Conformationally readdressed CCK-B/δ-opioid pepitide ligands

The sequence of a cholecystokinin (CCK) related peptide was modified to obtain analogues, which intereact selectively either with CCK-B, or with δ-opioid receptors. Two kinds of peptides were designed, namely, the cyclic peptides of the H-Tyr-cyclo(D -Pen-Gly-Trp-L -/D-3-transmecaptoproline)-Asp-Phe-NH₂ sequence (compounds 1a and 1b , respectively), and the linear peptides of the H-Tyr-D -Val-Gly-Trp-L /D -3-trans-methylmercaptoproline-Asp-Phe-NH₂ sequence (compounds 2a and 2b , respectively). The only difference between the chemical structures of the linear analogues compared to the cyclic ones is that one covalent bond has been eliminated and a sulfur atom is replaced by a methyl group. Molecular modeling showed that, among low-energy conformers of cyclic compounds 1 , there are three-dimensional structures compatible to the model for δ- receptor- bound conformer, suggested earlier[G. V. Nikiforovich. V. J. Hruby. O. Prakash, and C. A. Gehrig (1991) Biopolymers. vol. 31. pp. 941–955]. Results of binding assays fully supported the rationale for the design of compounds 1 and 2 . The cyclic analogue 1a has K_i values of 4.5 and > 5000 n M at δ- and μ-opioid receptors, respectively; IC₅₀ values of 3000 n M for both CCK-A and CCK-B receptors, whereas its linear counterpart 2a has k_i values of 462 and 229 nM at δ- and μ-opioid receptors, respectively; and IC₅₀ values of 1.6 and > 10.000 nM for CCK-A and CCK-B receptors, respectively. The results of this study demonstrate a possibility to redirect a peptide sequence that interacts with one type of receptors (CCK-B receptors) toward interaction with another type (δ-opioid receptors) belonging to a different physiological system. This redirection could be performed by changing the conformational properties of the peptide with very minimal changes in its chemical structure. © 1995 John Wiley & Sons, Inc.     

Characterization of the bioactive form of linear peptide antagonists at the δ-opioid receptor

The stereochemical requirements for δ-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The δ-opioid receptor analogues with significant affinity, Tyr-Tic-NH₂ (TI-NH₂), Tyr-Tic-Phe-OH (TIP), Tyr-Tic-Phe-NH₂(TIP-NH₂), Tyr-Tic-Phe-Phe-OH (TIPP), Tyr-Tic-Phe-Phe-NH₂) (TIPP-NH₂), and the low affinity δ-opioid peptides Tyr-Pro-Phe-Pro-NH₂ (morphiceptin) and Tyr-Phe-Phe-Phe-NH₂ (TPPP-NH₂), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic simulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH₂ and TIPP-NH₂, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr¹-D -Pen²-Gly³-Phe⁴-D -Pen⁵) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high δ-opioid affinity L -Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr¹ and Tic² residues. Moreover, both the Tic² and Phe³ residues could mimic the hydrophobic interactions with the receptor of the Phe⁴ moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di-and tripeptides. The low δ-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition. © 1996 John Wiley & Sons, Inc.     

Design and characterization of a model αβ peptide

CD and nmr spectroscopy were used to compare the conformational properties of two related peptides. One of the peptides, Model AB, was designed to adopt a helix-turn-extended strand (αβ) tertiary structure in water that might be stabilized by hydrophobic interactions between two leucine residues in the amino-terminal segment and two methionine residues in the carboxyl terminal segment. The other peptide, AB Helix, has the same amino acid sequence as Model AB except that it lacks the-Pro-Met-Thr-Met-Thr-Gly segment at the carboxyl-terminus. Although the carboxyl-terminal segment of Model AB was found to be unstructured, its presence increases the number of residues in a helical conformation, shifts the pKas of three ionizable side chains by 1 pH unit or more compared to an unstructured peptide, stabilizes the peptide as a monomer in high concentrations of ammonium sulfate, increases the conformational stability of residues at the terminal ends of the helix, and results in many slowly exchanging amide protons throughout the entire backbone of the peptide. These results suggest that interactions between adjacent segments in a small peptide can have significant structure organizing effects. Similar kinds of interactions may be important in determining the structure of early intermediates in protein folding and may be useful in the de novo design of independently folding peptides. © 1995 John Wiley & Sons, Inc.     

