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Elastin-like peptides (ELPs) exhibit a reversible phase transition, known as coacervation, triggered by temperature changes. This property makes them useful as stimuli-responsive molecular materials for various applications. Among ELPs, short peptide chain lengths have some advantages over long peptide chain lengths because short ELPs can be easily obtained by chemical synthesis, allowing the use of various amino acids, including D-type and unnatural amino acids, at any position in the sequence. Moreover, the incorporated amino acids readily affect the temperature-responsive behavior of ELPs. However, to be utilized in various applications, it is necessary to develop short ELPs and to investigate their temperature-responsive properties. To obtain further insights into the temperature-responsive behavior of the short ELPs, we investigated branched short ELP analogs composed of (FPGVG)

Elastin comprises hydrophobic repetitive sequences, such as Val-Pro-Gly-Val-Gly, which are thought to be important for the temperature-dependent reversible self-association (coacervation). Elastin and elastin-like peptides (ELPs), owing to their characteristics, are expected to be applied as base materials for the development of new molecular tools, such as drug-delivery system carrier and metal-scavenging agents. Recently, several studies have been reported on the dendritic or branching ELP analogues. Although the topological difference of the branched ELPs compared to their linear counterparts may lead to useful properties in biomaterials, the available information regarding the effect of branching on molecular architecture and thermoresponsive behavior of ELPs is scarce. To obtain further insight into the thermoresponsive behavior of branched ELPs, novel ELPs, such as nitrilotriacetic acid (NTA)-(FPGVG) n conjugates, that is, (NTA)-Fn analogues possessing 1-3 (FPGVG) n (n = 3, 5) molecule(s), were synthesized and investigated for their coacervation ability. Turbidity measurement of the synthesized peptide analogues revealed that (NTA)-Fn analogues showed strong coacervation ability with various strengths. The transition temperature of NTA-Fn analogues exponentially decreased with increasing number of residues. In the circular dichroism measurements, trimerization did not alter the secondary structure of each peptide chain of the NTA-Fn analogue. In addition, it was also revealed that the NTA-Fn analogue possesses one peptide chain that could be utilized as metal-scavenging agents. The study findings indicated that multimerization of short ELPs via NTA is a useful and powerful strategy to obtain thermoresponsive molecules.

Elastin-like peptides (ELPs) are thermoresponsive biopolymers inspired by the characteristic repetitive sequences of natural elastin. As ELPs exhibit temperature-dependent reversible self-assembly, they are expected to be biocompatible thermoresponsive materials for drug delivery carriers. One of the most widely studied ELPs in this field is the repetitive pentapeptide, (VPGXG)

A hyaluronic acid-degrading enzyme (hyaluronidase; HAase) is involved in tumor growth and inflammation, and consequently, HAase inhibitors have received recent attention as potential pharmaceuticals. Previous studies have discovered a wide range of inhibitors; however, unfortunately, most of them are dissimilar to the original ligand hyaluronic acid, and their mode of inhibition remains ambiguous or seems promiscuous. This situation presents an urgent need for readily available and highly reliable assay systems identifying the promiscuous inhibitory properties of HAase inhibitors. We have previously proposed a unique method to identify promiscuous nonspecific binding inhibitors of HAase by using the DMSO-perturbing effect. Here, to obtain mechanistic insights into the DMSO-perturbing assay, we studied the addition effect of 11 water-compatible chemicals on HAase inhibitory assay. Intriguingly, the perturbing property was found to be highly specific to DMSO. Furthermore, kinetic analyses described characteristic description of the perturbing property of DMSO: DMSO displayed entropy-driven interactions with HAase, whereas nonperturbing agents such as ethanol and urea exhibited enthalpy-driven interactions. The enthalpy-driven tight interactions of ethanol and urea with HAase would lead to the irreversible denaturation of the enzymes, while the entropy-driven weak interactions caused structural and catalytic perturbation, generating nonproductive but nondenatured states of enzymes, that are key species of the perturbation assay. With these mechanistic understandings in hand, the present assay will enable rapid and reliable identification of HAase inhibitors with certain pharmaceutical potential.

Elastin comprises hydrophobic repetitive sequences, such as Val-Pro-Gly-Val-Gly, which are thought to be important for the temperature-dependent reversible self-association (coacervation). Elastin and elastin-like peptides (ELPs), owing to their characteristics, are expected to be applied as base materials for the development of new molecular tools, such as drug-delivery system carrier and metal-scavenging agents. Recently, several studies have been reported on the dendritic or branching ELP analogues. Although the topological difference of the branched ELPs compared to their linear counterparts may lead to useful properties in biomaterials, the available information regarding the effect of branching on molecular architecture and thermoresponsive behavior of ELPs is scarce. To obtain further insight into the thermoresponsive behavior of branched ELPs, novel ELPs, such as nitrilotriacetic acid (NTA)–(FPGVG)n conjugates, that is, (NTA)-Fn analogues possessing 1–3 (FPGVG)n (n = 3, 5) molecule(s), were synthesized and investigated for their coacervation ability. Turbidity measurement of the synthesized peptide analogues revealed that (NTA)-Fn analogues showed strong coacervation ability with various strengths. The transition temperature of NTA-Fn analogues exponentially decreased with increasing number of residues. In the circular dichroism measurements, trimerization did not alter the secondary structure of each peptide chain of the NTA-Fn analogue. In addition, it was also revealed that the NTA-Fn analogue possesses one peptide chain that could be utilized as metal-scavenging agents. The study findings indicated that multimerization of short ELPs via NTA is a useful and powerful strategy to obtain thermoresponsive molecules.

