Furo[2,3‐d]pyrimidines and Oxazolo[5,4‐d]pyrimidines as Inhibitors of Receptor Tyrosine Kinases (RTK)

Receptor tyrosine kinases such as VEGFR2 (vascular endothelial growth factor receptor 2, KDR) or EGFR (epidermal growth factor receptor) play crucial roles in a variety of diseases, such as cancer. Recently, some pyrrolopyrimidines were shown to be potent EGFR inhibitors. Therefore, new types of oxazolo[5,4‐d]pyrimidines and furo[2,3‐d]pyrimidines were synthesized (Schemes 1 and 2). Appropriately substituted derivatives of these classes of compounds inhibited VEGFR2 and EGFR with IC₅₀ values in the low nanomolar range (see Table). Generally, the furopyrimidines were somewhat more active than the oxazolopyrimidines. The best inhibitors, 20m, 20p , and 20r , had an IC₅₀ of 3 nM towards EGFR and showed a good selectivity, being distinctly less active towards VEGFR2.     

Directed evolution of high-affinity antibody mimics using mRNA display

We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.     

Single‐Molecule Force Measurement Guides the Design of Multivalent Ligands with Picomolar Affinity

Interaction of multiple entities and receptors, or multivalency is widely applied to achieve high affinity ligands for diagnostic and therapeutic purposes. However, lack of knowledge on receptor distribution in living subjects remains a challenge for rational structure design. Herein, we develop a force measurement platform to probe the distribution and separation of the cell surface vascular endothelial growth factor receptors (VEGFR) in live cells, and use this to assess the geometry of appropriate linkers for distinct multivalent binding modes. A tetravalent lead ZD‐4, which was developed from an antitumor drug ZD6474 (Vandetanib) with combined hybrid binding effects, yielded a 2000‐fold improvement in the binding affinity to VEGFR with IC₅₀ value of 25 pm . We confirmed the improved affinity by the associated increase of tumor uptake in the VEGFR‐targeting positron emission tomography (PET) imaging using U87 tumor xenograft mouse model.     

Pristimerin, a triterpenoid, inhibits tumor angiogenesis by targeting VEGFR2 activation

Pristimerin is a triterpenoid isolated from Celastrus and Maytenus spp. that has been shown to possess a variety of biological activities, including anti-cancer activity. However, little is known about pristimerin's effects on tumor angiogenesis. In this study, we examined the function and the mechanism of this compound in tumor angiogenesis using multiple angiogenesis assays. We found that pristimerin significantly reduced both the volume and weight of solid tumors and decreased angiogenesis in a xenograft mouse tumor model in vivo. Pristimerin significantly inhibited the neovascularization of chicken chorioallantoic membrane (CAM) in vivo and abrogated vascular endothelial growth factor (VEGF)-induced microvessel sprouting in an ex vivo rat aortic ring assay. Furthermore, pristimerin inhibited the VEGF-induced proliferation, migration and capillary-like structure formation of human umbilical vascular endothelial cells (HUVECs) in a concentration-dependent manner. Mechanistic studies revealed that pristimerin suppressed the VEGF-induced phosphorylation of VEGF receptor 2 kinase (KDR/Flk-1) and the activity of AKT, ERK1/2, mTOR, and ribosomal protein S6 kinase. Taken together, our results provide evidence for the first time that pristimerin potently suppresses angiogenesis by targeting VEGFR2 activation. These results provide a novel mechanism of action for pristimerin which may be important in the treatment of cancer.     

