Using surface plasmon resonance to directly determine binding affinities of combinatorially selected cyclopeptides and their linear analogs to a streptavidin chip

In our recent report, several HPQ-containing streptavidin ligands were identified from a structurally constrained combinatorial library, and the relative affinities in IC(50) of these tight-binding ligands were revealed by a captured enzyme-linked immunosorbent assay. In the present work, surface plasmon resonance was employed to directly evaluate the binding affinities between immobilized streptavidin and combinatorially selected ligands. The equilibrium dissociation constants and kinetic on/off rates of a previously identified N-to-side chain and newly synthesized N-to-C cyclopeptides were readily deduced using Scatchard analysis and computational simulation. It was found that both cyclopeptides bound streptavidin far more tightly than its linear counterpart ( approximately 1000-fold), while the reversed (QPH) linear and cyclic peptidyl ligands were hardly recognized by streptavidin. Consequently, not only was the binding specificity of synthetic ligands distinguished qualitatively but also the entropic advantage of conformationally constrained cyclopeptides over their linear forms was demonstrated quantitatively by surface plasmon resonance.     

Different affinities and selectivities of endothelin-1 and endothelin-3 binding to various rat tissues

Inhibition of [125I]endothelin-1 ([125I]ET-1) binding to membrane fractions from various rat tissues by ET-1 and endothelin-3 (ET-3) was investigated. Brain tissue demonstrated a 37-fold higher affinity for ET-1 compared to lung, while a greater than 1000-fold difference in affinity for ET-3 was observed in these two tissues. Furthermore, the ratio of the IC50 value of ET-3 to that of ET-1 in each tissue varied from approximately 2 in brain, kidney and liver to greater than 100 in heart and spleen. These experiments show that the tissues examined have different affinities as well as different selectivities for ET-1 and ET-3.     

The VI international conference on prostaglandins and related compounds

The effects of a heme ligand, cyanide, on pure ovine prostaglandin H synthase have been examined in detail as one approach to elucidating the role of the heme cofactor in cyclooxygenase and peroxidase catalysis by the synthase. Cyanide bound to the synthase heme with an affinity (Kd) of 0.19 mM, and inhibited the peroxidase activity of the synthase, with a K_I value of 0.23 mM. Cyanide increased the sensitivity of the cyclooxygenase to inhibition by the peroxide scavenger, glutathione peroxidase. This increased sensitivity to inhibition reflect and increase in the level of peroxide required to activate the cyclooxygenase, from 21 nM in absence of cyanide to over 300 nM when 2.5 mM cyanide was present. The increase in peroxide activator requirement with increasing cyanide concentration closely paralleled the formation of the holoenzyme-cyanide complex. These effects of low levels of cyanide suggest that the heme prosthetic group of the synthase participates in the efficient activation of the cyclooxygenase by peroxide. Cyanide blocked the stimulation of cyclooxygenase velocity by phenol, but not the phenol-induced increase in overall oxygen consumption. This blockade by cyanide was noncompetitive with respect to phenol and was characterized by a K_I of 4 mM. The higher K_I value for this effect suggests that cyanide can also interact at a site other than the heme prosthetic group. The role of the heme prosthetic group in promoting efficient activation of the cyclooxygenase by peroxide appears to be central to the ability of the synthase to amplify the ambient peroxide concentration rapidly.     

Characterisation of the prostanoid receptors mediating contraction of guinea-pig isolated trachea

The receptors mediating prostanoid-induced contraction of guinea-pig isolated trachea have been characterised in terms of a recently proposed general classification of prostanoid receptors. Results obtained on the trachea were compared with those obtained on guinea-pig fundus, which contains a sub-type of PGE₂-sensitive (EP-) receptor termed the EP₁-receptor, and guinea-pig lung strip, which contains a thromboxane-sensitive or TP-receptor. The following agonists were studied, PGE₂, PGF₂α and the thromboxane-like agonists U-46619 and Wy17186. The antagonists studied were SC-19220 which selectivity blocks EP₁-receptors, and AH19437 which selectively blocks TP-receptors. On guinea-pig fundus the rank order of agonist potency was PGE₂ > PGF₂α > Wy17186 U-46619, and responses to all agonists were antagonised by SC-19220 but not by AH19437. On guinea-pig lung strip the rank order of potency was U-46619 > Wy17186 PGF₂α > PGE₂ and responses to all agonists tested were blocked by AH19437 but not by SC-19220. On the trachea, the rank order was PGE₂ = U-46619 > Wy17186 = PGF₂α. SC-19220 antagonised responses to PGE₂ and PGF₂α, but not those to U-46619 or Wy17186. Conversely, AH19437 antagonised responses to U-46619 and Wy17186 but not those to PGE₂ or PGF₂α. It is concluded that prostanoid-induced contractions of guinea-pig trachea can be mediated by both EP₁- and TP-receptors.     