We reported that bisphenol AF (BPAF) works as an agonist for estrogen receptor (ER) ERα but as an antagonist for ERβ. Similar results were observed for bisphenol E analogs (BPE-X) such as BPE-F, BPE-Cl, and BPE-Br, each consisting of a series of a tri-halogenated methyl group CX3 in the central alkyl moiety. It was demonstrated that the electrostatic halogen bond based on the dispersion force of halogen atoms is a major driving force in the activities of bifunctional ERα-agonist and ERβ-antagonist. Since the chlorine atoms present in bisphenol C (BPC) exist in a π-π conjugated system due to the presence of an adjacent C = C double bond, we intended to prove that BPC is also a bifunctional ERα-agonist and ERβ-antagonist exhibiting greatly enhanced agonist/antagonist activities. BPC was evaluated for its ability to activate ERα and ERβ in the luciferase reporter gene assay using HeLa cells. With high receptor-binding ability to both ERs, BPC was found to be fully active for ERα but inactive for ERβ. BPC’s definite antagonist activity in ERβ was revealed by its inhibitory activity against 17β-estradiol. Thus, BPC is a bifunctional ERα-agonist and ERβ-antagonist. These agonist/antagonist activities were discovered to be extremely high among series of halogen-containing bisphenol compounds. This comparative structure-activity study revealed that the ascending order of ERα-agonist and ERβ-antagonist activities was BPE-F ≪ BPE-Cl ≲ BPAF < BPE-Br ≪ BPC. The highly intensified receptor interaction of BPC is attributable to the presence of an n-π-π-n conjugation system mediated through the >C = CCl2 double bond.

Elastin-like peptides (ELPs) consist of distinctive repetitive sequences, such as (VPGVG)n, exhibit temperature-dependent reversible self-assembly (coacervation), and have been considered to be useful for the development of thermoresponsive materials. Further fundamental studies evaluating coacervative properties of novel nonlinear ELPs could present design concepts for new thermoresponsive materials. In this study, we prepared novel ELPs, cyclic (FPGVG)n (cyclo[FPGVG]n, n = 1–5), and analyzed their self-assembly properties and structural characteristics. Cyclo[FPGVG]n (n = 3–5) demonstrated stronger coacervation capacity than the corresponding linear peptides. The coacervate of cyclo[FPGVG]5 was able to retain water-soluble dye molecules at 40 °C, which implied that cyclo[FPGVG]5 could be employed as a base material of DDS (drug delivery system) matrices and other biomaterials. The results of molecular dynamics simulations and circular dichroism measurements suggested that a certain chain length was requ...

Elastin-like peptides (ELP) are distinct, repetitive, hydrophobic sequences, such as (VPGVG)n, that exhibit coacervation— the property of reversible, temperature-dependent self-association and dissociation. ELPs can be found in elastin and have been developed as new scaffold biomaterials. However, the detailed relationship between their amino acid sequences and coacervation properties remain obscure because of the structural flexibility of ELPs. In this study, we synthesized a novel, dimeric ELP analog (H-C(WPGVG)3-NH2)2, henceforth abbreviated to (CW3)2, and analyzed its self-assembly properties and structural factors as indicators of coacervation. Turbidity measurements showed that (CW3)2 demonstrated coacervation at a much lower concentration than its monomeric form and another ELP. In addition, the coacervate held water-soluble dye molecules. Thus, potent and distinct coacervation was obtained with a remarkably short sequence of (CW3)2. Furthermore, fluorescence microscopy, dynamic light scattering, a...

Heptapeptide SFLLRNP is a receptor−tethered ligand of protease-activated receptor 1 (PAR-1), and its Phe at position 2 is essential for the aggregation of human platelets. To validate the structural elements of the Phe-phenyl group in receptor activation, we have synthesized a complete set of S/Phe/LLRNP peptides comprising different series of fluorophenylalanine isomers (Fn)Phe, where n = 1, 2, 3, and 5. Phe-2-phenyl was strongly suggested to be involved in the edge-to-face CH/π interaction with the receptor aromatic group. In the present study, to prove this receptor interaction definitively, we synthesized another series of peptide analogs containing (F4)Phe-isomers, with the phenyl group of each isomer possessing only one hydrogen atom at the ortho, meta, or para position. When the peptides were assayed for their platelet aggregation activity, S/(2,3,4,6-F4)Phe/LLRNP and S/(2,3,4,5-F4)Phe/LLRNP exhibited noticeable activity (34% and 6% intensities of the native peptide, respectively), whereas S/(2,3,5,6-F4)Phe/LLRNP was completely inactive. The results indicated that, at the ortho and meta positions but not at the para position, benzene-hydrogen atoms are required for the CH/π interaction to activate the receptor. The results provided a decisive evidence of the molecular recognition property of Phe, the phenyl benzene-hydrogen atom of which participates directly in the interaction with the receptor aromatic π plane.

