Heat Shock Protein 90 (Hsp90) is a molecular chaperone that folds and assembles immature polypeptides to mature and functional proteins. Hsp90 has three distinct domains; the N-terminal ATP binding domain; the middle domain, which binds to most of Hsp90's client proteins; the C-terminal domain, which is involved in dimerization of Hsp90. There are over one hundred Hsp90 client and chaperone proteins, many of which play a role in cellular signaling. Hsp90 is directly involved in stabilization of many proteins as well as indirectly linked to numerous intracellular signaling pathways. Hsp90 is up-regulated in many cancer types, which makes it an attractive oncogenic target; it directly and indirectly regulates proteins that are involved in programmed cell death and cell survival. Recently, we have shown that Sansalvamide A (SanA), binds in a pocket between Hsp90's N-middle domain, and it inhibits binding between Hsp90 and several C-terminal client proteins. Defining the molecular interactions between Hsp90 and SanA derivatives, and how these affect Hsp90/chaperone proteins interactions, will facilitate the development of novel Hsp90 inhibitors. Currently, we have developed potent SanA compound 145. Described are our studies that use Surface Plasmon Resonance (SPR) to characterize the binding characteristics between these derivatives and Hsp90. The SPR technique eliminates the need to use labeled SanA derivatives and it allows binding interactions to be observed in real time. In using this technology, we confirmed that compound 145 bound to Hsp90 with a high affinity. Future studies need to be done to assess binding characteristics for new generation SanA derivatives, where the success of these studies will allow us to engineer potent Hsp90 inhibitors. Hsp90's relationship with certain client proteins plays a critical role in the development and progression of cancer. FKBP38 and FKBP52 are immunophilins that interact with Hsp90 and both contribute to the progression of cancer. Previous data have shown that the interaction of Hsp90 with FKBP38 and FKBP52 is allosterically disrupted by our lead compound, SanA 145. The latter half of my studies focuses on how the cytotoxicity of SanA 145 is affected when these two immunophilins are either up regulated or down regulated in HeLa cells. From our experiments we've concluded that down-regulation of both FKBP38 and FKBP52 leads to an increase in cell proliferation and therefore a decrease in the cytotoxicity of SanA compounds. We've also shown that up-regulation of both FKBP38 and FKBP52 does not cause an overall change in the cell survival of HeLa cells, which causes no change in cytotoxicity of SanA compounds compared to non-treated cell controls.