An experimental and molecular-modeling study of the binding of linked sulfated tetracyclitols to FGF-1 and FGF-2

The experimental binding affinities of a series of linked sulfated tetracyclitols [Cyc2N-R-NCyc2, where Cyc = C6H6(OSO3Na)3 and R = (CH2)n (n = 2-10), p-xylyl or (C2H4)2-Ncyc] for the fibroblast growth factors FGF-1 and FGF-2 have been measured by using a surface plasmon resonance assay. The KD values range from 7.0 nM to 1.1 microM for the alkyl-linked ligands. The binding affinity is independent of the flexibility of the linker, as replacement of the alkyl linker with a rigid p-xylyl group did not affect the KD. Calculations suggest that binding modes for the p-xylyl-linked ligand are similar to those calculated for the flexible alkyl-linked tetracyclitols. The possible formation of cross-linked FGF:cyclitol complexes was examined by determining KD values at increasing protein concentrations. No changes in KD were observed; this suggesting that only 1:1 complexes are formed under these assay conditions. Monte Carlo multiple-minima calculations of low-energy conformers of the FGF-bound ligands showed that all of the sulfated tetracyclitol ligands can bind effectively in the heparan sulfate-binding sites of FGF-1 and FGF-2. Binding affinities of these complexes were estimated by the Linear Interaction Energy (LIE) method to within a root-mean-square deviation of 1 kcal mol(-1) of the observed values. The effect of incorporating cations to balance the overall charge of the complexes during the LIE calculations was also explored.     

ELISA法间接检测血管内皮生长因子抑制剂的相对结合力

目的采用ELISA法间接检测血管内皮生长因子抑制剂(VEGF Trap)的相对结合力。方法以固定量的VEGF165与VEGF Trap结合,通过双抗体夹心ELISA法检测未结合的、游离的VEGF165,进而求得VEGFTrap的半数抑制浓度(IC50),计算其与反应体系中参比品的IC50之比,即为VEGFTrap的相对结合力。结果同一批次的3支VEGF Trap样品分别经3次测定,相对结合力分别为(100.86±9.43)%、(88.46±13.94)%和(82.66±9.60)%,均值为(90.66±12.60)%,符合该制品质量标准的要求。3次试验的变异系数分别为9.35%、15.76%和11.61%。结论 ELISA法精密性良好,结果客观,影响因素少,可用于VEGF Trap相对亲和力的常规检测。     

Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A₁₆₅ region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B₁₆₇ derived peptides were more effective than VEGF-A₁₆₅ peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.     

Single-Step Per-O-Sulfonation of Sugar Oligomers with Concomitant 1,6-Anhydro Bridge Formation for Binding Fibroblast Growth Factors

Many circulating cancer-related proteins, such as fibroblast growth factors (FGFs), associate with glycosaminoglycans—particularly heparan sulfate—at the cell surface. Disaccharide analogues of heparan sulfate had previously been identified as the shortest components out of the sugars that bind to FGF-1 and FGF-2. Taking note of the typical pose of l -iduronic acid, we conceived of per-O-sulfonated analogues of such disaccharides, and devised a single-step procedure for per-O-sulfonation of unprotected sugars with concomitant 1,6-anhydro bridge formation to achieve such compounds through direct use of SO₃ ⋅ Et₃N as sulfonation reagent and dimethylformamide as solvent. The synthesized sugars based on the oligomaltose backbone bound FGF-1 and FGF-2 mostly at the sub-micromolar level, although the tetrasaccharide analogue achieved low-nanomolar binding with FGF-2.     

Iterative Nonproteinogenic Residue Incorporation Yields α/β‐Peptides with a Helix–Loop–Helix Tertiary Structure and High Affinity for VEGF

Inhibition of specific protein–protein interactions is attractive for a range of therapeutic applications, but the large and irregularly shaped contact surfaces involved in many such interactions make it challenging to design synthetic antagonists. Here, we describe the development of backbone‐modified peptides containing both α‐ and β‐amino acid residues (α/β‐peptides) that target the receptor‐binding surface of vascular endothelial growth factor (VEGF). Our approach is based on the Z‐domain, which adopts a three‐helix bundle tertiary structure. We show how a two‐helix “mini‐Z‐domain” can be modified to contain β and other nonproteinogenic residues while retaining the target‐binding epitope by using iterative unnatural residue incorporation. The resulting α/β‐peptides are less susceptible to proteolysis than is their parent α‐peptide, and some of these α/β‐peptides match the full‐length Z‐domain in terms of affinity for receptor‐recognition surfaces on the VEGF homodimer.     