Bisphenol A and its derivatives are recognized as endocrine disruptors based on their complex effects on estrogen receptor (ER) signaling. While the effects of bisphenol derivatives on ERα have been thoroughly evaluated, how these chemicals affect ERβ signaling is less well understood. Herein, we sought to identify novel ERβ ligands using a radioligand competitive binding assay to screen a chemical library of bisphenol derivatives. Many of the compounds identified showed intriguing dual activities as both ERα agonists and ERβ antagonists. Docking simulations of these compounds and ERβ suggested that they bound not only to the canonical binding site of ERβ but also to the coactivator binding site located on the surface of the receptor, suggesting that they act as coactivator-binding inhibitors (CBIs). Receptor-ligand binding experiments using WT and mutated ERβ support the presence of a second ligand-interaction position at the coactivator-binding site in ERβ, and direct binding experiments of ERβ and a coactivator peptide confirmed that these compounds act as CBIs. Our study is the first to propose that bisphenol derivatives act as CBIs, presenting critical insight for the future development of ER signaling-based drugs and their potential to function as endocrine disruptors.

Many types of insects have been used as foods and protein sources. In this study, we investigated the usefulness of housefly larvae (Musca domestica) based on their amino acid composition and multifunctional biological activities. First, the utility of the amino acid composition of housefly larvae was evaluated by amino acid analysis. Notably, the housefly larvae contained sufficient amounts of all essential amino acids, and the amino acid composition was similar to that of hen eggs. Second, we prepared housefly larvae water extract (HLWE) using the decoction method and explored the biological activities of the extract for potential application of the extract as a functional food. HLWE showed significant antioxidant activity (75.4% at 5.00 mg/mL), angiotensin-I-converting enzyme (ACE) inhibitory activity (half-maximal inhibitory concentration [IC50] = 0.430 mg/mL), and dipeptidyl peptidase-IV (DPP-IV) inhibitory activity ([IC50] = 3.52 mg/mL). We found that the low-molecular-weight constituents ( 6 kDa) contributed to DPP-IV inhibition. Our results suggested that housefly larvae may provide a useful source of multifunctional protein.

17β-Estradiol (E2) is a natural steroid ligand for the structurally and physiologically independent estrogen receptors (ERs) ERα and ERβ. We recently observed that CF3-containing bisphenol AF (BPAF) works as an agonist for ERα but as an antagonist for ERβ. Similar results were also observed for the CCl3-containing bisphenol designated as HPTE. Both BPAF and HPTE are comprised of a tri-halogenated methyl group in the central alkyl moiety of their bisphenol structures, which strongly suggests that halogens contribute directly to the agonist/antagonist dual biological functions. We conducted this study to investigate the structure-activity relationships by assessing together newly synthesized CF3- and CBr3-containing bisphenol E analogs (BPE-X). We first tested bisphenols for their receptor binding ability and then for their transcriptional activities. Halogen-containing bisphenols were found to be fully active for ERα, but almost completely inactive for ERβ. When we examined these bisphenols for their inhibitory activities for E2 in ERβ, we observed that they worked as distinct antagonists. The ascending order of agonist/antagonist dual biological functions was BPE-F

[Image: see text] In search for enzyme inhibitors, we often encounter “promiscuous” enzyme inhibitors exhibiting nonspecific binding property toward enzyme active site. Therefore, inhibitory candidates should be mechanistically characterized as early as possible in discovery processes. However, there remains a lack of highly reliable and readily available methodology to evaluate specificity of initial hits inhibitors. The present study developed and established a novel DMSO-perturbing assay to identify promiscuous enzyme inhibitors. The assay successfully identified nonspecific binding inhibitors with a broad scope, typically by the attenuation of inhibitory activity by the influence of DMSO-addition. This attenuation would be attributed to the nonspecific binding property of inhibitors toward both productive and nonproductive (nondenatured) states of enzymes in perturbation solution. This working hypothesis was supported by spectroscopic analyses of enzyme conformations and analyses of solvent effects on perturbation. Overall, these results provided a novel concept of the DMSO-perturbing assay.

Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency.

Elastin, a core protein of the elastic fibers, exhibits the coacervation (temperature-dependent reversible association/dissociation) under physiological conditions. Because of this characteristic, elastin and elastin-derived peptides have been considered to be useful as base materials for developing various biomedical products, skin substitutes, synthetic vascular grafts, and drug delivery systems. Although elastin-derived polypeptide (Val-Pro-Gly-Val-Gly)n also has been known to demonstrate coacervation property, a sufficiently high (VPGVG)n repetition number (n>40) is required for coacervation. In the present study, a series of elastin-derived peptide (Phe-Pro-Gly-Val-Gly)5 dimers possessing high coacervation potential were newly developed. These novel dimeric peptides exhibited coacervation at significantly lower concentrations and temperatures than the commonly used elastin-derived peptide analogs; this result suggests that the coacervation ability of the peptides is enhanced by dimerization. Circular dichroism (CD) measurements indicate that the dimers undergo similar temperature-dependent and reversible conformational changes when coacervation occurs. The molecular dynamics calculation results reveal that the sheet-turn-sheet motif involving a type II β-turn-like structure commonly observed among the dimers and caused formation of globular conformation of them. These synthesized peptide dimers may be useful not only as model peptides for structural analysis of elastin and elastin-derived peptides, but also as base materials for developing various temperature-sensitive biomedical and industrial products.

