This report highlights the advantages of low-affinity, multivalent interactions to recognize one cell type over another. Our goal was to devise a strategy to mediate selective killing of tumor cells, which are often distinguished from normal cells by their higher levels of particular cell surface receptors. To test whether multivalent interactions could lead to highly specific cell targeting, we used a chemically synthesized small-molecule ligand composed of two distinct motifs: (1) an Arg-Gly-Asp (RGD) peptidomimetic that binds tightly (Kd approximately 10(-9)M) to alphavbeta3 integrins and (2) the galactosyl-alpha(1-3)galactose (alpha-Gal epitope), which is recognized by human anti-alpha-galactosyl antibodies (anti-Gal). Importantly, anti-Gal binding requires a multivalent presentation of carbohydrate residues; anti-Gal antibodies interact weakly with the monovalent oligosaccharide (Kd approximately 10(-5)M) but bind tightly (Kd approximately 10(-11) M) to multivalent displays of alpha-Gal epitopes. Such a display is generated when the bifunctional conjugate decorates a cell possessing a high level of alphavbeta3 integrin; the resulting cell surface, which presents many alpha-Gal epitopes, can recruit anti-Gal, thereby triggering complement-mediated lysis. Only those cells with high levels of the integrin receptor are killed. In contrast, doxorubicin tethered to the RGD-based ligand affords indiscriminate cell death. These results highlight the advantages of exploiting the type of the multivalent recognition processes used by physiological systems to discriminate between cells. The selectivity of this strategy is superior to traditional, abiotic, high-affinity targeting methods. Our results have implications for the treatment of cancer and other diseases characterized by the presence of deleterious cells.
Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance ( R ) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways. INTRODUCTIONThe molecular recognition of pathogens by plants is often characterized by a gene-for-gene relationship that requires a specific plant resistance ( R ) gene and a corresponding pathogen avirulence ( avr ) gene (Flor, 1971). Genetic evidence from a wide diversity of plant pathosystems suggests that when an appropriate R-avr gene pair is present, the result is host resistance, whereas absence or inactivation of either member of the gene pair results in susceptibility of the host to the pathogen. A common explanation for the molecular basis of this gene-for-gene relationship is an elicitorreceptor model (Gabriel and Rolfe, 1990). According to this model, avr genes directly or indirectly produce an elicitor that is recognized by the corresponding R gene-encoded receptor. This molecular interaction then triggers downstream signaling events that result in the activation of plant defenses and the limitation of pathogen growth.R genes have been cloned from several plant species (reviewed in Bent, 1996; Baker et al., 1997; Hammond-Kosack and Jones, 1997). These include R genes that mediate resistance to bacterial, fungal, oomycete, viral, and nematode pathogens. Many of these R gene products share structural motifs, which indicates that disease resistance to diverse pathogens may operate through similar pathways. For example, leucine-rich repeats (LRRs) are common to most of the R genes that have been characterized (Bent et al., 1994;Jones et al., 1994;Mindrinos et al., 1994; Whitham et al., 1994; Grant et al., 1995;Lawrence et al., 1995;Song et al., 1995; Dixon et al., 1996; Anderson et al., 1997;Parker et al., 1997). LRRs have been shown to play a role in protein-protein interactions (Kobe and Deisenhofer, 1994). This fact, along with the common occurrence of LRRs in R gene proteins, has led to speculation that LRRs serve as the binding domain for the pathogen-produced elicitor (Bent, 1996; Baker et al., 1997).Despite recent work in this area, it remains to be proven that LRR-containing R gene products function as receptors. In tomato, high-affinity binding sites from intact membranes have been found for an elicitor produced by races of Cladosporium fu...
Strategies to eliminate tumor cells have long been sought. We envisioned that a small molecule could be used to decorate the offending cells with immunogenic carbohydrates and evoke an immune response. To this end, we describe the synthesis of bifunctional ligands possessing two functional motifs: one binds a cell-surface protein and the other binds a naturally occurring human antibody. Our conjugates combine an RGD-based peptidomimetic, to target cells displaying the alpha v beta3 integrin, with the carbohydrate antigen galactosyl-alpha(1-3)galactose [Galalpha(1-3)Gal or alpha-Gal]. To generate such bifunctional ligands, we designed and synthesized RGD mimetics 1 b and 2 c, which possess a free amino group for modification. These compounds were used to generate bifunctional derivatives 1 c and 2 d, with dimethyl squarate serving as the linchpin; thus, our synthetic approach is modular. To evaluate the binding of our peptidomimetics to the target alpha v beta3-displaying cells, we implemented a cell-adhesion assay. Results from this assay indicate that the designed, small-molecule ligands inhibit alpha v beta3-dependent cell adhesion. Additionally, our most effective bifunctional ligand exhibits a high degree of selectivity (4000-fold) for alpha v beta3 over the related alpha v beta5 integrin, a result that augurs its utility in specific cell targeting. Finally, we demonstrate that the bifunctional ligands can bind to alpha v beta3-positive cells and recruit human anti-Gal antibodies. These results indicate that both the integrin-binding and the anti-Gal-binding moieties can act simultaneously. Bifunctional conjugates of this type can facilitate the development of new methods for targeting cancer cells by exploiting endogenous antibodies. We anticipate that our modifiable alpha v beta3-binding ligands will be valuable in a variety of applications, including drug delivery and tumor targeting.