4-Aminopiperidine ureas as potent selective agonists of the human β3-Adrenergic receptor

The preparation and structure–activity relationships (SARs) of potent agonists of the human β₃-adrenergic receptor (AR) derived from a 4-aminopiperidine scaffold are described. Examples combine human β₃-AR potency with selectivity over human β₁-AR and/or human β₂-AR agonism. Compound 29s was identified as a potent (EC₅₀=1 nM) and selective (greater than 400-fold over β₁- with no β₂-AR agonism) full β₃-AR agonist with in vivo activity in a transgenic mouse model of thermogenesis.     

Fluorine nmr studies of poly(N-acryloyl-β-alanine)–α-chymotrypsin conjugates

Fluorine nmr experiments carried out at 51.0 and 94.1 MHz have been used to explore the interaction of the probe molecule p-fluorocinnamate with conjugates formed from α-chymotrypsin and poly(N-acryloyl-β-alanine). The data obtained include enzyme-induced chemical-shift effects, spin-lattice (R₁) and transverse (R₂) relaxation rates, and the rate constant for dissociation of the fluorocinnamate–enzyme complexes. Analysis of the results indicates that while overall molecular tumbling of the enzyme molecule is not greatly changed by attachment of polymers of various sizes, conjugated polymer can appreciably affect the structure of the p-fluorocinnamate binding site. The important variable involved in such structural changes appears to be the amount of polymer present per mole of protein.     

Chronic exposure to antibodies directed against anti-opiate peptides alter δ-opioid receptor levels

The development of addictive states in response to chronic opioid use may be regulated partially by the release of endogenous peptides. These anti-opiate peptides (AOP) are secreted or released into the CNS and produce diverse actions that counterbalance the effects of prolonged opiate exposure. Though the mechanism(s) by which these peptides exert their physiological properties remain largely unknown, there is some indication that AOP’s modulate opioid receptor levels. In this study, we investigated the effects of chronically infused α-melanocyte stimulating hormone (α-MSH), dynorphin_1-8 (DYN_1-8), dynorphin A (DYNA), and NPFF antibodies on δ-opioid receptor expression in rat brains. Quantitative autoradiographic experiments revealed that antibodies directed against α-MSH and DYNA produced significant increases in delta receptor levels in the caudate, claustrum, and cingulate cortex of the rat brain. Conversely, NPFF monoclonal antibodies caused significant decreases in the caudate, nucleus accumbens, olfactory tubercle, and cingulate cortex. These results suggest that the density of δ-opioid receptors is affected by changes in the levels of the anti-opioid peptides in the extracelluar fluid in the rat brain.     

Discovery of highly potent and selective D4 ligands by interactive SAR study

A series of thienylmethylphenylpiperazins was synthesized and tested for affinity towards the five subtypes of dopaminergic receptors. Compound 5f showed more than 1000 folds selectivity to D4 receptors; analogue 5e showed the highest affinity to D4 receptors with K_i 3.9 nM. An interactive SAR approach was adopted and lead to compound 14a with K_i (D4) as low as 0.03 nM. Molecular docking studies showed a potential, first to report arene cation interaction between the D4 unique residue Arg-186 and the ligands’ arene moiety, explaining the importance of having a strong negative electrostatic potential at this area of the compound structure.     

Discovery of highly potent,selective, covalent inhibitors of JAK3

A useful and novel set of tool molecules have been identified which bind irreversibly to the JAK3 active site cysteine residue. The design was based on crystal structure information and a comparative study of several electrophilic warheads.     

