The first structure-activity relationship study of oxytocin as a positive allosteric modulator for the µ opioid receptor.

Positive allosteric modulators (PAMs) of G protein-coupled receptors (GPCRs) have drawn attention as novel drug candidates. PAMs can enhance the activities of endogenous agonists which are not only secreted at appropriate times and in parts of the body, but also are immediately metabolized. Therefore, they are expected to show fewer side effects than exogeneous orthosteric ligands. Recently, we have reported that oxytocin (OT) functioned as a PAM of the µ opioid receptor (MOR) which was one of the most potent targets for analgesics. OT is thus thought to be a useful compound for the development of novel analgesics. In this study, several OT analogs were synthesized and evaluated with an intact cell-based assay to investigate the crucial structures of OT for exerting the PAM activity. The assay results indicated that the cyclic structure formed by an intramolecular disulfide bond and the three C-terminal residues containing a small Gly residue of OT were essential for their function as a MOR-PAM. Intriguingly, two analogs having an amide or an ethylene tether instead of the intramolecular disulfide bridge did not have any PAM effects. The results suggested that the disulfide linkage of OT would be a key structure for exerting the PAM activity at the MOR.

Oxytocin Is a Positive Allosteric Modulator of κ-Opioid Receptors but Not δ-Opioid Receptors in the G Protein Signaling Pathway.

Oxytocin (OT) influences various physiological functions such as uterine contractions, maternal/social behavior, and analgesia. Opioid signaling pathways are involved in one of the analgesic mechanisms of OT. We previously showed that OT acts as a positive allosteric modulator (PAM) and enhances µ-opioid receptor (MOR) activity. In this study, which focused on other opioid receptor (OR) subtypes, we investigated whether OT influences opioid signaling pathways as a PAM for δ-OR (DOR) or κ-OR (KOR) using human embryonic kidney-293 cells expressing human DOR or KOR, respectively. The CellKeyTM results showed that OT enhanced impedance induced by endogenous/exogenous KOR agonists on KOR-expressing cells. OT did not affect DOR activity induced by endogenous/exogenous DOR agonists. OT potentiated the KOR agonist-induced Gi/o protein-mediated decrease in intracellular cAMP, but did not affect the increase in KOR internalization caused by the KOR agonists dynorphin A and (-)-U-50488 hydrochloride (U50488). OT did not bind to KOR orthosteric binding sites and did not affect the binding affinities of dynorphin A and U50488 for KOR. These results suggest that OT is a PAM of KOR and MOR and enhances G protein signaling without affecting ß-arrestin signaling. Thus, OT has potential as a specific signaling-biased PAM of KOR.

In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and ß-Arrestin-Mediated Pathways.

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the ß-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® ß-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® ß-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

Visible-Light-Induced Synthesis of 1,2,3,4-Tetrahydroquinolines through Formal [4+2] Cycloaddition of Acyclic α,ß-Unsaturated Amides and Imides with N,N-Dialkylanilines.

1,2,3,4-Tetrahydroquinolines should be applicable to the development of new pharmaceutical agents. A facile synthesis of 1,2,3,4-tetrahydroquinolines that is achieved by a photoinduced formal [4+2] cycloaddition reaction of acyclic α,ß-unsaturated amides and imides with N,N-dialkylanilines under visible-light irradiation, in which a new IrIII complex photosensitizer, a thiourea, and an oxidant act cooperatively in promoting the reaction, is reported. The photoreaction enables the synthesis of a wide variety of 1,2,3,4-tetrahydroquinolines, while controlling the trans/cis diastereoselectivity (>99:1) and constructing contiguous stereogenic centers. A chemoselective cleavage of an acyclic imide auxiliary is demonstrated.

Effects of replacement and addition of an amino acid contained in a cyclic peptide corresponding to a ß-hairpin loop sequence of human EGF receptor.

Effects of replacement and addition of an amino acid in a cyclic decapeptide 1 (cyclic-CYNPTTYQMC) for inhibitory activity to dimerization of human epidermal growth factor receptor (EGFR) were examined. By alanine scanning of 1 corresponding to the arm structure (residues 246-254) of a ß-hairpin loop sequence (residues 242-259) of EGFR, it was confirmed that replacement of any amino acid in the loop structure lowered the dimerization inhibitory activity of 1. Among the residues examined, Tyr at position 246 and Thr at 250 were found to be crucial for dimer formation. Addition of an amino acid to the N-terminus of 1 also affected the dimerization inhibitory activity. Addition of an amino acid containing a moderately hydrophilic side-chain increased the inhibitory activity. In contrast, an intramolecular hydrogen bond of 1 is not thought to be crucial for holding the dimer structure on the basis of the dimerization inhibitory activities of N-methylated analogues of 1. These results will be useful for the design and evaluation of a potent dimerization inhibitor as an anti-proliferation agent. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Development of anti-HIV peptides based on a viral capsid protein.

