CD在木脂素类化合物绝对构型测定中的应用

目的介绍圆二色谱(circular dichroism,CD)在木脂素类化合物绝对构型测定中的应用规律。方法在查阅国内外相关文献的基础上,对CD在芳基萘类、联苯环辛烯类、二苄基丁内酯类、双四氢呋喃类、苯并呋喃类等木脂素类化合物绝对构型测定中的应用规律进行综述。结果 CD在木脂素类化合物绝对构型测定中的应用具有一定的规律性。结论初步总结了CD在木脂素类化合物绝对构型测定中的应用规律,以期为该类化合物绝对构型测定提供有益的参考。     

Comments on the use of a dichromophoric circular dichroism assay for the identification of β-turns in peptides

Use of the dichromophoric CD assay for β-turn formation in peptide sequences has been investigated. The assay involves the observation of Cotton effects in CD spectra, originating from the approach of N- and C-terminal aromatic chromophores in tetrapeptides. The approach of the chromophores was believed to be brought about by a beta;-turn in the peptide structure. Our investigations were paralleled by NMR studies which revealed the presence of a previously unreported hydrogen bond in the β-turn conformers, which appears to play a role in the generation of the observed Cotton effects. This suggests caution in the use of the CD technique alone as an assay for β-turn conformers in peptides.     

A comparative study of cyclization strategies applied to the synthesis of head-to-tail cyclic analogs of a viral epitope

A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.     

Conformational study of vasoactive intestinal peptide by computational methods

The conformational profile of vasoactive intestinal peptide (VIP) was characterized using computational methods. The strategy devised included a close examination of the conformational profile of the first 11 residues fragment followed by a study that considered the compatibility of the different conformations found with a continuation of the polypeptide chain in a-helical conformation. Accordingly, a detailed analysis of the conformational preferences of the N-terminal fragment of VIP(1–11) was carried out within the framework of the molecular mechanics approach, using simulated annealing in an iterative fashion as the sampling technique. In a second step, low-energy structures of the fragment were fused to the remainder of the VIP chain in the form of two noninteracting α-helices, according to a model of the structure of the peptide proposed from NMR studies. After investigation for compatibility of each of the low-energy structures of VIP(1–11) with the two helical regions by energy minimization, only 5 of 35 structures were discarded. Analysis of the structures characterized indicates that most of the conformations of VIP(1–11), including the global minimum, can be described as bent conformations. Conformations exhibiting α-turns and ß-turns, previously proposed by NMR studies were also characterized. The conformational analysis also suggests that the common structural features found in VIP(1–11) should also be present in VIP. Finally, because of the sequence homology between VIP and Peptide T, and the fact that both are ligands of the CD4 receptor, both sets of low-energy conformations were compared for similarity. The relevance of these results as guidance of the design of new peptide analogs targeted to the CD4 receptor is also discussed.     

Structural study of the sodium channel inactivation gate peptide including an isoleucine–phenylalanine–methionine motif and its analogous peptide (phenylalanine/glutamine) in trifluoroethanol solutions and SDS micelles

In order to gain insight into the gating mechanisms of Na⁺ channels, in particular their inactivation mechanisms, we studied the structures of the Na⁺ channel inactivation gate related peptide which includes the IFM (Ile‐Phe‐Met) motif (Ac‐KKKFGGQDIFMTEEQKK‐NH₂; K1480–K1496 in rat brain type‐IIA Na⁺ channels, MP‐3A) and its F/Q(Gln) substituted one (MP‐4A) in trifluoroethanol (TFE) solutions and sodium dodecyl sulfate (SDS) micelles using circular dichroism (CD) and ¹H‐NMR spectroscopies. Based on observed nuclear Overhauser effect constraints, three‐dimensional structures of MP‐3A and MP‐4A were determined using simulated annealing molecular dynamics/energy minimization calculations. In TFE solutions, no appreciable differences in the structure were observed using either CD or NMR spectra. In SDS micelles, however, the two peptides exhibited definitely different structures from each other. It was found that in MP‐3A, residues I1488 and T1491 were spatially proximate with each other owing to hydrogen bonding between the amide proton of I1488 and the hydroxyl oxygen atom of T1491, whereas in MP‐4A, F/Q substitution separated them owing to conformational changes. The solvent‐accessible surfaces calculated for the structures of MP‐3A and MP‐4A showed that the former has a smoother interaction surface to the hydrophobic docking site than the latter. In conclusion, the conformational changes, as well as decreased hydrophobicity around the IFM motif owing to the F/Q mutation, may be one reason why F1489Q mutated channels cannot inactivate almost completely.     