Construction of ribosome display library based on lipocalin scaffold and screening anticalins with specificity for estradiol

A new anticalin against estradiol (E(2)), a kind of endocrine disruptor, was obtained in the present study to detect E(2) levels. A member of the lipocalin family from Pieris brassicae called bilin-binding protein (BBP) was employed for the preparation of a random library to specifically complex E(2). Sixteen amino acid residues at the center of the binding site, which were formed by four loops on top of an eight-stranded β-barrel, were subjected to targeted random mutagenesis. Estradiol-binding BBP variants so-called 'anticalins', which exhibit binding activity for compounds, such as E(2), were selected from the resulting library by combining both ribosome display and screening techniques. Four variants of complex E(2) with high affinity were identified. These variants exhibited dissociation constants (KDs) as low as 54.265 nM. ELISA showed that ribosome displayed anticalin (E(2)-A) specifically bound E(2). The 50% inhibition concentration (IC(50)) for E(2) was 50 ng mL(-1) and the limit of detection (LOD:IC(10)) was 0.071 ng mL(-1). The experimental results suggest that E(2)-A can be used as a potential anticalin to detect E(2) in animals.     

Monoclonal antibodies against human platelet membrane glycoprotein IIIa and collagen-induced platelet activation

Two monoclonal antibodies against human platelet membrane glycoprotein IIIa (GPIIIa) were obtained. One monoclonal antibody, designated as 1B1, was found to inhibit both collagen-induced platelet aggregation and release reactions. This antibody also inhibited the binding of 125I-labeled collagen to human platelets. On the other hand, the other antibody, designated as B10, had no effect on platelet activation induced by a number of physiological stimulants including collagen. Direct binding studies involving 125I-labeled 1B1 or B10 demonstrated that the binding sites for these antibodies on unstimulated platelets have dissociation constants of 4.2 and 14.0 nM, respectively. The binding of 125I-labeled 1B1 or B10 to platelets was not inhibited by the other antibody. Purified 1B1 and B10 were covalently coupled to Affi-Gel and then proteolytic fragments of GPIIIa were applied to the Affi-Gel immunoadsorbent columns. Of the several proteolytic fragments, the 56 kilodaltons (kDa) fragment obtained on digestion with V8 protease bound to both of the columns. The 69 and 55 kDa fragments obtained with BrCN bound to only the 1B1 Affi-Gel column, while the 63 kDa fragment obtained with chymotrypsin only bound to the B10-Affi-Gel column. Based on the partial amino acid sequences of these fragments and the amino acid sequence of GPIIIa (C. A. Fitzgerald, B. Steiner, S. C. Rall, Jr., S. Lo and D. R. Phillips, J. Biol. Chem., 262, 3936 (1987), the epitopes for 1B1 and B10 were concluded to be located at amino acids 335 to 582 and 206 to 335, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibition profiles of 4'-acylpyrrole–5-fluoroindolin-2-ones with a C-3' side chain for VEGFR2, PDGFR-β, and FGFR-1 protein kinases

In this study, we reported the inhibition profiles of 4′-acylpyrrole–5-fluoroindolin-2-one 3 with a C-3′ side chain for VEGFR2, PDGFR-β, and FGFR-1 protein kinases. The pyrrole-fused cyclohexanone moiety provided 3 with the best potency to inhibit the three kinases, and the C-3′ side chains contributed to the different inhibition profiles of 3 . Compound 3b with a C-3′ 2-carboxylethyl side chain showed good potency for the three kinase (IC₅₀: 25–260 nM), and compound 3g with a N,N-dialkyl-2-carbamoylethyl side chain was more active for VEGFR2 (IC₅₀: 59 nM) and PDGFR-β (IC₅₀: 16 nM) than FGFR-1 (IC₅₀: 1.7 μM). The C-3′ 3-(dialkylamino)propyl side chain accomplished 3h – j as selective PDGFR-β inhibitors (IC₅₀: 7.8–13 nM). Compound 3b was further investigated and found potent to inhibit VEGF- and FGF-dependent cell proliferation with moderate in vivo anticancer activity. Results from docking simulations revealed that the interactions of 3b with VEGFR2 and FGFR-1 which could account for the different inhibition profiles of 3 .     