Characterisation of the prostanoid receptors mediating contraction of guinea-pig isolated trachea

The receptors mediating prostanoid-induced contraction of guinea-pig isolated trachea have been characterised in terms of a recently proposed general classification of prostanoid receptors. Results obtained on the trachea were compared with those obtained on guinea-pig fundus, which contains a sub-type of PGE2-sensitive (EP-) receptor termed the EP1-receptor, and guinea-pig lung strip, which contains a thromboxane-sensitive or TP-receptor. The following agonists were studied, PGE2, PGF2 alpha and the thromboxane-like agonists U-46619 and Wy17186. The antagonists studied were SC-19220 which selectively blocks EP1-receptors, and AH19437 which selectively blocks TP-receptors. On guinea-pig fundus the rank order of agonist potency was PGE2 greater than PGF2 alpha greater than Wy-17186 approximately equal to U-46619, and responses to all agonists were antagonised by SC-19220 but not by AH19437. On guinea-pig lung strip the rank order of potency was U-46619 greater than Wy17186 much greater than PGF2 alpha greater than PGE2 and responses to all agonists tested were blocked by AH19437 but not by SC-19220. On the trachea, the rank order was PGE2 = U-46619 greater than Wy17186 = PGF2 alpha. SC-19220 antagonised responses to PGE2 and PGF2 alpha, but not those to U-46619 or Wy17186. Conversely, AH19437 antagonised responses to U-46619 and Wy17186 but not those to PGE2 or PGF2 alpha. It is concluded that prostanoid-induced contractions of guinea-pig trachea can be mediated by both EP1- and TP-receptors.     

Synthesis and binding affinities of methylvesamicol analogs for the acetylcholine transporter and sigma receptor

We synthesized methylvesamicol analogs 13-16 and investigated the binding characteristics of 2-[4-phenylpiperidino]cyclohexanol (vesamicol) and methylvesamicol analogs 13-16, with a methyl group introduced into the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (-)-o-methylvesamicol [(-)-OMV] (13) (Ki=6.7 nM), as well as (-)-vesamicol (Ki=4.4 nM), had a high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (Ki=3.0 nM), as well as SA4503 (Ki=4.4 nM), reported as a sigma-1 mapping agent for positron emission tomography (PET), had a high affinity for the sigma-1 receptor. The binding affinity of (+)-PMV (16) for the sigma-1 receptor (Ki=3.0 nM) was about 13 times higher than that for the sigma-2 (sigma-2) receptor (Ki=40.7 nM). (+)-PMV (16) (Ki=199 nM) had a much lower affinity for VAChT than SA4503 (Ki=50.2 nM) and haloperidol (Ki=41.4 nM). These results showed that the binding characteristics of (-)-OMV (13) to VAChT were similar to those of (-)-vesamicol and that (+)-PMV (16) bound to the sigma-1 receptor with high affinity. In conclusion, (-)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a methyl group for labeling with 11C, may become not only a new VAChT ligand and a new type of sigma receptor ligand, respectively, but may also become a new target compound of VAChT and the sigma-1 receptor radioligand for PET, respectively.     

Studies of the characterisation of prostanoid receptors: A proposed classification

Comparison of rank orders of agonist potency of the anturally occurring prostanoids. PGD₂, PGE₂, PGF₂α and PGI₂ as well as the stable TxA₂ mimetic, U-46619, on a range of smooth muscle preparations provides evidences evidence for the existence of distinct receptors for PGE₂. PGF₂α and TxA₂. Since others have provided evidence for the existence of distinct receptors for PGD₂ and PGI₂, we suggest that receptors exist for each of these naturally occurring 2-series prostanoids. Results obtained with two specific prostanoid receptor blocking drugs, SC-19220 and AH 19437, supported and extend these conclusions. SC-19220 selectively block some but not all PGE-sensitive receptors. While AH 19437 selectively blocks all U-46619/TxA₂-sensitive receptors. A nomenclature for prostanoid receptors is proposed,; in which each receptor is designated the letter P preceded by a letter signifying the most potent natural prostanoid agonist at than receptor, such that receptors sensitivity to PGs D₂, E₂, F₂α, I₂ and TxA₂ become DP-, EP-, FP- and TP- receptors respectively. Where some sub-division is required within a receptor group, e.g. EP-receptors (SC-19220-sensitive and SC-19220-insensitive), subcript numbers may be used such that these are EP₁ and EP₂ subtypes. The resulting scheme is a working hypothesis and its confirmation requires the development of potent selective prostanoid receptor blocking drugs for each postulated type.     