Peptide inhibitors with cell permeability targeting an HIV-1 capsid (CA) protein might make therapeutic by regulating HIV-1 replication. Overlapping fragment peptide libraries covering the whole sequence of an HIV-1 CA protein have been synthesized with the addition of an octa-arginyl moiety to increase their cell permeability. Amongst these peptides, several compounds which inhibit the HIV-1 replication cycle have been found. Conjugation of cell-penetrating functions such as an octa-arginyl group to individual peptides in combination with the addition of chloroquine in cell-based anti-HIV assays was previously proven to be a useful assay method with which to search for active peptides. Anti-HIV assays have been performed in the presence or absence of chloroquine and found that most of compounds have higher anti-HIV activity in the presence, rather than in the absence of chloroquine. Some potent seeds as anti-HIV agents might naturally lie hidden in CA proteins, and could become useful leads to HIV inhibitors.

Functional evaluation of fluorescein-labeled derivatives of a peptide inhibitor of the EGF receptor dimerization.

A cyclic decapeptide (1, ), which acts on the extracellular region of the EGF receptor, preventing it from dimerizing, has been developed. Peptide 2, which was labeled with fluorescein at the N-terminus of peptide 1, was synthesized based on structure-activity relationship studies. Peptide 2 essentially retained the inhibitory activity of peptide 1 against the receptor autophosphorylation. Confocal microscopy studies revealed that in carcinoma cells, the fluorescence of peptide 2 was localized inside some vesicles. Treatment of intact cells by peptide 1 in combination with peptide 2 decreased the fluorescence intensity significantly compared to treatment with only peptide 2. These results indicate that peptide 2 competes with peptide 1 for binding to the cellular surface. Six derivatives of peptide 2, in which constituent amino acids, with the exception of two cysteines and proline were randomized, were synthesized and used to treat the cells. Peptides 6 and 9 showed the highest fluorescence intensity in cells. From the results of the EGF receptor autophosphorylation assay, these two derivatives were proven to have higher inhibitory activity than peptide 2, which would therefore be a useful delivery peptide and fluorescent probe to find new inhibitors against the EGF receptor. Peptides 6 and 9 are promising leads for EGF receptor inhibitors.

Small-Molecule CD4 Mimics Containing Mono-cyclohexyl Moieties as HIV Entry Inhibitors.

CD4 mimics are small molecules that inhibit the protein-protein interaction between gp120 and CD4, which is a key interaction for the entry of human immunodeficiency virus (HIV) into host immune cells. In the present study, mono-cyclohexyl-type CD4 mimics were designed to form hydrophobic and electrostatic interactions with Val430 and Asp368 located in the entrance of the Phe43 cavity of gp120, the interaction site of CD4. YIR-329, a novel 1-azaspiro[5.5]undecane derivative with a cyclohexyl ring attached to the piperidine ring, exhibited only slightly weaker anti-HIV activity than a previously described lead HAR-171, and modeling results indicated the formation of advantageous interactions by the para-chlorophenyl moiety of YIR-329. To introduce an electrostatic interaction with Asp368, derivatives with a guanidino group on the piperidine nitrogen atom were synthesized. Mono-cyclohexyl-type CD4 mimics with a guanidino group, such as YIR-819 (N(1) -(4-chlorophenyl)-N(2) -(1-(2-(N-(amidino)glycinamide)ethyl)-2-cyclohexylpiperidin-4-yl)oxalamide) and YIR-821 (1-(2-(5-guanidinovaleramide)ethyl derivative of YIR-819), were identified that exhibit approximately fivefold more potent anti-HIV activity than YIR-329. In combination with a neutralizing antibody, their anti-HIV activities were augmenting. Modeling results suggest that these compounds interact effectively with Val430 and either Asp368 or Asp474 in the gp120 Phe43 cavity. YIR-819 and YIR-821 represent useful lead compounds for the further development of HIV-1 entry inhibitors and could potentially be useful for co-administration with neutralizing antibodies for the treatment of HIV infection and AIDS.

A minimally cytotoxic CD4 mimic as an HIV entry inhibitor.

