The Proteolytic Stability and Cytotoxicity Studies of l‐Aspartic Acid and l‐Diaminopropionic Acid derived β‐Peptides and a Mixed α/β‐Peptide

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β‐Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β ‐peptides, β ³‐ and β²‐peptides derived from l ‐aspartic acid and l ‐diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β‐peptidic compounds and a mixed α /β‐peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α‐peptide. Peptides containing β‐linkages were resistant to all conditions. The mixed α /β‐peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β‐peptide was slower than that would be expected for an α‐peptide. In addition, these β‐peptides were not toxic to HeLa and COS‐1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β‐peptides containing β‐amino acids or small β‐peptide fragments as therapeutic peptides.     

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

Involvement of NF‐κB in the upregulation of cystathionine‐γ‐lyase,a hydrogen sulfide‐forming enzyme,and bladder pain accompanying cystitis in mice

Hydrogen sulfide (H₂S) is generated from l ‐cysteine by multiple enzymes including cystathionine‐γ‐lyase (CSE), and promotes nociception by targeting multiple molecules such as Ca_v3.2 T‐type Ca²⁺ channels. Bladder pain accompanying cyclophosphamide (CPA)‐induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H₂S/Ca_v3.2 pathway. Therefore, we investigated whether NF‐κB, as an upstream signal of the CSE/H₂S system, contributes to bladder pain in mice with CPA‐induced cystitis. Bladder pain‐like nociceptive behaviour was observed in CPA‐treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. β‐Cyano‐l ‐alanine, a reversible selective CSE inhibitor, prevented CPA‐induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF‐κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF‐κB inhibitor. PDTC and curcumin, which inhibits NF‐κB signals, abolished CPA‐induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA‐induced activation of NF‐κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H₂S/Ca_v3.2 signaling. Therefore, NF‐κB‐inhibiting compounds including curcumin may be useful for the treatment of cystitis‐related bladder pain.     

Analogues of arginine vasopressin and its agonist and antagonist modified in the N‐terminal part of the molecule with l‐β‐homophenylalanine

Abstract: In continuation of our efforts to elucidate the role of positions 2 and 3 in arginine vasopressin (AVP) and its analogues, we designed and synthesized peptides modified in these positions with l ‐β‐homophenylalanine (β‐Hph). Two of them had just this single modification, the next two peptides are analogues of the V₂ agonist, namely [3‐mercaptopropionic acid (Mpa)¹]AVP (dAVP). The last two compounds were designed by substitution of positions 2 or 3 of a potent V_1a antagonist, [1‐mercaptocyclohexaneacetic acid (Cpa)¹]AVP, with β‐Hph. All the peptides were tested for their pressor and antidiuretic and uterotonic in vitro activities in the rat. All the activities tested have been found to be significantly decreased. Three analogues, i.e. [Mpa¹,β‐Hph²]AVP, [Cpa¹,β‐Hph²]AVP, [Cpa¹,β‐Hph³]AVP, turned out to be uterotonic antagonists with pA₂ = 6.3 ± 0.2, 6.3 ± 0.1, 6.0 ± 0.3 respectively. The last one exhibited antipressor properties also (pA₂ = 6.4 ± 0.1).     

Entrapping unusual folding characteristics of the β‐Ala residues in a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH3

Abstract: Crystal structure analysis of a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH₃ (β‐Ala: 3‐amino propionic acid; Aib: α‐aminoisobutyric acid) revealed distinct conformational preferences for folded [φ≈136°, µ ≈ ?62°, ψ ≈100°] and semifolded [φ ≈ 83°, µ ≈ ?177°, ψ ≈ ?117°] structures of the N‐ and C‐terminus β‐Ala residues, respectively. The overall folded conformation is stabilized by unusual N_i···H‐N_i+1 and nonconventional C–H···O intramolecular hydrogen bonding interactions.     

[Nle3,d‐Phe6]‐γ2‐melanocyte‐stimulating hormone possesses the renal excretory but not the cardiovascular actions of the native γ2‐melanocyte‐stimulating hormone in anaesthetized rats

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Transforming growth factor β‐activated kinase 1 inhibitor suppresses the proliferation in triple‐negative breast cancer through TGF‐β/TGFR pathway

Breast cancer is one of the most invasive cancer types in female population. The functional activity of Transforming growth factor β‐activated kinase 1 (TAK1) in breast cancer progression increasingly attracts attention as it provides a potential target for antibreast cancer drug development. However, the fundamental role of TAK1 for triple‐negative breast cancer (TNBC) progression and the effect of potential anti‐TAK1 drug candidate needs to be further evaluated. Herein, we focused on the role of TAK1 in human breast cancer cells, and we hypothesized that the inhibition of TAK1 activation can repress the growth of human TNBC cells. We found that the TAK1 is robustly activated within cancer cell population of clinic‐derived TNBC samples and the human breast cancer cell lines in culture. Furthermore, we determined the effect of 5Z‐7‐oxozeaenol (5Z‐O), a TAK1‐specific small molecule inhibitor, on proliferation of human TNBC cell line. 5Z‐O treatment significantly suppressed the proliferation of human TNBC cells. Collectively, these demonstrate the role of TAK1 in human breast cancer and the antiproliferate effect of TAK1 inhibitor. Our study sets the stage for further research on TAK1 as a promising target for development of anti‐TNBC drugs and therapeutic strategies.     