Identification of a new cell-penetrating peptide derived from the african swine fever virus CD2v protein

The African swine fever virus (ASFV) is a huge and complex DNA virus that can lead to the acute death of pigs and cause huge losses to the global swine industry. The CD2v protein is a transmembrane protein encoded by the ASFV’s EP402R gene, which can effectively inhibit the bystander lymphocyte proliferation in response to mitogens and mediate the absorption of red blood cells to ASFV-infected cells. The CD2v protein contains repetitive amino acid sequences ([KPCPPP]₃ labeled as RAAS), which is reported as a genetic marker and an epitope. However, the specific biological function of the RAAS is unknown. Here, we have found that the truncated CD2v protein with RAAS can enter Chinese hamster ovary cells, but the truncated CD2v protein without RAAS cannot enter the cells. Also, the RAAS can carry the macromolecular protein EGFP to enter various cells through multiple endocytic processes that are dependent on time, concentration, and location. Besides, the RAAS enter the cells via the macropinocytosis or the clathrin-mediated endocytosis. These results indicate that the RAAS can function as a cell-penetrating peptide that provides a new insight for ASFV research and has potential application value as a tool for drug delivery.     

A solid‐phase synthetic strategy for the preparation of peptide‐based affinity labels: synthesis of dynorphin A analogs

Abstract: Solid‐phase synthetic methodology was developed for the preparation of peptide‐based affinity labels. The initial peptides synthesized were dynorphin A (Dyn A) analogs [Phe(p‐X)⁴,d ‐Pro¹⁰]Dyn A(1–11)NH₂ containing isothiocyanate (X = –N=C=S) and bromoacetamide (X = –NHCOCH₂Br) groups. The peptides were assembled on solid supports using Fmoc‐protected amino acids, and the side chain amine to be functionalized, Phe(p‐NH₂), was protected by the Alloc (allyloxycarbonyl) group. Following removal of the Alloc group by palladium(0), the reactive isothiocyanate and bromoacetamide functionalities were successfully introduced while the peptides were still attached to the resin. Synthesis of these peptides was carried out on polystyrene (PS) and polyethylene glycol–polystyrene (PEG–PS) resins containing the PAL [peptide amide linker, 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid] linker. Both the rate of Alloc deprotection and the purity of the crude affinity‐labeled peptides obtained were found to be dependent on the resin used for peptide assembly.     

A study of critical functionality-related characteristics of HPMC for sustained-release tablets

Abstract

The drug release profile from hydrophilic matrix tablets can be crucially affected by the variability of physicochemical properties of the controlled release agent. This study investigates and seeks to understand the functionality-related characteristics (FRCs) of hydroxypropyl methylcellulose (HPMC) type 2208, K4M grade, that influence the release rate of the model drug carvedilol from hydrophilic matrix tablets during the entire dissolution profile. The following FRCs were examined: particle size distribution, degree of substitution, and viscosity. Eight different HPMC samples were used to create a suitable design space. Multiple linear regression (MLR) and partial least squares regression (PLSR) analyses were used to create models for each time point. The PLSR results show that the first part of the drug release profiles is mainly regulated by the HPMC particle size. Apparent viscosity and hydroxypropoxy content (%HP) become important in later stages of the drug release profile, when the influence of particle size distribution decreases. These findings make it possible to better understand the importance of FRCs. Larger HPMC particles increase drug release in the first part of the drug release profile, whereas decreased apparent viscosity and a higher degree of %HP increase the drug release rate in the later part of the drug release profile.     