Elastin and collagen are extracellular matrix proteins that are widely distributed in the body. Although elastin essentially functions as a skin moisturizer, there have been few reports on its other fundamental chemical and biological functions. In this study, we investigated the moisturizing and whitening (tyrosinase inhibition) effects of elastin to examine its usefulness as a cosmetic material. Water-soluble hot alkali pig aorta (HAPA)-elastin was prepared from pig aorta using the hot alkali method. HAPA-elastin showed a widely distributed molecular weight and had a coacervation property that mediated reversible self-assembly of its molecules with increasing temperature. Amino acid analysis of HAPA-elastin showed a high content (81.5%) of hydrophobic amino acids such as Gly, Ala, Val, and Pro. Des (desmosine) and Ide (isodesmosine), which are characteristic amino acids of elastin, accounted for more than 0.4% of the total amino acid content. HAPA-elastin showed a moisture-retaining property. The water content of skin samples treated with and without HAPA-elastin was 77.2% ± 7.8% and 49.4% ± 10.1%, respectively. HAPA-elastin also inhibited tyrosinase activity by 11.3% ± 3.9%. The results obtained indicate that elastin has a useful function as a cosmetic material.

Tropoelastin is the primary component of elastin, which forms the elastic fibers that make up connective tissues. The hydrophobic domains of tropoelastin are thought to mediate the self-assembly of elastin into fibers, and the temperature-mediated self-assembly (coacervation) of one such repetitive peptide sequence (VPGVG) has been utilized in various bio-applications. To elucidate a mechanism for coacervation activity enhancement and to develop more potent coacervatable elastin-derived peptides, we synthesized two series of peptide analogs containing an aromatic amino acid, Trp or Tyr, in addition to Phe-containing analogs and tested their functional characteristics. Thus, position 1 of the hydrophobic pentapeptide repeat of elastin (X(1)P(2)G(3)V(4)G(5)) was substituted by Trp or Tyr. Eventually, we acquired a novel, short Trp-containing elastin-derived peptide analog (WPGVG)3 with potent coacervation ability. From the results obtained during this process, we determined the importance of aromaticity and hydrophobicity for the coacervation potency of elastin-derived peptide analogs. Generally, however, the production of long-chain synthetic polypeptides in quantities sufficient for commercial use remain cost-prohibitive. Therefore, the identification of (WPGVG)3, which is a 15-mer short peptide consisting simply of five natural amino acids and shows temperature-dependent self-assembly activity, might serve as a foundation for the development of various kinds of biomaterials.

Aldose reductase (AR) inhibitors are used clinically to treat long-term diabetic complications. Previous studies reported a series of AR inhibitory candidates, but unfortunately the mode of inhibition was poorly described due mainly to the lack of readily available methods for evaluating the specificity. The present study examined the AR inhibitory effects of novel synthetic hydantoins and their structural relatives, some of which were obtained from chemically engineered extracts of natural plants, and discovered several novel AR inhibitors with moderate inhibitory activity. The identified inhibitors were then subjected to a two-step mechanistic characterization using a detergent-addition assay and our novel dimethyl sulfoxide (DMSO)-perturbation assay. The detergent-addition assay revealed aggregation-based inhibitors, and the subsequent DMSO-perturbation assay identified nonspecific binding inhibitors. Thus, the present study demonstrates the usefulness of the DMSO-perturbation screen for identifying nonspecific binding characteristics of AR inhibitors.

An endocrine-disrupting chemical Bisphenol A (BPA) binds specifically to a nuclear receptor (NR) named ERRγ. Although the importance of receptor-binding evaluation for human NRs is often stressed, the binding characteristics of so-called next-generation (NextGen) bisphenol compounds are still poorly understood. The ultimate objective of this investigation was to evaluate BPA and its NextGen analogs for their abilities to bind to 21 human NRs, the greatest members of NRs for which tritium-labeled specific ligands were available. After establishing the detailed assay conditions for each NR, the receptor binding affinities of total 11 bisphenols were evaluated in competitive binding assays. The results clearly revealed that BPA and the NextGen bisphenols of BPAF, BPAP, BPB, BPC, BPE, and BPZ were highly potent against one or more of NRs such as CAR, ERα, ERβ, ERRγ, and GR, with IC50 values of 3.3–73 nM. These bisphenols were suggested strongly to be disruptive to these NRs. BPM and BPP also appeared to be disruptive, but less potently. BPF exhibited only weak effects and only against estrogen-related NRs. Surprisingly, most doubtful bisphenol BPS was supposed not to be disruptive. The NRs to which BPA and NextGen bisphenols did not bind were RARα, RARβ, RARγ, and VDR. PPARγ, RORα, RORβ, RORγ, RXRα, RXRβ, and RXRγ, exhibited very weak interaction with these bisphenols. The ten remaining NRs, namely, ERRγ, ERβ, ERα, CAR, GR, PXR, PR, AR, LXRβ, and LXRα, showed distinctly strong binding to some bisphenols in this order, being likely to have consequential endocrine-disruption effects.

Spinoxin (SPX; α-KTx6.13), isolated from venom of the scorpion Heterometrus spinifer , is a K + channel-specific peptide toxin (KTx), which adopts a cysteine-stabilized α/β scaffold that is cross-linked by four disulfide bridges (Cys1–Cys5, Cys2–Cys6, Cys3–Cys7, and Cys4–Cys8). To investigate the role of the individual disulfide bonds in the structure-activity relationship of SPX, we synthesized four SPX analogs in which each pair of cysteine residues was replaced by alanine residues. The analysis of circular dichroism spectra and inhibitory activity against Kv1.3 channels showed that the SPX analogs lacking any of three specific disulfide bonds (Cys1–Cys5, Cys2–Cys6, and Cys3–Cys7) were unable to form the native secondary structure and completely lost inhibitory activities. Thus, we conclude that Cys1–Cys5, Cys2–Cys6, and Cys3–Cys7 are required for the inhibition of the Kv1.3 channel by SPX. In contrast, the analog lacking Cys4–Cys8 retained both native secondary structure and inhibitory activity. Interestingly, one of the isomers of the analog lacking Cys1–Cys5 also showed inhibitory activities, although its inhibition was ∼18-fold weaker than native SPX. This isomer had an atypical disulfide bond pairing (Cys3–Cys4 and Cys7–Cys8) that corresponds to that of maurotoxin (MTX), another α-KTx6 family member. These results indicate that the Cys1–Cys5 and Cys2–Cys6 bonds are important for restricting the toxin from forming an atypical (MTX-type) disulfide bond pairing among the remaining four cysteine residues (Cys3, Cys4, Cys7, and Cys8) in native SPX.