Permeability of a novel β‐lactamase inhibitor LK‐157 and its ester prodrugs across rat jejunum in vitro

Objectives LK‐157 is a novel 10‐ethylidene tricyclic carbapenem that resembles the structure of the broad‐spectrum antibiotic sanfetrinem and acts as a potent inactivator of β‐lactamases of classes A, C and D. LK‐157 is a highly soluble but poorly permeable drug. Since most of the β‐lactams are poorly absorbed, ester prodrugs LK‐159, LK‐157E1 and LK‐157E2 were designed to enhance membrane permeability. This study investigated the permeability of LK‐157 and the three ester prodrugs across rat intestine in vitro. The morpholinoethyl ester of sanfetrinem was also investigated. Method Permeability across rat jejunum was determined using EasyMount side‐by‐side diffusion chambers. Key findings The solubility and permeability of morpholinoethyl ester LK‐157E2 were superior to those of LK‐159 and LK‐157E1. The morpholinoethyl ester of sanfetrinem LK‐176E1 had the highest observed permeability coefficient and consequently the highest predicted absorption in humans. Conclusions These results suggest that the morpholinoethyl esters of LK‐157 and sanfetrinem could be further investigated to assess bioavailability in vivo.     

Combined use of ESI‐MS and UV diode‐array detection for localization of disulfide bonds in proteins: application to an α‐l‐fucosidase of pea

Abstract: A simplified strategy is described for the assignment of disulfide bonds in proteins of medium to high molecular mass (10–30 kDa). The method combines the use of high‐performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC‐ESI‐MS) and HPLC with UV diode‐array detection (HPLC diode array). The denatured protein is subjected to proteolysis and the peptide mixture is divided into three fractions: (i) underivatized peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyridylated peptides. The three peptide ensembles are then subjected to chromatographic and spectroscopic analysis. A systematic methodology is described to analyze the large amount of data obtained. The method was applied to the localization of disulfide bonds in α‐l ‐fucosidase from pea. The two disulfide bonds were located between residues Cys⁶⁴ and Cys¹⁰⁹ and between Cys¹⁶² and Cys¹⁶⁹, while Cys¹²⁷ was free.     

3‐butyl‐6‐fluoro‐1 (3H)‐isobenzofuranone,a derivative of dl‐n‐butylphthalide,attenuates hydrogen peroxide‐induced damage in PC12 cells

The anti‐cerebral ischemia agent, dl‐3‐n‐butylphthalide (NBP), is effective in models of vascular dementia in animals. The present study investigates the protective effect of 3‐butyl‐6‐fluoro‐1 (3H)‐isobenzofuranone (6‐F‐NBP), a fluoro derivative of dl‐nbutylphthalide, in hydrogen peroxide (H₂O₂)‐induced damage in PC12 cells. Exposure of PC12 cells to H₂O₂ for 24 h led to decreased cell survival, glutathione peroxidase (GSH‐PX), and mitochondrial membrane potential (MMP). In contrast, malondialdehyde (MDA) production, nitric oxide synthase (NOS) activity, nitric oxide (NO) formation, and intracellular reactive oxygen species (ROS) were increased, as was intracellular accumulation of [Ca²⁺]_i. However, pretreatment with 6‐F‐NBP markedly reversed the changes induced by H₂O₂, exhibiting a protective effect against H₂O₂‐induced cytotoxicity in PC12 cells. The compound may have therapeutic potential in the treatment of cerebral ischemia by inhibiting the oxidative damage. Drug Dev Res 72: 259–264, 2011. © 2010 Wiley‐Liss, Inc.     

A peptide derived from the C‐terminal part of a plant cysteine protease folds into a stack of two β‐hairpins,a scaffold present in the emerging family of granulin‐like growth factors

Abstract: A 35 amino acid residue peptide corresponding to the N‐terminal subdomain of the granulin‐like repeat from rice oryzain β was synthesized and regioselectively oxidized to produce a species with a [1–3, 2–4] disulfide‐pairing pattern. The resulting peptide was studied in solution using NMR and was shown to adopt the tertiary topology of a stack of two β‐hairpins found in the emerging family of granulin‐like growth factors. Because of the longer second β‐hairpin, the overall conformation of the peptide is somewhat more flexible than that of its well‐structured carp granulin‐1 analog. Except for the cysteine alignment, there is very little sequence homology between granulin‐like growth factors from the animal kingdom and the granulin‐like repeats at the C‐termini of plant cysteine proteases. Therefore, the stack of two β‐hairpins may be a conserved three‐dimensional organization of the granulin‐like repeats from evolutionary distant sources with a significant role in specific protein–protein interactions.     