The Structural and Functional Role of the B‐chain C‐terminal Arginine in the Relaxin‐3 Peptide Antagonist,R3(BΔ23‐27)R/I5

Relaxin‐3, a member of the insulin superfamily, is involved in regulating stress and feeding behavior. It is highly expressed in the brain and is the endogenous ligand for the receptor RXFP3. As relaxin‐3 also interacts with the relaxin receptor RXFP1, selective agonists and antagonists are crucial for studying the physiological function(s) of the relaxin‐3/RXFP3 pair. The analog R3(BΔ23‐27)R/I5, in which a C‐terminally truncated human relaxin‐3 (H3) B‐chain is combined with the INSL5 A‐chain, is a potent selective RXFP3 antagonist and has an Arg residue remaining on the B‐chain C‐terminus as a consequence of the recombinant protein production process. To investigate the role of this residue in the RXFP3 receptor binding and activation, the analogs R3(BΔ23‐27)R/I5 and R3(BΔ23‐27)R containing the B‐chain C‐terminal Arg as well as R3(BΔ23‐27)/I5 and R3(BΔ23‐27), both lacking the Arg, were chemically assembled and their secondary structure and receptor activity assessed. The peptides generally had a similar conformation but those with the extra Arg residue displayed a significantly increased affinity for the RXFP3. Interestingly, in contrast to R3(BΔ23‐27)R and R3(BΔ23‐27)R/I5, the peptide R3(BΔ23‐27) is a weak agonist. This suggests that the C‐terminal Arg, although increasing the affinity, alters the manner in which the peptide binds to the receptor and thereby prevents activation, giving R3(BΔ23‐27)R/I5 its potent antagonistic activity.     

A pangenotypic,single tablet regimen of sofosbuvir/velpatasvir for the treatment of chronic hepatitis C infection

Introduction: Hepatitis C virus (HCV) infects nearly 170 million people worldwide and is a leading cause of progressive liver damage, cirrhosis, and hepatocellular carcinoma. Curative therapies have historically relied on interferon-based treatments and were limited by significant toxicity and poor response rates, particularly among patients with prior treatment failure and advanced hepatic fibrosis. The recent advent of direct acting antiviral (DAA) agents which target key steps in the HCV viral life cycle has transformed the landscape of HCV treatment by offering highly effective and well tolerated interferon-free treatments. However, current therapies are genotype-specific and have variable efficacy amongst less prevalent HCV variants.

Areas covered: This review covers the preclinical and clinical development of sofosbuvir/velpatasvir (SOF/VEL), an interferon-free, once daily, pangenotypic treatment for the treatment of chronic hepatitis C virus (HCV) infection. All relevant literature from 2014 through September of 2016 is included.

Expert opinion: SOF/VEL offers the promise of a single tablet, interferon- and ribavirin-free treatment that has extremely high efficacy in persons with chronic HCV infection regardless of genotype, subtype, treatment history or fibrosis status. It is expected to play a major role on a global scale in the therapeutic armamentarium against this ubiquitous threat to human health.     

人乳头瘤病毒基因型检测在宫颈病变中的应用

目的研究HPV的基因亚型在不同程度的宫颈病变中的分布情况,以探讨不同基因型HPV感染的临床意义。方法采用HPV分型基因检测系统对妇科门诊就诊的1324例妇女进行宫颈细胞HPVDNA检测,对371例HPV感染的患者进行宫颈薄层液基细胞学检测(TCT)及阴道镜下宫颈多点活检,根据细胞和病理学诊断将其分为宫颈癌与高度鳞状上皮内瘤变(HSIL)组、低度鳞状上皮内瘤变(LSIL)组以及正常或炎症组,分析各组内HPV亚型的分布。结果HPV与宫颈癌变密切相关,随宫颈病变程度加重,高危型HPV基因型阳性率增加,且双重和多重感染有增加趋势;HPV-16、58、33、18、52和31在宫颈癌和HSIL组的感染率为59.8%、15.2%、12.2%、9.7%、9.1%和8.5%,说明这些亚型致癌型较强。结论HPV基因型的检测对预测宫颈病变进展、评估预后及指导治疗有重要意义。     