靶向VEGFR受体抑制剂齐墩果酸衍生物的合成及初步抗肿瘤活性研究

以天然产物齐墩果酸为母体,设计合成齐墩果酸衍生物,采用计算机辅助药物设计,对C-3、C-28位结构改造,设计合成12个未见文献报道的靶向VEGFR受体抑制剂; 采用噻唑蓝(MTT)法,用人肝癌细胞(HepG2)和乳腺癌细胞(MCF-7)对其进行初步体外抗肿瘤活性筛选;其结构经1H-NMR、13C-NMR谱确证。活性测试得出化合物I7、II1与阳性对照药相比有较强抑制作用,其抗肿瘤活性高于母体OA,分子对接结果显示I7和II1 与 VEGFR 受体具有较好的结合能力,值得进一步研究。     

Reconsideration of serum Ti(IV) transport: albumin and transferrin trafficking of Ti(IV) and its complexes

The trafficking of titanium(IV) by human serum transferrin (HsTf) has been implicated in the physiology of this hydrolysis-prone metal. The current work broadens to include the further interactions of Ti(IV) in serum that bear on this model. Ti2HsTf (2 equiv) binds the transferrin receptor TfR1 with Kd1 = 6.3 +/- 0.4 nM and Kd2 = 410 +/- 150 nM, values that are the tightest yet measured for a metal other than iron but weaker than the corresponding ones for Fe2HsTf due to both slightly slower on rates and slightly faster off rates. Comparing the affinities of metals for HsTf with the affinities of the resulting M2HsTf species for TfR1, we speculate that the formation of an M2HsTf complex of high affinity may predict a lobe-closed conformation that leads to a favorable interaction with TfR1. Human serum albumin (HSA), an important serum competitor for metal binding, can bind up to 20 equiv of Ti(IV) supplied in several forms. With some ligands, Ti(IV) may bind to the N-terminal metal binding site of albumin, forming a ternary complex. However, the dominant type of HSA binding is via Ti(IV) in complex form, probably at surface sites. Notably, HSA greatly stabilizes the titanocene moiety of the drug candidate Cp2TiCl2 with respect to hydrolysis and precipitation. HSA binds Ti(IV) citrate supplied as a hydrolyzed or unhydrolyzed source, with 1 equiv of citrate remaining bound. Titanium(IV) monocitrate neither competes with the binding of reporter molecules known to dock at canonical drug sites I or II nor binds at the N-terminus. HsTf outcompetes HSA for soluble Ti(IV) in a direct competition, but once bound to albumin, the transfer of Ti(IV) from HSA to HsTf is quite slow. Each of these findings has implications for the metabolism of Ti(IV) in human serum.     

VEGFR2活性腔性质以及与抑制剂的结合模式研究

VEGFR2介导肿瘤诱导的血管生成作用, 是抑制肿瘤生长和转移的新靶点. 为深入探讨VEGFR2活性腔性质以及与抑制剂的结合模式, 采用多拷贝同时搜寻法(MCSS)研究VEGFR2活性腔的性质, 然后用分子对接方法对5个已上临床的VEGFR抑制剂与VEGFR2活性腔进行对接计算, 讨论它们的结合模式, 确定与配体结合相关的关键残基. 研究发现: 疏水腔I, II是配体结合的关键区域, 残基Glu915, Cys917是关键的氢键作用位点, Lys866, Glu883和Asp1044形成的极性区域对提高配体亲合力很重要, 疏水腔III和极性腔IV是额外增强配体结合力的区域, IV区的Arg1030可提供额外的氢键作用位点. 本研究可为全新VEGFR2抑制剂的合理药物设计提供理论依据, 为寻找新的抗肿瘤药物奠定基础.     

