cADPR解离过程的拉伸分子动力学模拟

目的 研究cADPR从CD38活性口袋解离的过程中配体和受体间的相互作用,并寻找接近真实条件的蛋白结构模型.方法 采用拉伸分子动力学的方法模拟cADPR从CD38活性口袋解离的过程.在cGDPR和CD38晶体复合物基础上进行修改,得到cADPR和CD38的复合物;再用动力学优化得到平衡稳定的cAD-PR和CD38复合物...     

Discovery of 2‐(3,4‐dialkoxyphenyl)‐2‐(substituted pyridazin‐3‐yl)acetonitriles as phosphodiesterase 4 inhibitors with anti‐neuroinflammation potential based on three‐dimensional quantitative structure–activity relationship study

Phosphodiesterase 4 (PDE4) inhibitors with potential activities for CNS disorders provide a new therapeutic strategy for depression. To discover PDE4 inhibitors with anti‐neuroinflammation activities, reliable three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) models on our previous reported catecholic PDE4 inhibitors was built with a statistically significant cross‐validated coefficient (q²), conventional coefficient (r²), and good predictive capabilities based on the molecular docking results, using comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods. Based on the analysis of CoMFA and CoMSIA contour maps, a series of 2‐(3,4‐dialkoxyphenyl)‐2‐(substituted pyridazin‐3‐yl) acetonitriles 16a–i was designed and synthesized. Among these compounds, compound 16a exhibited good inhibitory activities toward PDE4B1 and PDE4D7 with mid‐nanomolar IC₅₀ values and potential anti‐neuroinflammation activity in BV‐2 cells. Docking simulation of compound 16a in the PDE4 catalytic domain activity pocket revealed that compound 16a maybe assumed a “V‐shaped” conformation, extending the side chain to S‐pocket.     

A facile automated synthesis of N‐succinimidyl 4‐[18F]fluorobenzoate ([18F]SFB) for 18F‐labeled cell‐penetrating peptide as PET tracer

A fully automated synthesis of N‐succinimidyl 4‐[¹⁸F]fluorobenzoate ([¹⁸F]SFB) was carried out by a convenient three‐step, one‐pot procedure on the modified TRACERlab FX_FN synthesizer, including [¹⁸F]fluorination of ethyl 4‐(trimethylammonium triflate)benzoate as the precursor, saponification of the ethyl 4‐[¹⁸F]fluorobenzoate with aqueous tetrapropylammonium hydroxide instead of sodium hydroxide, and conversion of 4‐[¹⁸F]fluorobenzoate salt ([¹⁸F]FBA) to [¹⁸F]SFB treated with N,N,N′,N′‐tetramethyl‐O‐(N‐succinimidyl)uranium tetrafluoroborate (TSTU). The purified [¹⁸F]SFB was used for the labeling of Tat membrane‐penetrating peptide (containing the Arg‐Lys‐Lys‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Pro‐Leu‐Gly‐Leu‐Ala‐Gly‐Glu‐Glu‐Glu‐Glu‐Glu‐Glu‐Glu sequence, [¹⁸F]CPP) through radiofluorination of lysine amino groups. The uncorrected radiochemical yields of [¹⁸F]SFB were as high as 25–35% (based on [¹⁸F]fluoride) (n=10) with a synthesis time of～40 min. [¹⁸F]CPP was produced in an uncorrected radiochemical yields of 10–20% (n=5) within 30 min (based on [¹⁸F]SFB). The radiochemical purities of [¹⁸F]SFB and [¹⁸F]CPP were greater than 95%. Copyright © 2010 John Wiley & Sons, Ltd.     

Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the κ‐opioid receptor

Abstract: A series of cyclic, disulfide‐ or dithioether‐containing tetrapeptides based on previously reported potent μ‐ and δ‐selective analogs has been explored with the aim of improving their poor affinity to the κ‐opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe³ replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo‐Arg), or aromatic sides chains (Trp, Tyr, p‐NH₂Phe), and with d ‐Pen⁴ replaced by d ‐Cys⁴, and binding affinities to stably expressed μ‐, δ‐, and κ‐receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the κ‐receptor, with the exception of the tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Cys]‐NH₂, cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Ki^μ = 1.26 nm , Ki^δ = 16.1 nm , Ki^κ = 38.7 nm ). Modeling of the κ‐receptor/ligand complex in the active state reveals that the receptor‐binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the μ‐receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d ‐Pen or d ‐Cys) and correlates well with the observed κ‐receptor binding affinity.     