The plastic chemical bisphenol A (BPA) has recently been suspected to be a base structure of endocrine disrupting chemicals, which achieve their adverse effects by interfering with human nuclear receptors. For instance, BPA, bisphenol AF, and tetrabromo- or tetrachloro-BPA (X 4 -BPA) have been characterized as binders for ERRγ, ER, and PPARγ, respectively. This ongoing string of findings has led to apprehension that some other BPA derivatives might also perturb important human nuclear receptors. The retinoid-related orphan receptor RORγ has been strongly suspected to be a target of highly hydrophobic chemical substances because of its extreme affinity for lipophilic sterols. In the present study, we tested a series of BPA derivatives for their ability to bind to RORγ, and identified two distinctly potent derivatives having isopropyl or sec -butyl groups at positions adjacent to the BPA-4-hydroxyl group. In particular, di- sec -butyl-BPA has emerged as a considerably potent ligand (IC 50 = 146 nM). In the reporter gene assay, these compounds suppressed the basal constitutive transcriptional activity originally induced by wild-type RORγ. The present results strongly suggested that RORγ, and perhaps also RORα and RORβ, binds highly hydrophobic and sterically hindered chemical substances, inducing some unspecified physiological and biochemical disruptions.

The elastin precursor tropoelastin possesses a number of polymeric peptides with repeating 3–9 mer sequences. One of these is the pentapeptide Val-Pro-Gly-Val-Gly (VPGVG) present in almost all animal species, and its polymer (VPGVG)n coacervates just as does tropoelastin. In the present study, in order to explore the structural requirements essential for coacervation, (VPGVG)n and its shortened repeat analogs (VPGV)n, (VPG)n, and (PGVG)n were synthesized and their structural properties were investigated. In our turbidity measurements, (VPGVG)n demonstrated complete reversible coacervation in agreement with previous findings. The Gly5-deleted polymer (VPGV)n also achieved self-association, though the onset of self-association occurred at a lower temperature. However, the dissociation of (VPGV)n upon temperature lowering was found to occur in a three-step process; the Vali4-Vali+11 structure arising in the VPGV polypeptide appeared to perturb the dissociation. No self-association was observed for (VPG)n or (PGVG)n repeats. Spectroscopic measurements by CD, FT-IR, and 1H-NMR showed that the (VPGV)n and (VPG)n both assumed ordered structures similar to that of (VPGVG)n. These results demonstrated that VPGVG is a structural element essential to achieving the β-spiral structure required for self-association followed by coacervation, probably due to the ideal spatial arrangement of the hydrophobic Val residues. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

A computer-aided docking study was carried out to quickly clarify the binding structure of the ligand-receptor complex between bisphenol A (BPA), a well-known endocrine disruptor, and estrogen-related receptor gamma (ERRgamma). The resulting complex indicated that BPA binds to the ligand-binding pocket of ERRgamma without any disruptions of the activation conformation.

Elastin, a core protein of the elastic fibers, exhibits the coacervation (temperature-dependent reversible association/dissociation) under physiological conditions. Because of this characteristic, elastin and elastin-derived peptides have been considered to be useful as base materials for developing various biomedical products, skin substitutes, synthetic vascular grafts, and drug delivery systems. Although elastin-derived polypeptide (Val-Pro-Gly-Val-Gly)n also has been known to demonstrate coacervation property, a sufficiently high (VPGVG)n repetition number (n40) is required for coacervation. In the present study, a series of elastin-derived peptide (Phe-Pro-Gly-Val-Gly)5 dimers possessing high coacervation potential were newly developed. These novel dimeric peptides exhibited coacervation at significantly lower concentrations and temperatures than the commonly used elastin-derived peptide analogs; this result suggests that the coacervation ability of the peptides is enhanced by dimerization. Circular dichroism (CD) measurements indicate that the dimers undergo similar temperature-dependent and reversible conformational changes when coacervation occurs. The molecular dynamics calculation results reveal that the sheet-turn-sheet motif involving a type II β-turn-like structure commonly observed among the dimers and caused formation of globular conformation of them. These synthesized peptide dimers may be useful not only as model peptides for structural analysis of elastin and elastin-derived peptides, but also as base materials for developing various temperature-sensitive biomedical and industrial products.

