Dynamics and Physics of Integrin Activation in Tumor Cells by Nano-Sized Extracellular Ligands and Electromagnetic Fields.

Integrins are stress-sensing proteins expressed on the surface of cells. They regulate bidirectional signal transduction during cell-cell or cell-extracellular matrix (ECM) contacts. Integrins link the ECM with the cytoplasm through interaction with their ligands. Biophysically, such interactions can be understood as changes in stress fields at specific integrin stress-sensing domains, such as the MIDAS and ADMIDAS domains. Stress changes between ligands and cytoskeletal structures are involved in cancer cell growth by altering signal transduction pathways dependent on integrin activation. In this chapter, previous results regarding integrin activation and tumor cell growth using nanoparticles (NPs) of different materials, sizes and shapes are placed within a framework of polarized NPs in the ECM by external electromagnetic fields, in which the synergic action between polarized NPs and electromagnetic fields activates the integrins. Small size NPs activate integrins via the polar component of the dipole force between NPs and integrin sensing stress sites, while large size NPs exercise a similar action via the radial component. A quantum electrodynamic model also accounts for ECM overstressing by electromagnetic mode trapping between coherent symmetric and antisymmetric quantum states.

Could Dissecting the Molecular Framework of ß-Lactam Integrin Ligands Enhance Selectivity?

By dissecting the structure of ß-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins αvß3, α5ß1, and α4ß1. New selective ligands with antagonist or agonist activities of cell adhesion in the nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells as a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseointegration and bone regeneration. Molecular modeling studies on prototypic compounds and αvß3 or α5ß1 integrin supported the notion that ligand carboxylate fixing to the metal ion-dependent adhesion site in the ß-subunit can be sufficient for binding the receptors, while the aryl side chains play a role in determining the selectivity as well as agonism versus antagonism.

Chiral ß-lactam-based integrin ligands through Lipase-catalysed kinetic resolution and their enantioselective receptor response.

Obtainment and testing of pure enantiomers are of great importance for bioactive compounds, because of the assessed implications of enantioselectivity in receptor-mediated responses. Herein we evaluated the use of biocatalysis to obtain enantiomerically pure ß-lactam intermediates further exploited in the synthesis of novel integrin ligands as single enantiomers. From a preliminary screening on a set of commercially available hydrolases, Burkholderia Cepacia Lipase (BCL) emerged as a suitable and highly performing enzyme for the kinetic resolution of a racemic azetidinone, key intermediate for the synthesis of novel agonists of integrins. Upon optimization of the biocatalytic protocol in terms of enzymes, acylating agents and procedures, the two ß-lactam enantiomers were obtained in excellent enantiomeric excesses (94% and 98% ee). Synthetic elaborations on the separated enantiomers allowed the synthesis of four chiral ß-lactams which were evaluated in cell adhesion assays on Jurkat cell line expressing α4ß1 integrin, and K562 cell line expressing α5ß1 integrin. Biological tests revealed that only (S)-enantiomers maintained the agonist activity of racemates with a nanomolar potency, and a specific enantio-recognition by integrin receptors was demonstrated.

On the contributing role of the transmembrane domain for subunit-specific sensitivity of integrin activation.

Integrins are α/ß heterodimeric transmembrane adhesion receptors. Evidence exists that their transmembrane domain (TMD) separates upon activation. Subunit-specific differences in activation sensitivity of integrins were reported. However, whether sequence variations in the TMD lead to differential TMD association has remained elusive. Here, we show by molecular dynamics simulations and association free energy calculations on TMDs of integrin αIIbß3, αvß3, and α5ß1 that αIIbß3 TMD is most stably associated; this difference is related to interaction differences across the TMDs. The order of TMD association stability is paralleled by the basal activity of these integrins, which suggests that TMD differences can have a decisive effect on integrin conformational free energies. We also identified a specific order of clasp disintegration upon TMD dissociation, which suggests that the closed state of integrins may comprise several microstates. Our results provide unprecedented insights into a possibly contributing role of TMD towards subunit-specific sensitivity of integrin activation.