Ensemble‐based ADME–Tox profiling and virtual screening for the discovery of new inhibitors of the Leishmania mexicana cysteine protease CPB2.8ΔCTE

In an effort to identify novel molecular warheads able to inhibit Leishmania mexicana cysteine protease CPB2.8ΔCTE, fused benzo[b]thiophenes and β,βʹ‐triketones emerged as covalent inhibitors binding the active site cysteine residue. Enzymatic screening showed a moderate‐to‐excellent activity (12%–90% inhibition of the target enzyme at 20 μm ). The most promising compounds were selected for further profiling including in vitro cell‐based assays and docking studies. Computational data suggest that benzo[b]thiophenes act immediately as non‐covalent inhibitors and then as irreversible covalent inhibitors, whereas a reversible covalent mechanism emerged for the 1,3,3ʹ‐triketones with a Y‐topology. Based on the predicted physicochemical and ADME–Tox properties, compound 2b has been identified as a new drug‐like, non‐mutagen, non‐carcinogen, and non‐neurotoxic lead candidate.     

Molecular basis for covalent inhibition of glyceraldehyde‐3‐phosphate dehydrogenase by a 2‐phenoxy‐1,4‐naphthoquinone small molecule

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2‐phenoxy‐1,4‐naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2‐phenoxy‐1,4‐naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2‐phenoxy‐1,4‐naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non‐covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2‐phenoxy‐1,4‐naphthoquinone displays selectivity for GAPDHs over two other cysteine‐dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.     

The effect of a side chain‐backbone swap on protein stability

Abstract: To assess the relative importance of backbone hydrogen bonding (H‐bonding) vs. side chain hydrophobicity in protein structural formation, a method called side chain‐backbone swap is proposed. Such a method swaps the side chain and backbone portions of certain amino acid residues, such as Asp, Glu, Asn, Gln, Lys, and Arg. Such a swap retains the sequence of a polypeptide and preserves the identity of the backbone linkage. On the other hand, the swap disrupts backbone H‐bonding geometry because of the introduction of extra methylene groups into the peptide backbone. In this project, we chose the two‐stranded α‐helical coiled‐coil to implement side chain‐backbone swap. A pair of 36‐residue peptides was designed. The two peptides have identical sequence with four residues in each heptad repeat occupied by glutamyl residues. Each glutamic acid was incorporated either as α‐glutamyl residue (the peptide is denoted as α‐Glu‐36) or as γ‐glutamyl residue (the peptide is denoted as γ‐Glu‐36). The inter‐conversion between the two peptides constitutes a side chain‐backbone swap. Residues constituting the hydrophobic core of the coiled‐coil, however, are left unchanged. The peptide pair was characterized by circular dichroism spectroscopy, reversed‐phase liquid chromatography (RPLC), and two‐dimensional nuclear magnetic resonance (NMR). The results indicate that α‐Glu‐36 is a two‐stranded α‐helical coiled‐coil while γ‐Glu‐36 lacks stable structural elements. It is concluded that, at least for coiled‐coils where hydrophobic interactions are predominantly long‐range, local backbone H‐bonding is a required for structural formation, consistent with a hierarchic folding mechanism. The methodological implication of side chain‐backbone swap is also discussed.     

Synthesis and Anti‐neuroinflammatory Activity of Lactone Benzoyl Hydrazine and 2‐nitro‐1‐phenyl‐1H‐Indole Derivatives as p38α MAPK Inhibitors

Inhibition of p38 mitogen‐activated protein kinases (MAPKs) would allow significant modulation of the neuroinflammation condition associated with Alzheimer's disease (AD). Inspired from the pharmacophore of natural NF‐κB and p38α MAPK inhibitor 5,6‐dehydrokawain and p38α MAPK inhibitors 1a, 1‐pyrazolyl‐3‐(4‐((2‐anilinopyrimidin‐4‐yl)oxy)napththalen‐1‐yl)ureas, and 1b , a class of indole–pyrimidinyl compounds which were patented respectively, we designed, de novo synthesized, and evaluated two kinds of novel series of lactone benzoyl hydrazine derivatives and 2‐nitro‐1‐phenyl‐1H‐indole derivatives in an effort to develop pharmacologically tractable agents to alleviate the progression of AD. Fourteen of the seventeen synthesized compounds exhibit significant inhibitory effect on the nitric oxide (NO) production induced by lipopolysaccharide (LPS)‐induced microglia activation with IC₅₀ less than the control 5,6‐dehydrokawain. Notably, compound 27 , 6‐methoxy‐2‐nitro‐1‐(1H‐1, 2, 3‐triazol‐1‐yl)‐1H‐indole, with IC₅₀ values of 1.6  μ m can markedly inhibit p38 α MAPK and NO release in BV‐2 microglial cells. The molecular dynamic (MD) simulations demonstrate that compound 27 inhibits p38 α MAPK through binding to the Glu71 and Asp168 residues. Moreover, in vitro study shows that all compounds can easily cross the blood–brain barrier (BBB) and did not exhibit any acute cellular toxicity checked by MTT assay. These investigations provide promising chemical lead candidate as anti‐neuroinflammatory agents for AD.     

