共查询到20条相似文献,搜索用时 31 毫秒
1.
Josan JS Handl HL Sankaranarayanan R Xu L Lynch RM Vagner J Mash EA Hruby VJ Gillies RJ 《Bioconjugate chemistry》2011,22(7):1270-1278
Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 ? is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging. 相似文献
2.
Multivalency - a way to enhance binding avidities and bioactivity - preliminary applications to EPO.
Multivalency has advantages over monovalency for binding interactions and even for activity. In particular, avidity is higher since the off-rate of a multivalent species is much slower than that of a monomer. This is particularly profitable for ligand-binding receptors that require dimerization for activity, such as the receptor of erythropoietin (EPOR). Peptides that mimic the action of erythropoietin (EPO) have been described with no sequence similarity with the human hormone: erythropoietin mimetic peptide (EMP) and EPO receptor peptide (ERP). These two peptides have similar activity but interact through different sites on the EPOR. Here, we describe the construction of several new synthetic homo- and hetero-dimers based on EMP-ERP sequences. To link the monomeric molecules together, several monodisperse polyamide linkers of different lengths were synthesized with dialdehyde functionalities. Chemoselective oxime chemistry was used to obtain homogeneous constructs. Certain chemical incompatibilities were dealt with via a protection approach. The oximes are stable under normal conditions and so lend themselves to biological testing. 相似文献
3.
Ramesh Alleti Josef Vagner Dilani Chathurika Dehigaspitiya Valerie E. Moberg N.G.R.D. Elshan Narges K. Tafreshi Nabila Brabez Craig S. Weber Ronald M. Lynch Victor J. Hruby Robert J. Gillies David L. Morse Eugene A. Mash 《Bioorganic & medicinal chemistry》2013,21(17):5029-5038
Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27 ± 3.9 nM and 4.2 ± 0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported. 相似文献
4.
《Bioorganic & medicinal chemistry》2014,22(22):6360-6365
Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25 ± 10 Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH2 was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-c[Asp-His-2′-D-Nal-Arg-Trp-Lys]-NH2 and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)3 units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands. 相似文献
5.
Increasing valence can enhance the ability of molecular targeting constructs to bind specifically to targeted cells for drug delivery. Here, we mathematically model the length and flexibility of a linker used to conjoin two peptide ligands of a divalent targeting construct and investigate the influence both on binding avidity and specificity. Four different models are used to approximate varying degrees of linker flexibility (random coil, rigid rod, jointed rods, and combined rod-random coil) and for each linker a binding enhancement factor (VR) is derived that quantifies the increased rate of each construct's second binding event over the first. Results indicate that the moderately flexible models can best reproduce experimentally measured avidities. Also, the magnitude of VR, in conjunction with receptor density and ligand concentration, significantly influences the achievable specificity. Thus, the model elucidates important considerations in designing multivalent targeting constructs for use in delivery of targeted therapy or imaging. 相似文献
6.
Effective targeting of drugs to cells requires that the drug reach the target cell and interact specifically with it. In this study, we synthesized a biomacromolecular, multivalent construct intended to target glioblastoma tumors. The construct was created by linking three dodecapeptides, reported to bind the alpha 6beta1 integrin, with poly(ethylene glycol) linkers. The construct is intended to be delivered locally, and it demonstrates a more homogeneous and more rapid perfusion profile in comparison with quantum dots. The binding specificity of the construct was investigated by using glioblastoma cells and normal human astrocyte cells. The results reveal qualitative differences in binding between glioma and normal human astrocyte cells, with a moderate increase in binding avidity due to multivalency (0.79 microM for the trivalent construct versus 4.28 microM for the dodecapeptide). Overall, biomacromolecular constructs appear to be a promising approach for targeting with high biocompatibility, good perfusion abilities, and specificity. 相似文献
7.