SU9516 Increases α7ß1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7ß1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7ß1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7ß1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD.

A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins.

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins αvß3, αvß5, αvß6, αvß8, α5ß1, αIIbß3, using homogenous ELISA-like solid phase binding assay.

New ß-Lactam Derivatives Modulate Cell Adhesion and Signaling Mediated by RGD-Binding and Leukocyte Integrins.

A novel series of ß-lactam derivatives that was designed and synthesized to target RGD-binding and leukocyte integrins is reported. The compound library was evaluated by investigating the effects on integrin-mediated cell adhesion and cell signaling in cell lines expressing αvß3, αvß5, αvß6, α5ß1, αIIbß3, α4ß1, and αLß2 integrins. SAR analysis of the new series of azetidinones enabled the recognition of structural elements associated with integrin selectivity. We obtained selective and potent agonists that could induce cell adhesion and promote cell signaling mediated by αvß3, αvß5, α5ß1, or α4ß1 integrin, and antagonists for the integrins αvß3 and α5ß1, as well as α4ß1 and αLß2, preventing the effects elicited by the respective endogenous agonists.

Downstream effect profiles discern different mechanisms of integrin αLß2 inhibition.

The integrin leucocyte function-associated antigen-1 (αLß2, LFA-1) plays crucial roles in T cell adhesion, migration and immunological synapse (IS) formation. Consequently, αLß2 is an important therapeutic target in autoimmunity. Three major classes of αLß2 inhibitors with distinct modes of action have been described to date: Monoclonal antibodies (mAbs), small molecule α/ß I allosteric and small molecule α I allosteric inhibitors. The objective of this study was to systematically compare these three modes of αLß2 inhibition for their αLß2 inhibitory as well as their potential agonist-like effects. All inhibitors assessed were found to potently block αLß2-mediated leucocyte adhesion. None of the inhibitors induced ZAP70 phosphorylation, indicating absence of agonistic outside-in signalling. Paradoxically, however, the α/ß I allosteric inhibitor XVA143 induced conformational changes within αLß2 characteristic for an intermediate affinity state. This effect was not observed with the α I allosteric inhibitor LFA878 or the anti-αLß2 mAb efalizumab. On the other hand, efalizumab triggered the unscheduled internalization of αLß2 in CD4+ and CD8+ T cells while LFA878 and XVA143 did not affect or only mildly reduced αLß2 surface expression, respectively. Moreover, efalizumab, in contrast to the small molecule inhibitors, disturbed the fine-tuned internalization/recycling of engaged TCR/CD3, concomitantly decreasing ZAP70 expression levels. In conclusion, different modes of αLß2 inhibition are associated with fundamentally different biologic effect profiles. The differential established here is expected to provide important translational guidance as novel αLß2 inhibitors will be advanced from bench to bedside.

Periostin and TGF-ß-induced protein: Two peas in a pod?

Periostin (PN) and TGF-ß-induced protein (ßig-h3) are paralogs that contain a single emilin and four fasciclin-1 modules and are secreted from cells. PN receives attention because of its up-regulation in cancer and degenerative and allergic diseases. ßig-h3 is highly enriched in cornea and best known for harboring mutations in humans associated with corneal dystrophies. Both proteins are expressed widely, and many functions, some over-lapping, have been attributed to PN and ßig-h3 based on biochemical, cell culture, and whole animal experiments. We attempt to organize this knowledge so as to facilitate research on these interesting and incompletely understood proteins. We focus particularly on whether PN and ßig-h3 are modified by vitamin K-dependent γ-glutamyl carboxylation, a question of considerable importance given the profound effects of γ-carboxylation on structure and function of other proteins. We consider the roles of PN and ßig-h3 in formation of extracellular matrix and as ligands for integrin receptors. We attempt to reconcile the contradictory results that have arisen concerning the role of PN, which has emerged as a marker of TH2 immunity, in murine models of allergic asthma. Finally, when possible we compare and contrast the structures and functions of the two proteins.

