycin (mTOR) activation. The treatment with analogs 3 (Tyr-Inp-Leu-Asp-Leu-Phe-OH), 5 (Dmt-Inp-Leu-Asp-Leu-Phe-OH) and 7 (Tyr-Inp-Leu-Asp-Leu-Phe-NH2) most effectively prevented neuronal death via attenuation of ROS, mitochondrial dysfunction and Caspase-3 activity. Peptides 5 and 7 significantly increased the protein expression of the phosphorylated-AKT (p-AKT) and phosphorylated-mTOR (p-mTOR). Additionally, selected analogs could also ameliorate LPS-mediated inflammation in macrophages via inhibition of intracellular generation of ROS and NO production. Our findings suggest that R-6 analogs exert protective effects, possibly related to an anti-oxidation mechanism in in vitro model of PD. The data shows that the most potent peptides can inhibit 6-OHDA injury by activating the PI3-K/AKT/mTOR pathway, thus playing a neuroprotective role and may provide a rational and robust approach in the design of new therapeutics or even functional foods.

Analgesic Peptides: From Natural Diversity to Rational Design.

Pain affects one-third of the global population and is a significant public health issue. The use of opioid drugs, which are the strongest painkillers, is associated with several side effects, such as tolerance, addiction, overdose, and even death. An increasing demand for novel, safer analgesic agents is a driving force for exploring natural sources of bioactive peptides with antinociceptive activity. Since the G protein-coupled receptors (GPCRs) play a crucial role in pain modulation, the discovery of new peptide ligands for GPCRs is a significant challenge for novel drug development. The aim of this review is to present peptides of human and animal origin with antinociceptive potential and to show the possibilities of their modification, as well as the design of novel structures. The study presents the current knowledge on structure-activity relationship in the design of peptide-based biomimetic compounds, the modification strategies directed at increasing the antinociceptive activity, and improvement of metabolic stability and pharmacodynamic profile. The procedures employed in prolonged drug delivery of emerging compounds are also discussed. The work summarizes the conditions leading to the development of potential morphine replacements.

Synthesis, Biological Activity and Molecular Docking of Chimeric Peptides Targeting Opioid and NOP Receptors.

Recently, mixed opioid/NOP agonists came to the spotlight for their favorable functional profiles and promising outcomes in clinical trials as novel analgesics. This study reports on two novel chimeric peptides incorporating the fragment Tyr-c[D-Lys-Phe-Phe]Asp-NH2 (RP-170), a cyclic peptide with high affinity for µ and κ opioid receptors (or MOP and KOP, respectively), conjugated with the peptide Ac-RYYRIK-NH2, a known ligand of the nociceptin/orphanin FQ receptor (NOP), yielding RP-170-RYYRIK-NH2 (KW-495) and RP-170-Gly3-RYYRIK-NH2 (KW-496). In vitro, the chimeric KW-496 gained affinity for KOP, hence becoming a dual KOP/MOP agonist, while KW-495 behaved as a mixed MOP/NOP agonist with low nM affinity. Hence, KW-495 was selected for further in vivo experiments. Intrathecal administration of this peptide in mice elicited antinociceptive effects in the hot-plate test; this action was sensitive to both the universal opioid receptor antagonist naloxone and the selective NOP antagonist SB-612111. The rotarod test revealed that KW-495 administration did not alter the mice motor coordination performance. Computational studies have been conducted on the two chimeras to investigate the structural determinants at the basis of the experimental activities, including any role of the Gly3 spacer.

Argireline: Needle-Free Botox as Analytical Challenge.

Argireline-containing cosmetics attract public interest due to their confirmed reduction of facial wrinkles. Argireline is a peptide that works by inhibiting the release of neurotransmitters in the neuromuscular junction, producing a botox-like effect. Therefore, it is used as a safe needle-free alternative to botox treatment. In this work we investigated the presence of Argireline in cosmetic creams and sera by application of reversed phase liquid chromatography and tandem mass spectrometry (RP-HPLC/MS and MS/MS). The analysis revealed the presence of argireline and its oxidized form in several different cosmetics. The methionine residue in Argireline sequence was indicated as oxidation point according to neutral loss MS studies. The developed sample preparation strategy minimizes and monitors methionine oxidation, bringing to our attention the question of impact of ingredients on the stability of cosmetic product.

