Differential scanning calorimetry studies of NaCl effect on the inverse temperature transition of some elastin-based polytetra-, polypenta-, and polynonapeptides

Differential scanning calorimetry studies of the effect of NaCl on protein-based polymer self-assembly has been carried out on six elastin-based synthetic sequential polypeptides- i.e., the polypentapeptide (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n and its more hydrophobic analogues (L -Leu¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n and (L -Val¹-L -Pro²-L -Ala³-L -Val⁴-Gly⁵)_n; the polytetrapeptide (L -Val¹-L -Pro²-Gly³-Gly⁴)_n and its more hydrophobic analogue (L -IIe¹-L -Pro²-Gly³-Gly⁴)_n; and the polynonapeptide (a pentatetra hybrid), (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵-L -Val⁶-L -Pro⁷-Gly⁸-Gly⁹)_n. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order of the polypentapeptide, as the temperature is raised from below to above that of the transition, has been repeatedly observed using different physical characterizations. In the present experiments, it is observed that the transition temperatures of the polypeptides studied are linearly dependent on NaCl concentration. The molar effectiveness of NaCl in shifting the transition temperature ΔT_m/[N], is about 14°C/[N], with the dependence on peptide hydrophobicity being fairly small. Interestingly, however, the δΔQ/ [N] does depend on the hydrophobicity of a polypeptide.     

Degradation of luteinizing hormone-releasing hormone by serum and plasma in vitro

Luteinizing hormone-releasing hormone (LH-RH) is degraded in vitro by serum and plasma from several species (human, rat, guinea-pig and cattle). Separation of the degradation products by high-performance liquid chromatography (HPLC) followed by amino acid analysis and radioimmunoassay showed that the main sites of cleavage are the Trp₃-Ser₄ and Tyr₅-Gly₆ bonds. Two peptidases are responsible since the cleavage at Trp₃-Ser₄ can be selectively inhibited by EDTA. In human plasma, the peptidase responsible for Trp₃-Ser₄ hydrolyssishas a K_m of 2.9 · 10^?4 M and V of 30 nmol/h per ml plasma. The half-life in vitro of LH-RH in serum and plasma from various species ranges from 3 h (guinea-pig) to 9.8 h (human). The peptidase cleaving LH-RH at Tyr₅-Gly₆ is present as an impurity in some commercial bovine serum and plasma albumins. Such contamination may have important practical implications for work involving peptide assays where albumins are used as carrier proteins.     

Conformation of CD4-derived cyclic hexapeptides by nmr and molecular dynamics

Two cyclic hexapeptides, cyclo[Ala¹-D -Ala²-Ser³-Phe⁴-Gly⁵-Ser⁶] and cyclo[Ala¹-Gly²-Ser³-Phe⁴-Gly⁵-Ser⁶], derived from the loop portion of the C′C″ ridge of CD4, were characterized by high-resolution nmr spectroscopy and simulated annealing studies. In DMSO-d₆ both of these peptides display a single conformer on the nmr time scale with two intramolecular H-bond (1 ← 4) stabilized β-turns at positions 2–3 and 5–6. The nmr derived distance constraints were used in simulated annealing calculations to generate the solution structures. These structures adopt energetically comparable conformational substates that are not resolvable on the nmr time scale. In aqueous solution, the H-bond stabilized β-turn conformation for cyclo [Ala-D -Ala-Ser-Phe-Gly-Ser] is no longer the predominant structural form. Structures generated using molecular dynamics simulations with no experimental constraints were compared with those from nmr analysis. The correlation between these two sets of structures allows the use of molecular simulations as a predictive tool for the conformational analysis of small peptides. © 1994 John Wiley & Sons, Inc.     

Nuclear overhauser enhancement demonstration of the type II β-turn in repeat peptides of tropoelastin

Nuclear Overhauser enhancement (NOE) experiments have been performed with the elastin peptides, namely; HCO-Val₁-Pro₂-Gly₃-Gly₄-OMe, t-Boc-Val₁-Pro₂-Gly₃-Val₄-Gly₅-OMe and t-Boc-Val₆-Ala₁-Pro₂-Gly₃-Val₄-Gly₅-OMe in DMSO-d₆. An NOE of approximately 10% was observed between the αCH of Pro₂ and the NH of Gly₃ involved in the β-turn of all three peptides. This finding shows the close proximity of two aforementioned protons and thus shows the occurrence of Type II β-turn in the repeat elastin peptides. The intermolecular distances are calculated and compared with the distances obtained from other model systems.     