New fluorometric enzyme immunoassay for 17beta-estradiol by homogeneous reaction using biotinylated estradiol

A new fluorometric enzyme immunoassay for 17β-estradiol (E2) using biotinylated estradiol (BE) as a probe ligand, is described. In this method, E2 is detected indirectly by a solid-phase avidin-biotin binding assay, in which the biotin is immobilized on a microtiter plate (biotin-plate). After the competitive reaction between E2 and BE for the anti-E2 antibody in solution, the free E2 and BE are separated from the bound forms by means of ultrafiltration. The concentration of BE in the solution is determined from the reaction between the biotin immobilized on the plate and the free BE for the limited biotin binding sites of avidin conjugated with horseradish peroxidase (avidin-HRP), which is added to the solution. The enzymatic reaction of HRP was measured by a fluorometric analysis with the QuantaBlu™ Fluorogenic Peroxidase Substrate (QFPS) in order to detect of the avidin-biotin binding with a high degree of sensitivity. The detection limit and linear range for the determination of E2 were 0.12 nM and from 0.12 to 25 nM, respectively. The relative standard deviations (R.S.D.) for the E2 assay were between 2.2 and 9.1% (n = 3). The cross-reactivity for several other estrogens was also evaluated.     

Src Kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells

Background  

The cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor) – dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1) and Flt-1 (Fms-like tyrosine kinase-1). However, to date, it has not been determined which VEGF receptor (VEGFR) is involved in binding to and activating Src kinase following VEGF stimulation of the receptors.     

Quantitative analysis of betulinic acid in mouse,rat and dog plasma using electrospray liquid chromatography/mass spectrometry

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.     

A novel class of small functional peptides that bind and inhibit human alpha-thrombin isolated by mRNA display

Here we report the in vitro selection of novel small peptide motifs that bind to human alpha-thrombin. We have applied mRNA display to select for thrombin binding peptides from an unbiased library of 1.2 x 10(11) different 35-mer peptides, each containing a random sequence of 15 amino acids. Two clones showed binding affinities ranging from 166 to 520 nM. A conserved motif of four amino acids, DPGR, was identified. Clot formation of human plasma is inhibited by the selected clones, and they downregulate the thrombin-mediated activation of protein C. The identified peptide motifs do not share primary sequence similarities to any of the known natural thrombin binding motifs. As new inhibitors for human thrombin open interesting possibilities in thrombosis research, our newly identified peptides may provide further insights into this field of investigation and may be possible candidates for the development of new anti-thrombotic agents.     

Characterization of DNA-protein complexes by capillary electrophoresis-single molecule fluorescence correlation spectroscopy

A high-speed capillary electrophoresis mobility shift assay (CEMSA) for determining the binding ratios of DNA-protein complexes in solution is demonstrated. Single molecule fluorescence correlation spectroscopy (FCS) was used to resolve the bound and unbound fluorescently labeled DNA molecules as they flowed continuously through a fused silica capillary under the influence of an applied electric field. Resolution of the bound and unbound complexes was based on the difference in their electrophoretic mobilities, and was accomplished without the need to perform a chemical separation. Data sufficient to perform the analysis was acquired in less than 10 s, compared to the minutes that are normally needed to carry out such measurement via CE separation. The binding ratios were determined with 5 to 10% precision and agreed with the results obtained by CE separation within experimental error. The resolution of the CEMSA based FCS analysis (CEMSA-FCS) was significantly higher than for the analysis performed by conventional diffusional FCS, due to the higher mass sensitivity of the electrophoretic mobility compared to the translational diffusion coefficient. Fluorescently labeled 39-mer single stranded DNA (ssDNA) and the single stranded binding protein (SSB) from Escherichia coli was used as the model system. The dissociation constant of the ssDNA-SSB complex was estimated to be approximately 2 nM based on the CEMSA-FCS analysis.     

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions     

Effect of the cell type and cell density on the binding of living cells to a silica particle: an atomic force microscope study

We used the atomic force microscope to study how the cell type and the density of cells adsorbed at a substrate can affect the adhesion between a living cell and a model drug delivery system (DDS) carrier nano-particle. We used three different anchorage-dependent cells, i.e., a living mouse fibroblast cell (L929), a living human colon cancer cell (Caco2), and a living mouse malignant melanoma cell (B16F10). For the DDS model nano-particle, we used a silica colloid. In order to correlate the adhesion force with the cell types, the growth curve of the cells were determined with a haemocytometer. The shapes of the cells at the different stages were monitored by light microscopy, and the morphology of their surfaces obtained by tapping mode atomic force microscopy.

