Overcoming drug resistance is one of the major challenges facing cancer therapies. This resistance can arise from tumour heterogeneity, where the innate immunity of a small population of cancer cells to the administered therapeutics results in their survival and proliferation. This challenge may be overcome by targeting a protein whose constitutive activity is essential for the survival of all cell types, such as the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). SERCA is a transmembrane protein which uses ATP hydrolysis to actively transport Ca 2+ from the cytosol into the sarco/endoplasmic reticulum lumen -an activity essential for maintenance of internal calcium stores, proper calcium signalling and regulation. Thapsigargin (TG, Figure ) is a potent inhibitor of SERCA that is effective at nanomolar concentrations. Inhibition of SERCA by TG causes a cascade of events that ultimately leads to cell death: the depletion of ER Ca 2+ stores is followed by a sustained unfolded protein response, resulting in the upregulation of proapoptotic factors, the activation of caspases, and apoptosis. The development of resistances against TG-induced apoptosis is an unlikely event due to the essential function of SERCA, which prohibits the downregulation of its activity. At the same time, however, the omnipresence of SERCA hinders the administration of an inhibitor such as TG for the treatment of cancer. An effective use of TG as a novel therapy requires modifying the compound in a manner that it is selective for the desired target cells. This concept was successfully employed for prostate cancer (PC) by incorporating a peptide substrate for the prostate specific antigen (expressed in PC tumours) into TG to bestow the construct with selectively towards malignant tissue in vivo. In this study, we explore a general possibility for controlling the TG-mediated SERCA inhibition by using azobenzenesphotoactive compounds, which can be switched between their cis-and trans-configuration (Figure ) with light. Photoisomerization of azobenzene results in significant change to its chemical structure and properties: the trans-configuration, which is accessed through thermal relaxation in the dark or active photoswitching with blue light, is planar and essentially non-polar, while the cis-configuration, which is accessed with UV light, adopts a bent conformation, resulting in a ~3.5 Å change in endto-end distance and a considerable increase (~3 Debye) in polarity. To develop compounds that possess large differences in their inhibitory efficacy in response to the structural isomerization of the photoswitch, a small library of azobenzene-TG (AzTG) derivates were synthesised (Figure ) and their inhibition of SERCA in both cis-and trans-configuration were assessed. For the synthesis, 8-O-debutanoyl thapsigargin was prepared according to a newly developed protocol and subjected to Steglich esterification conditions with the corresponding azobenzene carboxylic acid building blocks (2-4) which proceeded to give AzTG-2, AzTG-4 and AzTG-6 in good yields (see Supporting Information). In order to rationalize our findings, we aimed to complement the results of our functional assays with crystal structures of SERCA in complex with the AzTG derivates. These structures were envisaged to reveal the specific interaction site(s) of the azobenzene groups and to provide a guide for the design of more efficient, next-generation photoswitchable inhibitors. Photoswitchable SERCA inhibitors may also serve as valuable tool compounds in studies of calcium signalling pathways and mechanisms in molecular cell biology and physiology.