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A₂ treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [³H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A₂ from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by $\text{β-}\text{bungarotoxin}$. (2) Phospholipase A₂ from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A₂ treatment on [³H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

Acylated des-(Ala1-Gly2)-somatostatin analogs: prolonged inhibition of growth hormone secretion

The following eight analogs of somatostatin were synthesized by solid phase: des-[Ala¹-Gly²]-somatostatin (I); des-[Ala¹-Gly²]-H₂somatostatin (II); $\text{N}$-acetyl-Cys³-somatostatin (III); $\text{N}$-acetyl-Cys³-H₂somatostatin (IV); $\text{N}$-pyvalyl-Cys³-H₂somatostatin (V); $\text{N}$-acrylyl-Cys³-H₂somatostatin (VI); $\text{N}$-benzoyl-Cys³-H₂somatostatin (VII); $\text{N}$-hexanoyl-Cys³-H₂somatostatin (VIII). Deletion of the N-terminal dipeptide Ala¹-Gly² is compatible with high biological activity. A single s.c. injection of these analogs as a microsuspension in saline inhibits for 24–72 hours (depending on the compound) the secretion of growth hormone normally stimulated in rats by pentobarbital.     

Characterization of kappa1 and kappa2 opioid binding sites in frog (rana esculenta) brain membrane preparation

The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa₁ is the dominat receptor subtype, frog brain contains mainly the kappa₂ subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa₂ ligands (Met⁵)enkephalin-Arg⁶-Phe⁷ and etorphine also show high affinities to the frog brain. Kappa₁ binding sites measured in the presence of 5 M /D-Ala²-Leu⁵/enkephalin represent 25–30% of [³H]ethylketocyclazocine binding in frog brain membranes. The kappa₂ subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala²-(Me)Phe⁴-Gly⁵-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.Abbreviations used DAGO /D-Ala²-(Me)Phe⁴-Gly⁵-ol/enkephalin - DALE /D-Ala²-L-Leu⁵/-enkephalin - EKC ethylketocyclazocine - DHM dihydromorphine - CAM N-cyclopropylmethylnorazidomorphine - nor-BNI nor-binaltorphimine - MR2034 (-)-(1R,5R,9R)-5, 9-dimethyl-2 (L-tetrahydrofuryl-2'-hydroxy-6,7benzomorphan) - MR2035 (+)-(1R,5R,9R)-5,9-dimethyl-2 (L-tetrahydrofuryl-2'-hydroxy-6,7-benzomorphan), U50488H=3,4-dichloro-N-/2-(1-pyrrolidinyl) —cyclohexo/-benzene-acetamide - PD117302 trans-N-methyl-N-/2-(1-pyrrolidinyl) — cyclohexyl/-benzo (b) thiophene-4-acetamide     

Proton and carbon magnetic resonance studies of the synthetic polypentapeptide of elastin.

Proton and ¹³C magnetic resonance studies are reported on the synthetic polypentapeptide of elastin, HCO-(Val₍₁₎-Pro₍₂₎-Gly₍₃₎-Val₍₄₎-Gly₍₅₎)_n-Val-OMe, where n ∼- 18. Temperature and solvent dependence of peptide N$\text{H}$ chemical shift and solvent dependence of peptide carbonyl chemical shift were used to delineate these moieties preliminary to identification of secondary structure.Based on these studies it is proposed, for the organic solvents of dimethyl sulfoxide, methanol, and low-temperature trifluoroethanol, that dynamic hydrogen bonds form in order of decreasing frequency of occurrence between the Val₍₁₎$\text{C}$O and the Val₍₄₎ N$\text{H}$ (a β-turn), between the Gly₍₃₎ N$\text{H}$ and the Gly₍₅₎$\text{C}$O (an 11-atom, hydrogen-bonded ring), and a more limited interaction between the Gly₍₃₎$\text{C}$O and the Gly₍₅₎ N$\text{H}$ (a γ-turn).Arguments are presented that relate the conformational features proposed above to the coacervate, which is a filamentous state.     

Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies

The temperature dependence of the composition of coacervate and equilibrium phases is examined for the polypentapeptide of elastin (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n in water. This provides for the development of a phase diagram. CD data is presented that provides information on associated polypeptide structure changes that, when added to previous CD, nmr, and dielectric relaxation data at lower water composition, allow construction of a phase-structure diagram of the polypentapeptide–water system. The molecular-weight dependence of phase change (coacervation) is included. The volume–composition studies as a function of temperature also provide temperature coefficients of expansion and of composition important in analyzing the mechanism of elasticity.     

Synthesis and evaluation of N6-substituted apioadenosines as potential adenosine A3 receptor modulators

Adenosine receptors (ARs) trigger signal transduction pathways inside the cell when activated by extracellular adenosine. Selective modulation of the A₃AR subtype may be beneficial in controlling diseases such as colorectal cancer and rheumatoid arthritis. Here, we report the synthesis and evaluation of β-d-apio-d-furano- and α-d-apio-l-furanoadenosines and derivatives thereof. Introduction of a 2-methoxy-5-chlorobenzyl group at N⁶ of β-d-apio-d-furanoadenosine afforded an A₃AR antagonist (10c, K_i = 0.98 μM), while a similar modification of an α-d-apio-l-furanoadenosine gave rise to a partial agonist (11c, K_i = 3.07 μM). The structural basis for this difference was examined by docking to an A₃AR model; the antagonist lacked a crucial interaction with Thr94.     

Extended N6 substitution of rigid C2-arylethynyl nucleosides for exploring the role of extracellular loops in ligand recognition at the A3 adenosine receptor

2-Arylethynyl-(N)-methanocarba adenosine 5′-methyluronamides containing rigid N⁶-(trans-2-phenylcyclopropyl) and 2-phenylethynyl groups were synthesized as agonists for probing structural features of the A₃ adenosine receptor (AR). Radioligand binding confirmed A₃AR selectivity and N⁶-1S,2R stereoselectivity for one diastereomeric pair. The environment of receptor-bound, conformationally constrained N⁶ groups was explored by docking to an A₃AR homology model, indicating specific hydrophobic interactions with the second extracellular loop able to modulate the affinity profile. 2-Pyridylethynyl derivative 18 was administered orally in mice to reduce chronic neuropathic pain in the chronic constriction injury model.     

Peptide substrates for G protein-coupled receptor kinase 2

G protein-coupled receptor kinases (GRKs) control the signaling and activation of G protein-coupled receptors through phosphorylation. In this study, consensus substrate motifs for GRK2 were identified from the sequences of GRK2 protein substrates, and 17 candidate peptides were synthesized to identify peptide substrates with high affinity for GRK2. GRK2 appears to require an acidic amino acid at the −2, −3, or −4 positions and its consensus phosphorylation site motifs were identified as (D/E)X_1–3(S/T), (D/E)X_1–3(S/T)(D/E), or (D/E)X_0–2(D/E)(S/T). Among the 17 peptide substrates examined, a 13-amino-acid peptide fragment of β-tubulin (DEMEFTEAESNMN) showed the highest affinity for GRK2 (K_m, 33.9 μM; V_max, 0.35 pmol min⁻¹ mg⁻¹), but very low affinity for GRK5. This peptide may be a useful tool for investigating cellular signaling pathways regulated by GRK2.     

Design,synthesis and evaluation of [3H]PF-7191, a highly specific nociceptin opioid peptide (NOP) receptor radiotracer for in vivo receptor occupancy (RO) studies

Herein we report the identification of (+)-N-(2-((1H-pyrazol-1-yl)methyl)-3-((1R,3r,5S)-6′-fluoro-8-azaspiro[bicyclo[3.2.1]octane-3,1′-isochroman]-8-yl)propyl)-N-[³H]-methylacetamide {[³H]PF-7191 [(+)-11]} as a promising radiotracer for the nociceptin opioid peptide (NOP) receptor. (+)-11 demonstrated high NOP binding affinity (K_i = 0.1 nM), excellent selectivity over other opioid receptors (>1000×) and good brain permeability in rats (C_b,u/C_p,u = 0.29). Subsequent characterization of [³H](+)-11 showed a high level of specific binding and a brain bio-distribution pattern consistent with known NOP receptor expression. Furthermore, the in vivo brain binding of [³H](+)-11 in rats was inhibited by a selective NOP receptor antagonist in a dose–responsive manner. This overall favorable profile indicated that [³H](+)-11 is a robust radiotracer for pre-clinical in vivo receptor occupancy (RO) measurements and a possible substrate for carbon-11 labeling for positron emission tomography (PET) imaging in higher species.     