In this study, we developed a series of Phe-containing elastin-derived peptide-analogs, (Phe-Pro-Gly-Val-Gly)n (n = 1-5) and analyzed their reversible coacervation properties. Compared to the native elastin-derived repeating peptide sequence ((Val-Pro-Gly-Val-Gly)10), one of the Phecontaining 5-mer repeating peptide sequences ((Phe-Pro-Gly-Val-Gly)5) clearly exhibited stronger coacervation properties. The coacervation of (Phe-Pro-Gly-Val-Gly)5 is nearly the same as that of polypeptides (Val-Pro-Gly-Val-Gly)n (n > 40). Although large molecular weights (>10,000 Da) are generally required for the coacervation of elastin-derived peptides, (Phe-Pro-Gly-Val-Gly)5 exhibited reversible coacervation properties despite its low molecular weight (MW = 2,305 Da). High performance liquid chromatography (HPLC) and circular dichroism (CD) analysis revealed that (Phe-Pro-Gly-Val-Gly)5 has high hydrophobicity and an ordered structure with a type II β-turn, which contributes to the strong coacervation ability of the peptide. In addition, (Phe-Pro-Gly-Val-Gly)5 exhibited an effective particle size distribution (60-70 nm) at body temperature (37°C) and a dispersed small particle size similar to that of the monomer peptides at low temperatures. These properties, along with its small size and simple design, render the peptide suitable for use in biomaterials, including drug-delivery carriers.

The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [-Ala2, Leu5]-enkephalyl-Cys(Npys)6 and [-Ala2,Leu(CH2SNpys)5]enkephalin, and dynorphin A analogues of [-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide and [-Ala2,Cys(Npys)8]dynorphin A-(1-9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide.

Pigment-dispersing factor PDF is an 18-amino acid insect neuropeptide that mediates a circadian rhythmicity in locomotor activity. PDF is coded in a precursor protein together with another neuropeptide named PDF-associated peptide, PAP. PDF is highly conserved among insects, whereas the homology of PAPs is very low with considerably varied amino acid sequences. Since such dissimilarity has suggested that the function of PAP peptide is not associated with that of PDF, we have attempted to analyze the sequences of PDF precursor proteins among a series of species of insects and hypothesized that PDF precursors are classified into at least three different classes: Drosophila-Musca, Meimuna-Romalea, and Gryllus. In order to exemplify this hypothesis, we here describe the molecular cloning of the pdf-gene of the black blowfly Phormia regina and an in silico screening for the pdf-gene in the genome databank of the mosquito Anopheles gambie, both species belonging to the Diptera. It was found that deduced amino acid sequences of PDF peptides are almost completely conserved among all Dipterans and also the amino acid sequences of PAPs are considerably highly preserved (55–82 similarity) among the species of Diptera. The results confirmed the validity of grouping the PDF precursor proteins. In situ hybridization was carried out in fly brains to identify the precise locations of pdf-expressing cells and to examine any daily cycling of pdf mRNA. Intense signals for pdf mRNA were identified in the medulla, but not in the pars lateralis where PDF neurons were strongly immunostained by the antibody raised against PDF peptide. Hybridization was also performed for the brain samples at two hour intervals throughout the day. Although very intense hybridization signals were observed at ZT8 even in some neurites, no prominent rhythmicity of pdf mRNA expression was observed.

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neurotransmitter for the circadian rhythms of the locomotor activity in flies. Recently, two completely different types of PDF precursor were clarified; that of the cricket Gryllus bimaculatus and that of the last-summer cicada Meimuna opalifera. The G. bimaculatus PDF precursor is extraordinarily short and comprises a nuclear localization signal (NLS), while the M. opalifera PDF precursor is of ordinary length, comparable to that seen for the precursors of crustacean β-PDH homologues. Although their PDF peptide regions were exactly the same, the regions containing a signal peptide combined with a PDF-associated peptide (PAP) were remarkably different from each other. Such a grouping suggested a fundamental role for the PAP peptide in the circadian clock, perhaps associated with PDF function. In the present study, the cDNA cloning of PDF from the adult brains of the housefly Musca domestica was carried out and it was found that an isolated clone (527 bp) encodes a PDF precursor protein of ordinary length. The PDF peptide shows a high sequence identity (78%–94%) and similarity (89%–100%) to insect PDFs and also to the crustacean β-PDH peptides. In particular, there is only a single amino acid difference between the PDFs of Musca and Drosophila; at position 14 Ser for Musca PDF and Asn for Drosophila PDF. A characteristic Ser10 in Drosophila was retained in Musca, indicating the presence of a structural profile unique to these PDFs. The results of sequence analyses suggest that Musca and Drosophila PDFs are to be considered members of a single group that has evolved structurally. When the primary structure of the PAP regions was compared, the Musca PDF precursor also belonged to the same group as that to which the Drosophila PDF precursor belongs. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.

TheS-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [d-Ala2, Leu5]-enkephalyl-Cys(Npys)6 and [d-Ala2,Leu(CH2SNpys)5]enkephalin, and dynorphin A analogues of [d-Ala2,Cys(Npys)12]dynorphin A-(1–13) amide and [d-Ala2,Cys(Npys)8]dynorphin A-(1–9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [d-Ala2, Cys(Npys)12]dynorphin A-(1–13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [d-Ala2,Cys(Npys)12]dynorphin A-(1–13) amide.