Integrin-targeted pH-responsive micelles for enhanced efficiency of anticancer treatment in vitro and in vivo.

The key to developing more nanocarriers for the delivery of drugs in clinical applications is to consider the route of the carrier from the administration site to the target tissue and to look for a simple design to complete this whole journey. We synthesized the amphiphilic copolymer cRGDfK-poly(ethylene glycol)-b-poly(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl) ethane methacrylate) (cRGD-PETM) to construct multifunctional micelles. These micelles combined enhanced drug-loading efficiency with tumor-targeting properties, visual detection and controllable intracellular drug release, resulting in an improved chemotherapeutic effect in vivo. Doxorubicin (DOX) was encapsulated within the cRGD-PETM micelles as a model drug (termed as cRGD-PETM/DOX Ms). The size and morphology of the micelles were characterized systematically. As a result of the hydrophobic interaction and the π-π conjugation between the DOX molecules and the PTTMA copolymers, the cRGD-PETM/DOX Ms showed an excellent drug-loading capacity. The results of in vitro drug-release studies indicated that the cumulative release of DOX from cRGD-PETM/DOX Ms at pH 5.0 was twice that at pH 7.4. The results of fluorescent microscopic analysis showed that the cRGD-PETM/DOX Ms could be internalized by 4T1 and HepG2 cells via receptor-mediated endocytosis with rapid intracellular drug release, which resulted in increased cytotoxicity compared with free DOX. Ex vivo imaging studies showed that the cRGD-PETM/DOX Ms improved the accumulation and retention of the drug in tumor tissues. Studies of the in vivo anticancer effects showed that the cRGD-PETM/DOX Ms had a significantly higher therapeutic efficacy with lower side-effects than free DOX and PETM/DOX Ms. These results show that the multifunctional cRGD-PETM/DOX Ms have great potential as vehicles for the delivery of hydrophobic anticancer drugs.

Integrin modulators: a patent review.

INTRODUCTION: Integrins are heterodimeric cell surface receptors, which enable adhesion, proliferation, and migration of cells by recognizing binding motifs in extracellular matrix (ECM) proteins. As transmembrane linkers between the cytoskeleton and the ECM, they are able to recruit a huge variety of proteins and to influence signaling pathways bidirectionally, thereby regulating gene expression and cell survival. Hence, integrins play a key role in various physiological as well as pathological processes, which has turned them into an attractive target for pharmaceutical research. AREAS COVERED: In this review, the latest therapeutic developments of drug candidates and recently patented integrin ligands are summarized. EXPERT OPINION: Integrins have been proven to be valuable therapeutic targets in the treatment of several inflammatory and autoimmune diseases, where leukocyte adhesion processes are regulated by them. Furthermore, they play an important role in pathological angiogenesis and tumor metastasis, being a promising target for cancer therapy.

Defining single molecular forces required to activate integrin and notch signaling.

Cell-cell and cell-matrix mechanical interactions through membrane receptors direct a wide range of cellular functions and orchestrate the development of multicellular organisms. To define the single molecular forces required to activate signaling through a ligand-receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor-ligand tension. Using a range of tethers with tunable tension tolerances, we show that cells apply a universal peak tension of about 40 piconewtons (pN) to single integrin-ligand bonds during initial adhesion. We find that less than 12 pN is required to activate Notch receptors. TGT can also provide a defined molecular mechanical cue to regulate cellular functions.

Integrin bi-directional signaling across the plasma membrane.