Several CD4 mimics have been reported as HIV-1 entry inhibitors which can block the interaction between the viral envelope glycoprotein gp120 and the cell surface protein CD4. We previously found a lead compound 2 (YYA-021) with high anti-HIV activity and low cytotoxicity. Pharmacokinetic analysis however showed compound 2 to have wide tissue distribution and relatively high distribution volumes in rats and rhesus macaques. In the present study we searched for more hydrophilic CD4 mimics with a view to reducing tissue distribution. A new compound (5) with a 1,3-benzodioxolyl moiety was found to have relatively high anti-HIV activity and no significant cytotoxicity. Compound 5 is more hydrophilic than compound 2 and the pharmacokinetics of the intravenous administration of compound 5 in a rhesus macaque showed that compound 5 has lower tissue distribution than compound 2, suggesting that compound 5 possesses a better profile.

Multimerized HIV-gp41-derived peptides as fusion inhibitors and vaccines.

To date, several antigens based on the amino-terminal leucine/isoleucine heptad repeat (NHR) region of an HIV-1 envelope protein gp41 and fusion inhibitors based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of gp41 have been reported. We have developed a synthetic antigen targeting the membrane-fusion mechanism of HIV-1. This uses a template designed with C3-symmetric linkers and mimics the trimeric form of the NHR-derived peptide N36. The antiserum obtained by immunization of the N36 trimeric antigen binds preferentially to the N36 trimer and blocks HIV-1 infection effectively, compared with the antiserum obtained by immunization of the N36 monomer. Using another template designed with different C3-symmetric linkers, we have also developed a synthetic peptide mimicking the trimeric form of the CHR-derived peptide C34, with ∼100 times the inhibitory activity against the HIV-1 fusion mechanism than that of the monomer C34 peptide. A dimeric derivative of C34 has potent inhibitory activity at almost the same levels as this C34 trimer mimic, suggesting that presence of a dimeric form of C34 is structurally critical for fusion inhibitors. As examples of rising mid-size drugs, this review describes an effective strategy for the design of HIV vaccines and fusion inhibitors based on a relationship with the native structure of proteins involved in HIV fusion mechanisms. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 622-628, 2016.

Exploration of labeling by near infrared dyes of the polyproline linker for bivalent-type CXCR4 ligands.

We have previously used poly-L-proline linkers for the development of bivalent-type ligands for the chemokine receptor, CXCR4. The bivalent ligands with optimum linkers showed specific binding to CXCR4, suggesting the existence of CXCR4 possibly as a dimer on the cell membrane, and enabled definition of the amount of CXCR4 expressed. This paper reports the synthesis by a copper-catalyzed azide-alkyne cycloaddition reaction as the key reaction, of bivalent CXCR4 ligands with near infrared (NIR) dyes at the terminus or the center of the poly-L-proline linker. Some of the NIR-labeled ligands, which would be valuable probes useful in studies of the behavior of cells expressing CXCR4, have been obtained. The information concerning the effects of the labeling positions of NIR dyes on their binding properties is useful for the design of modified bivalent-type CXCR4 ligands.

Trivalent ligands for CXCR4 bearing polyproline linkers show specific recognition for cells with increased CXCR4 expression.

The assembly status of G protein-coupled receptors (GPCR) on the cell surface is of interest because the multimerization of GPCR could play pivotal roles in cellular functions. A bivalent ligand with polyproline linkers for CXCR4 has been shown to serve as a "molecular ruler" as a result of the rigid structure of polyproline helices. To expand the utility of the ligands with rigid linkers and explore the possible multimeric forms of GPCR, trivalent ligands with polyproline helices were newly designed and synthesized. The binding affinities of the trivalent ligands for CXCR4 suggested that the ligands recognize the dimeric form of CXCR4 on the cell surface. The fluorescence imaging and analysis by flow cytometry revealed that the ligand with 9 proline linkers binds to CXCR4 with remarkable specificity. The results of the present study suggest that the ligand design with rigid linkers is useful in the multimeric form, but the design of trivalent ligands requires different strategic approaches.

Anti-HIV screening for cell-penetrating peptides using chloroquine and identification of anti-HIV peptides derived from matrix proteins.

Previously, compounds which inhibit the HIV-1 replication cycle were found in overlapping peptide libraries covering the whole sequence of an HIV-1 matrix (MA) protein constructed with the addition of an octa-arginyl group. The two top lead compounds are sequential fragments MA-8L and MA-9L. In the present study, the addition of chloroquine in cell-based anti-HIV assays was proven to be an efficient method with which to find anti-HIV compounds among several peptides conjugated by cell-penetrating signals such as an octa-arginyl group: the conjugation of an octa-arginyl group to individual peptides contained in whole proteins in combination with the addition of chloroquine in cells is a useful assay method to search active peptides. To find more potent fragment peptides, individual peptides between MA-8L and MA-9L, having the same peptide chain length but with sequences shifted by one amino acid residue, were synthesized in this paper and their anti-HIV activity was evaluated with an anti-HIV assay using chloroquine. As a result, the peptides in the C-terminal side of the series, which are relatively close to MA-9L, showed more potent inhibitory activity against both X4-HIV-1 and R5-HIV-1 than the peptides in the N-terminal side.

Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, ß-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with Kd values between 8-147 µM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein.

Evaluation of dimerization-inhibitory activities of cyclic peptides containing a ß-hairpin loop sequence of the EGF receptor.

Structure-activity relationships of cyclic peptides mimicking the ß-hairpin structure of the 'dimerization arm' at residues 242-259 of the EGF receptor are examined. Cyclic peptides containing the arm head of the ß-hairpin loop showed inhibitory activity toward the EGF receptor's dimerization. Cyclic peptides containing a Retro-Inverso sequence of the dimerization arm showed clear inhibitory effects on the dimerization in vitro and efficiently suppressed the proliferation of A431 cells, which abundantly express the EGF receptor on their surface. The effects at a specific hydrophobic site of the loop structure were expected to enhance the interactions with the receptor.

Intact-cell-based surface plasmon resonance measurements for ligand affinity evaluation of a membrane receptor.

Toward future applications to the discovery of drugs against membrane receptors on pathological cells, an intact-cell-based surface plasmon resonance (SPR) methodology has been developed. The injection of a suspension of epidermal carcinoma A431 cells (5×10(7)cells/ml), as an analyte, generated clear SPR responses to epidermal growth factor (EGF) immobilized on the sensor chip. Because the responses were competitively reduced by the free ligand EGF, added to the analyte cell suspension, they certainly reflect the specific interaction of the immobilized EGF with the extracellular region of its receptor, which is highly expressed on the surface of the A431 cells.

Quantitative evaluation of refolding conditions for a disulfide-bond-containing protein using a concise ¹8O-labeling technique.

A concise method was developed for quantifying native disulfide-bond formation in proteins using isotopically labeled internal standards, which were easily prepared with proteolytic ¹8O-labeling. As the method has much higher throughput to estimate the amounts of fragments possessing native disulfide arrangements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) than the conventional high performance liquid chromatography (HPLC) analyses, it allows many different experimental conditions to be assessed in a short time. The method was applied to refolding experiments of a recombinant neuregulin 1-ß1 EGF-like motif (NRG1-ß1), and the optimum conditions for preparing native NRG1-ß1 were obtained by quantitative comparisons. Protein disulfide isomerase (PDI) was most effective at the reduced/oxidized glutathione ratio of 2:1 for refolding the denatured sample NRG1-ß1 with the native disulfide bonds.

Verification of protein disulfide bond arrangement by in-gel tryptic digestion under entirely neutral pH conditions.

To develop a concise proteomic procedure to verify the protein disulfide bond arrangement, non-reductive trypsin digestion of neuregulin 1-beta1 (176-246), a model disulfide-containing protein, was assessed by a proteolytic (18)O-labeling analysis. As a result, the commonly used in-gel tryptic digestion method has been improved for use entirely under neutral pH conditions. With this procedure, the disulfide arrangement of proteins could represent a clinical index candidate in pathological proteomic studies.

Synthesis of amyloid beta peptide 1-42 (E22Delta) click peptide: pH-triggered in situ production of its native form.

Amyloid beta peptide (Abeta) 1-42 is known to be involved in the onset of Alzheimer's disease (AD). We developed a click peptide of Abeta1-42 as a useful tool for AD research on the basis of an O-acyl isopeptide method. The click peptide quickly produced intact Abeta1-42 via a pH-dependent O-to-N intramolecular acyl migration (pH-click). Herein, a click peptide (26-O-acyl isoAbeta1-42 (E22Delta)) of a new mutant Abeta1-42 (E22Delta) was synthesized. The mutant click peptide was more water-soluble than Abeta1-42 (E22Delta). Moreover it quantitatively converted to the native peptide under physiological conditions (pH 7.4, 37 degrees C). CD analyses showed a conformational change from a random-coil structure of the click peptide to a beta-sheet structure of the in situ produced Abeta1-42 (E22Delta). This click peptide is a useful precursor of a mutant Abeta1-42 to establish an experiment system for investigating the properties of the mutant.

Inhibitory effect of a dimerization-arm-mimetic peptide on EGF receptor activation.

A cyclic decapeptide was chemically synthesized that mimics the loop structure of a beta-hairpin arm of the EGF receptor, which is highly involved in receptor dimerization upon activation by ligand binding. This peptide was revealed to reduce dimer formation of the receptor in a detergent-solubilized extract of epidermoid carcinoma A431 cells and to inhibit receptor autophosphorylation at less than 10 microM in the intact cells.