Recognition of pathogens by plants is mediated by several distinct families of functionally variable but structurally related disease resistance ( R ) genes. The largest family is defined by the presence of a putative nucleotide binding domain and 12 to 21 leucine-rich repeats (LRRs). The function of these LRRs has not been defined, but they are speculated to bind pathogen-derived ligands. We have isolated a mutation in the Arabidopsis RPS5 gene that indicates that the LRR region may interact with other plant proteins. The rps5-1 mutation causes a glutamate-to-lysine substitution in the third LRR and partially compromises the function of several R genes that confer bacterial and downy mildew resistance. The third LRR is relatively well conserved, and we speculate that it may interact with a signal transduction component shared by multiple R gene pathways. INTRODUCTIONThe molecular recognition of pathogens by plants is often characterized by a gene-for-gene relationship that requires a specific plant resistance ( R ) gene and a corresponding pathogen avirulence ( avr ) gene (Flor, 1971). Genetic evidence from a wide diversity of plant pathosystems suggests that when an appropriate R-avr gene pair is present, the result is host resistance, whereas absence or inactivation of either member of the gene pair results in susceptibility of the host to the pathogen. A common explanation for the molecular basis of this gene-for-gene relationship is an elicitorreceptor model (Gabriel and Rolfe, 1990). According to this model, avr genes directly or indirectly produce an elicitor that is recognized by the corresponding R gene-encoded receptor. This molecular interaction then triggers downstream signaling events that result in the activation of plant defenses and the limitation of pathogen growth.R genes have been cloned from several plant species (reviewed in Bent, 1996; Baker et al., 1997; Hammond-Kosack and Jones, 1997). These include R genes that mediate resistance to bacterial, fungal, oomycete, viral, and nematode pathogens. Many of these R gene products share structural motifs, which indicates that disease resistance to diverse pathogens may operate through similar pathways. For example, leucine-rich repeats (LRRs) are common to most of the R genes that have been characterized (Bent et al., 1994;Jones et al., 1994;Mindrinos et al., 1994; Whitham et al., 1994; Grant et al., 1995;Lawrence et al., 1995;Song et al., 1995; Dixon et al., 1996; Anderson et al., 1997;Parker et al., 1997). LRRs have been shown to play a role in protein-protein interactions (Kobe and Deisenhofer, 1994). This fact, along with the common occurrence of LRRs in R gene proteins, has led to speculation that LRRs serve as the binding domain for the pathogen-produced elicitor (Bent, 1996; Baker et al., 1997).Despite recent work in this area, it remains to be proven that LRR-containing R gene products function as receptors. In tomato, high-affinity binding sites from intact membranes have been found for an elicitor produced by races of Cladosporium fu...
L-selectin is a leukocyte cell-surface protein that facilitates the rolling of leukocytes along the endothelium, a process that leads to leukocyte migration to a site of infection. Preventing L-selectin-mediated rolling minimizes leukocyte adhesion and extravasation; therefore, compounds that inhibit rolling may act as anti-inflammatory agents. To investigate the potential role of multivalent ligands as rolling inhibitors, compounds termed neoglycopolymers were synthesized that possess key structural features of physiological L-selectin ligands. Sulfated neoglycopolymers substituted with sialyl Lewis x derivatives (3',6-disulfo Lewis x or 6-sulfo sialyl Lewis x) or a sulfatide analog (3,6-disulfo galactose) inhibited L-selectin-mediated rolling of lymphoid cells. Functional analysis of the inhibitory ligands indicates that they also induce proteolytic release of L-selectin. Thus, their inhibitory potency may arise from their ability to induce shedding. Our data indicate that screening for compounds that promote L-selectin release can identify ligands that inhibit rolling.
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