A collagen IV-derived peptide disrupts α5β1 integrin and potentiates Ang2/Tie2 signaling

The angiopoietin (Ang)/Tie2 signaling pathway is essential for maintaining vascular homeostasis, and its dysregulation is associated with several diseases. Interactions between Tie2 and α5 β1 integrin have emerged as part of this control; however, the mechanism is incompletely understood. AXT107, a collagen IV–derived peptide, has strong antipermeability activity and has enabled the elucidation of this previously undetermined mechanism. Previously, AXT107 was shown to inhibit VEGFR2 and other growth factor signaling via receptor tyrosine kinase association with specific integrins. AXT107 disrupts α5 β1 and stimulates the relocation of Tie2 and α5 to cell junctions. In the presence of Ang2 and AXT107, junctional Tie2 is activated, downstream survival signals are upregulated, F-actin is rearranged to strengthen junctions, and, as a result, endothelial junctional permeability is reduced. These data suggest that α5 β1 sequesters Tie2 in nonjunctional locations in endothelial cell membranes and that AXT107-induced disruption of α5 β1 promotes clustering of Tie2 at junctions and converts Ang2 into a strong agonist, similar to responses observed when Ang1 levels greatly exceed those of Ang2. The potentiation of Tie2 activation by Ang2 even extended to mouse models in which AXT107 induced Tie2 phosphorylation in a model of hypoxia and inhibited vascular leakage in an Ang2-overexpression transgenic model and an LPS-induced inflammation model. Because Ang2 levels are very high in ischemic diseases, such as diabetic macular edema, neovascular age-related macular degeneration, uveitis, and cancer, targeting α5 β1 with AXT107 provides a potentially more effective approach to treat these diseases.Fil: Mirando, Adam C.. University Johns Hopkins; Estados UnidosFil: Shen, Jikui. University Johns Hopkins; Estados UnidosFil: Silva, Raquel Lima E.. University Johns Hopkins; Estados UnidosFil: Chu, Zenny. University Johns Hopkins; Estados UnidosFil: Sass, Nicholas C.. University Johns Hopkins; Estados UnidosFil: Lorenc, Valeria Erika. University Johns Hopkins; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Green, Jordan J.. University Johns Hopkins; Estados Unidos. AsclepiX Therapeutics; Estados UnidosFil: Campochiaro, Peter A.. University Johns Hopkins; Estados UnidosFil: Popel, Aleksander S.. University Johns Hopkins; Estados UnidosFil: Pandey, Niranjan B.. University Johns Hopkins; Estados Unidos. AsclepiX Therapeutics; Estados Unido

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.Human Frontier Science Program (Strasbourg, France) (Postdoctoral Fellowship LT000307/2013

Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases

In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs) have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis

A mechanistic integrative computational model of macrophage polarization: Implications in human pathophysiology.

Macrophages respond to signals in the microenvironment by changing their functional phenotypes, a process known as polarization. Depending on the context, they acquire different patterns of transcriptional activation, cytokine expression and cellular metabolism which collectively constitute a continuous spectrum of phenotypes, of which the two extremes are denoted as classical (M1) and alternative (M2) activation. To quantitatively decode the underlying principles governing macrophage phenotypic polarization and thereby harness its therapeutic potential in human diseases, a systems-level approach is needed given the multitude of signaling pathways and intracellular regulation involved. Here we develop the first mechanism-based, multi-pathway computational model that describes the integrated signal transduction and macrophage programming under M1 (IFN-γ), M2 (IL-4) and cell stress (hypoxia) stimulation. Our model was calibrated extensively against experimental data, and we mechanistically elucidated several signature feedbacks behind the M1-M2 antagonism and investigated the dynamical shaping of macrophage phenotypes within the M1-M2 spectrum. Model sensitivity analysis also revealed key molecular nodes and interactions as targets with potential therapeutic values for the pathophysiology of peripheral arterial disease and cancer. Through simulations that dynamically capture the signal integration and phenotypic marker expression in the differential macrophage polarization responses, our model provides an important computational basis toward a more quantitative and network-centric understanding of the complex physiology and versatile functions of macrophages in human diseases

Suppression of Ocular Vascular Inflammation through Peptide-Mediated Activation of Angiopoietin-Tie2 Signaling

Persistent inflammation is a complication associated with many ocular diseases. Changes in ocular vessels can amplify disease responses and contribute to vision loss by influencing the delivery of leukocytes to the eye, vascular leakage, and perfusion. Here, we report the anti-inflammatory activity for AXT107, a non-RGD, 20-mer αvβ3 and α5β1 integrin-binding peptide that blocks vascular endothelial growth factor (VEGF)-signaling and activates tyrosine kinase with immunoglobulin and EGF-like domains 2 (Tie2) using the normally inhibitory ligand angiopoietin 2 (Ang2). Tumor necrosis factor α (TNFα), a central inflammation mediator, induces Ang2 release from endothelial cells to enhance its stimulation of inflammation and vascular leakage. AXT107 resolves TNFα-induced vascular inflammation in endothelial cells by converting the endogenously released Ang2 into an agonist of Tie2 signaling, thereby disrupting both the synergism between TNFα and Ang2 while also preventing inhibitor of nuclear factor-κB α (IκBα) degradation directly through Tie2 signaling. This recovery of IκBα prevents nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear localization, thereby blocking NF-κB-induced inflammatory responses, including the production of VCAM-1 and ICAM-1, leukostasis, and vascular leakage in cell and mouse models. AXT107 also decreased the levels of pro-inflammatory TNF receptor 1 (TNFR1) without affecting levels of the more protective TNFR2. These data suggest that AXT107 may provide multiple benefits in the treatment of retinal/choroidal and other vascular diseases by suppressing inflammation and promoting vascular stabilization

Regulation of the tumor immune microenvironment and vascular normalization in TNBC murine models by a novel peptide

Triple-negative breast cancer (TNBC) is a highly metastatic and aggressive disease with limited treatment options. Recently, the combination of the immune checkpoint inhibitor (ICI) atezolizumab (anti-PD-L1) with nab-paclitaxel was approved following a clinical trial that showed response rates in at least 43% of patients. While this approval marks a major advance in the treatment of TNBC it may be possible to improve the efficacy of ICI therapies through further modulation of the suppressive tumor immune microenvironment (TIME). Several factors may limit immune response in TNBC including aberrant growth factor signaling, such as VEGFR2 and cMet signaling, inefficient vascularization, poor delivery of drugs and immune cells, and the skewing of immune cell populations toward immunosuppressive phenotypes. Here we investigate the immune-modulating properties of AXT201, a novel 20 amino-acid integrin-binding peptide in two syngeneic mouse TNBC models: 4T1-BALB/c and NT4-FVB. AXT201 treatment improved survival in the NT4 model by 20% and inhibited the growth of 4T1 tumors by 47% over 22 days post-inoculation. Subsequent immunohistochemical analyses of 4T1 tumors also showed a 53% reduction in vascular density and a 184% increase in pericyte coverage following peptide treatment. Flow cytometry analyses demonstrated evidence of a more favorable anti-tumor immune microenvironment following treatment with AXT201, including significant decreases in the populations of T regulatory cells, monocytic myeloid-derived suppressor cells, and PD-L1 expressing cells and increased expression of T cell functional markers. Together, these findings demonstrate immune-activating properties of AXT201 that could be developed in combination with other immunomodulatory agents in the treatment of TNBC