Synthesis, Pharmacological Evaluation, and Computational Studies of Cyclic Opioid Peptidomimetics Containing ß3-Lysine.

Our formerly described pentapeptide opioid analog Tyr-c[D-Lys-Phe-Phe-Asp]NH2 (designated RP-170), showing high affinity for the mu (MOR) and kappa (KOR) opioid receptors, was much more stable than endomorphine-2 (EM-2) in the rat brain homogenate and displayed remarkable antinociceptive activity after central (intracerebroventricular) and peripheral (intravenous ) administration. In this report, we describe the further modification of this analog, which includes the incorporation of a ß3-amino acid, (R)- and (S)-ß3-Lys, instead of D-Lys in position 2. The influence of such replacement on the biological properties of the obtained analogs, Tyr-c[(R)-ß3-Lys-Phe-Phe-Asp]NH2 (RP-171) and Tyr-c[(S)-ß3-Lys-Phe-Phe-Asp]NH2, (RP-172), was investigated in vitro. Receptor radiolabeled displacement and functional calcium mobilization assays were performed to measure binding affinity and receptor activation of the new analogs. The obtained data revealed that only one of the diastereoisomeric peptides, RP-171, was able to selectively bind and activate MOR. Molecular modeling (docking and molecular dynamics (MD) simulations) suggests that both compounds should be accommodated in the MOR binding site. However, in the case of the inactive isomer RP-172, fewer hydrogen bonds, as well as instability of the canonical ionic interaction to Asp147, could explain its very low MOR affinity.

Design, Synthesis and Functional Analysis of Cyclic Opioid Peptides with Dmt-Tic Pharmacophore.

The opioid receptors are members of the G-protein-coupled receptor (GPCR) family and are known to modulate a variety of biological functions, including pain perception. Despite considerable advances, the mechanisms by which opioid agonists and antagonists interact with their receptors and exert their effect are still not completely understood. In this report, six new hybrids of the Dmt-Tic pharmacophore and cyclic peptides, which were shown before to have a high affinity for the µ-opioid receptor (MOR) were synthesized and characterized pharmacologically in calcium mobilization functional assays. All obtained ligands turned out to be selective antagonists of the δ-opioid receptor (DOR) and did not activate or block the MOR. The three-dimensional structural determinants responsible for the DOR antagonist properties of these analogs were further investigated by docking studies. The results indicate that these compounds attach to the DOR in a slightly different orientation with respect to the Dmt-Tic pharmacophore than Dmt-TicΨ[CH2-NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]), a prototypical DOR antagonist peptide. Key pharmacophoric contacts between the DOR and the ligands were maintained through an analogous spatial arrangement of pharmacophores, which could provide an explanation for the predicted high-affinity binding and the experimentally observed functional properties of the novel synthetic ligands.

Pharmacological Characterization of µ-Opioid Receptor Agonists with Biased G Protein or ß-Arrestin Signaling, and Computational Study of Conformational Changes during Receptor Activation.

In recent years, G protein vs. ß-arrestin biased agonism at opioid receptors has been proposed as an opportunity to produce antinociception with reduced adverse effects. However, at present this approach is highly debated, a reason why more information about biased ligands is required. While the practical relevance of bias in the case of µ-opioid receptors (MOP) still needs to be validated, it remains important to understand the basis of this bias of MOP (and other GPCRs). Recently, we reported two cyclopeptides with high affinity for MOP, the G protein biased Dmt-c[d-Lys-Phe-pCF3-Phe-Asp]NH2 (F-81), and the ß-arrestin 2 biased Dmt-c[d-Lys-Phe-Asp]NH2 (C-33), as determined by calcium mobilization assay and bioluminescence resonance energy transfer-based assay. The biased character of F-81 and C-33 has been further analyzed in the [35S]GTPγS binding assay in human MOP-expressing cells, and the PathHunter enzyme complementation assay, used to measure ß-arrestin 2 recruitment. To investigate the structural features of peptide-MOP complexes, we performed conformational analysis by NMR spectroscopy, molecular docking, and molecular dynamics simulation. These studies predicted that the two ligands form alternative complexes with MOP, engaging specific ligand-receptor contacts. This would induce different displays of the cytosolic side of the seven-helices bundle, in particular by stabilizing different angulations of helix 6, that could favor intracellular coupling to either G protein or ß-arrestin.