Integrins are heterodimeric cell adhesion molecules that are important in many biological functions, such as cell migration, proliferation, differentiation, and survival. They can transmit bi-directional signals across the plasma membrane. Inside-out activating signal from some cell surface receptors bound with soluble agonists triggers integrins conformational change leading to high affinity for extracellular ligands. Then binding of ligands to integrins results in outside-in signaling, leading to formation of focal adhesion complex at the integrin cytoplasmic tail and activation of downstream signal pathways. This bi-directional signaling is essential for rapid response of cell to surrounding environmental changes. During this process, the conformational change of integrin extracellular and transmembrane/cytoplasmic domains is particularly important. In this review, we will summarize recent progress in both inside-out and outside-in signaling with specific focus on the mechanism how integrins transmit bi-directional signals through transmembrane/cytoplasmic domains.

Integrin agonists as adjuvants in chemotherapy for melanoma.

PURPOSE: Metastatic melanomas are generally resistant to chemotherapy and radiation, even when wild-type for p53. These tumors often grow in small nests where many of the cells have little contact with extracellular matrix (ECM). Previous work showed that M21 melanomas undergo apoptosis in response to chemotherapy when cells are adherent to ECM but not in suspension. Thus, reduced integrin-dependent adhesion to ECM could mediate therapy resistance. The goal of this study was to test whether stimulation of integrin signaling could increase chemotherapeutic efficacy. EXPERIMENTAL DESIGN: Colony forming assays and survival assays were used to test the responses of melanoma lines in vitro. Severe combined immunodeficient mice with subcutaneous human melanomas received chemotherapy with or without reagents that stimulate integrin signaling; tumor volume was then monitored over time. RESULTS: Clonal growth assays confirmed that M21 cells showed reduced sensitivity to the chemotherapeutic drug 1-beta-D-arabinofuranosylcytosine (araC). When five additional primary melanoma lines were screened, 80% showed higher sensitivity when adherent compared with suspended. Subcutaneous M21 tumors in vivo showed minimal ECM between tumor cells. To evaluate the importance of integrin signaling in chemoresistance in this model, mice were treated with araC, with or without the multivalent snake venom disintegrin contortrostatin or the activating anti-beta1 integrin antibody TS2/16. Although araC, TS2/16, or contortrostatin alone had little effect on M21 tumor growth, combining araC with either integrin signaling reagents strongly reduced growth (P = 0001). CONCLUSIONS: Loss of integrin-mediated adhesion is rate limiting for therapeutic response in this model. Combining chemotherapy with reagents that stimulate integrin signaling may therefore provide a new approach to therapy.

Quantification of integrin receptor agonism by fluorescence lifetime imaging.

Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to antagonists. Indeed, anti-integrin compounds have failed in the clinic because of secondary side effects resulting from agonistic activity. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Association of talin with beta1 integrin and paxillin with alpha4 integrin was dependent on both the ligand and receptor activation state, and was sensitive to inhibition with small molecule RGD and LDV mimetics, respectively. An adaptation of the assay revealed the agonistic activity of these small molecules, thus demonstrating that these compounds may induce secondary effects in vivo via integrin activation. This study provides insight into the dependence of the activity of small molecule anti-integrin compounds upon receptor conformation, and provides a novel quantitative assay for the validation of potential integrin antagonists.

Release of cardiac troponin I from viable cardiomyocytes is mediated by integrin stimulation.

