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A prevailing dogma is that inhibition of vascular thrombosis by antagonizing platelet integrin αIIbβ3 cannot be achieved without compromising hemostasis, thus causing serious bleeding and increased morbidity and mortality. It is speculated that these adverse outcomes result from drug-induced activating conformational changes in αIIbβ3 but direct proof is lacking. Here, we report the structure-guided design of peptide Hr10 and a modified form of the partial agonist drug tirofiban that act as “pure” antagonists of αIIbβ3, i.e., they no longer induce the conformational changes in αIIbβ3. Both agents inhibit human platelet aggregation but preserve clot retraction. Hr10 and modified tirofiban are as effective as partial agonist drugs in inhibiting vascular thrombosis in humanized mice, but neither causes serious bleeding, establishing a causal link between partial agonism and impaired hemostasis. Pure orthosteric inhibitors of αIIbβ3 may thus provide safer alternatives for human therapy, and valuable tools to probe structure–activity relationships in integrins., Current inhibitors of platelet integrin αIIbβ3 cause excessive bleeding, which limited their clinical use in cardiac patients. Here the authors design pure orthosteric αIIbβ3 inhibitors that prevent platelet aggregation and thrombosis without causing bleeding in humanized mouse models of thrombosis

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding β3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin β3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for β3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.

Copyright © 2020 American Chemical Society. RGD is a prolific example of a tripeptide used in biomaterials for cell adhesion, but the potency of free or surface-bound RGD tripeptide is orders-of-magnitude less than the RGD domain within natural proteins. We designed a set of peptides with varying lengths, composed of fragments of fibronectin protein whose central three residues are RGD, in order to vary their conformational behavior without changing the binding site's chemical environment. With these peptides, we measure the conformational dynamics and transient structure of the active site. Our studies reveal how flanking residues affect conformational behavior and integrin binding. We find that disorder of the binding site is important to the potency of RGD peptides and that transient hydrogen bonding near the RGD site affects both the energy landscape roughness of the peptides and peptide binding. This phenomenon is independent of longer-range folding interactions and helps explain why short binding sequences, including RGD itself, do not fully replicate the integrin-targeting properties of extracellular matrix proteins. Our studies reinforce that peptide binding is a holistic event and fragments larger than those directly involved in binding should be considered in the design of peptide epitopes for functional biomaterials.

[Image: see text] The integrin αVβ3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVβ3-mediated cell adhesion to αVβ3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVβ3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC(50), which correlates with cilengitide’s enhancement of tumor growth in vivo.

Integrins αVβ3 and α5β1 play critical roles in tumor survival, invasion, metastasis, and angiogenesis and are validated targets for cancer therapy and molecular imaging. Increasing evidence suggests that targeting both integrins simultaneously with antagonists is more effective in cancer therapy because of concerns about resistance and paradoxical promotion of tumor growth with use of agents highly selective for a single integrin. Engineered Arg-Gly-Asp (RGD)-containing 3.5 kDa cysteine-knot proteins (knottins 2.5F and 2.5D) are attractive drug candidates due to their exceptional structural stability and high affinity binding to certain integrins. 2.5F binds both αVβ3 and α5β1, whereas 2.5D is αVβ3-selective. To elucidate the structural basis of integrin selection, we determined the structures of 2.5F and 2.5D both as apo-proteins and in complex with αVβ3. These data, combined with MD simulations and mutational studies, revealed a critical role of two αVβ3-specific residues in the vicinity of the metal ion dependent adhesion site (MIDAS) in promoting an αVβ3-induced fit of 2.5D. In contrast, conformational selection accounted for the specificity of 2.5F to both integrins. These data provide new insights into the structural basis of integrin-ligand binding specificity, and could help in development of integrin-targeted therapeutics.

A prevailing dogma is that effective inhibition of vascular thrombosis by therapeutic targeting of platelet integrin αIIbβ3 cannot be achieved without compromising hemostasis. The resulting serious bleeding and increased morbidity and mortality have limited use of current anti-αIIbβ3 drugs to high-risk cardiac patients. It is speculated that these adverse outcomes result from drug-induced conformational changes in αIIbβ3 but direct proof is lacking. We used structure-guided design to generate the ligand-mimetic peptide Hr10 and a modified form of the partial agonist drug Tirofiban that now act as “pure” orthosteric antagonists of αIIbβ3, i.e. they no longer induce the conformational changes in αIIbβ3 and also suppress these changes in presence of agonists. Both agents inhibited human platelet aggregation effectively but without interfering with clot retraction. When tested in a humanized mouse model of thrombosis predictive of clinical efficacy, Hr10 was as effective as the αIIbβ3 partial agonist peptide drug Eptifibatide in inhibiting arteriolar thrombosis, but in sharp contrast to Eptifibatide, Hr10 did not cause serious bleeding, establishing a causal link between partial agonism and impaired hemostasis. Pure orthosteric inhibitors of αIIbβ3 may thus offer safer alternatives for human therapy. Our structure-guided approach may also find utility in designing similar drug candidates targeting other integrins and in providing vital tools for further probing structure-activity relationships in integrins.

