Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis

Abstract

Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds ðLOPACÞ 1280 collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.high-content screening | small compound screening | lymphatic endothelium | chemical genetics

Ecto‐5′‐nucleotidase/CD73 enhances endothelial barrier function and sprouting in blood but not lymphatic vasculature

“…It has been shown that adenosine and other purines promote endothelial proliferation and migration in vitro [24,25], although results in more physiological models of sprouting angiogenesis have been contradictory [7,[26][27][28]. Therefore, we analyzed the role of extracellular purine metabolism on angio-and lymphangiogenesis using a quantitative model of sprouting [29]. Cultured human LECs used in the sprouting assays expressed higher levels of CD73 on their surface than the BECs and showed clear induction of sprouting after vascular endothelial growth factor A (VEGF-A) and bFGF stimulation ( Fig.…”

Boronic-aurone Derivatives as Anti-Tubercular Agents: Design, Synthesis and Biological Evaluation

“…particularly during the early phases of the preclinical drug discovery process [31,32]. To underscore the importance of this approach, the first FDA approved antitubercular drug in over 40 years, bedaquiline [33], and others in advanced clinical development (Delamanid, Sutezolid and SQ109) were discovered through phenotypic screening [34,35].…”

“…Whole-cell screening is getting renewed attention in the drug discovery process compared to target-based method [31,32]. Adopting whole-cell phenotypic screening approach, even for rationally designed ligands, has the unique advantage that it is able to interrogate all biochemical targets simultaneously in a specific physiological environment; Table 1: Synthetic yield for boronic-aurone derivatives.…”

An In Vivo Antilymphatic Screen in Zebrafish Identifies Novel Inhibitors of Mammalian Lymphangiogenesis and Lymphatic-Mediated Metastasis

“…S1). The identification of statins as antilymphatic was expected as both compounds were recently shown to have antilymphatic properties (33). The secondary screen using fli1:egfp embryos identified kaempferol, leflunomide, cinnarizine, and flunarizine as agents that prevented lymphangiogenesis in the zebrafish without disrupting blood vessel development, whereas lovastatin and simvastatin disrupted ISV development at the screening dose of 5 mg/ mL.…”