The natural product withaferin A (WFA) is a potent angiogenesis inhibitor and it targets the ubiquitin-proteasome pathway in vascular endothelial cells. We generated a biotinylated affinity analog WFA-LC 2 B for use as a probe to study angiogenesis. WFA-LC 2 B inhibits angiogenic sprouting in vitro and it causes levels of ubiquitinated proteins to increase in tumor necrosis factor-α-treated human umbilical vein endothelial cells, confirming the retention of WFA's biological activity. We show that WFA-LC 2 B forms protein adducts in endothelial cells which are competed by free WFA in vivo. This WFA-LC 2 B analog will be useful to isolate the biological target of WFA. KeywordsBiotinylated analog; Natural product; Binding protein; Ubiquitin; Angiogenesis inhibitor Withaferin A (WFA), an important prototype of the withanolide class of natural products ( Fig. 1), is a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera and its related solanaceas species. 1 The withanolides are known to exert very potent and diverse cytotoxic, anti-stress, cardioactive, central nervous system, and immunomodulatory activities. 2 Since the early discovery of WFA during the 1960s, the major interest has been on its anti-tumor cytotoxic activities. 3,4 However, the non-cytotoxic anti-inflammatory 5 and immunomodulatory mechanisms 6 of WFA have thus far remained rather poorly characterized. These latter disease-altering activities are highly pertinent to the practice of ayurveda, a traditional form of Indian medicine, which has borne out many effective formulations from W. somnifera, especially for the treatment of chronic human diseases such as arthritis and female bleeding disorders. 2 Angiogenesis, which is the growth of new blood vessels from preexisting vasculature, is a pathogenic manifestation in cancers, 7 and it is also widely recognized to be critically involved in the pathogenesis of arthritis, endometriosis, age-related macular degeneration, diabetic retinopathy, etc. 7 Since these non-malignant inflammatory diseases could also benefit from anti-angiogenic therapeutics, 8 that such extracts could possess heretofore unrecognized inhibitors of angiogenesis. In fact, we demonstrated that W. somnifera extracts containing non-cytotoxic levels of withanolides, and also WFA, the derived active principle of these extracts, exert potent anti-angiogenic activity in vivo at very low doses. 10 Furthermore, at low nanomolar concentrations, we showed that WFA directly targets endothelial cell proliferation and exerts cytostatic cell cycle G 1 arrest in human umbilical vein endothelial cells (HUVECs). Interestingly, noncytotoxic sub-to-low micromolar concentrations of WFA also inhibit in vitro vessel formation 10,11 in the three-dimensional endothelial cell sprouting assay (3D-ECSA). At such doses, WFA potently inhibits TNF-α-induced NF-κB-DNA-binding activity, a mechanism which is associated with stabilization of phosphorylated IκB-α in the cytoplasm. Our findings suggest that WFA does not in...

HCECs share with HUVECs a similar response to UPP inhibitors, suggesting that this well-conserved pathway that regulates angioinflammatory mechanisms could be exploited for drug-targeting in the development of novel agents for CNV treatment.