Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

Immunohistochemical screening for beta6-integrin subunit expression in adenocarcinomas using a novel monoclonal antibody reveals strong up-regulation in pancreatic ductal adenocarcinomas in vivo and in vitro.

AIMS: To analyse the expression of alphavbeta6, an epithelial integrin involved in wound healing and tumorigenesis, in various human carcinoma types. METHODS AND RESULTS: A new monoclonal antibody to the human beta6 subunit, 5C4, was used to locate alphavbeta6 in 157 cancers of gastroenteropancreatic and 21 of lung origin. The data were validated by analysis of alphavbeta6 extracted from histological sections. Alphavbeta6 integrin showed strongest expression in 34 pancreatic ductal adenocarcinomas (mean score 2.88 +/- 0.52), followed by 24 intestinal-type gastric carcinomas (1.45 +/- 1.06) and eight lung adenocarcinomas (1.37 +/- 1.1). Moderate expression was found in 31 diffuse-type gastric carcinomas (0.94 +/- 0.83), seven duodenal adenocarcinomas (0.8 +/- 1.34) and 26 colorectal adenocarcinomas (0.76 +/- 0.71). Little alphavbeta6 was seen in seven liver cell carcinomas and six neuroendocrine tumours. Well-differentiated carcinomas expressed more beta6 than poorly differentiated tumours. Peritumoral epithelial tissues where alphavbeta6-expressing tumours arose also expressed alphavbeta6. There was no correlation between expression of alphavbeta6 and its ligands tenascin and fibronectin in pancreatic and gastric carcinomas. Spheroid formation by pancreatic carcinoma cell lines led to alphavbeta6 up-regulation, but appeared independent of classical ligand binding to alphavbeta6. CONCLUSIONS: Our findings indicate that: (i) alphavbeta6 is overexpressed in pancreatic adenocarcinomas; (ii) alphavbeta6-positive carcinomas originate from alphavbeta6-expressing tissues; (iii) alphavbeta6 expression in tumours seems to be regulated independently from that of its ligands tenascin and fibronectin; and (iv) in-vitro overexpression of alphavbeta6 in pancreatic carcinoma cell lines accompanies spheroid formation.

RGD-mutants of B-lymphotropic polyomavirus capsids specifically bind to alpha(v)beta3 integrin.

Integrins are a family of cell surface proteins that function as receptors for extracellular matrix ligands and for some viruses. A subset of integrins recognises peptide sequences containing arginine-glycine-aspartic acid (RGD) motifs as ligands. The B-lymphotropic polyomavirus (LPV) has a non-enveloped capsid that recognises a sialylated cell surface receptor. To change the receptor binding specificity we have replaced sets of three amino acids in three predicted surface loops of the major capsid protein VP1 of the B-lymphotropic polyomavirus LPV by RGD. Ten mutants gave rise to the expected 40 kDa VP1 protein upon expression from a baculovirus vector in insect cells. Five of the VP1 mutants representing all three surface loops have retained the ability to spontaneously assemble to capsids in the nuclei of the insect cells. Structural changes of the mutant capsid surface were shown by differential reactivity with a set of 7 neutralising monoclonal antibodies that recognise conformational surface epitopes of wildtype LPV virions. In addition all mutant capsids had lost specific binding to the LPV receptor. Three mutant capsids of one loop (BC) showed specific binding to alpha(v)beta3 integrin but not to integrins alpha(v)beta5, alpha(v)beta6, or to alpha(IIb)beta3 known also to recognise RGD containing peptide sequences. This selective binding of the mutant capsids could be inhibited by synthetic peptides that specifically bind to alpha(v)beta3 integrin with IC50 values between 10 and 40 nM.

Integrins, cations and ligands: making the connection.

Integrins are cell adhesion receptors that couple extracellular divalent cation-dependent recognition events with intracellular mechanical and biochemical responses and vice versa, thus affecting every function of nucleated cells. The structural basis of this bidirectional signaling and its dependency on cations has been the focus of intensive study over the past three decades. Significant progress made recently in elucidating the three-dimensional structure of the extracellular and cytoplasmic segments of integrins is giving valuable new insights into the tertiary and quaternary changes that underlie activation, ligand recognition and signaling by these receptors.

Crystal structure of the extracellular segment of integrin alpha Vbeta3.

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

Solid-phase synthesis of a nonpeptide RGD mimetic library: new selective alphavbeta3 integrin antagonists.

