Effect of linker and spacer on the design of a fibronectin-mimetic peptide evaluated via cell studies and AFM adhesion forces

The design of a fibronectin-mimetic peptide that specifically binds to the alpha 5beta 1 integrin has been widely studied because of this integrin's participation in many physiological and pathological processes. A promising design for such a peptide includes both the primary binding site RGD and the synergy site PHSRN connected by a linker and extended off of a surface by a spacer. Our original hypothesis was that the degree of hydrophobicity/hydrophilicity between the two sequences (RGD and PHSRN) in fibronectin is an important parameter in designing a fibronectin-mimetic peptide (Mardilovich, A.; Kokkoli, E. Biomacromolecules 2004, 5, 950-957). A peptide-amphiphile, PR_b, that was previously designed in our laboratory employed a hydrophobic tail connected to the N terminus of a peptide headgroup that was composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. Even though our previous work (Mardilovich, A.; Craig, J. A.; McCammon, M. Q.; Garg, A.; Kokkoli, E. Langmuir 2006, 22, 3259-3264) demonstrated that PR_b is a promising sequence compared to fibronectin, this is the first study that tests our hypothesis by comparing PR_b to other peptides with hydrophobic or hydrophilic linkers. Furthermore, different peptide-amphiphiles were designed that could be used to study the effect of building blocks systematically, such as the peptide headgroup linker length and hydrophobicity/hydrophilicity as well as the headgroup spacer length on integrin adhesion. Circular dichroism spectroscopy was first employed, and the collected spectra demonstrated that only one peptide-amphiphile exhibited a secondary structure. Their surface topography was evaluated by taking atomic force microscopy (AFM) images of Langmuir-Blodgett peptide-amphiphile membranes supported on mica. Their adhesion was first evaluated with AFM force measurements between the different sequences and an AFM tip functionalized with purified integrins. The amphiphiles were further characterized via 1-12 h cell studies that examined human umbilical vein endothelial cell adhesion and extracellular matrix fibronectin production. The AFM studies were in good agreement with the cell studies. Overall, the adhesion studies validated our hypothesis and demonstrated for the first time that a "neutral" linker, which more closely mimics the cell adhesion domain of fibronectin, supports higher levels of adhesion compared to other peptide designs with a hydrophobic or hydrophilic linker or even fibronectin. Neutral linker lengths that were within the distance found between PHSRN and RGD in fibronectin performed equally well. However, the 10 amino acid neutral linker gave slightly better cell adhesion than did the control fibronectin at all times. Also, a short spacer was shown to give higher adhesion than other sequences with no spacer or a longer spacer, suggesting that a short spacer is necessary to extend the sequence further away from the interface. In conclusion, this work outlines a logical approach that can be applied for the rational design of any protein-mimetic peptide with two binding sites.     

PR_b-targeted PEGylated liposomes for prostate cancer therapy

In recent years, there has been considerable effort in designing improved delivery systems by including site-directed surface ligands to further enhance their selective targeting. The goal of this study is to engineer alpha5beta1-targeted stealth liposomes (nanoparticles covered with poly(ethylene glycol) (PEG)) that will bind to alpha5beta1-expressing LNCaP human prostate cancer cells and efficiently release the encapsulated load intracellularly. For this purpose, liposomes (with and without PEG2000) were functionalized with a fibronectin-mimetic peptide (PR_b) and delivered to LNCaPs. The amount of PEG2000 and other liposomal components were characterized by 1H NMR, and the amount of peptide by the bicinchoninic acid protein assay. Fibronectin is the natural ligand for alpha5beta1, and a promising design for a fibronectinmimetic peptide includes both the primary binding site (RGD) and the synergy site (PHSRN) connected by a linker and extended off a surface by a spacer. We have previously designed a peptide-amphiphile, PRb, that employed a hydrophobic tail, connected to the N-terminus of a peptide headgroup composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. We have examined different liposomal formulations, functionalized only with PR_b or with PR_b and PEG2000. For PR_b-targeted PEGylated liposomes, efficient cell binding was observed for peptide concentrations of 2 mol % and higher. When compared to GRGDSP-targeted stealth liposomes, PR_b functionalization was superior to that of GRGDSP as shown by increased LNCaP binding, internalization efficiency, as well as cytotoxicity after incubation of LNCaPs with tumor necrosis factor-alpha (TNFalpha)-encapsulated liposomes. More importantly, PR_b is alpha5beta1-specific, whereas many integrins bind to small RGD peptides. Thus, the proposed PR_b-targeted delivery system has the potential to deliver a therapeutic payload to prostate cancer cells in an efficient and specific manner.     

