Alterations in brain Protein Kinase A activity and reversal of morphine tolerance by two fragments of native Protein Kinase A inhibitor peptide (PKI)

Two peptide fragments of native Protein Kinase A inhibitor (PKI), PKI-(6-22)-amide and PKI-(Myr-14-22)-amide, significantly reversed low-level morphine antinociceptive tolerance in mice. The inhibition of Protein Kinase A (PKA) activity by both peptide fragments was then measured in specific brain regions (thalamus, periaqueductal gray (PAG), and medulla) and in lumbar spinal cord (LSC), which in previous studies have been shown to play a role in morphine-induced analgesia. In drug naive animals, cytosolic PKA activity was greater than particulate PKA activity in each region, while cytosolic and particulate PKA activities were greater in thalamus and PAG compared to medulla and LSC. The addition of both peptides to homogenates from each region completely abolished cytosolic and particulate PKA activities in vitro. Following injection into the lateral ventricle of the brain of drug naive mice and morphine-tolerant mice, both peptides inhibited PKA activity in the cytosolic, but not the particulate fraction of LSC. In addition, cytosolic and particulate PKA activities were inhibited by both peptides in thalamus. These results demonstrate that the inhibition of PKA reverses morphine tolerance. Moreover, the inhibition of PKA activity in specific brain regions and LSC from morphine-tolerant mice by PKI analogs administered i.c.v. is evidence that PKA plays a role in morphine tolerance.     

基于HDAC抑制剂设计的多靶点抗癌药物的研究进展

肿瘤的发生和发展涉及多个信号传导通路。研究表明,多靶点抗癌药物可提高单靶点抗癌药物的治疗效果,并降低耐药性,是抗癌药物研发的重要研究方向。目前,多靶点抗癌药物的设计是其研发的主要挑战之一。组蛋白去乙酰化酶（histone deacetylases,HDACs）与肿瘤的发生密切相关,其抑制剂可以降低肿瘤细胞凋亡的阈值,具有广泛的抗肿瘤活性,并且可与多种抗肿瘤药物联合使用发挥协同作用。目前,已有2个HDAC抑制剂被美国FDA批准用于治疗皮肤T-细胞淋巴瘤,分别是Vorinostat（SAHA）和Romidepsin（FK228）,还有多个HDAC抑制剂如PXD-101（Phase II）、LBH589（Phase III）、MS-275（Phase II）等尚处于临床研究阶段。本文主要对基于HDAC抑制剂的多靶点抗癌药物的设计思路和生物活性进行综述。     

Conformational induction is the key process for activation of the AT1 receptor

It is currently unclear whether activation of the AT1 receptor by agonists involves conformational selection or induction. We evaluated the pharmacological properties of wild type and N111G CAM human AT1 receptors stably expressed in HEK293 cells. Although [Sar1]-Ang II and Ang IV were full agonists at both receptors, the potency of Ang IV was 280-fold lower at the wild type receptor. [Sar1, Ile8]-Ang II was only a full agonist at the N111G CAM AT1 receptor. [Sar1]-Ang II and [Sar1, Ile8]-Ang II displayed similar high affinity binding to both receptors. In contrast, Ang IV displayed low affinity binding to the wild type and high affinity binding to the N111G CAM AT1 receptor. Based on these observations we provide strong evidence that conformational induction is the key process for activation of the AT1 receptor. Only by the creation of CAMs can conformational selection be envisaged to take place.     

Conformational aspects of the antifibrillatory activity of procaine

The effects of procaine and foursemi-rigid conformational analogs (compounds 1, 2, 3 and 4) were tested and compared on isolated rabbit atria. Procaine and the four analogs produced positive inotropic effects at all dose levels tested.

The antifibrillatory activity of procaine and its analogs arranges in decreasing order of activity was compound 4 > 3 > 2 > 1 procaine. The antifibrillatory activity of the compounds correlated to the distance between the ring nitrogen and the ester oxygen; that is, as the N-O distance increased the concentration required to reduce the following frequency decreased. However, the compound became more toxic as the N-O distance increased. Our data do not confirm the commonly regarded direct relationship between local anesthetic activity and antifibrillatory activity of procaine. Differences in activity displayed by the isomers of procaine could reflect differences in the ability of these analogs to bind to receptors responsible for the respective actions.     