The transition from leukocyte rolling to firm adhesion is called arrest. β2integrins are required for neutrophil arrest1. Chemokines can trigger neutrophil arrest in vivo2and in vitro3. Resting integrins4exist in a “bent-closed” conformation, i.e., not extended (E−) and not high affinity (H−), unable to bind ligand. Electron microscopic images of isolated β2integrins in “open” and “closed” conformations5inspired the switchblade model of integrin activation from E−H−to E+H−to E+H+67. Recently8, we discovered an alternative pathway of integrin activation from E−H−to E−H+to E+H+. Spatial patterning of activated integrins is thought to be required for effective arrest, but so far only diffraction-limited localization maps of activated integrins exist8. Here, we combine superresolution microscopy with molecular modeling to identify the molecular patterns of H+E−, H−E+, and H+E+activated integrins on primary human neutrophils. At the time of neutrophil arrest, E+H+integrins form oriented (non-random) nanoclusters that contain a total of 4,625±369 E+H+β2integrin molecules.

The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments. The distal segment consists of the distal-early (DE) and distal-late (DL) segments. How the proximal and distal segments develop along the anteroposterior axis is poorly understood. Here we show that knockdown of taz in zebrafish caused shortening and a significant reduction in the number of principal cells of the PST-DE segment, and proximalization of the pronephric tubule in 24 hpf embryos. RA treatment expanded the pronephric proximal domain in normal embryos as in taz morphants, an effect that was further enhanced upon exposure of taz morphants to RA. The early pronephric defects in 24 hpf taz morphants led to the failure of anterior pronephric tubule migration and convolution, and to PCT dilation and cyst formation in older embryos. In situ hybridization showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors. The present findings show that Taz is required in the anteroposterior patterning of the pronephric progenitor domain in the intermediate mesoderm, acting in part by regulating RA signaling in the pronephric progenitor field in the intermediate mesoderm.

Talin (tln) binds and activates integrins to couple extracellular matrix–bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1fl02k mutants. The ethylnitrosourea-induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1, but neither tln2 nor -2a, was dominantly expressed in the heart and vessels. Unlike tln1 and -2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity, partly through regulating F-actin networks in zebrafish. The similar expression profiles of tln1 and integrin β1b (itgb1b) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z-disks and vessel lumens. Taken together, the results of this work suggest that Tln1-mediated Itgβ1b plays a crucial role in maintaining cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.—Wu, Q., Zhang, J., Koh, W., Yu, Q., Zhu, X., Amsterdam, A., Davis, G. E., Arnaout, M. A., Xiong, J.-W. Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish.

Summary Targeting both integrins αVβ3 and α5β1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.5F binds selectively to αVβ3 and α5β1, whereas knottin 2.5D is αVβ3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with αVβ3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages αVβ3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.

In the kidney, the renal proximal tubule (PT) reabsorbs solutes into the peritubular capillaries through active transport. Here, we replicate this reabsorptive function in vitro by engineering a microfluidic PT. The microfluidic PT architecture comprises a porous membrane with user-defined submicron surface topography separating two microchannels representing a PT filtrate lumen and a peritubular capillary lumen. Human PT epithelial cells and microvascular endothelial cells in respective microchannels created a PT-like reabsorptive barrier. Co-culturing epithelial and endothelial cells in the microfluidic architecture enhanced viability, metabolic activity, and compactness of the epithelial layer. The resulting tissue expressed tight junctions, kidney-specific morphology, and polarized expression of kidney markers. The microfluidic PT actively performed sodium-coupled glucose transport, which could be modulated by administration of a sodium-transport inhibiting drug. The microfluidic PT reproduces human physiology at the cellular and tissue levels, and measurable tissue function which can quantify kidney pharmaceutical efficacy and toxicity.

Ischaemic acute kidney injury (AKI), an inflammatory disease process, often progresses to chronic kidney disease (CKD), with no available effective prophylaxis. This is in part due to lack of clinically relevant CKD models in non-human primates. Here we demonstrate that inhibition of the archetypal innate immune receptor CD11b/CD18 prevents progression of AKI to CKD in cynomolgus monkeys. Severe ischaemia-reperfusion injury of the right kidney, with subsequent periods of the left ureter ligation, causes irreversible right kidney failure 3, 6 or 9 months after AKI. Moreover, prophylactic inactivation of CD11b/CD18, using the orthosteric CD11b/CD18 inhibitor mAb107, improves microvascular perfusion and histopathology, reduces intrarenal pro-inflammatory mediators and salvages kidney function long term. These studies reveal an important early role of CD11b+ leukocytes in post-ischaemic kidney fibrosis and failure, and suggest a potential early therapeutic intervention to mitigate progression of ischaemic AKI to CKD in humans., Acute kidney injury can progress to chronic kidney disease. Here Dehnadi et al. develop a post-ischaemic chronic kidney disease model in cynomolgus monkeys and show that prophylactic inhibition of CD11b/CD18 leukocyte receptor via a monoclonal antibody inhibits progression of kidney disease and fibrosis.

SUMMARY Leukocyte adhesion requires β2-integrin activation. Resting integrins exist in a bent-closed conformation—i.e., not extended (E−) and not high affinity (H−)—unable to bind ligand. Fully activated E+H+ integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans. E−H− transitions to E+H+ through E+H− or through EH+, which binds to ICAMs on the same cell in cis. Spatial patterning of activated integrins is thought to be required for effective arrest, but no high-resolution cell surface localization maps of activated integrins exist. Here, we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest, E−H+ integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern, we blocked integrin binding to ICAMs in cis, which significantly relieved the face-to-face orientation., Graphical Abstract

Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1), commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI), as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFβ receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFβ receptor-mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFβ-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.