Design of fully active FGF-1 variants with increased stability

Fibroblast growth factor 1 is a powerful mitogen playing an important role in morphogenesis, angiogenesis and wound healing and is therefore of potential medical interest. Using homologous sequence and structure comparisons, we designed and constructed 16 mutants of FGF-1 with increased thermodynamic stability, as determined by chemical and heat denaturation. For multiple mutants, additive effects on stability were observed, providing mutants up to 7.8 degrees C more stable than the wild-type. None of the introduced mutations affected any FGF-1 biological activities, such as stimulation of DNA synthesis, MAP kinase activation and binding to the FGF receptor on the cell surface. Our study provides a good starting point to improve the stability of FGF-1 in the context of its wide potential therapeutic applications. We showed that a homology approach is an effective method to change the thermodynamic properties of the protein without altering its function.     

The activation of fibroblast growth factors (FGFs) by glycosaminoglycans: influence of the sulfation pattern on the biological activity of FGF-1

Six synthetic heparin-like oligosaccharides have been used to investigate the effect of the oligosaccharide sulfation pattern on the stimulation of acidic fibroblast growth factor (FGF-1) induced mitogenesis signaling and the biological significance of FGF-1 trans dimerization in the FGF-1 activation process. It has been found that some molecules with a sulfation pattern that does not contain the internal trisaccharide motif, which has been proposed for high affinity for FGF-1, stimulate FGF-1 more efficiently than those with the structure of the regular region of heparin. In contrast to regular region oligosaccharides, in which the sulfate groups are distributed on both sides of their helical three-dimensional structures, the molecules containing this particular sulfation pattern display the sulfate groups only on one side of the helix. These results and the fact that these oligosaccharides do not promote FGF-1 dimerization according to sedimentation-equilibrium analysis, confirm the importance of negative-charge distribution in the activation process and strongly suggest that FGF dimerization is not a general and absolute requirement for biological activity.     

Defining the Potential of Aglycone Modifications for Affinity/Selectivity Enhancement against Medically Relevant Lectins: Synthesis,Activity Screening,and HSQC‐Based NMR Analysis

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N‐linked‐lactosides and of eight O‐linked N‐acetyllactosamines, each substituted with a 1,2,3‐triazole unit, prepared by copper‐catalyzed azide–alkyne cycloaddition (CuAAC). The totally regioselective β‐D ‐(1→4) galactosylation of a 6‐O‐TBDPSi‐protected N‐acetylglucosamine acceptor provided efficient access to the N‐acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface‐presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth‐regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5‐fold enhancement for prototype proteins, with further increases for galectins‐3 and ‐4. Moreover, the availability of ¹⁵N‐labeled proteins and full assignments enabled ¹H,¹⁵N HSQC‐based measurements for hu‐ man galectins‐1, ‐3, and ‐7 against p‐nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin‐3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure–activity correlations at other bioinspired sites in glycans and beyond the tested lectin types.     

Preparation and recognition performance of cholic acid-imprinted material prepared with novel surface-imprinting technique

