Highly Selective Binding and Inhibition of Pyr-His-Pro-NH2 (TRH) Function using a Polypyridinyl Macrocyclic Receptor with an Amphiphilic Cavity.

Macrocycle, cyclo[4] [(1,3-(4,6)-dimethylbezene)[4](2,6-(3,5)-dimethylpyridine (B4P4), shows highly selective binding affinity with protirelin (Pyr-His-Pro-NH2 ; TRH) among the tested 26 drug or drug adductive substrates. The stable complexation in a 1:1 manner was fully characterized in solution, gas phase, and solid state study. Furthermore, B4P4 acts as an efficient TRH inhibitor even at [macrocycle]:[drug] <1:300, both in membrane transport and cellar incubation. The current work provides an unprecedented strategy for macrocycles to be efficiently used in drug target therapy.

Characterization and distribution of GHRH, PACAP, TRH, SST and IGF1 mRNAs in the green iguana.

The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH2), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These results show that the main SA components in reptiles of the Squamata Order maintain a good structural conservation among vertebrate phylogeny, and suggest important physiological interactions (endocrine, autocrine and/or paracrine) between them due to their wide peripheral tissue expression.

Effect of L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 against in-vitro and in-vivo models of cerebral ischemia and associated neurological disorders.

Central nervous system plays a vital role in regulation of most of biological functions which are abnormally affected in various disorders including cerebral ischemia, Alzheimer's and Parkinson's (AD and PD) worldwide. Cerebral stroke is an extremely fatal and one of the least comprehensible neurological disorders due to limited availability of prospective clinical approaches and therapeutics. Since, some endogenous peptides like thyrotropin-releasing hormone have shown substantial neuroprotective potential, hence present study evaluates the newer thyrotropin-releasing hormone (TRH) analogue L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 for its neuroprotective effects against oxygen glucose deprivation (OGD), glutamate and H2O2 induced injury in pheochromocytoma cell lines (PC-12 cells) and in-vivo ischemic injury in mice. Additionally, the treatment was further analyzed with respect to models of AD and PD in mice. Cerebral ischemia was induced by clamping both bilateral common carotid arteries for ten minutes. Treatment was administered to the mice five minute after restoration of blood supply to brain. Consequential changes in neurobehavioural, biochemical and histological parameters were assessed after a week. L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 showed significant reduction in glutamate, H2O2 and OGD -induced cell death in concentration and time dependent manner. Moreover, L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 resulted in a substantial reduction in CA1 (Cornus Ammonis 1) hippocampal neuronal cell death, inflammatory cytokines, TNF-α, IL-6 and oxidative stress in hippocampus. In addition, L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 was found to be protective in two acute models of AD and PD as well these findings demonstrate the neuroprotective potential of L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 in cerebral ischemia and other diseases, which may be mediated through reduction of excitotoxicity, oxidative stress and inflammation.

Histidine-Directed Arylation/Alkenylation of Backbone N-H Bonds Mediated by Copper(II).

Chemical modification of proteins and peptides represents a challenge of reaction design as well as an important biological tool. In contrast to side-chain modification, synthetic methods to alter backbone structure are extremely limited. In this communication, copper-mediated backbone N-alkenylation or N-arylation of peptides and proteins by direct modification of natural sequences is described. Histidine residues direct oxidative coupling of boronic acids at the backbone NH of a neighboring amino acid. The mild reaction conditions in common physiological buffers, at ambient temperature, are compatible with proteins and biological systems. This simple reaction demonstrates the potential for directed reactions in complex systems to allow modification of N-H bonds that directly affect polypeptide structure, stability, and function.

Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.

The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 µM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.

Prodrugs of thyrotropin-releasing hormone and related peptides as central nervous system agents.

Prodrug design for brain delivery of small- and medium-sized neuropeptides was reviewed, focusing on thyrotropin-releasing hormone and structurally related peptides as examples. We have summarized our most important advances in methodology, as well as assessed the benefits and limitations of bioreversible chemical manipulation techniques to achieve targeting of the parent molecules into the central nervous system. The value of prodrug-amenable analogues as potential drug-like central nervous systems agents was highlighted.

Lipopolysaccharide modulation of thyrotropin-releasing hormone (TRH) and TRH-like peptide levels in rat brain and endocrine organs.