Computational Modeling of Kinase Inhibitor Selectivity

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Evaluation of a Raman Chemometric Method for Detecting Protein Structural Conformational Changes in Solution

Raman scattering shows promise as a powerful routine tool, to determine both secondary and the smaller tertiary structural changes that precede aggregation in both solutions and solids. A method was developed utilizing principal component analysis (PCA) of Raman spectra for detection of small, but meaningful, pH induced changes in tertiary protein structure linked to aggregate formation using α-lactalbumin solutions as a model. The sample preparation and spectral parameters, were optimized for a bulk Raman probe. Analysis of large regions (600–1850 cm^?1) yielded principal component (PC) scores useful for semi-quantitative comparison of protein conformation between formulations. PC loadings corresponded to specific structural peaks known to change with solution pH. PCA of circular dichroism (CD) spectra of dilute solutions yielded similar results. Sucrose is a common formulation excipient with a Raman spectrum that overlaps many protein peaks. With sucrose in the protein solution, the ability of PCA to discern protein structural changes from the Raman spectra was somewhat reduced. Analysis of a more limited spectral region (1530–1780 cm^?1) with negligible sucrose spectral contribution improved the discrimination of protein conformational states. The new Raman method accurately distinguished differences in protein structure in concentrated solutions. The long-term goal is to explore Raman characterization as a routine monitoring tool of protein stability in both solution and solid states.     

Conformational study of the neurotensin and Substance P fragments: Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro

The conformational behaviour of the basic hydrophilic Pro-Arg-Arg-Pro and Arg-Pro-Lys-Pro peptides, neurotensin (NT) and Substance P fragments, has been taken up by semi-empirical calculations. The presence of two Pro residues prevents these peptides from giving any folded structure (α helix, β turn.). In both peptides the most stable conformations are essentially relative to more or less stretched structures; structures involving one or more residues in a γ turn form are often encountered in Pro-Arg-Arg-Pro peptide while mixed structures involving residues in very different conformations are found for the Arg-Pro-Lys-Pro-peptide. In both peptides, positively charged Lys and Arg side-chains most often point in opposite directions. The Pro-Arg-Arg-Pro peptide is part of the active NT (7–13) fragment where both Arg residues are necessary to the activity. A tentative study shows that the hydrophilic tetrapeptide induces NT (7–13) stretched conformations.     

Conformational analysis: a tool for the elucidation of the antioxidant properties of ferulic acid derivatives in membrane models

With the aim to search and design more effective and safe antioxidant molecules to be used as functional ingredients in cosmetic formulations for UV protection, we evaluated the antioxidant/radical scavenging activities of ferulic acid and of some alkyl ferulates in both acellular and cellular systems. Ferulic acid esters, equipotent as antioxidant in homogeneous phase, showed when tested in membranous systems (rat liver microsomes, rat erythrocytes) marked differences in antioxidant potency. The n-C₁₂ derivative was the most potent, followed by n-C₈, n-C₁₆ and branched C₈, and then by ferulic acid.

A conformational study carried out by NMR and modelling, indicates that the different antioxidant activity of ferulates in membrane models is due to the different spatial conformation and arrangement of the side chain of the molecule, which governs the access and binding to the phospholipid bilayer, the modality of orientation of the scavenging/quenching nucleus (phenol moiety), and hence the overall antioxidant potency of the derivative.

These results emphasize the need of analytical studies (NMR and molecular modelling) addressed to the knowledge of the conformational parameters in combination with conventional antioxidant testings for understanding the antioxidant behaviour of a molecule in a biological membrane/system.     