The Responsiveness of Bee Venom Phospholipase A2 on Regulatory T Cells Correlates with the CD11c+CD206+Population in Human Peripheral Blood Mononuclear Cells

Bee venom phospholipase A2 (bvPLA2) has been reported to have therapeutic effects such as neuroprotection, anti-inflammation, anti-nociception, anti-cancer properties, caused by increasing regulatory T cells (Tregs). The mechanism of Tregs modulation by bvPLA2 has been demonstrated by binding with the mannose receptor, CD206 in experimental models of several diseases. However, it remains unknown whether this mechanism can also be applied in human blood. In this study, we collected peripheral blood samples from healthy donors and analyzed the percentages of monocyte-derived dendritic cells with CD206 (CD206⁺ DCs) before expansion, the proportion of Tregs, and the subpopulations after expansion treated with bvPLA2 or PBS using flow cytometry and the correlations among them. The percentage of Tregs tended to be higher in the bvPLA2 group than in the control group. There were significant positive correlations between the CD206 population in hPBMC and the proportions of Tregs treated with bvPLA2, especially in the Treg fold change comparing the increase ratio of Tregs in bvPLA2 and in PBS. These findings indicate that bvPLA2 increased the proportion of Tregs in healthy human peripheral blood and the number of CD206⁺ DCs could be a predictor of the bvPLA2 response of different individuals.     

Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13) — A novel approach for the treatment of inflammatory bowel disease

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-β1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD).The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])₂]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p < 0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p < 0.0001). TGF-β mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals.In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.     

Chlorophyll a, an active anti-proliferative compound of Ludwigia octovalvis, activates the CD95 (APO-1/CD95) system and AMPK pathway in 3T3-L1 cells

Ludwigia octovalvis is an aquatic plant widely distributed in Taiwan. It is traditionally used as a diuretic and is consumed as health drink. In this study, we evaluated the anti-proliferative activity of extracts and active constituent (chlorophyll a; CHL-a) of L. octovalvis in 3T3-L1 adipocytes; its mode of action on apoptosis was also investigated. Results showed that, among the different extracts and fractions, the ethylacetate layer (EAL) possessed the most potent anti-proliferative activity. Activity guided fractionation of the EAL obtained the bioactive constituent CHL-a (IC₅₀: 24.10 ± 0.83 nM). At concentrations 5–30 nM, CHL-a exhibited a dose-dependent accumulation of the Sub-G1 peak and caused cell cycle arrest at the G0/G1 phase. At 30 nM, it significantly reduced the cell viability, induced the appearance of DNA fragments, and enhanced the activation of caspase-3. Western blot data revealed that CHL-a decreased the level of Bcl-2, and increased the expression of CD95 (APO-1/CD95) and Bax. Furthermore, CHL-a up-regulated the AMPK and p-AMPK levels, and down-regulated the expression of PPAR-γ. These results conclude that CHL-a possesses potent anti-proliferative activity, and its apoptotic effects on 3T3-L1 adipocytes are mediated through the activation of CD95 (APO-1/CD95) system and the AMPK signaling pathway.     

let-7c-5p通过靶向 CD74基因在糖尿病视网膜病变中的致病作用

目的探索微 RNA(miRNA)let-7c-5p通过靶向 Ⅱ型穿膜蛋白 CD74基因在糖尿病视网膜病变( DR)中的作用机制。方法 2021年 8月至 2022年 3月，基于基因表达综合数据库( GEO)数据集中 43例正常对照和 80例糖尿病视网膜病变病人的 miRNA表达谱数据，通过加权基因共表达网络分析( WGCNA)获得 miRNA和核心基因，通过实时荧光定量逆转录聚合酶链反应( qRT-PCR)和双萤光素酶实验验证其调节机制。结果生物信息学分析显示 miRNA let-7c-5p与 CD74均是与 DR进展相关的核心基因。与正常培养的人类视网膜细胞( 1.01±0.02，1.00±0.01)相比，高糖处理人类视网膜细胞会显著下调 let-7c-5p的水(0.46±0.08，P<0.001)和显著上调 CD74的水平( 3.62±0.13，P<0.001)；且高糖处理的人类视网膜细胞 let-7c-5p与 CD74表达呈负相关关系( r=-0.99，P=0.012)。在 ENCORI数据库中预测到 let-7c-5p与 CD74的结合位点，且 let-7c-5p模拟物降低了细胞中 CD74-WT的相对萤光素酶活性(1.00±0.01比 0.43±0.06，P<0.001)。结论 let-7c-5p通过靶向抑制 CD74表达的减弱促进了 DR进展。现平     