《MABS-AUSTIN》2013,5(8):1157-1167
ABSTRACTSecreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8. We then used combinatorial antibody engineering by phage display to develop a variant antibody F2.A with specificity broadened to include FZD4. We confirmed that F2.A blocked binding of Wnt ligands, but not binding of Norrin, a ligand that also activates FZD4. Importantly, F2.A proved to be much more efficacious than either OMP-18R5 or F2 in inhibiting the growth of multiple RNF43-mutant pancreatic ductal adenocarcinoma cell lines, including patient-derived cells. 相似文献
8.
Michael Reynolds Marco Marradi Anne Imberty Soledad Penadés Serge Pérez 《Glycoconjugate journal》2013,30(8):747-757
Polyvalent carbohydrate-protein interactions play a key role in bio- and pathological processes, including cell-cell communication and pathogen invasion. In order to study, control and manipulate these interactions gold nanoparticles have been employed as a 3D scaffold, presenting carbohydrate ligands in a multivalent fashion for use as high affinity binding partners and a model system for oligosaccharide presentation at biomacromolecular surfaces. In this study, the binding of a series of mannose-functionalised gold nanoparticles to the dimeric BC2L-A lectin from Burkholderia cenocepacia has been evaluated. BC2L-A is known to exhibit a high specificity for (oligo)mannosides. Due to the unique structure and binding nature of this lectin, it provides a useful tool to study (oligo)saccharides presented on multivalent scaffolds. Surface plasmon resonance and isothermal titration calorimetric assays were used to investigate the effect of ligand presentation density towards binding to the bacterial lectin. We show how a combination of structural complementarities between ligand presentation and lectin architecture and statistical re-binding effects are important for increasing the avidity of multivalent ligands for recognition by their protein receptors; further demonstrating the application of glyconanotechnology towards fundamental glycobiology research as well as a potential towards biomedical diagnostics and therapeutic treatments. 相似文献
9.
Laura E. Ruff Ajay A. Sapre Justin S. Plaut Elisabeth De?Maere Charlotte Mortier Valerie Nguyen Kevin Separa Sofie Vandenbogaerde Laura Vandewalle Sadik C. Esener Bradley T. Messmer 《Nucleic acids research》2016,44(10):e96
High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life''s own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions. 相似文献
10.
Vagner J Handl HL Monguchi Y Jana U Begay LJ Mash EA Hruby VJ Gillies RJ 《Bioconjugate chemistry》2006,17(6):1545-1550
Rigid linkers of variable length were used to connect two high-affinity Nle4-D-Phe7-alpha-melanocyte stimulating hormone (NDP-alpha-MSH) or two low-affinity MSH(4) ligands. The linked peptides were synthesized by solid-phase methods. Control experiments indicate there is little or no effect of these linkers on NDP-alpha-MSH or MSH(4) binding to the human melanocortin 4 receptor (hMC4R). Tethering two high-affinity ligands gave no binding enhancement, while tethering two low-affinity ligands resulted in binding enhancement that decreased with increased linker length. Furthermore, for the low-affinity ligands, the enhancement of affinity is inversely proportional to the estimated molecular moments of inertia. These results are consistent with a model wherein binding is enhanced when the rate of ligand reattachment to the receptor is fast relative to the rate of ligand diffusion. 相似文献
11.
Human placental cytosol contains an androgen binding protein which binds the synthetic androgen methyltrienolone (R 1881) with high affinity (Kd 8.7 nM) and with an average binding capacity of 518 fmol/mg cytosol protein. This study provides further evidence that this protein is distinguishable from classical androgen receptors on the basis of steroid specificity and sulphydryl group sensitivity. Covalent labeling studies have shown this protein, which we have called "the methyltrienolone binding protein", to have a mol. wt of 67,000 daltons. 相似文献
12.