Pigment-dispersing factor PDF is an 18-amino acid insect neuropeptide that mediates a circadian rhythmicity in locomotor activity. PDF is coded in a precursor protein together with another neuropeptide named PDF-associated peptide, PAP. PDF is highly conserved among insects, whereas the homology of PAPs is very low with considerably varied amino acid sequences. Since such dissimilarity has suggested that the function of PAP peptide is not associated with that of PDF, we have attempted to analyze the sequences of PDF precursor proteins among a series of species of insects and hypothesized that PDF precursors are classified into at least three different classes: Drosophila-Musca, Meimuna-Romalea, and Gryllus. In order to exemplify this hypothesis, we here describe the molecular cloning of the pdf-gene of the black blowfly Phormia regina and an in silico screening for the pdf-gene in the genome databank of the mosquito Anopheles gambie, both species belonging to the Diptera. It was found that deduced amino acid sequences of PDF peptides are almost completely conserved among all Dipterans and also the amino acid sequences of PAPs are considerably highly preserved (55–82 similarity) among the species of Diptera. The results confirmed the validity of grouping the PDF precursor proteins. In situ hybridization was carried out in fly brains to identify the precise locations of pdf-expressing cells and to examine any daily cycling of pdf mRNA. Intense signals for pdf mRNA were identified in the medulla, but not in the pars lateralis where PDF neurons were strongly immunostained by the antibody raised against PDF peptide. Hybridization was also performed for the brain samples at two hour intervals throughout the day. Although very intense hybridization signals were observed at ZT8 even in some neurites, no prominent rhythmicity of pdf mRNA expression was observed.

X-Ray crystal structures of four different fluorine-substituted phenylalanines (two mono- and two tri-substitutions) were analyzed to investigate the effect of fluorine atom on the association pattern of benzene rings. Although respective structures showed similar molecular packing in such a way that the layers of hydrophobic benzene rings and hydrophilic amino/carboxyl groups were alternately running along a crystallographic axis, the association patterns of benzene rings were different depending on the substitution position and number of fluorine atoms. The general features could be that the partially displaced face-to-face interactions are increased with increase in the number of fluorine atoms, whereas the edge-to-face interactions are decreased. The C-H bond next to a fluorine-substituted carbon atom could serve as a donor of an intermolecular C-H.F hydrogen bond.

Xenoestrogen dialkyl phthalates, C(6)H(4)(COOC(n)H(m))(2), lack the phenolic hydroxyl group that is an essential structural component of the steroid A ring of 17 beta-estradiol. In order to examine whether dialkyl phthalates imitate the steroid structure, we have synthesized a series of 4-hydroxyl derivatives of dialkyl phthalates. The compounds were examined for their ability to displace [(3)H]17 beta-estradiol from the recombinant human estrogen receptor, which was expressed on Sf9 cells using the vaculovirus expression system. Dialkyl 4-hydroxyl phthalates were found to exhibit several-fold higher binding affinities compared to phthalates without the 4-hydroxyl group. From the analyses of receptor binding modes of dialkyl phthalates with and without the 4-hydroxyl group, it was deduced that the phthalic benzene ring mimics the steroid A ring. A biphasic binding curve observed for dicyclohexyl phthalate was also depicted by its 4-hydroxyl derivative, but it increased binding affinity only at the high affinity binding site. These data suggest that the phthalate benzene moiety recognizes the core of the estrogen receptor binding site and the hydrophobic interaction of the dialkyl moiety substantiates the binding characteristics of the phthalates. The present data indicate that even chemicals with slight structural analogy and weak receptor affinity can perturb the endocrine system when administered in high concentrations.

A clottable protein coagulogen of the horseshoe crab Tachypleus tridentatus is proteolytically converted into an insoluble coagulin gel through non-covalent self-polymerization. Here we identified binding sites for the polymerization. A tryptic fragment, derived from the coagulin polymer chemically cross-linked by a bifunctional cross-linker, was isolated. Amino acid sequence analysis indicated that the fragment consists of two peptides cross-linked between Lys(85) and Lys(156). The two lysine residues are oppositely located at the head and tail regions of the elongated molecule separated by a much greater distance than the length of the cross-linker, which suggests that the cross-linking occurs intermolecularly. Based on the x-ray structural analysis, exposure of a hydrophobic cove on the head in response to the release of peptide C has been postulated (Bergner, A., Oganessyan, V., Muta, T., Iwanaga, S., Typke, D., Huber, R., and Bode, W. (1996) EMBO J. 15, 6789-6797). An octapeptide containing Tyr(136), which occupies the tail end of coagulin, was found to inhibit the polymerization. Replacement of Tyr(136) of the peptide with Ala resulted in loss of the inhibitory activity. These results indicated that the polymerization of coagulin proceeds through the interaction between the newly exposed hydrophobic cove on the head and the wedge-shaped hydrophobic tail.

The thrombin receptor-tethered ligand SFLLRNP (abbreviation formed by one letter amino acid codes expressing Ser-Phe-Leu-Leu-Arg-Asn-Pro) consists of the Phe-2 residue essential for the receptor activation. In order to explore the molecular characteristics of this Phe-2-phenyl, a series of trifluorophenylalanines [(F3)Phe] was incorporated into this S/Phe/LLRNP for evaluation of their ability to induce the human platelet aggregation. A complete set of (F3)Phe in the L-configuration, namely, (2,3,4-F3)Phe, (2,3,5-F3)Phe, (2,3,6-F3)Phe, (2,4,5-F3)Phe, (2,4,6-F3)Phe, and (3,4,5-F3)Phe, was prepared from trifluorobenzyl bromides and diethyl acetamidomalonate. S/(2,3,4-F3)Phe/LLRNP was equipotent to S/Phe/LLRNP, while (2,4,5-F3)Phe-containing analog was almost twice as potent as those. (2,4,6-F3)Phe-analog exhibited about a half of the activity of S/Phe/LLRNP. (3,4,5-F3)Phe-, (2,3,5-F3)Phe-, and (2,3,6-F3)Phe-analogs were very weak. The analysis of these assay results suggested that Phe-2-phenyl of SFLLRNP is ...