Impact of HEC72702 chirality on the selective inhibition of hepatitis B virus capsid dimer: A dynamics–structure–energetics perspective

Chirality in drug design has been attracting wide interests and attention over the years based on its innate potentials of enhancing the selectivity and prowess of therapeutic molecules. This approach was fundamental to the recent design of two inhibitors, where (R,R)‐HEC72702 exhibited higher potency inhibition against hepatitis B virus capsid (HBVC) than (R,S)‐HEC72702. Nevertheless, the detailed molecular mechanism has remained unresolved. Here, we apply multiple computational approaches to explore, validate, and differentiate the binding modes of (R,R) and (R,S) ‐ HEC72702 and to explain the systematic roles mediated by chirality on the distinctive inhibition of HBVC dimer (HBVCd). Our findings revealed that chirality change from R,S to R,R engenders variations in the position of the propanoic acid group of HEC72702 toward the α5′ and C‐TER′ region of HBVCd chain B which could explain the higher inhibitory affinity of (R,R) ‐ HEC72702. Estimated binding free energies revealed a good correlation with bioactivity data. Moreover, analysis of energy decomposition revealed the prominent effects of van der Waals interactions in the binding process of both compounds to HBVCd. Furthermore, hierarchical clustering of residue‐based energetic contributions suggested two hot‐spot residues W125´ and F156´ play crucial roles in the systematic motions of the propanoic acid group toward chain B.     

ABT-450/ritonavir and ABT-267 in combination with ABT-333 for the treatment of hepatitis C virus

Introduction: The global prevalence of chronic hepatitis C virus (HCV) is estimated to be 80 – 115 million and currently viremic infections account for 350,000 deaths annually. As the knowledge about HCV evolves, new anti-viral treatments have been developed. The primary goal of antiviral therapies has been to eradicate HCV virus from serum and achieve sustained virologic response (SVR). Historically, interferon has been a staple of nearly all HCV treatment regimens, despite significant toxic effects.

Areas covered: In recent years, HCV treatment has changed rapidly and significantly. All-oral treatment regimens show promise for treatment with shorter duration and more manageable side effects. New antivirals aimed at improving SVR may provide a cure to nearly all HCV-infected patients. The unique combination of ABT-450 (paritaprevir) and ABT-267 (ombitasvir) provides highly effective treatment for patients with genotype 1 HCV. This review will examine the antiviral properties, pharmacokinetics, pharmacodynamics, and side effects of these agents.

Expert opinion: The combination of ABT-450/r and ABT-267 has improved potency, favorable side effect profile, and low risk of resistance compared to the first-generation protease inhibitors. This combination is likely to be a major part of novel upcoming HCV treatment regimens and is likely to be widely used by clinicians. Additional data is awaited in additional patient populations, and with possible shorter treatment durations.     

A Raman spectroscopic study of the diclofenac sodium–β-cyclodextrin interaction

The inclusion complex of diclofenac sodium (DCFNa) and β-cyclodextrin (βCD) was prepared and analyzed by using Raman and SERS spectroscopy. The interaction between DCFNa and βCD molecules was evidenced by monitoring the change in the peak positions and the widths of some guest molecule bands relative to those observed in the spectra of the free compound and 1:1 DCFNa–βCD physical mixture. Raman data have shown that the interaction between the guest and host molecules in the 1:1 DCFNa–βCD complex is maintained both through the dichlorophenyl ring and the phenylacetate group. SER spectra revealed that depending on the pH value of the solution different isomeric forms of the 1:1 guest–host complex are preferentially adsorbed on the silver surface via the nonbonding electrons of the oxygen atom. The orientation of the adsorbed species with respect to the metal surface was also elucidated.