The solid-phase synthesis of a low molecular weight RGD mimetic library is described. Activities of the compounds in inhibiting the interaction of ligands, vitronectin and fibrinogen, with isolated immobilized integrins alphavbeta3 and alphaIIbbeta3 were determined in a screening assay. Highly active and selective nonpeptide alphavbeta3 integrin antagonists with regard to orally bioavailability were developed, based on the aza-glycine containing lead compound 1. An important variation is the substitution of the aspartic amide of 1 by an aromatic residue. Furthermore, different guanidine mimetics have been incorporated to improve the pharmacokinetic profile. Exchange of the beta-amino acid NH by a methylene moiety in one set of RGD mimetics leads to the azacarba analogue compounds representing a novel peptidomimetic approach, which should increase the metabolic stability.

Noninvasive imaging of alpha(v)beta3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography.

The alpha(v)beta3 integrin is an important cell adhesion receptor involved in tumor-induced angiogenesis and tumor metastasis. Here we describe the 18F-labeling of the RGD-containing glycopeptide cyclo(-Arg-Gly-Asp-D-Phe-Lys(sugar amino acid)-) with 4-nitrophenyl 2-[18F]fluoropropionate and the evaluation of this compound in vitro and in tumor mouse models. Binding assays with isolated immobilized alpha(v)beta3, alpha(v)beta5, and alpha(IIb)beta3 as well as in vivo studies using alpha(v)beta3-positive and -negative murine and xenotransplanted human tumors demonstrated receptor-specific binding of the radiolabeled glycopeptide yielding high tumor:background ratios (e.g., 120 min postinjection: tumor:blood, 27.5; tumor:muscle, 10.2). First imaging results using a small animal positron emission tomograph suggest that this compound is suitable for noninvasive determination of the alpha(v)beta3 integrin status and therapy monitoring.

Glycosylated RGD-containing peptides: tracer for tumor targeting and angiogenesis imaging with improved biokinetics.

UNLABELLED: The alpha(v)beta3 integrin plays an important role in metastasis and tumor-induced angiogenesis. Targeting with radiolabeled ligands of the alpha(v)beta3 integrin may provide information about the receptor status and enable specific therapeutic planning. Previous studies from our group resulted in tracers that showed alpha(v)beta3-selective tumor uptake. However, these first-generation compounds predominantly revealed hepatobiliary excretion with high radioactivity found in the liver. In this report, the synthesis and biological evaluation of the first glycosylated RGD-containing peptide (RGD-peptide) for the noninvasive imaging of alpha(v)beta3 expression are described. METHODS: Peptides were assembled on a solid support using fluorenylmethoxycarbonyl-coupling protocols. The precursor cyclo(-Arg-Gly-Asp-D-Tyr-Lys(SAA)-) GP1 was synthesized by coupling 3-acetamido-2,6-anhydro-4,5,7-tri-O-benzyl-3-deoxy-beta-D-glycero-D-gulo-heptonic acid (SAA(Bn3)) with cyclo(-Arg(Mtr)-Gly-Asp(OtBu)-D-Tyr(tBu)-Lys-) and subsequent removal of the protection groups. Iodine labeling was performed by the Iodo-Gen method (radiochemical yield > 50%). The in vitro binding assays were performed using purified immobilized alpha(IIb)beta3, alpha(v)beta5, and alpha(v)beta3 integrins. For in vivo experiments, nude mice bearing xenotransplanted melanomas and mice with osteosarcomas were used. RESULTS: The glycosylated peptide 3-iodo-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Lys(SAA)-) GP2 showed high affinity and selectivity for alpha(v)beta3 in vitro (50% inhibitory concentration = 40 nmol/L). Pretreatment studies indicate specific binding of [125I]GP2 on alpha(v)beta3-expressing tumors in vivo. Comparison of the pharmacokinetics of [125I]GP2 and [125I]-3-iodo-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) [125I]P2 revealed for [125I]GP2 an increased activity concentration in the blood (e.g., 3.59 +/- 0.35 percentage injected dose [%ID]/g vs. 1.72 +/- 0.44 %ID/g at 10 min postinjection) and a significantly reduced uptake in the liver (e.g., 2.59 +/- 0.24 %ID/g vs. 21.96 +/- 2.78 %ID/g at 10 min postinjection). Furthermore, a clearly increased activity accumulation in the tumor was found (e.g., 3.05 +/- 0.31 %ID/g vs. 0.92 +/- 0.16 %ID/g at 240 min postinjection), which remained almost constant between 60 and 240 min postinjection. This resulted in good tumor-to-organ ratios for the glycosylated tracer (e.g., 240-min postinjection osteosarcoma model: tumor-to-blood = 16; tumor-to-muscle = 7; tumor-to-liver = 2.5), which were confirmed by the first gamma-camera images of osteosarcoma-bearing mice at 240 min postinjection. CONCLUSION: This study demonstrates that the introduction of a sugar moiety improves the pharmakokinetic behavior of a hydrophobic peptide-based tracer. Additionally, this alpha(v)beta3-selective glycosylated radioiodinated second-generation tracer GP2 shows high tumor uptake and good tumor-to-organ ratios that allow noninvasive visualization of alpha(v)beta3-expressing tumors and monitoring therapy with alpha(v)beta3 antagonists. Finally, the favorable biokinetics make the glycosylated RGD-peptide a promising lead structure for tracers to quantify the alpha(v)beta3 expression using PET.