Collective and single-molecule interactions of alpha5beta1 integrins

A novel biomimetic system was used to study collective and single-molecule interactions of the alpha5beta1 receptor-GRGDSP ligand system with an atomic force microscope (AFM). Bioartificial membranes, which display peptides that mimic the cell adhesion domain of the extracellular matrix protein fibronectin, are constructed from peptide-amphiphiles. The interaction measured with the immobilized alpha5beta1 integrins and GRGDSP peptide-amphiphiles is specifically related to the integrin-peptide binding. It is affected by divalent cations in a way that accurately mimics the adhesion function of the alpha5beta1 receptor. The recognition of the immobilized receptor was significantly increased for a surface that presented both the primary recognition site (GRGDSP) and the synergy site (PHSRN) compared to the adhesion measured with surfaces that displayed only the GRGDSP peptide. At the collective level, the separation process of the receptor-ligand pairs is a combination of multiple unbinding and stretching events that can accurately be described by the wormlike chain (WLC) model of polymer elasticity. In contrast, stretching was not observed at the single-molecule level. The dissociation of single alpha5beta1-GRGDSP pairs under loading rates of 1-305 nN/s revealed the presence of two activation energy barriers in the unbinding process. The high-strength regime above 59 nN/s maps the inner barrier at a distance of 0.09 nm along the direction of the force. Below 59 nN/s a low-strength regime appears with an outer barrier at 2.77 nm and a much slower transition rate that defines the dissociation rate (off-rate) in the absence of force (k(off) degrees = 0.015 s(-1)).     

Enhanced integrin mediated signaling and cell cycle progression on fibronectin mimetic peptide amphiphile monolayers

In recent years, a variety of biomimetic constructs have emerged which mimic the bioactive sequences found in the natural extracellular matrix (ECM) proteins such as fibronectin (FN) that promote cell adhesion as well as proliferation on artificially functionalized interfaces. Much interest lies in investigating the ability of the ECM mimetic materials in regulating a number of vital cell functions including differentiation, gene expression, migration, and proliferation. A peptide amphiphile PR_b containing both the cell adhesive GRGDSP and synergistic PHSRN peptide sequences was developed in our group that was shown to support enhanced cell proliferation and ECM FN secretion as compared to GRGDSP and FN functionalized interfaces. In this study, we have investigated the binding affinity of the PR_b peptide ligand with the FN cell surface receptor, the α(5)β(1) integrin. We compared PR_b functionalized surfaces with FN and BSA coated surfaces and GRGDSP functionalized surfaces in terms of promoting intracellular signaling cascades that are essential for enhanced cellular activity. Specifically, we studied the phosphorylation of focal adhesion kinase (FAK) at tyrosine residues Y397 and Y576 and the formation of cyclin D1, both of which are intracellular markers of integrin mediated attachment of cells, signaling pathways, and progression of cell cycle. FAK and cyclin D1 encourage enhanced cell proliferation, differentiation, and gene expression. Our results show that the PR_b peptide ligand has a specific and strong binding affinity for the α(5)β(1) integrin with a dissociation constant of 76.3 ± 6.3 nM. The PR_b peptide ligands supported enhanced FAK phosphorylation activity and increased cyclin D1 formation as compared to the widely used GRGDSP ligand, the native protein FN (positive control), and BSA nonadhesive surfaces (negative control). These results encourage the use of the FN mimetic PR_b peptide in functionalizing biomaterials for potential tissue engineering and therapeutic applications.     

