A major challenge in drug discovery is to identify small molecule inhibitors for the challenging targets including protein-protein interactions and multi-pass transmembrane proteins. Nature has evolved an ingenious solution to this problem as exemplified by the immunophilin ligand family of macrocyclic natural products, including rapamycin and FK506. Aside from their own larger sizes that enable more extensive interactions with proteins, these natural products also recruit the FKBP-family of chaperones to form much larger dimeric complexes before associating with their respective targets, mTOR and calcineurin. Inspired by this unique mode of action, we generated a 45,000-compound library of rapamycin-like macrocycles, named rapafucins, by fusing the FKBP-binding domain of rapamycin with a combinatorial tetrapeptide library. Screening of this library has led to the discovery of multiple members of the solute carrier (SLC) transporter superfamily, including members of the glucose transporter (GLUT) family. Two distinct inhibitors of GLUT were identified, rapaglutin A (RgA) and rapaglutin E (RgE). Whereas RgE is highly specific for GLUT1, RgA seems to inhibit multiple GLUT isoforms. RgA has been shown to be efficacious in a xenograft model of breast cancer while RgE was found to inhibit intracellular T cell receptor signaling by blocking the DNA- binding activity of NFAT. We will further characterize the antitumor activity of RgA by systematically assessing its effect on cellular metabolomic profiles and the AMPK-mTOR signaling pathway. In preliminary studies, we have found that RgA and phenformin have synergy in inhibiting triple negative breast cancer cells (TNBC). We will further delineate the mechanism of synergy and the effect of the RgA-phenfomin combination for inhibiting TNBC tumor growth in vivo. That RgE inhibited calcium stimulated DNA-binding activity of NFAT suggests a potential role of GLUT1 in regulating intracellular T cell receptor signaling. We will elucidate the mechanism underlying the immunsuppressive activity of RgE and investigate the potential of RgE as an immunosuppressant in vivo. Lastly, using a newly developed microarray platform, we will screen rapafucin libraries against each of the 14 isoforms of GLUTs in search of specific inhibitors of different isoforms of GLUT, which can become new chemical tools for studying the biology of GLUT.