Combined 3D‐QSAR Modeling and Molecular Docking Study on Quinoline Derivatives as Inhibitors of P‐selectin

P‐selectin is a promising target for developing novel atherosclerosis drugs. To understand the structure–activity correlation of quinolines‐based P‐selectin inhibitors, we have carried out a combined molecular docking and three‐dimensional quantitative structure–activity relationship (3D‐QSAR) modeling study. The study has resulted in two types of satisfactory 3D‐QSAR models, including the CoMFA model (r², 0.863; q², 0.589) and CoMSIA model (r², 0.866; q², 0.636), to predict the biological activity of new compounds. The detailed microscopic structures of P‐selectin binding with inhibitors have been studied by molecular docking. We have also developed docking based 3D‐QSAR models (CoMFA with r², 0.934; q², 0.591; CoMSIA with r², 0.896; q², 0.573). The contour maps obtained from the 3D‐QSAR models in combination with the docked binding structures help to better interpret the structure–activity relationship. All of the structural insights obtained from both the 3D‐QSAR contour maps and molecular docking are consistent with the available experimental activity data. The satisfactory results strongly suggest that the developed 3D‐QSAR models and the obtained P‐selectin‐inhibitor binding structures are reasonable for the prediction of the activity of new inhibitors and in future drug design.     

The conformational and biological analysis of a cyclic anti‐obesity peptide from the C‐terminal domain of human growth hormone

Abstract: The three‐dimensional solution structure of anti‐obesity drug (AOD), a 15‐residue, disulfide‐bonded, cyclic peptide, cyclo(6,13)‐H₂N‐Leu‐Arg‐Ile‐Val‐Gln‐Cys‐Arg‐Ser‐Val‐Glu‐Gly‐Ser‐Cys‐Gly‐Phe‐OH, derived from the C‐terminal domain of the human growth hormone (hGH) (residues 177–191) was determined using two‐dimensional ¹H NMR spectroscopy. AOD stimulates lipolysis and inhibits lipogenesis, in vitro, in rodent, porcine and human adipose tissues. These biological effects suggest that AOD is a potential therapeutic candidate for the treatment of obesity. Conformational studies of AOD were conducted in aqueous solution and in water/dimethylsulfoxide mixtures. In general, spectral quality was superior in the water/dimethylsulfoxide mixtures. The cyclic region of AOD in water/dimethylsulfoxide adopts type I β‐turns at residues Ser⁸‐Val⁹‐Glu¹⁰‐Gly¹¹ and Ser¹²‐Cys¹³‐Gly¹⁴‐Phe¹⁵, each preceded by loop‐like structures. Comparison of the conformation of this peptide with residues 177–191 in the native hGH protein X‐ray crystal structure indicates that the synthetic peptide retains some structural similarity to the intact protein. This study provides evidence that the C‐terminal region of hGH is a specific functional domain of the multifunctional hGH protein.     

噻唑类衍生物二氢乳清酸脱氢酶抑制剂的三维定量构效关系研究

目的 应用三维定量构效关系（3D-QSAR）研究噻唑类衍生物结构的二氢乳清酸脱氢酶抑制活性,为该类药物的设计和筛选提供可靠的理论依据。方法 针对38个以噻唑为基本骨架的二氢乳清酸脱氢酶抑制剂,分别应用分子力场分析（CoMFA）和比较分子相似性指数分析（CoMSIA）2种经典的方法进行了三维定量构效关系（3D-QSAR）研究,建立相关模型,验证模型的预测能力,三维等势图分析噻唑类衍生物结构与活性的关系。结果 CoMFA模型的交叉验证系数q²为0.796,相关系数r²为0.978;CoMSIA模型的q²以及r²分别为0.721和0.976;2种模型对化合物的活性预测与实际值接近;三维等势图可以全面直观的分析化合物结构对其活性的影响。结论 该3D-QSAR模型三维等势图揭示了结构特征与抑制活性的关系,模型具有较好的预测能力和较强的稳定性,为进一步开发研究打下了较好的基础。     

In vivo structure–activity studies on the dark‐color‐inducing neurohormone of locusts