Preparation and biological activities of a bivalent poly(ethylene glycol) hybrid containing an active site and its synergistic site of fibronectin

A bivalent poly(ethylene glycol) or PEG hybrid of fibronectin-related peptides was prepared. An active site peptide (RGD) and its synergistic site peptide (PHSRN) of fibronectin were conjugated with an amino acid-type PEG (aaPEG) to form PHSRN-aaPEG-RGD. A moderate spatial array between RGD and PHSRN in fibronectin may be required for synergic activity. The bivalent hybrid exhibited potent cell spreading activity and exhibited potent anti-metastatic activity in a model of experimental metastasis with B16-BL6 cells in mice. PEG may serve as a spacer for maintaining the desired spatial array.     

Inhibition of cell adhesion to fibronectin by oligopeptide-substituted polynorbornenes.

Polynorbornenes substituted with two different peptide sequences from the RGD-containing integrin cell-binding domain of fibronectin are potent inhibitors of human foreskin fibroblast cell adhesion to fibronectin-coated surfaces. Ring-opening metathesis polymerization (ROMP) using Ru==CHPh(Cl)(2)(PCy(3))(DHIMes) (1) as an initiator produced polymers substituted with GRGDS and PHSRN peptide sequences. The inhibitory activity was quantified for these polymers and compared to the free peptides and GRGES-containing controls. A homopolymer substituted with GRGDS peptides was significantly more active than the free GRGDS peptide (IC(50) of 0.18 +/- 0.03 and 1.33 +/- 0.20 mM respectively), and the copolymer containing both GRGDS and PHSRN is the most potent inhibitor (IC(50) of 0.04 +/- 0.01 mM). These results demonstrate that significant enhancements of observed biological activity can be obtained from polymeric materials containing more than one type of multivalent ligand and that ROMP is a useful method to synthesize such well-defined copolymers.     

Multimeric cyclic RGD peptides as potential tools for tumor targeting: solid-phase peptide synthesis and chemoselective oxime ligation

The alpha v beta 3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. Targeting this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Solid-phase peptide synthesis of multimeric cyclo(-RGDfE-)-peptides is described, which offer the possibility of enhanced integrin targeting due to polyvalency effects. These peptides contain an aminooxy group for versatile chemoselective oxime ligation. Conjugation with para-trimethylstannylbenzaldehyde results in a precursor for radioiododestannylation, which would allow them to be used as potential tools for targeting and imaging alpha v beta 3-expressing tumor cells. The conjugates were obtained in good yield without the need of a protection strategy and under mild conditions.     

Extracellular SH3 domain containing proteins--features of a new protein family

In the year 1994, the protein MIA (melanoma inhibitory activity) was found to be strongly expressed and secreted by malignant melanomas and subsequent studies revealed that MIA has an important function in melanoma development and invasion. Multidimensional NMR-spectroscopy and x-ray crystallography revealed that recombinant human MIA adopts a Src homology 3 (SH3) domain-like fold in solution, a structure with two perpendicular antiparallel three- and five-stranded beta-sheets. SH3 domains are protein modules that are found in many intracellular signalling proteins and mediate protein-protein interactions by binding to proline-rich peptide sequences. Unlike previously described protein structures with SH3 domain folds, MIA is a secreted single-domain protein of 12 kDa that contains an additional antiparallel beta-sheet and two disulfide bonds. Furthermore, the charge surrounding the canonical binding site differs from that of classical SH3 domains. The two disulfide bonds are crucial for correct folding and function as revealed by mutation analysis. Therefore, MIA appears to be the first extracellular protein adopting an SH3 domain-like fold. MIA was shown to interact with fibronectin, and MIA-interacting peptide ligands identified by phage display screening are similar to the consensus sequence of type III human fibronectin repeats, especially FN14. Interestingly, recent data revealed that MIA can also directly bind to integrin alpha 4 beta 1 and alpha 5 beta1 and that it modulates integrin activity negatively. These findings suggest an interesting role of the SH3-domain proteins in the extracellular compartment. Recently, MIA homologous proteins with a sequence identity of 44% and a sequence homology of approximately 80% were determined (TANGO, MIA-2, OTOR). This clearly suggests that this structural device is used more frequently, in processes ranging from developmental changes to the interference of cell attachment in the extracellular matrix. Detailed studies are necessary to determine the exact function of the MIA homologous proteins. It will be interesting to know whether additional protein families can be identified which are secreted and carry SH3 domain-like modules, in addition to elucidate what the specific physiological targets of this protein family are.     