Structural and conformational modifications of α-MSH/ACTH4–10 provide melanotropin analogues with highly potent behavioral activities

Previous studies have identified the (4–10) heptapeptide sequence as the central core of α-MSH/ACTH peptides required for mediation of important biological activities. In the present study, the structure-activity relationships of Nle⁴-substituted and -bridged cyclic α-MSH analogues, which were previously shown to exhibit a wide range of melanotropic potencies from weak agonism to super potency, were examined for grooming behavioral activity in the rat following intracerebroventricular injections. The results showed that stepwise C-terminal elongation of the linear Nle⁴-substituted Ac-α-MSH_4–10-NH₂ increased grooming potencies of the peptides in a manner similar to their actions on melanocytes. The most interesting finding was the observation that cyclization of the inactive linear “central (4–10) core” of α-MSH (Ac-α-MSH_4–10) to form Ac-[ ]-α-MSH_4–10-NH₂ resulted in a super potent agonist in the grooming assay. However, while cyclization of the (4–10) heptapeptide produced potent agonists on grooming behavior, the structure-activity relationships were different than the frog skin bioassay. These findings support the hypothesis that appropriate structural and confirmational modifications of α-MSH-related peptides can produce profound effects on the bioactivities of the peptides, and suggest that different structural-conformational requirements exist for α-MSH interactions with its various receptors.     

Ent-16α-methylamino-3-methoxy-1,3,5(10)-estratrien-17β-ol hydrochloride, a selective anti-arrhytmic enantiosteroid

The title compound and its antipode with natural steroid configuration were synthesized and tested. Both compounds showed equal potency as anti-arrhythmic compounds. An additional effect found for the steroid with natural configuration was its plasma cholesterol lowering activity in the rat. This suggests that enantiosteroids may have a more selective action than the steroids with natural configuration     

The legacy of enhanced N and S deposition as revealed by the combined analysis of δ13C, δ18O and δ15N in tree rings

This study aimed to evaluate the effects of long‐term repeated aerial nitrogen (N) and sulphur (S) misting over tree canopies of a Sitka spruce plantation in Scotland. We combined δ¹³C and δ¹⁸O in tree rings to evaluate the changes in CO₂ assimilation (A) and stomatal conductance (g_s) and to assess their contribution to variations in the intrinsic water‐use efficiency (WUE_i, i.e., the A/g_s ratio). Measurements of δ¹⁵N enabled shifts in the ecosystem N cycling following misting to be assessed. We found that: (i) N applications, with or without S, increased the ratio between A and g_s in favour of A, thus supporting a fertilizer effect of added N. (ii) After the treatments ceased, the trees quickly adjusted to the reductions of N deposition, but not to the reduction in S deposition, which had a negative effect on WUE_i by reducing A. This indicates that the beneficial role of N deposition may be negated in forests that previously received a high load of acid rain. (iii) δ¹⁵N in tree rings reflected the N dynamics caused by canopy retention, with the fingerprint also present in the litter, after the experiment stopped. (iv) Both our results (obtained using canopy applications) and a collection of published data (obtained using soil applications) showed that generally WUE_i increased in response to an increase of N applications, with the magnitude of the changes related to soil conditions and the availability of other nutrients. The shifts observed in δ¹⁵N in tree rings also suggest that both the quantity of the applied N and its quality, mediated by processes occurring during canopy N retention, are important determinants of the interactions between N and C cycles. Stable isotopes are useful probes to understand these processes and to put the results of short‐term experiments into context.     

Blood Platelets Contain and Secrete Laminin-8 (α4β1γ1) and Adhere to Laminin-8 via α6β1 Integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin γ1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to γ1 and β1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin β1 antibody–Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to β1 and γ1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin α4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20–35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to β1 and α6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (α4β1γ1) and that the cells adhere to the protein by using α6β1 integrin.     