Differential IgG-binding characteristics of staphylococcal protein A, streptococcal protein G, and a chimeric protein AG 总被引:4,自引:0,他引:4
M Eliasson R Andersson A Olsson H Wigzell M Uhlén 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(2):575-581
Various Gram-positive bacteria express different types of IgG-binding receptors, each of which displaying certain unique binding properties. To evaluate specificity and avidity aspects of the differential binding pattern, a set of competitive binding assays was employed, by using staphylococcal protein A (SPA), streptococcal protein G (SPG), and a chimeric protein AG. These receptors were analyzed, in a reciprocal fashion, for binding and inhibition of binding to a selected panel of polyclonal and monoclonal Ig. Results of the study reveal that a majority of the determinants on human and bovine IgG, recognized by SPA and SPG, are either coextensive or closely overlapping. Accordingly, a minor portion of the determinants appear to be unique in the sense that a particular determinant(s) is selectively identified by one of the two receptors. Binding assays involving purified Fc fragments from human IgG, suggest that SPG shows exclusive specificity for an Fab region determinant(s) not recognized by SPA, whereas the Fc determinants for SPA and SPG are identical or overlapping. Furthermore, one of the IgG subclasses of bovine origin appears to be seen by the SPG receptor only. The competition study also demonstrates that the novel chimeric protein AG receptor shows higher or equal avidity for variants of human IgG molecules compared to the best of its parental constituents. It can thus be deduced that chimeric receptors might be useful as optimized tools for immunologic applications. 相似文献
13.
Jagadish B Sankaranarayanan R Xu L Richards R Vagner J Hruby VJ Gillies RJ Mash EA 《Bioorganic & medicinal chemistry letters》2007,17(12):3310-3313
A regiochemical and stereochemical mixture of flexible linkers bearing terminal azide functionality was synthesized in two steps from squalene and was used to connect two high affinity NDP-alpha-MSH ligands or two low affinity MSH(4) ligands. The ligands were N-terminally acylated using N-hydroxysuccinimidoyl 5-hexynoate and were subsequently attached to the linker via copper-catalyzed 'click' 3+2 cyclization of the azide and alkyne moieties. In vitro biological evaluations showed that the binding affinity to the human melanocortin 4 receptor was not diminished for most linker-ligand combinations relative to the corresponding parental ligand. Statistical and cooperative binding effects were observed for dimeric constructs containing the low affinity ligand MSH(4), but not for dimeric NDP-alpha-MSH constructs, presumably due to slow off rates for this high affinity ligand. 相似文献
14.
Mechanism of Selective VEGF-A Binding by Neuropilin-1 Reveals a Basis for Specific Ligand Inhibition
Neuropilin (Nrp) receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF) family of proangiogenic cytokines and the semaphorin 3 (Sema3) family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A164/5 isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3. 相似文献
15.
《Journal of molecular biology》2019,431(24):4784-4795
Multidomain proteins often interact through several independent binding sites connected by disordered linkers. The architecture of such linkers affects avidity by modulating the effective concentration of intramolecular binding. The linker dependence of avidity has been estimated theoretically using simple physical models, but such models have not been tested experimentally because the effective concentrations could not be measured directly. We have developed a model system for bivalent protein interactions connected by disordered linkers, where the effective concentration can be measured using a competition experiment. We characterized the bivalent protein interactions kinetically and thermodynamically for a variety of linker lengths and interaction strengths. In total, this allowed us to critically assess the existing theoretical models of avidity in disordered, multivalent interactions. As expected, the onset of avidity occurs when the effective concentration reached the dissociation constant of the weakest interaction. Avidity decreased monotonously with linker length, but only by a third of what is predicted by theoretical models. We suggest that the length dependence of avidity is attenuated by compensating mechanisms such as linker interactions or entanglement. The direct role of linkers in avidity suggests they provide a generic mechanism for allosteric regulation of disordered, multivalent proteins. 相似文献
16.
Jane C. Spetzler Morten Meldal Ernst Meinjohanns Lucilla Steinaa Sren Mouritsen Klaus Bock 《Journal of peptide science》1997,3(6):397-414
The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid, Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides. © 1997 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
17.
L Savini G Campiani A Gaeta C Pellerano C Fattorusso L Chiasserini J M Fedorko A Saxena 《Bioorganic & medicinal chemistry letters》2001,11(13):1779-1782
Based upon synthetic and biochemical results, a novel and potent tacrine analogue and heterobivalent analogues of tacrine, were designed. The role played by the amino groups of homo- and heterobivalent ligands in the interaction with the peripheral and catalytic sites of AChE and BuChE were investigated. The syntheses of these materials together with the results of AChE/BuChE inhibition assays are detailed. 相似文献
18.