Conformational stability of the endothelin/sarafotoxin family of peptides

There are significant differences between the structures reported for members of the endothelin/sarafotoxin family of peptides, but also for the same peptides studied by different groups, raising the possibility that some of the differences are attributable to variation in solution conditions rather than intrinsic structural heterogeneity. We have shown, using circular dichroism spectroscopy and equilibrium sedimentation, that the secondary structures of these peptides are little affected by wide variations in pH, or by self-association. Although acetonitrile has a pronounced effect on the extent of peptide self-association it does not appear to alter the backbone structure of sarafotoxin SRTb, and has only minor effects on endothelin-1 andendothelin-3. The observed conformational variation thus appears largely to reflect sequence-dependent differences. © Munksgaard 1994.     

Protective Mechanism of Stabilizing Excipients Against Dehydration in the Freeze-Drying of Proteins

Purpose. To investigate the influence of type and amount of excipient on the preservation of the native structure and the biologic activity of freeze-dried lysozyme and catalase. Methods. The secondary structure of protein in the dried form and in aqueous solution was obtained using second derivative infrared spectroscopy and circular dichroism spectra respectively whilst the activity was determined using bioassay. Results. Small molecular excipients (glycerol, sorbitol, 1,6-anhydroglucose, sucrose, and trehalose) were found to stabilize the activity and/or the native structure of freeze-dried lysozyme and catalase, despite the processing temperatures being above Tg of excipient-protein mixtures. The preservation of catalase activity required excipient to be present at a lower excipient to enzyme mass ratio than that necessary to preserve native structure in the dried form. Combining dextran with sucrose synergistically protected the native structure of catalase but preserved the activity in an additive manner. Conclusion. The results indicate that the stabilization of catalase and lysozyme by excipients during dehydration was mainly due to water substitution rather than the formation of glass; the latter appearing not to be a prerequisite during freeze-drying.     

Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptors

Models of μ- and δ-receptor-bound backbone conformations of enkephalin cyclic analogues containing Phe⁴ were determined by comparing geometrical similarity among the previously found low-energy, backbone structures of -enkephalinamide, -enkephalinamide, -enkephalin and -enkephalin. The present μ-receptor-bound conformation resembles a β-I bend in the peptide backbone centred on the Gly³-Phe⁴ region. Two slightly different models were found for the δ-receptor-bound conformation; both of them are more extended than the μ-receptor-bound conformation and include a γ-turn (or a γ-like turn) on the Gly³ residue. Energetically favourable rotamers of Tyr and Phc side chains were also determined for the μ- and δ-conformations. The present models of μ- and δ-conformations share geometrical similarity with the low-energy structures of Leu-enkephalin and the analogue.     

Conformational study of cyclosporin A in acetone at low temperature

The conformation of cyclosporin A (CsA), an undecapeptide with seven N-methylated amino acids, was studied in acetone at 193 K. Previous studies of the conformation of CsA in different solvents, in the cyclosporin-cyclophilin complex and in complexes with LiCl showed that the conformation of the free and the bound CsA are different. Differences were observed at the conformation of the MeLeu⁹-MeLeu¹⁰ peptide bond, which is cis in solution and trans in the complex, and in the orientation of the amide protons and the N-Me groups. By using acetone, which is a proton acceptor, we wanted to influence the orientation of the amide protons. In the conditions used in this study a new conformation is found, which differs as well from the one previously observed in solution as from the Conformation observed in the complex. This conformation has a cis peptide bond between MeLeu⁹ and MeLeu¹⁰. The trans conformation of the peptide bond MeLeu⁹-MeLeu¹⁰, which is necessary for biological activity, was not induced. One of the amide protons is involved in an intramolecular H-bridge stabilising a β-turn around Sar³MeLeu⁴, and three of the seven NMe groups are oriented to the centre of the molecule. © Munksgaard 1994.     