Acrylamide (AM) was first graft-polymerized on the surface of crosslinked polyvinyl alcohol (CPVA) microspheres by initiating of cerium salt, and then the grafted polyacrylamide (PAM) was transformed to polyvinylamine (PVAm) via Hofmann degradation reaction, resulting in the grafted microspheres PVAm/CPVA. By adopting the novel surface molecular imprinting technique put forward by us, cholic acid molecule-imprinted material MIP-PVAm/CPVA was prepared with glutaraldehyde as crosslinking agent The binding character of MIP-PVAm/CPVA towards cholic acid molecules was studied in depth with both batch and column methods and using cholesterol as a contrast compound whose chemical structure is similar with cholic acid to a certain extent. The experimental results show that the surface-imprinted material MIP-PVAm/CPVA has excellent binding affinity and recognition selectivity for the template molecule, cholic acid. The selectivity coefficient of PVAm/CPVA microspheres (non-imprinting material) for cholic acid relative to cholesterol is only 1.314, displaying very poor recognition selectivity for cholic acid. However, after imprinting, the selectivity coefficient of MIP-PVAm/CPVA for cholic acid in respect to cholesterol is remarkably enhanced to 11.231, displaying the excellent recognition selectivity and binding affinity towards cholic acid molecules. Besides, MIP-PVAm/CPVA microspheres have fine desorption property, and by using a mixture of ethanol and NaOH aqueous solution as an eluent, the desorption ratios can reach 99.73% as the effluent amount gets up to 20 bed volumes (BV).     

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation

Vascular endothelial growth factor-A₁₆₅ (VEGF-A₁₆₅) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A₁₆₅ and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A₁₆₅ stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A₁₆₅ on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A₁₆₅ have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.     

Computer modelling of the receptor-binding domains of VEGF and PIGF

Models of the platelet-derived growth factor (PDGF)-like domains of vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) were built based on their homology to PDGF. These domains contain most of the determinants for receptor binding. The sequences of these proteins exhibit limited but significant homology to that of platelet-derived growth factor (PDGF), a member of the cystine knot growth factor family. The eight cysteine residues that are involved in intra- and interchain disulphide bonds are conserved. Two high affinity receptors for VEGF have been identified, only one of which binds PIGF. The models presented here are consistent with results that show that VEGF receptor binding is mediated by charged residues in the loops. A comparison of the models suggests that the difference in receptor- binding specificity between VEGF and PIGF may be due to differences in the distribution of positively charged residues and the exposure of hydrophobic residues in the loops.      

Development of Selective Bisubstrate‐Based Inhibitors Against Protein Kinase C (PKC) Isozymes By Using Dynamic Peptide Microarrays

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP‐binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate‐based inhibitors that target the more selective peptide‐binding site in addition to the ATP‐binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate‐based leads. Dynamic peptide microarrays were used to find peptide‐binding site inhibitors. These were linked with chemoselective click chemistry to an ATP‐binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate‐based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCθ amongst three isozymes.     

Cytokines and Venous Leg Ulcer Healing—A Systematic Review

Venous leg ulcers (VLUs) are the most common type of leg ulcers with a significant socioeconomic burden due to slow healing. Cytokines may be involved in the pathogenesis of VLUs. In this systematic review, our objective was to investigate the association between cytokine levels, including growth factors, with the healing of VLUs. PubMed, Embase, Web of Science and Cochrane Library were searched from their inception to August 2021. We retrieved 28 articles investigating 38 different cytokines in 790 patients. Cytokines were most commonly investigated in wound fluid and less frequently in biopsies and serum. The studies were judged as having a moderate to high risk of bias, and the results were often inconsistent and sometimes conflicting. A meta-analysis was not performed due to clinical and methodological heterogeneities. We found weak evidence for elevated IL-1α, IL-6, IL-8, TNF-α and VEGF levels in non-healing VLUs, an elevation that declined with healing. TGF-β1 levels tended to increase with VLU healing. Other cytokines warranting further investigations include EGF, FGF-2, GM-CSF, IL-1β, IL-1Ra and PDGF-AA/PDGF-BB. We conclude that non-healing VLUs may be associated with an elevation of a palette of pro-inflammatory cytokines, possibly reflecting activated innate immunity in these wounds. There is a paucity of reliable longitudinal studies monitoring the dynamic changes in cytokine levels during wound healing.     