A chemically stable peptide agonist to neuromedin U receptor type 2

Neuromedin U (NMU) is a peptide with appetite suppressive activity and other physiological activities via activation of the NMU receptors NMUR1 and NMUR2. In 2014, we reported the first NMUR2 selective agonist, 3-cyclohexylpropionyl-Leu-Leu-Dap-Pro-Arg-Asn-NH₂ (CPN-116). However, we found that CPN-116 in phosphate buffer is unstable because of Nα-to-N^β acyl migration at the Dap residue. In this study, the chemical stability of CPN-116 was evaluated under various conditions, and it was found to be relatively stable in buffers such as HEPES and MES. We also performed a structure-activity relationship study to obtain an NMUR2-selective agonist with improved chemical stability. Consequently, CPN-219 bearing a Dab residue in place of Dap emerged as a next-generation hexapeptidic NMUR2 agonist.     

Adenosine A2A receptor blocks the A 1 receptor inhibition of renal Na + transport and oxygen consumption

A high renal oxygen (O₂) need is primarily associated with the renal tubular O₂ consumption (VO₂) necessary for a high rate of sodium (Na⁺) transport. Limited O₂ availability leads to increased levels of adenosine, which regulates the kidney via activation of both A₁ and A_2A adenosine receptors (A1R and A2AR, respectively). The relative contributions of A1R and A2AR to the regulation of renal Na⁺ transport and VO₂ have not been determined. We demonstrated that A1R activation has a dose-dependent biphasic effect on both renal Na⁺/H⁺ exchanger-3 (NHE3), a major player in Na⁺ transport, and VO₂. Here, we report concentration-dependent effects of adenosine: less than 5 × 10⁻⁷ M adenosine-stimulated NHE3 activity; between 5 × 10⁻⁷ M and 10⁻⁵ M adenosine-inhibited NHE3 activity; and greater than 10⁻⁵ M adenosine reversed the change in NHE3 activity (returned to baseline). A1R activation mediated the activation and inhibition of NHE3 activity, whereas 10⁻⁴ M adenosine had no effect on the NHE3 activity due to A2AR activation. The following occurred when A1R and A2AR were activated: (a) Blockade of the A2AR receptor restored the NHE3 inhibition mediated by A1R activation, (b) the NHE-dependent effect on VO₂ mediated by A1R activation became NHE independent, and (c) A2AR bound to A1R. In summary, A1R affects VO₂ via NHE-dependent mechanisms, whereas A2AR acts via NHE-independent mechanisms. When both A1R and A2AR are activated, the A2AR effect on NHE3 and VO₂ predominates, possibly via an A1R–A2AR protein interaction. A2AR–A1R heterodimerization is proposed as the molecular mechanism enabling the NHE-independent control of renal VO₂.     

Design,synthesis, and biological evaluation of scaffold-based tripeptidomimetic antagonists for CXC chemokine receptor 4 (CXCR4)

Structure–activity relationship studies of the cyclopentapeptide CXCR4 antagonists (cyclo(-l-/d-Arg¹-Arg²-2-Nal³-Gly⁴-d-Tyr⁵-)) suggest that the l-/d-Arg¹-Arg²-2-Nal³ tripeptide sequence contained within these cyclopentapeptides serves as a recognition motif for peptidic CXCR4 antagonists. Starting by dissecting the cyclopentapeptide structure and reintroducing cyclic constraints in a stepwise manner, we here report a novel class of scaffold-based tripeptidomimetic CXCR4 antagonists based on the d-Arg-Arg-2-Nal motif. Biological testing of the prototype compounds showed that they represent new peptidomimetic hits; importantly, the modular nature of the scaffold provides an interesting starting point for future ligand optimization.     