The galectin family of lectins regulates multiple biologic functions, such as development, inflammation, immunity, and cancer. One common function of several galectins is the ability to trigger T cell death. However, differences among the death pathways triggered by various galectins with regard to glycoprotein receptors, intracellular death pathways, and target cell specificity are not well understood. Specifically, galectin-9 and galectin-1 both kill thymocytes, peripheral T cells, and T cell lines; however, we have found that galectin-9 and galectin-1 require different glycan ligands and glycoprotein receptors to trigger T cell death. The two galectins also utilize different intracellular death pathways, as galectin-9, but not galectin-1, T cell death was blocked by intracellular Bcl-2, whereas galectin-1, but not galectin-9, T cell death was blocked by intracellular galectin-3. Target cell susceptibility also differed between the two galectins, as galectin-9 and galectin-1 killed different subsets of murine thymocytes. To define structural features responsible for distinct activities of the tandem repeat galectin-9 and dimeric galectin-1, we created a series of bivalent constructs with galectin-9 and galectin-1 carbohydrate recognition domains connected by different peptide linkers. We found that the N-terminal carbohydrate recognition domain and linker peptide contributed to the potency of these constructs. However, we found that the C-terminal carbohydrate recognition domain was the primary determinant of receptor recognition, death pathway signaling, and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency make distinct contributions to the specific effects of different galectins in initiating T cell death. 相似文献
19.
Newman AH Cao J Bennett CJ Robarge MJ Freeman RA Luedtke RR 《Bioorganic & medicinal chemistry letters》2003,13(13):2179-2183
The dopamine D(3) receptor subtype has been targeted as a potential neurochemical modulator of the behavioral actions of psychomotor stimulants, such as cocaine. Previous synthetic studies provided structural requirements for high affinity binding to D(3) receptors which included a 2,3-dichloro-phenylpiperazine linked to an arylamido function via a butyl chain. To reduce lipophilicity of these agents and further investigate optimal conformation, a second series of 15 novel ligands was designed that included heteroaromatic substitution and unsaturated alkyl linkers. These compounds were synthesized and evaluated for binding at rat D(3) and D(2) receptors stably expressed in Sf9 cells. D(3) binding affinities ranged from K(i)=0.6-1080 nM, with a broad range of D(3)/D(2) selectivities (2-97). The discovery of potent, selective and bioavailable D(3) receptor ligands will provide essential molecular probes to elucidate the role D(3) receptors play in the psychomotor stimulant and reinforcing effects of cocaine. 相似文献
20.
Abstract Atheoretical study is presented of complex formation between DNA fragments of different base sequences and isolexins, “isohelical base reading polymers”, formed of heteroaromatic pentagonal rings joined by appropriate linkers. Extensive computations are performed for the isolexin composed of the furan-pyrrole-furan sequence. They involve charged ligands with propioamidinium groups at both ends as well eis neutral molecules with terminal methyl, carbonyl and amino groups. Two different groups (C=O and NH) are used as linkers between the base reading moieties. The role of these elements on the binding preference of the ligands has been examined. The results show that the mere possibility of formation of hydrogen bonds between a ligand and the nucleic acid bases is not sufficient to ensure its binding specificity which is determined largely by the interplay of electrostatic factors. Thus the dicationic isolexins uniformly prefer AT sequences. For the neutral isolexins the nature of the groups forming the linkers is a major factor in defining the specificity, although these groups do not participate directly in the interaction with DNA The C=O linkers favour binding to AT sequence while the N-H linkers permit preferential binding to the GAG sequence. Finally, for the first time in theoretical computations, a ligand is proposed which should bind preferentially to the minor groove of GC sequences: this ligand is a neutral isolexin composed of three furan rings linked by two N-H groups. This ligand is considered as an improvable prototype. Altogether the results presented open the path for the designing of minor groove ligands specific for any desirable DNA base sequence. 相似文献