Lipopolysaccharide (LPS) is a proinflammatory and depressogenic agent whereas thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2) is an endogenous antidepressant and neuroprotective peptide. LPS and TRH also have opposing effects on K+ channel conductivity. We hypothesized that LPS can modulate the expression and release of not only TRH but also TRH-like peptides with the general structure pGlu-X-Pro-NH2, where "X" can be any amino acid residue. The response might be "homeostatic," that is, LPS might increase TRH and TRH-like peptide release, thereby moderating the cell damaging effects of this bacterial cell wall constituent. On the other hand, LPS might impair the synthesis and release of these neuropeptides, thus facilitating the induction of early response genes, cytokines, and other downstream biochemical changes that contribute to the "sickness syndrome." Sprague-Dawley rats (300 g) received a single intraperitoneal injection of 100 microg/kg LPS. Animals were then decapitated 0, 2, 4, 8, and 24 h later. Serum cytokines and corticosterone peaked 2 h after intraperitoneal LPS along with a transient decrease in serum T3. TRH and TRH-like peptides were measured by a combination of high-performance liquid chromatography and radioimmunoassay. TRH declined in the nucleus accumbens and amygdala in a manner consistent with LPS-accelerated release and degradation. Various TRH-like peptide levels increased at 2 h in the anterior cingulate, hippocampus, striatum, entorhinal cortex, posterior cingulate, and cerebellum, indicating decreased release and clearance of these peptides. These brain regions are part of a neuroimmunomodulatory system that coordinates the behavioral, endocrine, and immune responses to the stresses of sickness, injury, and danger. A sustained rise in TRH levels in pancreatic beta-cells accompanied LPS-impaired insulin secretion. TRH and Leu-TRH in prostate and TRH in epididymis remained elevated 2-24 h after intraperitoneal LPS. We conclude that these endogenous neuroprotective and antidepressant-like peptides both mediate and moderate some of the behavioral and toxic effects of LPS.

Rapid modulation of TRH-like peptides in rat brain by thyroid hormones.

Recent identification of membrane receptors for T4, T3, 3,5-T2, and 3-iodothyronamine that mediate rapid physiologic effects of thyroid hormones suggested that such receptors may supplement the regulation of TRH and TRH-like peptides by nuclear T3 receptors. For this reason 200 g male Sprague-Dawley rats received daily i.p. injections of PTU or T4. Levels of TRH and TRH-like peptides were measured 0, 2 h or 1, 2, 3, or 4 days later. Rapid increases or decreases in TRH and TRH-like peptide levels were observed in response to PTU and T4 treatments in various brain regions involved in mood regulation. Significant effects were measured within 2 h of T4 injection. Nuclear T3 receptor-mediated changes in gene expression altering translation, post-translational processing and constitutive release of peptides require more than 2 h. We conclude that non-genomic mechanisms may contribute to the psychiatric effects of thyroid disease and thyroid hormone adjuvant treatment for major depression.

Prohormone-convertase 1 processing enhances post-Golgi sorting of prothyrotropin-releasing hormone-derived peptides.

Rat prothyrotropin-releasing hormone (pro-TRH) is endoproteolyzed within the regulated secretory pathway of neuroendocrine cells yielding five TRH peptides and seven to nine other unique peptides. Endoproteolysis is performed by two prohormone convertases, PC1 and PC2. Proteolysis of pro-TRH begins in the trans-Golgi network and forms two intermediates that are then differentially processed as they exit the Golgi and are packaged into immature secretory granules. We hypothesized that this initial endoproteolysis may be necessary for downstream sorting of pro-TRH-derived peptides as it occurs before Golgi exit and thus entry into the regulated secretory pathway. We now report that when pro-TRH is transiently expressed in GH4C1 cells, a neuroendocrine cell line lacking PC1, under pulse-chase conditions release is constitutive and composed of more immature processing intermediates. This is also observed by radioimmunoassay under steady-state conditions. When a mutant form of pro-TRH, which has the dibasic sites of initial processing mutated to glycines, is expressed in AtT20 cells, a neuroendocrine cell line endogenously expressing PC1, both steady-state and pulse-chase experiments revealed that peptides derived from this mutant precursor are secreted in a constitutive fashion. A constitutively secreted form of PC1 does not target pro-TRH peptides to the constitutive secretory pathway but results in sorting to the regulated secretory pathway. These results indicated that initial processing action of PC1 on pro-TRH in the trans-Golgi network, and not a cargo-receptor relationship, is important for the downstream sorting events that result in storage of pro-TRH-derived peptides in mature secretory granules.