A complete set of difluorophenylalanines in the l -configuration [ l -(F 2 )Phe] (namely, l -(2,3-F 2 )Phe, l -(2,4-F 2 )Phe, l -(2,5-F 2 )Phe, l -(2,6-F 2 )Phe, l -(3,4-F 2 )Phe, l -(3,5-F 2 )Phe) was prepared and incorporated into the thrombin receptor-tethered ligand peptide SFLLRNP to identify the phenyl hydrogens of the Phe-2 residue involved in the CH/π receptor interaction.

Nociceptin is a neuropeptide that binds to and activates the opioid receptor-like ORL1 receptor. In order to explore the structural elements necessary for receptor recognition and activation, we designed and synthesized a series of nociceptin analogues, in which nonpolar amino acid residues such as Gly6, Ala7, Ala11, Leu14, and Ala15were substituted respectively by Trp. [Trp6]- and [Trp7]nociceptins exhibited rather weak activities (5—15% of nociceptin), and [Trp11]- and [Trp15]nociceptins also showed reduced activities (40—50%). These results suggested that the space available for these particular residues are relatively restricted. By contrast, the Trp/Leu-substitution at position 14 retained full receptor binding activity, and the resulting [Trp14]nociceptin exhibited an increased biological activity in the functional assay using [35S]GTPγS. This suggested that the receptor residue interacting with nociceptin-Leu14 is the aromatic amino acid.

An antagonist specific for the thrombin receptor is expected to be a remedy for thrombosis. Structure-activity studies of thrombin receptor-tethered ligand SFLLRNP have revealed the importance of the Phe-2-phenyl group in receptor recognition and the replacement of the Phe-2 by para-fluorophenylalanine [(p-F)Phe] was found to enhance its activity [Nose, T. et al. (1993) Biochem. Biophys. Res. Commun. 193, 694-699]. In order to obtain a small sized antagonist, a series of (p-F)Phe derivatives was designed and synthesized novel structural elements essential for receptor interactions being introduced at both the N and C-termini. beta-Mercaptopropionyl (betaMp) or its derivative activated by S-3-nitro-2-pyridinesulphenyl (Npys) was introduced at the N-terminus, and phenylmethyl amines were coupled to the C-terminus. All compounds were inactive when assayed for human platelet aggregation, indicating that they are not agonists. beta-Mercaptopropionyl derivatives were also inactive as antagonists. However, Npys-containing analogs were found to inhibit the agonist activity of SFLLRNP. In particular, SNpys-betaMp-(p-F)Phe-NH-R [R = -CH(C6H5)2 and -CH2-CH-(C6H5)2] potently suppressed platelet aggregation. The results suggested that (p-F)Phe can be used as a structural core to construct an effective antagonist conformation.

A novel type of conformationally restricted peptides with the structure of H-D-Xaa-Phe-NH-CH2-C6H5 has been developed as inhibitors of serine proteinase chymotrypsin. The D-Xaa-alkyl and Phe-phenyl groups resulted in a formation of the hydrophobic core due to the side-chain-side-chain CH/pie interaction. Their spatial proximity was evidenced by 400 MHz 1H-nmr measurements, observing large upfield shifts of proton signals of D-Xaa-alkyl and nuclear Over-hauser effect (NOE) enhancements between the D-Xaa-alkyl and Phe-phenyl groups. This conformational restriction brought by CH/pie interaction produced an inhibitory structure, in which the C-terminal amide-benzyl group fits the chymotrypsin S1 site and the hydrophobic core binds to the S2 site. The inhibitory conformation was demonstrated crystallographically for the complex between the dipeptide H-D-Leu-Phe-NH-CH2-C6H4(p-F) and gamma-chymotrypsin. Detailed structure-activity studies have substantiated the structure of dipeptides in the active center of the enzyme.

Nociceptin or orphanin FQ is a novel neuropeptide that activates an opioid-like G protein-coupled receptor ORL1. This heptadecapeptide FGGFTGARKSARKLANQ resembles kappa-opioid peptide dynorphin A but exhibits an opposite effect to make animals hyperreactive to nociceptive stimulations (Meunier, J.-C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.-L., Guillemot, J.-C., Ferrara, P., Monsarrat, B., Mazarguil, H., Vassart, G., Parmentier, M., and Costentin, J. (1995) Nature 377, 532-535; Reinscheid, R. K., Nothacker, H.-P., Bourson, A., Ardati, A., Henningsen, R. A., Bunzow, J. R., Grandy, D. K., Langen, H., Monsma, F. J., Jr., and Civelli, O. (1995) Science 270, 792-794). In the present study, it was found that guinea pig brain contains receptors to which nociceptin binds much more strongly than to ORL1 receptors expressed in human 293 cells. Although the Tyr1 --Phe substitution for dynorphin A eliminates almost completely an ability to bind to opioid receptors, the Phe1 --Tyr substitution in nociceptin was found to retain almost fully both receptor binding affinity and in vivo hyperalgesic activity in tail-flick assay. Nociceptin was extremely weak to bind to opioid receptors, while Tyr1-nociceptin exhibited 10-40 times increased affinity, especially for mu receptors, due to its N-terminal sequential identity to opioid peptides. Shortened analogs of dynorphin A are known to retain receptor binding ability and analgesic activity, whereas the removal of C-terminal hexa- or decapeptide from nociceptin totally abolished the affinity for the ORL1 receptor. These results indicated that the mode of interaction between nociceptin and ORL1 receptor is quite different from that between dynorphin and opioid receptor and that the C-terminal portion of nociceptin is crucial for receptor recognition.