Synthesis and Pharmacological Evaluation of Hybrids Targeting Opioid and Neurokinin Receptors.

Morphine, which acts through opioid receptors, is one of the most efficient analgesics for the alleviation of severe pain. However, its usefulness is limited by serious side effects, including analgesic tolerance, constipation, and dependence liability. The growing awareness that multifunctional ligands which simultaneously activate two or more targets may produce a more desirable drug profile than selectively targeted compounds has created an opportunity for a new approach to developing more effective medications. Here, in order to better understand the role of the neurokinin system in opioid-induced antinociception, we report the synthesis, structure-activity relationship, and pharmacological characterization of a series of hybrids combining opioid pharmacophores with either substance P (SP) fragments or neurokinin receptor (NK1) antagonist fragments. On the bases of the in vitro biological activities of the hybrids, two analogs, opioid agonist/NK1 antagonist Tyr-[d-Lys-Phe-Phe-Asp]-Asn-d-Trp-Phe-d-Trp-Leu-Nle-NH2 (2) and opioid agonist/NK1 agonist Tyr-[d-Lys-Phe-Phe-Asp]-Gln-Phe-Phe-Gly-Leu-Met-NH2 (4), were selected for in vivo tests. In the writhing test, both hybrids showed significant an antinociceptive effect in mice, while neither of them triggered the development of tolerance, nor did they produce constipation. No statistically significant differences in in vivo activity profiles were observed between opioid/NK1 agonist and opioid/NK1 antagonist hybrids.

Cyclic mu-opioid receptor ligands containing multiple N-methylated amino acid residues.

In this study we report the in vitro activities of four cyclic opioid peptides with various sequence length/macrocycle size and N-methylamino acid residue content. N-Methylated amino acids were incorporated and cyclization was employed to enhance conformational rigidity to various extent. The effect of such modifications on ligand structure and binding properties were studied. The pentapeptide containing one endocyclic and one exocyclic N-methylated amino acid displayed the highest affinity to the mu-opioid receptor. This peptide was also shown to be a full agonist, while the other analogs failed to activate the mu opioid receptor. Results of molecular docking studies provided rationale for the explanation of binding properties on a structural basis.

Design and characterization of opioid ligands based on cycle-in-macrocycle scaffold.

The study reports on a series of novel cyclopeptides based on the structure Tyr-[d-Lys-Phe-Phe-Asp]NH2, a mixed mu and kappa opioid receptor agonist with low nanomolar affinity, in which Phe4 residue was substituted by cyclic amino acids, such as Pro or its six-membered surrogates, piperidine-2-, 3- or 4-carboxylic acids (Pip, Nip and Inp, respectively). All derivatives exhibited high mu- and moderate delta-opioid receptor affinity, and almost no binding to the kappa-opioid receptor. Conformational analysis suggested that the cis conformation of the peptide bond Phe3-Xaa4 influences receptor selectivity through the control of the position of Phe3 side chain. The results substantiate the use of the cycle-macrocyle scaffolds for fine-tuning receptor selectivity.

Synthesis, receptor binding studies, optical spectroscopic and in silico structural characterization of morphiceptin analogs with cis-4-amino-L-proline residues.

Three novel morphiceptin analogs, in which Pro in position 2 and/or 4 was replaced by cis-4-aminoproline connected with the preceding amino acid through the primary amino group, were synthesized. The opioid receptor affinities, functional assay results, enzymatic degradation studies and experimental and in silico structural analysis of such analogs are presented. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis, biological evaluation and structural analysis of novel peripherally active morphiceptin analogs.