First messenger regulation of mammalian sperm function via adenylyl cyclase/cAMP.

When released into an appropriate environment, mammalian spermatozoa begin to capacitate and then continue until fully capacitated and able to fertilize. During capacitation in vitro, some cells 'over-capacitate' and undergo spontaneous acrosome reactions; this would be highly undesirable in vivo since already acrosome-reacted spermatozoa are non-fertilizing. Recent studies have revealed that seminal plasma contains several small molecules that bind to specific receptors on the sperm plasma membrane and act as 'first messengers', causing biologically important changes in availability of the 'second messenger' cAMP. Fertilization promoting peptide (FPP), calcitonin and adenosine all regulate cAMP production, stimulating it in uncapacitated spermatozoa and then inhibiting it in capacitated cells; in contrast, angiotensin II stimulates cAMP throughout capacitation. The molecules that regulate cAMP appear to do so via G protein-modulated changes in membrane associated adenylyl cyclases (mACs). Both mouse and human spermatozoa have been shown to have Galphas and Galphai2, as well as several isoforms of mAC, located in the same regions as the specific receptors. Thus spermatozoa possess the required elements for several separate signal transduction pathways, many of which regulate mAC/cAMP and so maintain sperm fertilizing ability. In vivo, such responses could increase the chances of successful fertilization.

Agonist-induced conformational changes in thyrotropin-releasing hormone receptor type I: disulfide cross-linking and molecular modeling approaches.

The conformational changes at the cytoplasmic ends of transmembrane helices 5 and 6 (TMH5 and TMH6) of thyrotropin-releasing hormone (TRH) receptor type I (TRH-R1) during activation were analyzed by cysteine-scanning mutagenesis followed by disulfide cross-linking and molecular modeling. Sixteen double cysteine mutants were constructed by substitution of one residue at the cytoplasmic end of TMH5 and the other at that of TMH6. The cross-linking experiments indicate that four mutants, Q263C/G212C, Q263C/Y211C, T265C/G212C, and T265C/Y211C, exhibited disulfide bond formation that was sensitive to TRH occupancy. We refined our previous TRH-R1 models by embedding them into a hydrated explicit lipid bilayer. Molecular dynamics simulations of the models, as well as in silico double cysteine models, generated trajectories that were in agreement with experimental results. Our findings suggest that TRH binding induces a separation of the cytoplasmic ends of TMH5 and TMH6 and a rotation of TMH6. These changes likely increase the surface accessible area at the juxtamembrane region of intracellular loop 3 that could promote interactions between G proteins and key residues within the receptor.

Pressure/temperature combined treatments of precursors yield hormone-like peptides with pyroglutamate at the N terminus.

Peptides containing the cyclic product of glutamine at the N terminus are usually biologically active. If the cyclization of glutamine was associated with a volume reduction, pressure should displace the equilibrium in the direction of the lower volume. Here, results in model solutions and in whey are discussed, showing that the theorized cyclization of glutamine in Gln-His-ProNH(2) or Gln-Leu-ProNH(2) is significantly accelerated during the application of heat and even more strongly when elevated temperature and pressure combinations are used. The reaction rate depended on the intensity of the pressure treatment, the pH, and the nature of the amino acids adjacent to glutamine. The products of the reaction were identified as thyrotropin-releasing hormone (TRH) and [Leu(2)]TRH. The reported reactions could affect the naturally balanced concentration of short-chain peptides in foods and therefore induce unpredictable biological effects.

A study of the binding between polymers and peptides, using affinity capillary electrophoresis, applied to polymeric drug delivery systems.