Elevated cardiac troponin-I (cTnI) levels have been demonstrated in serum of patients without acute coronary syndromes, potentially via a stretch-related process. We hypothesize that this cTnI release from viable cardiomyocytes is mediated by stimulation of stretch-responsive integrins. Cultured cardiomyocytes were treated with (1) Gly-Arg-Gly-Asp-Ser (GRGDS, n = 22) to stimulate integrins, (2) Ser-Asp-Gly-Arg-Gly (SDGRG, n = 8) that does not stimulate integrins, or (3) phosphate-buffered saline (control, n = 38). Cells and media were analyzed for intact cTnI, cTnI degradation products, and matrix metalloproteinase (MMP)-2. Cell viability was examined by assay of lactate dehydrogenase (LDH) activity and by nuclear staining with propidium iodide. GRGDS-induced integrin stimulation caused increased release of intact cTnI (9.6 +/- 3.0%) as compared to SDGRG-treated cardiomyocytes (4.5 +/- 0.8%, p < 0.001) and control (3.0 +/- 3.4%, p < 0.001). LDH release from GRGDS-treated cardiomyocytes (15.9 +/- 3.8%) equalled that from controls (15.2 +/- 2.3%, p = n.s.), indicating that the GRGDS-induced release of cTnI is not due to cell necrosis. This result was confirmed by nuclear staining with propidium iodide. Integrin stimulation increased the intracellular and extracellular MMP2 activity as compared to controls (both p < 0.05). However, despite the ability of active MMP2 to degrade cTnI in vitro, integrin stimulation in cardiomyocytes was not associated with cTnI degradation. The present study demonstrates that intact cTnI can be released from viable cardiomyocytes by stimulation of stretch-responsive integrins.

Integrin alpha9beta1 directly binds to vascular endothelial growth factor (VEGF)-A and contributes to VEGF-A-induced angiogenesis.

Vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human endothelial cells are dependent on the integrin alpha9beta1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and 121 isoforms of VEGF-A depend on cooperative input from alpha9beta1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike alpha3beta1or alphavbeta3 integrins, alpha9beta1 was also found to bind the 121 isoform of VEGF-A. This interaction appears to be biologically significant, because alpha9beta1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -121. Together with our previous findings that alpha9beta1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin alpha9beta1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.

Effects of amino acid substitutions in and around the arginine-glycine-aspartic acid (RGD) sequence on fertilization and parthenogenetic development in mature bovine oocytes.

Integrins have been shown to be involved in the process of fertilization and many integrin-ligand interactions are mediated through the recognition of an arginine-glycine-aspartic acid (RGD) sequence. Despite the fact the RGD domain is a principal player in determining the functional characteristics of an adhesive protein, increasing evidence has accumulated implicating the amino acids flanking the RGD sequence in determining the functional properties of the RGD-containing protein. A set of linear peptides in which the amino acid sequence in and around the RGD tri-peptide was modified was synthesized to better understand the specificity of the RGD-receptor interaction. Mature oocytes were fertilized in vitro in the presence of RGD-containing and RGD-modified peptides. Both the RGD-containing and RGD-modified peptides impaired the ability of sperm to fertilize bovine oocytes, illustrated by a reduction in cleavage. The linear modified RGD containing peptides were also examined for their ability to induce parthenogenetic development with the objective of providing a linear RGD peptide with greater biological activity than the one (GRGDSPK) used previously (Campbell et al., 2000). The data demonstrate the specificity of the receptor for the RGD sequence, further implicate the involvement of integrins in the process of bovine fertilization, and illustrate the importance of the amino acids surrounding the RGD sequence in determining the binding and functional properties of RGD-containing peptides. The data support the findings that a linear RGD peptide can block fertilization and that amino acids around the RGD sequence have an impact on the biological activity of the receptor.

Integrins, cancer and snake toxins (mini-review)

Integrins encompass a family of transmembrane heterodimeric proteins of adhesion that maintain cells attached to other cells and to the extracellular matrix (ECM). Integrins work as bi-directional mechanotransducers, conveying mechanical signal from outside to inside the cell through a cascade of phosphorylation signals. On the other hand, the signal from inside to outside controls the strength and affinity of integrin adhesion. As proteins of focal contact, integrins are involved in diverse cell functions, such as cell activation, migration, growth, and survival. In the development of neoplastic disease and metastatic tumor, integrins can influence cancer invasiveness and progression, as well as mediate the formation of new blood vessels (angiogenesis). Diverse snake venom toxins have the ability to interact with multiple integrins, what results in inhibition of cell attachment, inhibition of angiogenesis, and induction of apoptotic death of tumor and vascular endothelial cells. The aim of this review is to present data about snake venom toxins that bind to integrins and evoke antiangiogenesis and antitumoral effects