Probing the Conformational Dynamics of the Bioactive Peptide TLQP‐21 in Solution: A Molecular Dynamics Study

VGF‐derived peptide, TLQP‐21, is a physiologically active neuropeptide exhibiting important roles in energy expenditure and balance, gastric contractility, reproduction, pain modulation, and stress. Although the physiological functions of the peptide constitute a research area of considerable interest, structural information is clearly lacking. Here, using extensive 550 nanoseconds molecular dynamics simulation in explicit water model, we have explored the folding energy landscape of the peptide. Principal component analysis and cluster analysis have been used to identify highly populated conformational states of the peptide in solution. The most populated structure of the peptide adopts a highly compact globular form stabilized by several hydrogen‐bonding interactions and π‐cationic interactions. Strong surface complementarity of hydrophobic residues allows tighter spatial fit of the residues within the core region of the peptide. Our simulation also predicts that the peptide is highly flexible in solution and that the region A₇‐R₉ and three C‐terminal residues, P₁₉‐R₂₁, possess strong helical propensity.     

Conformational changes of bovine plasma albumin prior to the salting-out of the protein in concentrated salt solution

By working at very low protein concentration (ca. 0.003%), it is possible to measure tryptophyl fluorescence intensity at 350nm (F₃₅₀) of bovine plasma albumin (BPA) as a function of pH under precipitating conditions (acidic concentrated salt solutions). Under such conditions, distinct changes in F₃₅₀ were seen before the starting of precipitation of BPA and no further changes in F₃₅₀ over the precipitating pH range. Comparison of pH-profiles monitored by F₃₅₀ with those by solubility in the presence of various salts at various concentrations indicated that the change of solubility is observed after definite changes in conformation of the protein.     

激酶变构抑制剂: 一种新的高效和高选择性调节激酶活性的方式(英文)

研究证明, 激酶活性失控是导致癌症的原因之一, 癌细胞存活和增殖与相应激酶活性的失控密切相关。选择性的抑制激酶活性已经成为开发安全有效的抗癌药的重点研究领域。到目前为止, 已有15种激酶抑制剂得到美国食品药品监督管理局的批准用于治疗多种癌症。其中, 激酶的变构抑制剂在临床上显示出更好的安全性和有效性。本文将总结这些激酶的变构构象、它们对应的抑制剂及其作用方式。     

Conformational influences of N2-methylation in chemotactic peptides

Physical and structural data on two of a series of related N-formyl methionine peptides are reported. The effects of peptide bond N-methylation on chemotactic response and on conformational properties observed in solution, in the solid state and by conformational energy calculations are described.     

人血清白蛋白与硫代寡核苷酸的结合研究

目的研究二价阳离子对白蛋白与硫代寡核苷酸结合的影响以及结合硫代寡核苷酸后白蛋白构象的改变。方法应用表面等离子体共振、圆二色散以及荧光光谱法对白蛋白与硫代寡核苷酸的结合行为进行了表征。结果（1）随着pH值升高,结合能力降低;（2）锌离子能显著促进白蛋白与硫代寡核苷酸的结合, 镍离子也促进结合,但与锌离子相比作用稍弱;（3）与硫代寡核苷酸结合后,白蛋白结构发生变化,产生β-折叠构造。结论通过实验方法证明硫代寡核苷酸结合于白蛋白的正电荷密集区;表明白蛋白可能介导硫代寡核苷酸的胞吞行为。     

Conformational studies of N-Tyr-MIF-1 in aqueous solution by 1H nuclear magnetic resonance spectroscopy

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH₂) is an endogenous brain peptide with multiple effects on animal behavior. However, there have been no studies on the conformation of this tetrapeptide. In this report, we studied the conformation of N-Tyr-MIF-1 in aqueous solution by conventional one-dimensional and two-dimensional (COSY and NOESY) ¹H nuclear magnetic resonance spectroscopy at 300 MHz. A complete set of assignments for the resolved resonances and approximate assignments for the overlapping resonances were made. The results demonstrate that N-Tyr-MIF-1 is in slow exchange between two conformers, most likely determined by the cis and trans states of the proline residue. The minor conformation represents 30 ± 3% of the population over the temperature range from 3° to 73°. In the major conformation, the tyrosine aromatic ring appears to be close enough to interact directly with the proline pyrrolidine ring, as indicated by a strong temperature dependence of the proline CβH, CδH and CδH′ chemical shifts. In contrast, this interaction of the tyrosine and proline rings is not present in the minor conformation.