Characterization of the bioactive form and molecular determinants of recognition of cyclic enkephalin peptides at the δ-opioid receptor

An extensive and systematic search strategy to determine the conformational profile of 12 cyclic disulfide-bridged opioid peptides with varying affinities at the δ receptor has been carried out to identify the structure that is recognized by the δ receptor for each analogue. The methods and procedures used here for the conformational search have already been validated for [D -Pen2, D -Pen5] enkephalin (DPDPE), one member of this family. Use of these methods led to a low-energy solution conformation of DPDPE in excellent agreement with all the geometric properties deduced from its solution nmr spectra. Each of the analogue was subjected to the same procedure, involving a combination of molecular dynamics simulations at high and low temperature. The study was repeated in two environmental conditions, an apolar environment, simulated by using a distance-dependent dielectric constant, and a polar environment by embedding the peptides in a high constant dielectric ( ε = 80). An automated comparison of the different conformers based on their backbone rms and average distance between the key aromatic moieties was followed by graphic analysis using maximum structural overlap. The cross-comparison of the conformations for each analogue revealed a unique conformer that may be recognized by the δ receptor for each high-affinity analogue that permitted maintaining the critical elements required for recognition in a simple spatial orientation, while maximizing similarity in other regions. © 1993 John Wiley & Sons, Inc.     

The α6β4 Integrin Is a Receptor for both Laminin and Kalinin

Previously, we have established K562 transfectants that express either α6Aβ1 or α6Bβ1 (Kα6A or Kα6B) on their surface. Both cell lines bind to laminin and kalinin after treatment with the β1-stimulatory antibody TS2/16. Here we introduce the full-length β4 cDNA into the α6A- and α6B-expressing K562 cells and selected stably transfected cells. The β4 subunit was expressed on the surface of both transfectants and it formed dimers with the α6A or α6B subunits. Immunoprecipitation and preclearing analyses revealed that both transfectants expressed α6β1, in addition to α6β4. While Kα6A and Kβ6B cells required TS2/16 stimulation for binding to laminin or kalinin, adhesion of the unstimulated β4-transfected Kα6A and Kα6B cells to these matrix components was already substantial. This adhesion was mediated by both α6β1 and α6β4 since it was completely blocked by an α6-specific antibody or by a combination of anti-β1 and anti-β4 antibodies, but only partially by either of these latter two antibodies alone. Adhesion to laminin was completely blocked by an antiserum to laminin fragment E8 as was the adhesion to kalinin by an antibody to kalinin, demonstrating the specificity of adhesion. Both transfectants always adhered more strongly to kalinin than to laminin. Furthermore, binding to kalinin was less well blocked by antibodies to β4 than binding to laminin, indicating that the affinity of α6β4 for kalinin is higher than that for laminin. The fact that α6β1 mediated adhesion without TS2/16 stimulation on the β4-transfected Kα6A and Kα6B cells suggests that some activation of α6β1 had occurred in these cells, even though binding was increased when they were actively stimulated by the antibody TS2/16. Finally, we show that Mn²⁺ induced binding of solubilized α6β4 to matrix containing kalinin, deposited by the murine cell line RAC-11P/SD. This binding was inhibited by the anti-α6 mAb GoH3. Together, these results indicate that both α6β1 and α6β4 are receptors for laminin and kalinin and that there are no differences in ligand specificity between the A and B variants of the α6 subunit when associated with either β1 or β4.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

m-Alkyl, α,α,α-trifluoroacetophenones: A new class of potent transition state analog inhibitors of acetylcholinesterase

A series of m-alkyl α,α,α-trifluoroacetophenones (1–5) was synthesized and evaluated as inhibitors of acetylcholinesterase from Torpedo california. All ketones (1–5) were found to be potent inhibitors of the enzyme; m-t-butyl α,α,α-trifluoroacetophenone (4) was the most potent inhibitor with a K_i value of 3.7 pM.     

3-Hydroxy-4-methyl-5-pentyl-2-iminopyrrolidine: a potent and highly selective inducible nitric oxide synthase inhibitor

(3S,4S,5R)-2-Imino-4-methyl-5-pentyl-3-pyrrolidinol hydrochloride (1) is a potent inducible nitric oxide synthase (i-NOS) inhibitor that has three times the selectivity of its parent, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine hydrochloride (2).