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity

Potent and selective adenosine A₁ receptor (A₁AR) antagonists with favourable pharmacokinetic properties used as novel diuretics and antihypertensives are desirable. Thus, we designed and synthesized a series of novel 4-alkylamino substitution-2-arylpyrazolo[4,3-c]quinolin-3-one derivatives. The aim of the present study is to characterize the biological profiles of the optimized compound, PQ-69. In vitro binding assay revealed a K_i value of 0.96 nM for PQ-69 in cloned hA₁ receptor, which was 217-fold more selective compared with hA_2A receptors and >1,000-fold selectivity for hA₁ over hA₃ receptor. The results obtained from [³⁵S]-GTPγS binding and cAMP concentration assays indicated that PQ-69 might be an A₁AR antagonist with inverse agonist activity. In addition, PQ-69 displayed highly inhibitory activities on isolated guinea pig contraction (pA2 value of 8.99) induced by an A₁AR agonist, 2-chloro-N6-cyclopentyl adenosine. Systemic administration of PQ-69 (0.03, 0.3, 3 mg/kg) increased urine flow and sodium excretion in normal rats. Furthermore, PQ-69 displayed better metabolic stability in vitro and longer terminal elimination half-life (t_1/2) in vivo compared with 1,3-dipropyl-8-cyclopentylxanthine. These findings suggest that PQ-69 exhibits potent antagonist effects on A₁AR in vitro, ex vivo and in vivo, it might be a useful research tool for investigating A₁AR function, and it could be developed as a potential therapeutic agent.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9424-5) contains supplementary material, which is available to authorized users.     

Synthesis and structure–activity relationships of pyrazolodiazepine derivatives as human P2X7 receptor antagonists

Screening of library compounds has yielded pyrazolodiazepine derivatives with P2X₇ receptor antagonist activity. To explore the structure–activity relationships (SAR) of these pyrazolodiazepines as human P2X₇ receptor antagonists, derivatives were synthesized by substitutions at positions R² and R³ of the pyrazolodiazepine skeleton. Using a 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP)-induced fluorescent ethidium uptake assay, the activities of these derivatives were tested in HEK-293 cells stably expressing human P2X₇ receptors. Moreover, the effect of these derivatives was assessed by measuring their effect on IL-1β release induced by BzATP-induced activation of differentiated THP-1 cells. A 2-phenethyl pyrazolodiazepine derivative with a 1-methyl-1H-3-indolyl group at position R² had fivefold greater activity than the derivative with a 5-isoquinolinyl at R². Moreover, a benzyl moiety at R³ had fivefold greater activity than a bicyclic moiety. The stereochemical effect at C-6 showed a preference for the (R)-isomer. Among the series of active derivatives, compound 23b, with a phenethyl group at R¹, a 3-methyl indole at R², and a benzyl at R³, exhibited activity similar to that of the positive control, KN-62, as shown by the inhibitory effects of IL-1β release.     

Radiosynthesis and evaluation of new PET ligands for peripheral cannabinoid receptor type 1 imaging

Cannabinoid receptor type 1 (CB1) is mainly expressed in the brain, as well as being expressed in functional relevant concentrations in various peripheral tissues. 1-(4-Chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea (PSNCBAM-1, 1) was developed as a potent allosteric antagonist for CB1 and its oral administration led to reductions in the appetite and body weight of rats. Several analogs of 1 (compounds 2 and 3) were recently identified through a series of structure-activity relationship studies. Herein, we report the synthesis of radiolabeled analogs of these compounds using [¹¹C]COCl₂ and an evaluation of their potential as PET ligands for CB1 imaging using in vitro and in vivo techniques. [¹¹C]2 and [¹¹C]3 were successfully synthesized in two steps using [¹¹C]COCl₂. The radiochemical yields of [¹¹C]2 and [¹¹C]3 were 17 ± 8% and 20 ± 9% (decay-corrected to the end of bombardment, based on [¹¹C]CO₂). The specific activities of [¹¹C]2 and [¹¹C]3 were 42 ± 36 and 37 ± 13 GBq/μmol, respectively. The results of an in vitro binding assay using brown adipose tissue (BAT) homogenate showed that the binding affinity of 2 for CB1 (K_D = 15.3 µM) was much higher than that of 3 (K_D = 26.0 µM). PET studies with [¹¹C]2 showed a high uptake of radioactivity in BAT, which decreased in animals pretreated with AM281 (a selective antagonist for CB1). In conclusion, [¹¹C]2 may be a useful PET ligand for imaging peripheral CB1 in BAT.