In the 11‐residue long dark‐color‐inducing neurohormone (DCIN = [His⁷]‐corazonin), of locusts, from residue 2 to residue 11, one amino acid at each time was substituted by d ‐phenylalanine (d ‐Phe). The dark‐color‐inducing effect of these peptides was investigated in comparison with unaltered DCIN by a bioassay based on nymphs of a DCIN‐deficient albino mutant of the migratory locust, Locusta migratoria. Substitution of any single amino acid by d ‐Phe always reduced the activity, but did not abolish it completely. Maximum inactivation was obtained after substitution of Gln4, Ser6, or Trp9. The latter two residues are within the partial sequence ‐Ser‐Xxx‐Gly‐Trp‐ (Xxx = His in the DCIN) that seems to be important for the dark‐color‐inducing activity, as found also in another study (Insect Biochem. Mol. Biol. 32, 2002, 909). Gln4, however, is outside of this partial sequence. Minimal, although still considerable, inactivation occurred after substitution of Gly8, Phe3, or Asn11, despite the fact that Gly8 is within the ‐Ser‐Xxx‐Gly‐Trp‐ partial sequence. In conclusion, no single active core was found, indicating that the whole sequence of the DCIN is necessary to induce maximum darkening effect. No difference was found in the activity of the peptides in which Gly8 was substituted by d ‐Phe or by L ‐Phe. Therefore the ‐Ser‐Xxx‐Gly‐Trp‐ partial sequence does not seem to be stabilized by a type II β‐turn. Nevertheless, existence of another kind of turn that includes this partial sequence is feasible. A single unsuccessful attempt was made to discover an antagonist to the DCIN.     

In vivo structure–activity studies on the dark‐color‐inducing neurohormone of locusts

Abstract: In the 11‐residue long dark‐color‐inducing neurohormone (DCIN = [His⁷]‐corazonin), of locusts, from residue 2 to residue 11, one amino acid at each time was substituted by d ‐phenylalanine (d ‐Phe). The dark‐color‐inducing effect of these peptides was investigated in comparison with unaltered DCIN by a bioassay based on nymphs of a DCIN‐deficient albino mutant of the migratory locust, Locusta migratoria. Substitution of any single amino acid by d ‐Phe always reduced the activity, but did not abolish it completely. Maximum inactivation was obtained after substitution of Gln4, Ser6, or Trp9. The latter two residues are within the partial sequence ‐Ser‐Xxx‐Gly‐Trp‐ (Xxx = His in the DCIN) that seems to be important for the dark‐color‐inducing activity, as found also in another study (Insect Biochem. Mol. Biol. 32, 2002, 909). Gln4, however, is outside of this partial sequence. Minimal, although still considerable, inactivation occurred after substitution of Gly8, Phe3, or Asn11, despite the fact that Gly8 is within the ‐Ser‐Xxx‐Gly‐Trp‐ partial sequence. In conclusion, no single active core was found, indicating that the whole sequence of the DCIN is necessary to induce maximum darkening effect. No difference was found in the activity of the peptides in which Gly8 was substituted by d ‐Phe or by l ‐Phe. Therefore the ‐Ser‐Xxx‐Gly‐Trp‐ partial sequence does not seem to be stabilized by a type II β‐turn. Nevertheless, existence of another kind of turn that includes this partial sequence is feasible. A single unsuccessful attempt was made to discover an antagonist to the DCIN.     

Glutaraldehyde cross‐linking alters the environment around Trp29 of cobrotoxin and the pathway for regaining its fine structure during refolding

Abstract: Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra‐ and intermolecule cross‐linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross‐linking was accompanied by a change in the gross conformation of cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with cobrotoxin, Trp²⁹ of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross‐linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde‐modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme‐linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp²⁹ of cobrotoxin and glutaraldehyde‐modified derivatives was formed differently. These results suggest that glutaraldehyde cross‐linking leads to a change in the microenvironment of cobrotoxin Trp²⁹ and alters the pathway of its fine structure formation during the refolding of cobrotoxin.     

N‐locking stabilization of covalent helical peptides: Application to Bfl‐1 antagonists

Recently, it was reported that tetrapeptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N‐lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N‐locked covalent BH3 peptides that were designed to selectively target the anti‐apoptotic protein Bfl‐1. The resulting agents were soluble in aqueous buffer and displayed a remarkable (low nanomolar) affinity for Bfl‐1 and cellular activity. The crystal structure of the complex between such N‐locked covalent peptide and Bfl‐1 provided insights on the geometry of the N‐locking strategy and of the covalent bond between the agent and Bfl‐1.     

Structure–activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin

Abstract: Peptides derived from the third α‐helix of the homeodomain (residues 43–58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N‐ and C‐terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C‐terminal segment ⁵²Arg‐Arg‐Met‐Lys‐Trp‐Lys‐Lys⁵⁸ of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide’s basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.     

New indolicidin analogues with potent antibacterial activity*

Abstract: Indolicidin is a 13‐residue antimicrobial peptide amide, ILPWKWPWWPWRR‐NH₂, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two‐fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR‐NH₂ in which Trp‐residues 4,6,8,9,11 were replaced in all positions with X = a single non‐natural building block; N‐substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non‐natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N‐terminus with a hydrophobic non‐natural building block. We prepared 22 analogues of indolicidin and [Phe^4,6,8,9,11] indolicidin, 11 of each, carrying a hydrophobic non‐natural building block attached to the N‐terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non‐natural building blocks, are promising lead structures for developing future therapeutics.