Zanthoxylum avicennae extract enhances GSK‐3β to attenuate β‐catenin via phosphatase 2A to block metastatic effects of HA22T cells and hepatocellular carcinoma xenografted nude mice

Hepatocellular carcinoma (HCC) metastasis is often associated with the activation of Wnt/β‐catenin signaling pathway. Zanthoxylum avicennae (Ying Bu Bo, YBB), a traditional herb with hepatoprotective effect, has been proven to inhibit human HCC in in vivo models however, the in vitro and in vivo effect of YBB on tumor metastasis is not clear yet. To determine whether YBB could inhibit HA22T human HCC cell by acting on β‐catenin metastatic signaling in vitro and in vivo, HA22T cells were treated with different concentrations of YBB extracts (YBBE) and analyzed by Immunofluorescence staining assay, western blot analysis, siRNA mediated gene knock‐down assays and co‐immunoprecipitation assay. Additionally, the HA22T‐implanted xenograft nude mice were used to confirm the assessed cellular effects. Mice treated with YBBEs showed a strong increasing trend in PP2Acα, GSK‐3β, APC, and β‐TrCP/HOS levels, however the expression of β‐catenin, p‐GSK‐3β, TBX 3, and IL8 proteins showed a decreasing trend. YBBE significantly downregulated the nuclear and cytosolic β‐catenin levels by facilitating the proteosomal degradation of β‐catenin. Moreover, as observed by co‐immunoprecipitation assay, YBBE directly promoted the protein interactions between GSK‐3β, β‐TrCP, APC, PP2A, and β‐catenin. In conclusion, both in vitro and in vivo models clearly demonstrated that YBBE inhibits β‐catenin involved metastatic signaling in highly metastatic HA22T cells through PP2A activation.     

MiR‐146a regulates PM1‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells

Exposure to particulate matter (PM) leads to kinds of cardiopulmonary diseases, such as asthma, COPD, arrhythmias, lung cancer, etc., which are related to PM‐induced inflammation. We have found that PM_2.5 (aerodynamics diameter <2.5 µm) exposure induces inflammatory response both in vivo and in vitro. Since the toxicity of PM is tightly associated with its size and components, PM₁ (aerodynamics diameter <1.0 µm) is supposed to be more toxic than PM_2.5. However, the mechanism of PM₁‐induced inflammation is not clear. Recently, emerging evidences prove that microRNAs play a vital role in regulating inflammation. Therefore, we studied the regulation of miR‐146a in PM₁‐induced inflammation in human lung bronchial epithelial BEAS‐2B cells. The results show that PM₁ induces the increase of IL‐6 and IL‐8 in BEAS‐2B cells and up‐regulates the miR‐146a expression by activating NF‐κB signaling pathway. Overexpressed miR‐146a prevents the nuclear translocation of p65 through inhibiting the IRAK1/TRAF6 expression, and downregulates the expression of IL‐6 and IL‐8. Taken together, these results demonstrate that miR‐146a can negatively feedback regulate PM₁‐induced inflammation via NF‐κB signaling pathway in BEAS‐2B cells.     

β‐turn formation by a six‐residue linear peptide in solution

Abstract: A model peptide AAGDYY‐NH₂ (B1), which is found to adopt a β‐turn conformation in the TEM‐1 β‐lactamase inhibitor protein (BLIP) in the TEM‐1/BLIP co‐crystal, was synthesized to elucidate the mechanism of its β‐turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT‐IR and ¹H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT‐IR spectroscopy confirmed the formation of a β‐turn in solution by the model peptide. The dihedral angles [(φ₃, ?₃) (φ₄, ?₄)] of [(?52°, ?32°) (?38°, ?44°)] of Gly‐Asp fragment in the model peptide are consistent with those of a type III β‐turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of β‐turn formation and stability.     

S1P facilitates IL‐1β production in osteoblasts via the JAK and STAT3 signaling pathways

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease, in which the immune system attacks synovial joint tissues. Interleukin (IL)‐1β is a critical proinflammatory cytokine in RA progression. Sphingosine‐1‐phosphate (S1P), a platelet‐derived lysophospholipid mediator, reportedly regulates osteoimmunology. Here, we investigated how S1P mediates IL‐1β expression in osteoblasts. Our analysis of records from the Gene Expression Omnibus (GEO) database demonstrate higher levels of IL‐1β in patients with RA compared with those with osteoarthritis. Stimulation of osteoblasts with S1P concentration dependently increased mRNA and protein expression of IL‐1β. Elevations in IL‐1β mRNA expression induced by S1P were reduced by the small interfering RNA (siRNA) against the S1P₁ receptor. S1P also augmented JAK and STAT3 molecular cascades. We also found that JAK and STAT3 inhibitors and their siRNAs antagonized S1P‐promoted IL‐1β expression. Our results indicate that S1P promotes the expression of IL‐1β in osteoblasts via the S1P₁ receptor and the JAK and STAT3 signaling pathways.