We have investigated the potential of affinity capillary electrophoresis (ACE) to evaluate binding constants between an anionic polydispersed polymer and four peptides. Nonlinear regression and three current linearization methods, the y-reciprocal, the x-reciprocal and the double-reciprocal, were employed for the estimation of the binding constants. The x-reciprocal and the double-reciprocal plots indicated the presence of two portions of straight lines for angiopeptin, triptorelin and the thyrotropin releasing hormone (TRH), and therefore the probable existence of a second-order interaction which causes the deviation from the 1:1 model. Peptide 1 exhibited a unique binding constant of 2.4 x 10(6)M(-1). In contrast, angiopeptin, triptorelin and TRH exhibited a K(1) of 4.0 x 10(6), 5.3 x 10(6) and 20.2 x 10(6)M(-1), respectively, and a K(2) of 0.4 x 10(6), 0.5 x 10(6) and 1.4 x 10(6)M(-1), respectively. The origin of the high scattering of the data points was further investigated. Neither the viscosity, nor the adsorption of the peptides to the capillary wall appeared to be the determining factor of data scattering. Finally, a possible adsorption of the polymer leading to the electroosmotic flow instability was supposed.

Inhibition of prolactin secretion from the male rat anterior pituitary by cryptic sequences of prothyrotropin releasing hormone, ProTRH178-199 and ProTRH186-199.

Previous studies have shown that intronic peptide sequences in the prohormone for thyrotropin-releasing hormone (TRH) have physiological actions on pituitary hormone secretion. The aim of this investigation was to examine the effect of the cryptic peptides, prothyrotropin- releasing hormone(178-199) (ProTRH(178-199)) and ProTRH(186-199), on prolactin (PRL) release from the anterior pituitary. Perifusion studies were performed with anterior pituitaries obtained from individual adult male Sprague Dawley rats at 70 90 d of age. Perifusate was collected in 5-min fractions for 25 min prior to peptide administration and for 60 min afterward. Pituitaries were perifused with a single 5 min pulse of either 2, 10, or 40 nM concentrations (peak pulse) of each peptide or the vehicle. Sixty minutes after peptide administration, a 200 mM pulse of potassium chloride was delivered to check tissue viability. Prolactin was measured in the perifusate by radioimmunoassay. Results showed that both peptides induced a significant long-term suppression of prolactin secretion that was still evident at 60 min after peptide exposure. ProTRH(186-199) was similar to ProTRH(178-199) in suppressing prolactin release at the 2 and 40 nM dose, suggesting that the amino acid sequence necessary for prolactin inhibition is contained within the smaller peptide fragment. These data indicate that a cryptic sequence within the proTRH peptide can have biological activity at the level of the anterior pituitary gland in regulating prolactin secretion.

Fertilization promoting peptide--a possible regulator of sperm function in vivo.

Fertilization promoting peptide (FPP), a tripeptide related to thyrotrophin releasing hormone (TRH), is found in seminal plasma. Recent evidence obtained in vitro suggests that FPP may play an important role in regulating sperm fertility in vivo. Specifically, FPP initially stimulates nonfertilizing (uncapacitated) spermatozoa to "switch on" and become fertile more quickly, but then arrests capacitation so that spermatozoa do not undergo spontaneous acrosome loss and therefore do not lose fertilizing potential. These responses are mimicked, and indeed augmented, by adenosine, known to regulate the adenylyl cyclase (AC)/cAMP signal transduction pathway. Both FPP and adenosine have been shown to stimulate cAMP production in uncapacitated cells but inhibit it in capacitated cells, with FPP receptors somehow interacting with adenosine receptors and G proteins to achieve regulation of AC. These events affect the tyrosine phosphorylation state of various proteins, some being important in the initial "switching on," others possibly being involved in the acrosome reaction itself. Calcitonin and angiotensin II, also found in seminal plasma, have similar effects in vitro on uncapacitated spermatozoa and can augment responses to FPP, suggesting that all four molecules may be involved in regulating availability of cAMP. It is plausible that these molecules have similar effects in vivo, affecting fertility by stimulating and then maintaining fertilizing potential. Either reductions in the availability of FPP, adenosine, calcitonin, and angiotensin II or defects in their receptors could contribute to male infertility. These exciting results may provide new approaches for diagnostic tests and treatments of certain categories of male infertility.

Kinetic investigation of the specificity of porcine brain thyrotropin-releasing hormone-degrading ectoenzyme for thyrotropin-releasing hormone-like peptides.

