Blocking neutrophil integrin activation prevents ischemia-reperfusion injury.

Neutrophil recruitment, mediated by β2 integrins, combats pyogenic infections but also plays a key role in ischemia-reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin's capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia-reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 integrin-mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 integrin-mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia-reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable

Identification of integrin drug targets for 17 solid tumor types.

Integrins are contributors to remodeling of the extracellular matrix and cell migration. Integrins participate in the assembly of the actin cytoskeleton, regulate growth factor signaling pathways, cell proliferation, and control cell motility. In solid tumors, integrins are involved in promoting metastasis to distant sites, and angiogenesis. Integrins are a key target in cancer therapy and imaging. Integrin antagonists have proven successful in halting invasion and migration of tumors. Overexpressed integrins are prime anti-cancer drug targets. To streamline the development of specific integrin cancer therapeutics, we curated data to predict which integrin heterodimers are pausible therapeutic targets against 17 different solid tumors. Computational analysis of The Cancer Genome Atlas (TCGA) gene expression data revealed a set of integrin targets that are differentially expressed in tumors. Filtered by FPKM (Fragments Per Kilobase of transcript per Million mapped reads) expression level, overexpressed subunits were paired into heterodimeric protein targets. By comparing the RNA-seq differential expression results with immunohistochemistry (IHC) data, overexpressed integrin subunits were validated. Biologics and small molecule drug compounds against these identified overexpressed subunits and heterodimeric receptors are potential therapeutics against these cancers. In addition, high-affinity and high-specificity ligands against these integrins can serve as efficient vehicles for delivery of cancer drugs, nanotherapeutics, or imaging probes against cancer

Pharmacologic inhibition of RGD-binding integrins ameliorates fibrosis and improves function following kidney injury

Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-β at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease

Integrin activation.

Integrin-mediated cell adhesion is important for development, immune responses, hemostasis and wound healing. Integrins also function as signal transducing receptors that can control intracellular pathways that regulate cell survival, proliferation, and cell fate. Conversely, cells can modulate the affinity of integrins for their ligands a process operationally defined as integrin activation. Analysis of activation of integrins has now provided a detailed molecular understanding of this unique form of "inside-out" signal transduction and revealed new paradigms of how transmembrane domains (TMD) can transmit long range allosteric changes in transmembrane proteins. Here, we will review how talin and mediates integrin activation and how the integrin TMD can transmit these inside out signals

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 avß3, avß5, avß6, avß8, a5ß1, aIIbß3, using homogenous ELISA-like solid phase binding assay.Postprint (published version

A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration.

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions

Using Cystine Knot Proteins as a Novel Approach to Retarget Oncolytic Measles Virus.

Modified measles virus (MV) has effective oncolytic activity preclinically and is currently being investigated in clinical trials for various types of cancer. We investigated the use of cystine knot proteins (CKPs) to direct MV activity. CKPs are short polypeptides that bind their targets with high affinity. We used a CKP that binds αvβ3, αvβ5, and α5β1 integrins with single-digit nanomolar affinity to retarget MV to the integrins (MV-CKPint). MV-CKPint infected, replicated in, and killed human glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma (DIPG), and melanoma cancer cells in vitro, all of which express the target integrins. MV-CKPint activity was competitively blocked by echistatin, an integrin binding peptide. When the CKP was cleaved from the viral H protein at an included protease site, virus activity was abrogated. When delivered intravenously (i.v.), the retargeted virus reached a subcutaneous glioblastoma tumor bed and produced cytopathic effects similar to that shown by intratumoral injection of the virus. Because these target integrins are overexpressed by tumor vascular endothelium, MV-CKPint may allow for effective therapy with i.v. injection. These results indicate for the first time that CKPs can be used to retarget MV for a receptor of choice. In addition, MV-CKPint provides proof of principle for the use of a CKP of interest to retarget any enveloped virus for both oncolytic and gene therapy purposes