The site-specific installation of methyl-lysine analogs into recombinant histones

Histone lysine residues can be mono-, di-, or trimethylated. These posttranslational modifications regulate the affinity of effector proteins and may also impact chromatin structure independent of their role as adaptors. In order to study histone lysine methylation, particularly in the context of chromatin, we have developed a chemical approach to install analogs of methyl lysine into recombinant proteins. This approach allows for the rapid generation of large quantities of histones in which the site and degree of methylation can be specified. We demonstrate that these methyl-lysine analogs (MLAs) are functionally similar to their natural counterparts. These methylated histones were used to examine the influence of specific lysine methylation on the binding of effecter proteins and the rates of nucleosome remodeling. This simple method of introducing site-specific and degree-specific methylation into recombinant histones provides a powerful tool to investigate the biochemical mechanisms by which lysine methylation influences chromatin structure and function.     

Design of peptide oxytocin antagonists with strikingly higher affinities and selectivities for the human oxytocin receptor than atosiban.

The peptide oxytocin (OT) antagonist atosiban, approved for tocolytic use in Europe (under the tradename Tractocile), represents an important new therapeutic advance for the treatment of premature labor. This paper presents some new peptide OT antagonists which offer promise as superior tocolytics. The solid phase synthesis is reported of four pairs of L and D-2-naphthylalanine (L/D-2Nal) position-2 modified analogs of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly-NH(2),d(CH(2))(5)[Tyr(Me)(2),Thr(4)]OVT) (A); the Tyr-NH(2) (9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2) (9)]OVT (B); the Eda(9) analog of (A), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)]OVT (C); and the retro COCH(2)Ph(4-0H)(10) modified analog of (C), d(CH(2))(5)[Tyr(Me)(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT (D). The eight new analogs of A-D are (1) desGly-NH(2),d(CH(2))(5)[D-2Nal(2),Thr(4)]OVT, (2) desGly-NH(2),d(CH(2))(5)[2-Nal(2),Thr(4)]OVT, (3) d(CH(2))(5)[D-2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (4) d(CH(2))(5)[2Nal(2),Thr(4),Tyr-NH(2) (9)]OVT, (5) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)]OVT, (6) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)]OVT, (7) d(CH(2))(5)[D-2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-0H)(10)]OVT, (8) d(CH(2))(5)[2Nal(2),Thr(4),Eda(9)<-- COCH(2)Ph(4-OH)(10)]OVT. Peptides 1-8 were evaluated for agonistic and antagonistic activities in in vitro and in vivo rat bioassays, in rat OT receptor (rOTR) binding assays and in human OT receptor (hOTR) and human vasopressin (VP) vasopressor (V(1a)) receptor (hV(1a)R) binding assays. Also reported are the hOTR and hV(1a)R affinity data for atosiban and for B. None of the eight peptides exhibit oxytocic or vasopressor agonism. Peptides 1-8 exhibit weak antidiuretic agonism (activities in the range 0.014-0.21 U/mg). Peptides 1-6 exhibit potent in vitro (no Mg(2+)) OT antagonism (anti-OT pA(2) values range from 7.63 to 8.08). Peptides 7 and 8 are weaker OT antagonists. Peptides 1-6 are all OT antagonists in vivo (estimated in vivo anti-OT pA(2) values in the range 6.94-7.23). Peptides 1-8 exhibit vasopressor antagonism, anti-V(1a) pA(2) values in the range 5.1-7.65. Peptides 1-8 exhibit high affinities for the rOTR (K(i) values = 0.3-7.8 nM). Peptides 1-4 and B exhibit surprisingly very high affinities for the hOTR; their K(i) values are 0.17, 0.29, 0.07, 0.14 and 0.59 nM, respectively. Peptides 1-4 and B exhibit respectively 449, 263, 1091, 546 and 129 times greater affinity for the hOTR than atosiban (K(i) = 76.4 nM). Peptides 1-4 exhibit high affinities for the hV(1a)R (K(i)s = 1.1 nM, 1.3 nM, 0.19 nM and 0.54 nM, all higher than the hV1(a)R affinities exhibited by atosiban (K(i) = 5.1 nM) and by B (K(i) = 5.26 nM). Because of their strikingly higher affinities for the hOTR than atosiban, peptides 1-4 and B exhibit gains in anti hOT/anti hV(1a) receptor selectivity compared with atosiban of 93, 64, 39, 56 and 127, respectively. These OT antagonists are thus promising candidates for development as potential new tocolytic agents.     