HUVEC cell affinity evaluation and integrin-mediated mechanism study on PHSRN-modified polymer

To investigate the role of the peptide Pro-His-Ser-Arg-Asn (PHSRN) in cell adhesion and growth, PHSRN- and Gly-Arg-Gly-Asp-Ser (GRGDS)-containing polymers (P-PN5 and P-GS5, respectively) were synthesized by modification of poly(D,L-lactide-co-beta-malic acid) (PLMA) with the corresponding peptides. The cell affinities of the modified polymers were evaluated by adhesion and proliferation experiments with human umbilical vein endothelial cells (HUVECs). The results showed that P-PN5 had comparable ability to that of P-GS5 in supporting HUVEC adhesion and growth. Furthermore, the integrin-mediated mechanism of cell-substrate interaction was investigated. The results showed that P-PN5 had similar binding affinity and binding strength towards α(5)β(1) compared to those of P-GS5. The findings suggest that PHSRN is capable of mediating the adhesion and growth of HUVECs independently and that PHSRN-modified polymers might be used as biologically compatible materials.     

Surface immobilization of fibronectin-derived PHSRN peptide on functionalized polymer films – Effects on fibroblast spreading

The Pro-His-Ser-Arg-Asn (PHSRN) sequence in fibronectin is a second cell-binding site that synergistically affects Arg-Gly-Asp (RGD). The PHSRN peptide also induces cell invasion and accelerates wound healing. We report on the surface immobilization of PHSRN by spontaneous adsorption on polysiloxane thin films which have different surface free energy characteristics. Low-surface energy (hydrophobic) polysiloxane and the corresponding high-surface energy (hydrophilic) surfaces obtained by UV–ozone treatments were used as adsorbing substrates. The peptide adsorption process was investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both adsorption kinetics and peptide rearrangement dynamics at the solid interface were significantly different on the surface-modified films compared to the untreated ones. Fibroblast cells cultures at short times and in a simplified environment, i.e., a medium-free solution, were prepared to distinguish interaction events at the interface between cell membrane and surface-immobilized peptide for the two cases. It turned out that the cell-adhesive effect of immobilized PHSRN was different for hydrophobic compared to hydrophilic ones. Early signatures of cell spreading were only observed on the hydrophilic substrates. These effects are explained in terms of different spatial arrangements of PHSRN molecules immobilized on the two types of surfaces.     

Photocontrol of cell adhesion processes: model studies with cyclic azobenzene-RGD peptides

A photoresponsive integrin ligand was synthesized by backbone-cyclization of a heptapeptide containing the integrin binding motif Arg-Gly-Asp (RGD) with 4-(aminomethyl)phenylazobenzoic acid (AMPB). Surface plasmon enhanced fluorescence spectroscopy showed that binding of the azobenzene peptide to alpha(v)beta(3) integrin depends on the photoisomeric state of the peptide chromophore. The higher affinity of the trans isomer could be rationalized by comparing the NMR conformations of the cis and trans isomers with the recently solved X-ray structure of a cyclic RGD-pentapeptide bound to integrin.     

Photoactive peptides for light-initiated tyrosyl radical generation and transport into ribonucleotide reductase

The mechanism of radical transport in the alpha2 (R1) subunit of class I E. coli ribonucleotide reductase (RNR) has been investigated by the phototriggered generation of a tyrosyl radical, *Y356, on a 20-mer peptide bound to alpha2. This peptide, Y-R2C19, is identical to the C-terminal peptide tail of the beta2 (R2) subunit and is a known competitive inhibitor of binding of the native beta2 protein to alpha2. *Y356 radical initiation is prompted by excitation (lambda >or= 300 nm) of a proximal anthraquinone, Anq, or benzophenone, BPA, chromophore on the peptide. Transient absorption spectroscopy has been employed to kinetically characterize the radical-producing step by time resolving the semiquinone anion (Anq*-), ketyl radical (*-BPA), and Y* photoproducts on (i) BPA-Y and Anq-Y dipeptides and (ii) BPA/Anq-Y-R2C19 peptides. Light-initiated, single-turnover assays have been carried out with the peptide/alpha2 complex in the presence of [14C]-labeled cytidine 5'-diphosphate substrate and ATP allosteric effector. We show that both the Anq- and BPA-containing peptides are competent in deoxycytidine diphosphate formation and turnover occurs via Y731 to Y730 to C439 pathway-dependent radical transport in alpha2. Experiments with the Y730F mutant exclude a direct superexchange mechanism between C439 and Y731 and are consistent with a PCET model for radical transport in which there is a unidirectional transport of the electron and proton transport among residues of alpha2.     