Morphiceptin (Tyr-Pro-Phe-Pro-NH2), a tetrapeptide amide, is a selective ligand of the µ-opioid receptor (MOR). This study reports the synthesis and biological evaluation of a series of novel morphiceptin analogs modified in positions 2 or/and 4 by introduction of 4,4-difluoroproline (F2Pro) in l or d configuration. Depending on the fluorinated amino acid configuration and its position in the sequence, new analogs behaved as selective full MOR agonists showing high, moderate, or relatively low potency. The most potent analog, Tyr-F2Pro-Phe-D-F2Pro-NH2, was also able to activate the κ-opioid receptor (KOR), although with low potency. Docking studies and the comparison of results with the high resolution crystallographic structure of a MOR-agonist complex revealed possible structure-activity relationships of this compound family.

Cyclodimerization of immunosuppressive fragment of HLA-DR molecule. Design, synthesis and ESI-MS/MS analysis.

The nonapeptide fragment of the HLA-DR molecule, located in the exposed loop of the alpha-chain (164-172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three-dimensional structure of the HLA-DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C-termini by spacer of (Gly5 )2 -Lys-NH2 and the N-termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG5 )2 K-resin analog was synthesized using solid-phase peptide synthesis protocols. The cyclization was achieved by cross-linking the N-terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega-bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Ring size in cyclic endomorphin-2 analogs modulates receptor binding affinity and selectivity.

The study reports the solid-phase synthesis and biological evaluation of a series of new side chain-to-side chain cyclized opioid peptide analogs of the general structure Tyr-[D-Xaa-Phe-Phe-Asp]NH2, where Xaa = Lys (1), Orn (2), Dab (3), or Dap (4) (Dab = 2,4-diaminobutyric acid, Dap = 2,3-diaminopropionic acid), containing 17- to 14-membered rings. The influence of the ring size on binding to the MOP, DOP and KOP opioid receptors was studied. In general, the reduction of the size of the macrocyclic ring increased the selectivity for the MOP receptor. The cyclopeptide incorporating Xaa = Lys displayed subnanomolar MOP affinity but modest selectivity over the KOP receptor, while the analog with the Orn residue showed increased affinity and selectivity for MOP. The analog with Dab was a weak MOP agonist and did not bind to the other two opioid receptors. Finally, the peptide with Xaa = Dap was completely MOP receptor-selective with subnanomolar affinity. Interestingly, the deletion of one Phe residue from 1 led to the 14-membered Tyr-c[D-Lys-Phe-Asp]NH2 (5), a potent and selective MOP receptor ligand. The in vitro potencies of the new analogs were determined in a calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. A good correlation between binding and the functional test results was observed. The influence of the ring size, solid support and the N-terminal protecting group on the formation of cyclodimers was studied.

Synthesis and mass spectrometry analysis of quaternary cryptando-peptidic conjugates.

The bicyclic amines in the form of cryptands, the crown ether analogs, were used in the synthesis of cryptando-peptidic conjugates with simultaneous formation of quaternary ammonium nitrogen moiety. A series of model cryptando-peptidic conjugates at the peptide N-terminus was efficiently prepared by the standard Fmoc solid phase synthesis. Tandem mass spectrometric analysis of the obtained conjugates has shown the specific fragmentation pattern during MS/MS experiment. The obtained cryptandic quaternary ammonium group undergoes the Hofmann elimination during collision-induced dissociation fragmentation followed by the ethoxyl group elimination. The presented quaternization of cryptands by iodoacetylated peptides is relatively easy and compatible with standard solid-phase peptide synthesis. Additionally, the applicability of such peptide derivatives and their isotopologues selectively deuterated at the α-carbon in the quantitative LC-MS analysis was analyzed.

Synthesis of linear and cyclic opioid-based peptide analogs containing multiple N-methylated amino acid residues.

A series of six novel opioid peptide analogs containing one to three N-methylamino acid residues, and six cyclic counterparts of these peptides were prepared by the solid-phase method. Introduction of two consecutive N-methylated amino acids, as well as cyclization of such conformationally constrained sequences, turned out to be challenging. The use of a recently reported triazine-based coupling reagent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate, enabled the synthesis and cyclization of the designed analogs in acceptable yields and with a lesser amount of by-products than observed with the standard coupling reagents such as TBTU or HATU.

Facile synthesis of deuterium-labeled denatonium cation and its application in the quantitative analysis of Bitrex by liquid chromatography-mass spectrometry.

Quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) is frequently based on the application of isotopically labeled standards which usually have to be specially synthesized. Although the synthesis of deuterated isotopologues is relatively inexpensive, they are not considered as good internal standards due to the possible deuterium effect on the retention time during LC-MS analysis. We developed a method of deuterium labeling of denatonium benzoate (Bitrex) via H/D exchange of its α-carbon hydrogen atoms in CH2 group situated between carbonyl and quaternary ammonium groups. The proposed strategy is rapid, cost-efficient, and does not require derivatization reagents or further purification. The LC-MS analysis of isotopologues revealed that the introduced deuterons do not undergo back exchange under acidic conditions, and the co-elution of deuterated and non-deuterated forms is observed. The obtained deuterated standard was applied in the quantitative LC-MS analysis of Bitrex in commercially available household products.

Comparison of modification sites in glycated crystallin in vitro and in vivo.

Glycation of α-crystallin is responsible for age- and diabetic-related cataracts, which are the main cause of blindness worldwide. We optimized the method of identification of lysine residues prone to glycation using the combination of LC-MS, isotopic labeling, and modified synthetic peptide standards with the glycated lysine derivative (Fmoc-Lys(i,i-Fru,Boc)-OH). The in vitro glycation of bovine lens α-crystallin was conducted by optimized method with the equimolar mixture of [(12)C6]- and [(13)C6]D-glucose. The in vivo glycation was studied on human lens crystallin. The glycated protein was subjected to proteolysis and analyzed using LC-MS. The results of in vitro and in vivo glycation of α-crystallin reveal a different distribution of the modified lysine residues. More Amadori products were detected as a result of the in vitro reaction due to forced glycation conditions. The developed method allowed us to identify the glycation sites in crystallin from eye lenses obtained from patients suffering from the cataract. We identified K166 in the A chain and K166 in the B chain of α-crystallin as major glycation sites during the in vitro reaction. We found also two in vivo glycated lysine residues: K92 in the B chain and K166 in the A chain, which are known as locations for Amadori products. These modification sites were confirmed by the LC-MS experiment using two synthetic standards. This study demonstrates the applicability of the LC-MS methods combined with the isotopic labeling and synthetic peptide standards for analysis of post-translational modifications in the biological material.

Microwave-assisted 18O labeling of Fmoc-protected amino acids.

Recently, there has been an increased interest in isotopical labeling of peptides. Although there are several techniques allowing for a complete labeling of all carboxyl groups in peptides, regioselective labeling would be beneficial in many situations. Such labeling requires the use of (18)O-labeled Fmoc amino acids. We have designed a method for such labeling that is an improvement on a technique proposed earlier. The new procedure is suitable for microscale synthesis and could be used in peptide and proteomics laboratories. Although for the majority of tested amino acids our method gives good labeling efficiency, it is time consuming. Therefore, we have decided to use microwave-assisted procedure. This approach resulted in reduction of reaction time to 15 min and increased reaction efficiency.

New method of peptide cleavage based on Edman degradation.

A straightforward cleavage method for N- acylated peptides based on the phenylthiohydantoin (PTH) formation is presented. The procedure could be applied to acid-stable resins, such as TentaGel HL-NH[Formula: see text]. We designed a cleavable linker that consists of a lysine residue with the [Formula: see text]-amino group blocked by Boc, whereas the [Formula: see text]-amino group is used for peptide synthesis. After the peptide assembly is completed, the protecting groups in peptide side chains are removed using trifluoroacetic acid, thus liberating also the [Formula: see text]-amino group of the lysine in the linker. Then the reaction with phenyl isothiocyanate followed by acidolysis causes an efficient peptide release from the resin as a stable PTH derivative. Furthermore, the application of a fixed charge tag in the form of 2-(4-aza-1-azoniabicyclo[2.2.2]octylammonium)acetyl group increases ionization efficiency and reduces the detection limit, allowing ESI-MS/MS sequencing of peptides in the subfemtomolar range. The proposed strategy is compatible with standard conditions during one-bead-one-compound peptide library synthesis. The applicability of the developed strategy in combinatorial chemistry was confirmed using a small training library of [Formula: see text]-chymotrypsin substrates.