新型层状粘土结构F-T合成催化剂的研制

用FeCl₃·6H₂O和Na₂SiO₃水玻璃共沉淀法制备蒙脱土结构的新型F-T合成催化剂。用XRD、SEM和N₂低温吸附-脱附等方法对催化剂的物相结构进行测定,确认催化剂具有层状结构并有较高的比表面积。催化剂经过预处理,F-T合成在浆态床反应器中进行,反应条件：270 ℃,0.5 MPa,气流量34.5 mL·min^-1,n(H₂)∶n（CO）=2∶1,反应时间6 h。产物分析得知,C₉～C₂₅烃类的选择性达到90%以上,使ASF分布规律发生正偏移。     

Effect of Guibi-Tang,a Traditional Herbal Formula,on Retinal Neovascularization in a Mouse Model of Proliferative Retinopathy

Ocular pathologic angiogenesis is an important causative risk factor of blindness in retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular macular degeneration. Guibi-tang (GBT) is a frequently used oriental herbal formula in East Asian countries, and is also called Qui-pi-tang in Chinese and Kihi-To in Japanese. In the present study, we investigated the preventive effect of GBT on retinal pathogenic neovascularization in a mouse model of oxygen-induced retinopathy (OIR). C57BL/6 mice were exposed to 75% hyperoxia for five days on postnatal day 7 (P7). The mice were then exposed to room air from P12 to P17 to induce ischemic proliferative retinopathy. GBT (50 or 100 mg/kg/day) was intraperitoneally administered daily for five days (from P12 to P16). On P17, Retinal neovascularization was measured on P17, and the expression levels of 55 angiogenesis-related factors were analyzed using protein arrays. GBT significantly decreased retinal pathogenic angiogenesis in OIR mice, and protein arrays revealed that GBT decreased PAI-1 protein expression levels. Quantitative real-time PCR revealed that GBT reduced vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and plasminogen activator inhibitor 1 (PAI-1) mRNA levels in OIR mice. GBT promotes potent inhibitory activity for retinal neovascularization by decreasing VEGF, FGF2, and PAI-1 levels.     

反义核酸药物与抗肿瘤研究

现在各国正在利用反义技术发展一类由核酸组成的新型药物,即反义核酸药物。我们合成了5种血管内皮生长因子(VEGF)的反义寡聚脱氧核苷酸(ODN_S)。测定了它们在S180肿瘤细胞中对VEGF表达的抑制作用,还测定了它们在鼠角膜肿瘤模型上抑制由肿瘤引起的新生血管形成和肿瘤的生长。结果表明,部分硫代的或发夹结构修饰的ODNs均可有效抑制VEGF的表达、肿瘤的生长和新生血管的形成。线性的ODN效果不好。两种作用于不同位点的ODN联合使用可得到最好的效果。因此反义VEGF寡聚脱氧核苷酸有望成为抗肿瘤和抗新生血管形成的新型药物。     

Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.     

可溶性血管内皮生长因子受体2片段的表达及其对血管内皮细胞增殖的影响

目的探讨可溶性血管内皮生长因子受体2(sKDR)抑制血管内皮细胞增殖及在血管生成中的作用。方法提取脐静脉内皮细胞(HUVEC)总RNA,扩增KDR基因膜外1~4结构域,构建原核表达载体pQE40-KDR,转化E.coli M15,经IPTG诱导表达,镍离子柱亲和层析纯化后复性,用Western blot检测sKDR蛋白的表达,MTT比色法和鸡胚尿囊膜(CAM)试验分别检测其对HUVEC增殖的影响及其对血管生成的作用。结果经RT-PCR扩增得到了1150 bp左右的sKDR片段,并在pQE40原核表达系统中表达了sKDR蛋白,以包涵体形式存在。纯化后蛋白电泳呈现相对分子质量50000左右的单一条带,纯化蛋白占总蛋白的98%,蛋白含量为80μg/ml。Western blot证实其为重组sKDR蛋白。MTT检测结果显示,sKDR可抑制血管内皮生长因子(VEGF)刺激的HUVEC增殖,并阻滞VEGF诱导的CAM血管增生。结论已成功构建sKDR原核表达载体,并在大肠杆菌M15中获得表达,纯化的sKDR片段具有与VEGF结合的生物学功能,有望成为基因治疗肿瘤血管形成的理想靶点。     

Structure activity relationships of monocyte chemoattractant proteins in complex with a blocking antibody

Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 A structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody-chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.