Lipopeptides incorporated into supported phospholipid monolayers have high specific activity at low incorporation levels

The ability to present cell adhesion molecule (CAM) ligands in controlled amounts on a culture surface would greatly facilitate the control of cell growth and differentiation. Supported lipid monolayer/bilayer systems have previously been developed that allow for presentation of CAM ligands for cell interaction; however, these systems have employed peptide loadings much higher than those used in poly(ethylene glycol) (PEG)-based immobilization systems. We report the development of synthetic methods that can be used for the efficient and versatile creation of many linear and cyclic lipid-linked peptide moieties. Using RGD-based peptides for the alpha5beta1 integrin as a model system, we have demonstrated that these lipopeptides support efficient cell binding and spreading at CAM ligand loadings as low as 0.1 mol %, which is well below that previously reported for supported lipid systems. Engineered lipopeptide-based surfaces offer unique presentation options not possible with other immobilization systems, and the high activity at low loadings we have shown here may be extremely useful in presenting multiple CAM ligands for studying cell growth, differentiation, and signaling.     

Liquid chromatography/tandem mass spectrometry characterization of oxidized amyloid beta peptides as potential biomarkers of Alzheimer's disease

Alzheimer's disease is characterized by the deposition of senile plaques that consist primarily of amyloid beta peptides. There is substantial evidence that amyloid beta is oxidized in vivo, which has led to the suggestion that oxidative stress is an important mediator of Alzheimer's disease. Metal-catalyzed oxidation can mimic in vivo oxidation of amyloid beta because the metal ion binds to the amino acid residues at the site of oxidation, which then deliver reactive oxygen species to that site. Based on electrospray mass spectrometry, it has been suggested that metal-catalyzed oxidation occurs on histidines-13 and -14. Unfortunately, the amyloid beta peptides provide complex spectra, so it is difficult to definitively characterize the sites of oxidation. Trypsin digestion of both native and oxidized amyloid beta1-16 and amyloid beta1-40 resulted in the formation of tryptic peptides corresponding to amyloid beta6-16, which could be separated by liquid chromatography (LC). Sites of oxidation were then unequivocally characterized as histidine-13 and histidine-14 by LC/tandem mass spectrometric (MS/MS) analysis of the tryptic peptides. The ability to analyze the specific amyloid beta6-16 tryptic fragments derived from full-length amyloid beta peptides will make it possible to determine whether oxidation in vivo occurs at specific histidine residues and/or at other amino acid residues such as methionine-35. Using methodology based on LC/MS/MS it will also be possible to analyze the relative amounts of oxidized peptides and native peptide in cerebrospinal fluid from patients with Alzheimer's disease as biomarkers of oxidative stress.     

Receptor-bound conformation of an alpha(5)beta(1) integrin antagonist by (15)N-edited 2D transferred nuclear overhauser effects

We report the results of (15)N-edited 2D transferred NOE experiments of the partially (15)N-labeled alpha(5)beta(1) antagonist c[Mpa(15)N-Arg-(15)N-Gly-(15)N-Asp-(15)N-Asp-(15)N-Val-Cys]-NH(2) (Mpa denotes mercaptopropionic acid) in the presence of the native alpha(5)beta(1) receptor. The alpha(5)beta(1) integrin receptor is believed to be involved in tumor metastasis and the rational design of alpha(5)beta(1) integrin antagonist is therefore of considerable interest. Our experiments provide insight into the alpha(5)beta(1) receptor-bound conformation of the antagonist c[MpaRGDDVC]-NH2 and will be important for the design of novel antagonists.     