Synthesis and biological evaluation of integrin antagonists containing trans- and cis-2,5-disubstituted THF rings.

The synthesis of a series of RGD mimetics is described. All compounds consist of a central 2,5-disubstituted tetrahydrofuran core, a variable linker to a guanidino group, and a beta-amino alanine unit to mimic the carboxylic acid. Three types of linkers were investigated: a simple four-atom methylene chain (type A, compounds 14, 15, 16, and 17), a four-atom methylene chain with an additional chiral center, and a nitrogen substituent (type B, compounds 38, 39, and 40), and an amide linker of different length with an additional chiral center (type C, compounds 59, 60, 61, and 62). A variety of compounds were tested as potential integrin antagonists in a receptor binding assay (alphaIIbbeta3, alphavbeta3, and alphavbeta5). The relative and absolute configuration of the chiral centers at the THF ring had a pronounced effect on the binding activity and selectivity. Compound 14 proved to be a selective inhibitor of alphaIIbbeta3 (IC50=20nM), whereas compound 40 exhibited high activity for binding of alphaIIbbeta3 (IC50=67nM) and alphavbeta3 (IC50=52nM).

Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy.

Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor.

Nanoscale topography of the basement membrane underlying the corneal epithelium of the rhesus macaque.

This paper quantitatively defines the nanoscale topography of the basement membrane underlying the anterior corneal epithelium of the macaque. Excised corneal buttons from macaques were placed in 2.5 mM ethylenediaminetetraacetate (EDTA) for 2.5 h, after which the epithelium was carefully removed to expose the underlying basement membrane. The integrity of the remaining basement membrane was verified using fluorescent microscopy in conjunction with antibody staining directed against laminin and collagen type IV as well as transmission electron microscopy. Characterization of the surface of the basement membrane was performed using transmission electron microscopy, high-resolution, low-voltage scanning electron microscopy, and atomic force microscopy. Quantitative data were obtained with all three imaging techniques and compared. The basement membrane has a complex topography consisting of tightly cross-linked fibers intermingled with pores. The mean elevation of features measured by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy was 149 +/- 60 nm, 191 +/- 72 nm, and 147 +/- 73 nm, respectively. Mean fiber diameter as measured by SEM was 77 +/- 44 nm and pore diameter was 72 +/- 40 nm, with pores occupying approximately 15% of the total surface area. Similar feature types and dimensions were also found for Matrigel, a commercially available basement membrane-like complex, supporting that a minimum of artifact was introduced by corneal preparative procedures to remove the overlying epithelium. Topographic features amplified the surface area over which cell-substratum interactions occur by an estimated 400%. The three-dimensional structure of the basement membrane exhibits a rich complex topography of individual features, consisting of pores and fibers with dimensions ranging from 30 to 400 nm. These nanoscale substratum features may modulate fundamental cell behaviors such as adhesion, migration, proliferation, and differentiation.

Nanoscale topography of the corneal epithelial basement membrane and Descemet's membrane of the human.

