Nelfinavir Blocks Export of Newly Synthesized Protein from the ER and Interacts with ER-Resident and Mitochondrial Proteins in an Activity-Dependent Fashion

Abstract

The majority of multiple myeloma (MM) patients initially respond to proteasome inhibitor (PI)-based therapy, but later develop PI resistance and ultimately dies from PI-refractory MM. PI sensitivity of MM cells correlates with the activation status of the unfolded protein response (UPR) and its major activating axis IRE1/XBP1. PI-refractory MM cells downregulate IRE1/XBP1, and high IRE1/XBP1 expression correlates with clinical sensitivity to PI treatment. The anti-HIV drug nelfinavir induces IRE1/XBP1 and overcomes PI-resistance of MM in vitro . Nelfinavir showed unprecedented clinical activity in PI-refractory, heavily pretreated MM with ORR 65% in combination with bortezomib and dexamethasone. The molecular target of nelfinavir and its mechanism of action in MM cells are unknown. We synthesized structurally modified nelfinavir analogues equipped with a covalently protein-binding, photo-reactive moiety and functional groups that allow affinity purification of the nelfinavir analogue bound to its interacting cellular protein (SC-441), together with appropriate controls where the active center of nelfinavir was sterically occupied (SC-451). Isolation and identification of nelfinavir-interacting proteins in AMO-1 MM cells and cells adapted to carfilzomib (AMO-CFZ) was achieved with these tools by photoactivation in living cells, affinity purification and tandem mass spectrometry. We identified a small set of proteins that specifically bound to active nelfinavir. The majority of these were endoplasmic reticulum (ER)-resident proteins involved in protein quality control and transport from ER to Golgi apparatus. In addition, individual proteins from mitochondria or the nucleus were identified. We thus hypothesized that nelfinavir interferes with intracellular protein transport, in line with its UPR-activating properties. Using a construct of ST-SBP-TNFa-EGFP engineered with an ER-retention signal (KDEL) that can be released upon biotin treatment of cells (RUSH-system), in conjunction with confocal microscopy and flow cytometry in U2OS cells, we show that nelfinavir, but not its analogue SC-451, which lacks proteasome inhibitor-sensitizing activity, retains TNFa-EGFP in the ER and prevents its transport to the Golgi and into the extracellular space. We confirmed in MM cells that active nelfinavir, but not SC-451, likewise retains newly synthesized IgA by preventing its secretion, and similarly decreases MHC class I export to the cell surface.Functionally depletion of the putative ER-targets of nelfinavir by siRNA in U2OS cells sensitized cells to nelfinavir and proteasome inhibitors. The mitochondrial targets of nelfinavir were identified as constituents of the mitochondria permeability transition pore (mPTP), a multimeric protein complex that regulates the permeability of mitochondrial membranes. The formation of reactive oxygen species (ROS) and inhibition of ABCB1 efflux, known for nelfinavir, can be functionally linked to mPTP modulation.In conclusion, we demonstrate that nelfinavir blocks export of newly synthetized protein from the ER, consistent with its activity-dependent binding to ER proteins. The block in protein export from the ER by nelfinavir is consistent with its ER stress-inducing, IRE1/XBP1-activating, and PI-sensitizing effect in the clinic. We further identify mPTP as an additional protein target of nelfinavir, consistent with its ROS-inducing and ABCB1-modulating activity. The block in ER protein export represents a novel mechanism of action to sensitize MM cells for proteasome inhibitor treatment. DisclosuresNo relevant conflicts of interest to declare.