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides.

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.     

Template assembled cyclopeptides as multimeric system for integrin targeting and endocytosis

The alpha(V)beta(3) integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. c[-RGDfV-] peptide represents a selective alpha(V)beta(3) integrin ligand that has been extensively used for research, therapy, and diagnosis of neoangiogenesis. We report here the modular synthesis and biological characterization of template assembled cyclopeptides as a multimeric system for targeting and endocytosis of cells expressing alpha(V)beta(3) integrin. c[-RGDfK-] was cleanly assembled in a multivalent mode by chemoselective oxime bond formation to a cyclodecapeptides template labeled by different reporter groups. Binding propensity to the alpha(V)beta(3) receptor and the associated good uptake property displayed by the multivalent molecules demonstrated the interest in the RAFT molecule to design new multimeric system with hitherto unreported properties. These compounds offer an interesting perspective for the reevaluation of integrins as angiogenesis regulators (Hynes, R. O. Nature Med. 2003, 9, 918-921) as well as for the design of more sophisticated systems such as molecular conjugate vectors.     

Building a novel vitronectin assay by immobilization of integrin on calixarene monolayer

Membrane proteins possess significant hydrophobic domains and are likely to deplete their native activity immobilized on the solid surface relative to those occurring in a membrane environment. To investigate an efficient immobilization method, calix[4]crown-ether monolayer as an artificial protein linker system was constructed on the gold surface and characterized by Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS), atomic force microscopy (AFM) and cyclic voltammetry (CV). Integrin alpha(v)beta3 was functionally immobilized onto the monolayer and the integrin-vitronectin interaction was investigated by surface plasmon resonance (SPR). It was found that calix[4]crown-ether was assembled as a monolayer on the gold surface. Orientation and accessibility of integrin alpha(v)beta3 was assessed by sensitive binding of its natural ligand, vitronectin at pg mL(-1) level. Moreover, surface coverage of integrin layer and thickness calculated through SPR curve simulation verified that integrin layer was a monolayer in activated form. In combination with the SPR method, this calix[4]crown monolayer provided a reliable and simple experimental platform for the investigation of isolated membrane proteins under experimental conditions resembling those of their native properties.     

Peptide-Hypervalent Iodine Reagent Chimeras: Enabling Peptide Functionalization and Macrocyclization**

Herein, we report a novel strategy for the modification of peptides based on the introduction of highly reactive hypervalent iodine reagents—ethynylbenziodoxolones (EBXs)—onto peptides. These peptide-EBXs can be readily accessed, by both solution- and solid-phase peptide synthesis (SPPS). They can be used to couple the peptide to other peptides or a protein through reaction with Cys, leading to thioalkynes in organic solvents and hypervalent iodine adducts in water buffer. Furthermore, a photocatalytic decarboxylative coupling to the C-terminus of peptides was developed using an organic dye and was also successful in an intramolecular fashion, leading to macrocyclic peptides with unprecedented crosslinking. A rigid linear aryl alkyne linker was essential to achieve high affinity for Keap1 at the Nrf2 binding site with potential protein-protein interaction inhibition.     

The role of the alpha4 integrin-paxillin interaction in regulating leukocyte trafficking

The movement of leukocytes from the blood into peripheral tissues is a central feature of immune surveillance, but also contributes to the pathogenesis of inflammatory and autoimmune diseases. Integrins are a family of adhesion and signaling molecules made up of paired a and beta subunits, and the integrin alpha4beta1 plays a prominent role in the trafficking of mononuclear leukocytes. We have previously described the direct interaction of the signaling adaptor molecule paxillin with the cytoplasmic domain of the alpha4 integrin subunit. This interaction is critical for alpha4beta1 integrin dependent cell adhesion under shear flow conditions as it provides a needed connection to the actin cytoskeleton. Furthermore, the alpha4-paxillin interaction is required for effective alpha4beta1 dependent leukocyte migration and does so through the temporal and spatial regulation of the small GTPase Rac. These findings make the alpha4-paxillin interaction a potentially attractive therapeutic target in controlling leukocyte trafficking.