A series of 3'-substituted deschloroepibatidine analogues (3a-g and 4) showed high affinity for alpha4beta2 binding and relatively weak affinity for alpha7 nAChRs. The 3'-ethynyl (3g) and 3'-fluoro (3a) analogues with K(i) values of 0.02 and 0.037 nM, respectively, were the most potent. Even though the alpha4beta2 binding affinity of several of the analogues were equal to that of epibatidine, all of the compounds were weak agonists in the antinociceptive, hypothermia, and spontaneous activity test in mice. In contrast, all of the compounds were functional antagonists of nicotine-induced antinociception. In general, compounds 3a-g and 4 were more potent in the tail-flick assay than the hot-plate test. For example, the 3'-fluoro analogue 3a and the N-methyl-3'-iodo analogue 4 showed AD(50) values of 0.07 and 0.04 microg/kg, respectively, in the tail flick test and only 35 and 0% inhibition at 20 and 10 microg/kg in the hot-plate assay, respectively. These results suggest that these compounds will be highly useful for identifying which specific receptor subtypes are involved in each of nicotine's pharmacological effects. The high affinity of the N-methyl-3'-iodo analogue 4 combined with its weak agonist and potent antagonist activity suggests that carbon-11 and iodine-123 analogues may be useful as PET and SPECT ligands, respectively, for studying nAChRs in vivo.

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...