PURPOSE: Quantitatively define and compare the nanoscale topography of the corneal epithelial basement membrane (anterior basement membrane) and Descemet's membrane (posterior basement membrane) of the human. METHODS: Human corneas not suitable for transplantation were obtained from the Wisconsin Eye Bank. The corneas were placed in 2.5 mM EDTA for 2.5 h or 30 min. for removal of the epithelium or endothelium, respectively. After removal of the overlying cells, specimens were fixed in 2% glutaraldehyde and either examined in this state by atomic force microscopy only or dehydrated through an ethanol series and prepared for transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). RESULTS: The subepithelial and subendothelial basement membrane surfaces have a similar appearance that consists of an interwoven meshwork of fibers and pores. Topographic feature sizes were found to be in the nanometer size range with the epithelial basement membrane features larger and less densely packed than Descemet's membrane features. The topographic features are fractile in nature and increase surface area for cell contact. CONCLUSION: With the use of the TEM, SEM, and AFM, a detailed description of the surface topography of corneal epithelial basement membrane and Descemet's membrane of the human cornea are provided. The significance of differences in corneal basement membrane topography may reflect differences in function of the overlying cells or may be related to differences in cell migration and turnover patterns between the epithelium and endothelium.

Surface coating with cyclic RGD peptides stimulates osteoblast adhesion and proliferation as well as bone formation.

The physiological inertness of synthetic implant materials often results in insufficient implant integration and limited acceptance of implants in tissues. After implantation the implant surface is often separated from the surrounding healthy and regenerating tissue, for example by a fibrous capsule. To avoid this host-versus-graft reaction, a strong mechanical contact between tissue and implant must be ensured. An enhanced contact between graft and the surrounding tissue can be provided by coating the implant with cell-adhesive molecules. The highly active and alpha(v)beta(3)- and alpha(v)beta(5)-integrin-selective peptide c(-RGDfK-) (f=D-phenylalanine) was functionalized with various linker molecules containing an acrylamide end group by using the lysine side chain of c(-RGDfK-). The acrylamide group can be used to bind the peptide covalently to poly(methyl methacrylate) (PMMA) surfaces. The coated surfaces effectively bind to murine osteoblasts as well as human osteoblasts in vitro when a minimum distance of 3.5 nm between surface and the constrained RGD sequence is provided. In contrast to osteoblasts in cell suspension, surface-bound osteoblasts show no apoptosis but proliferate by a factor of 10 over a 22 d period. Coating of inert implant surfaces with highly active and alpha(v)-selective peptides affords a marked improvement in osteoblast binding over current technologies. In vivo studies show that peptide-coated PMMA pellets implanted into the patella groove of rabbits are integrated into the regenerating bone tissue faster and more strongly than uncoated pellets.

N-Methylated cyclic RGD peptides as highly active and selective alpha(V)beta(3) integrin antagonists.

The alpha(V)beta(3) integrin receptor plays an important role in human tumor metastasis and tumor-induced angiogenesis. The in vivo inhibition of this receptor by antibodies or by cyclic peptides containing the RGD sequence may in the future be used to selectively suppress these diseases. Here we investigate the influence of N-methylation of the active and selective alpha(V)beta(3) antagonist cyclo(RGDfV) (L1) on biological activity. Cyclo(RGDf-N(Me)V-) (P5) was found to be even more active than L1 and is one of the most active and selective compounds in inhibiting vitronectin binding to the alpha(V)beta(3) integrin. Its high-resolution, three-dimensional structure in water was determined by NMR techniques, distance geometry calculations, and molecular dynamics calculations, providing more insight into the structure-activity relationship.

Sheep, pig, and human platelet-material interactions with model cardiovascular biomaterials.

The relationship between cardiovascular device performance in animals and humans is not straightforward. As the principal formed element in a thrombus, platelets play a major role in determining the hemocompatibility of mechanical heart valves and other high-shear-rate cardiovascular devices. Since larger animals are required to test many such devices, sheep and porcine platelet responses were compared to humans. Adhesion, spreading, and the formation of thrombilike structures were examined in vitro on pyrolytic carbon mechanical heart valve leaflets, National Institutes of Health-reference polyethylene and silicone rubber, and Formvar. Principal findings were that platelet responses are strongly dependent upon the biomaterial and the species: Porcine and human platelets spread extensively on pyrolytic carbon, formed thrombuslike structures on Formvar, and were least active on silicone rubber. Human and porcine platelets responded differently to polyethylene: Human platelets spread extensively, while porcine platelets remained pseudopodial. In contrast, sheep platelets attached much less, never reached fully spread shapes, and were far less active overall. Since porcine responses were generally similar to humans, pigs may be a useful predictor of in vivo platelet-biomaterial interaction in humans. Conversely, as ovine platelets were much less active, this must be accounted for in the evaluation of cardiovascular devices tested in sheep.