Force measurements showed that the Caco2 cell bound little to a silica particle, regardless of the cell density. The L929 cell bound well to a silica particle for low and high cell densities. The B16F10 cell bound little to a silica particle for low cell densities, but bound well for high cell densities. AFM images showed that the L929 cell did not contain folds. The B16F10 cells, however, displayed folds in the cell surface for low cell densities, but no folds in the cell for high cell densities. As literature also reported that the Caco2 cell contains folds, these results suggested that cells with folds showed less adhesion to a silica particle than cells without folds. The presence of folds in the cell presumably decreased the number of sites on the cell that could hydrogen bond or undergo van der Waals binding with the silanol groups of the silica particle.     

Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionisation tandem mass spectrometric (LC/ESI-MS/MS) method was developed for the analysis of acetylcholine and choline in microdialysis samples. A Ringer's solution that contains high (150 mM) concentrations of inorganic salts was used to extract acetylcholine and choline from a rat or mouse brain. The separation of acetylcholine, choline, an internal standard acetyl-beta-methylcholine, endogenous compounds and inorganic cations was achieved with hydrophilic interaction chromatography using a diol column. The eluent consisted of 20 mM ammonium formate (pH 3.3) and acetonitrile (20:80) which is favourable for the ESI process. Limits of detection (signal-to-noise (S/N) ratio = 3) of 0.02 nM (0.2 fmol) for acetylcholine and 1 nM (10 fmol) for choline were observed using standards diluted in Ringer's solution. A good linearity was obtained from the limit of quantitation: 0.1 nM (S/N ratio = 10) to 50 nM (r = 0.999) for acetylcholine and within the concentration range of 100-3500 nM (r = 0.998) for choline. The between-day repeatability of the method was good; RSD was 3.1% at 1 nM level of acetylcholine and 3.5% at 1000 nM level of choline. The recoveries for addition of 1 or 2.5 nM acetylcholine and 0.2 or 1 microM choline in microdialysis balancing samples were between 93 and 101% indicating that no suppressing endogenous compounds were co-eluting with acetylcholine or choline. The developed method was applied to the analysis of microdialysis balancing samples collected from rat and mouse brains.     

Capillary electrophoresis coupled with laser-induced fluorescence polarization as a hybrid approach to ultrasensitive immunoassays.

Immunoassays using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a powerful approach to the determination of trace amounts of analytes in a complex biological matrix. However, its applicability is limited by the requirement that the free and bound tracer (fluorescently labeled compound) be resolved for their identification and quantitation. Here we show that replacing LIF with laser-induced fluorescence polarization (LIFP) permits ultrasensitive immunoassays to be performed with or without the separation of the free and bound tracer. A binding system involving cyclosporin A (CyA) and monoclonal antibody to CyA was chosen to demonstrate both homogeneous and heterogeneous immunoassay approaches. In the homogeneous scheme where the free and bound tracer were not separated, the fluorescence polarization of the mixture was a quantitative measure of the antibody-bound tracer. The concentration and mass detection limits for CyA using the homogeneous competitive assay were found to be 1 nM and 1 amol (10(-18) mol), respectively. The heterogeneous assay involved a nearly baseline separation of the free and bound tracer using CE with a phosphate running buffer of pH 7.0. The complex of the tracer with the antibody had a fluorescence polarization of approximately 0.24 whereas the free tracer had negligible polarization. The fluorescence polarization was independent of analyte concentration, and the fluorescence intensity of either the free or bound tracer was used for quantitation. Results from both assays suggest that the CE-LIFP approaches may have a wider application than the immunoassays based on either CE-LIF or fluorescence polarization alone.