Prostaglandin E2 mediates IL-1beta-related fibroblast mitogenic effects in acute lung injury through differential utilization of prostanoid receptors

The fibroproliferative response to acute lung injury (ALI) results in severe, persistent respiratory dysfunction. We have reported that IL-1beta is elevated in pulmonary edema fluid in those with ALI and mediates an autocrine-acting, fibroblast mitogenic pathway. In this study, we examine the role of IL-1beta-mediated induction of cyclooxygenase-2 and PGE2, and evaluate the significance of individual E prostanoid (EP) receptors in mediating the fibroproliferative effects of IL-1beta in ALI. Blocking studies on human lung fibroblasts indicate that IL-1beta is the major cyclooxygenase-2 mRNA and PGE2-inducing factor in pulmonary edema fluid and accounts for the differential PGE2 induction noted in samples from ALI patients. Surprisingly, we found that PGE2 produced by IL-1beta-stimulated fibroblasts enhances fibroblast proliferation. Further studies revealed that the effect of fibroblast proliferation is biphasic, with the promitogenic effect of PGE2 noted at concentrations close to that detected in pulmonary edema fluid from ALI patients. The suppressive effects of PGE2 were mimicked by the EP2-selective receptor agonist, butaprost, by cAMP activation, and were lost in murine lung fibroblasts that lack EP2. Conversely, the promitogenic effects of mid-range concentrations of PGE2 were mimicked by the EP3-selective agent, sulprostone, by cAMP reduction, and lost upon inhibition of Gi-mediated signaling with pertussis toxin. Taken together, these data demonstrate that PGE2 can stimulate or inhibit fibroblast proliferation at clinically relevant concentrations, via preferential signaling through EP3 or EP2 receptors, respectively. Such mechanisms may drive the fibroproliferative response to ALI.     

Distribution and function of prostanoid receptors: studies from knockout mice

Recent developments in the molecular biology of the prostanoid receptors has allowed the investigation of the physiological roles of each individual receptor type and subtype. The following article reports the prostanoid receptor distributions deduced from Northern blot and in situ hybridization analyses, summarizes the phenotypes of each receptor knockout mice, and discusses recent studies investigating the effects of each receptor deficiency on the inflammatory response and female reproductive processes. The combination of expression pattern and knockout analyses enabled us to determine which receptor expressed in a particular cell is important for the maintenance of normal and/or pathological physiology. The results from these analyses may be useful in the development of novel therapeutics that can selectively manipulate prostanoid-mediated actions.     

Chemical data related to the tribal affinities of Hulsea and Arnica

The taxonomic placement of Hulsea is discussed in light of the distribution of pyrrolizidine alkaloids, sesquiterpene lactones, and polyacetylenic compounds in the Asteraceae.     

Cellular distribution of prostanoid EP receptors mRNA in the rat gastrointestinal tract

The inhibition of PGE(2) synthesis resulting from sustained NSAIDs therapy has been linked to gastrointestinal irritations and ulceration. The multiple physiological effects of PGE(2) in the gut are mediated through the activation of four receptors termed EP(1-4). The aim of the study was to determine the precise distribution of the four prostaglandin E(2) receptors in the rat stomach, small intestine, and colon. We used non-radioactive in situ hybridization techniques on paraffin-embedded tissue. Mucous cells of the stomach and goblet cells of the small intestine and colon were found to express mRNA for all four EP subtypes. A positive hybridization signal for EP(1), EP(3), and EP(4) was detected in the parietal cells of the stomach whereas the chief cells expressed low levels of EP(1) and EP(3). The EP(1) and EP(3) receptor mRNA could also be detected in the muscularis mucosa, longitudinal muscle and enteric ganglias of the stomach and small intestine. However, close examination of the enteric ganglias indicated that most of the positive labeling was localized to the glial cells, although some neurons did express EP(3). In conclusion, we have detailed the distribution of prostanoid EP receptors in the gut at the cellular level, giving new insights to the role of prostaglandins in gastrointestinal functions.     

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development. We describe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic α-helical peptide, melittin, and selected structural analogs. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.