Effects of synthetic micro- and nano-structured surfaces on cell behavior.

Topographical cues, independent of biochemistry, generated by the extracellular matrix may have significant effects upon cellular behavior. Studies have documented that substratum topography has direct effects on the ability of cells to orient themselves, migrate, and produce organized cytoskeletal arrangements. Basement membranes are composed of extracellular matrix proteins and found throughout the vertebrate body, serving as substrata for overlying cellular structures. The topography of basement membranes is a complex meshwork of pores, fibers, ridges, and other features of nanometer sized dimensions. Synthetic surfaces with topographical features have been shown to influence cell behavior. These facts lead to the hypothesis that the topography of the basement membrane plays an important role in regulating cellular behavior in a manner distinct from that of the chemistry of the basement membrane. This paper describes the topography of the basement membrane and reviews the fabrication of synthetic micro- and nano-structured surfaces and the effects of such textured surfaces on cell behavior.

Decreased angiogenesis and arthritic disease in rabbits treated with an alphavbeta3 antagonist.

Rheumatoid arthritis (RA) is an inflammatory disease associated with intense angiogenesis and vascular expression of integrin alphavbeta3. Intra-articular administration of a cyclic peptide antagonist of integrin alphavbeta3 to rabbits with antigen-induced arthritis early in disease resulted in inhibition of synovial angiogenesis and reduced synovial cell infiltrate, pannus formation, and cartilage erosions. These effects were not associated with lymphopenia or impairment of leukocyte function. Furthermore, when administered in chronic, preexisting disease, the alphavbeta3 antagonist effectively diminished arthritis severity and was associated with a quantitative increase in apoptosis of the angiogenic blood vessels. Therefore, angiogenesis appears to be a central factor in the initiation and persistence of arthritic disease, and antagonists of integrin alphavbeta3 may represent a novel therapeutic strategy for RA.

Definition of an unexpected ligand recognition motif for alphav beta6 integrin.

Integrin interactions with extracellular matrix proteins are mediated by brief oligopeptide recognition sequences, and synthetic peptides containing such sequences can inhibit integrin binding to the matrix. The RGD peptide motif is recognized by many integrins including alphav beta6, a specific receptor for fibronectin thought to support epithelial cell proliferation during wound healing and carcinoma progression. We report here the discovery of an unexpected non-RGD recognition motif for integrin alphav beta6. We compared the recognition profiles of recombinant alphav beta6 and alphav beta3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to alphav beta3 contained ubiquitous RGD sequences. However, on alphav beta6, in addition to RGD- containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL, selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to alphav beta6 in isolated receptor assays (IC50 = 20 nM), and in cell adhesion assays (IC50 = 50 microM). DLXXL peptides were highly specific inhibitors of alphav beta6-fibronectin interaction as synthetic scrambled or reversed DLXXL peptides were inactive. NH2- and COOH-terminal modifications of the flanking amino acids suggested that the preceding two and a single trailing amino acid were also involved in interaction with alphav beta6. The DLXXL sequence is present in several matrix components and in the beta chain of many integrins. Although there is as yet no precise biological role known for DLXXL, it is clearly a specific inhibitory sequence for integrin alphav beta6 which has been unrecognized previously.

Design, synthesis and biological evaluation of nonpeptide integrin antagonists.

Recent studies demonstrated that peptide and antibody antagonists of integrin alpha v beta 3 block angiogenesis and tumor growth. In this article, the design, synthesis and biological evaluation of a series of nitroaryl ether-based, nonpeptide mimetics are described. The design of these compounds was based on Merck's arylether/alpha-aminoacid/guanidine framework and incorporates a novel nitroaryl system. The synthesized mimetics were tested against a variety of integrins (alpha v beta 3, alpha IIb beta 3, and alpha v beta 5) in order to determine their binding selectivity and ability to inhibit cell adhesion. Selected compounds were also tested for their ability to inhibit angiogenesis in vivo in the CAM (chick chorioallantoic membrane) assay. From the generated compound library, compounds 16 and 19 proved to be potent and selective inhibitors of alpha IIb beta 3 (IC50 = 14 nM) whereas compound 11 showed excellent in vivo inhibition of angiogenesis (at 30 micrograms/embryo).