Evidence indicates that neuronally released thyrotropin-releasing hormone (TRH) is selectively inactivated by TRH-degrading ectoenzyme (TRH-DE) (EC ). TRH-DE inhibitors may be used to enhance the therapeutic actions of TRH and to investigate the functions of TRH and TRH-DE in the central nervous system. Although TRH-DE appears to exhibit a high degree of specificity toward TRH, systematic specificity studies, which would facilitate inhibitor design, have not been previously conducted for this enzyme. In this paper we present the first description of TRH-DE specificity across a directed peptide library in which the histidyl (P(1)') residue of TRH was replaced by a series of amino acids. Peptides were synthesized using standard solid phase chemistry. Kinetic parameters were measured either by continuous or discontinuous fluorometric assays or by quantitative high pressure liquid chromatography. The P(1)' residue was found to influence significantly both the ability of the peptides to bind to TRH-DE, as measured by their K(i) values, and the ability of TRH-DE to catalyze their hydrolysis. Moderately bulky, uncharged P(1)' residues were found to bind preferentially to TRH-DE. Results from this screen provide valuable information for the development of TRH-DE inhibitors and have led to the identification of two potent, reversible TRH-DE inhibitors, l-pyroglutamyl-l-asparaginyl-l-prolineamide (K(i) = 17.5 micrometer) and Glp-Asn-Pro-7-amido-4-methyl coumarin (K(i) = 0.97 micrometer).

Quantitative analysis of pyroglutamic acid in peptides.

A simplified and rapid procedure for the determination of pyroglutamic acid in peptides was developed. The method involves the enzymatic cleavage of an N-terminal pyroglutamate residue using a thermostable pyroglutamate aminopeptidase and isocratic HPLC separation of the resulting enzymatic hydrolysate using a column switching technique. Pyroglutamate aminopeptidase from a thermophilic archaebacteria, Pyrococcus furiosus, cleaves N-terminal pyroglutamic acid residue independent of the molecular weight of the substrate. It cleaves more than 85% of pyroglutamate from peptides whose molecular weight ranges from 362.4 to 4599.4 Da. Thus, a new method is presented that quantitatively estimates N-terminal pyroglutamic acid residue in peptides.

Effect of chain length on absorption of biologically active peptides from the gastrointestinal tract.

OBJECTIVES: Protein digestion generates many peptides in the gut lumen. Some of these peptides possess biological effects when tested using in vitro systems. It is clear that dipeptides and tripeptides can be absorbed intact from the gastrointestinal tract. However, the fate of larger peptides and small proteins remains unclear. Equally unclear are the biologic potencies of absorbed peptides and the quantity of peptide that must be administered into the gut to produce a biologic effect. Thus, the purpose of this study was to determine the effect of amino acid chain length on the ability of enterally administered peptides to produce biologic effects. METHODS: Small bowel feeding tubes, jugular catheters, and arterial lines were placed into adult male Sprague-Dawley rats. Rats were administered intravenous (50 microg) and enteral (125 and 500 microg) thyrotropin-releasing hormone (TRH, a tripeptide), intravenous (100 microg) and enteral (100 and 500 microg) luteinizing hormone-releasing hormone (LHRH, a decapeptide), and intravenous (0.5 mg) and enteral (0.5 and 25 mg) insulin (a 51-amino acid polypeptide). The quantity of peptide administered represented less than 0.5% of a rat's normal daily protein intake. The biologic effect of TRH, LHRH, and insulin were assessed using thyroid-stimulating hormone (TSH) response, follicle-stimulating hormone (FSH) response, and glucose. We also measured serum levels of insulin in the rats following enteral insulin administration. RESULTS: The results indicate that enteral TRH (125 and 500 microg) produced the same TSH response as intravenous TRH. The response to 500 microg enteral LHRH was 50% of the response to intravenous LHRH and the response to 25 mg enteral insulin was 30% of the response to 0.5 mg intravenous insulin. Serum insulin levels increased significantly following both 0.5 and 25 mg enteral insulin. CONCLUSIONS: These results support the concept that small (di- and tripeptides) and large (10-51 amino acids) peptides generated in the diet can be absorbed intact through the intestines and produce biologic effects at the tissue level. The potency of the enterally administered peptides decreases as the chain length increases. We postulate that absorbed dietary peptides play a role in the modulation of organ function and disease progression.