Inhibition of FicD-mediated AMPylation and deAMPylation by Isoprenoid Diphosphates

FicD regulates Unfolded Protein Response (UPR) through reversible AMPylation and deAMPylation of BiP, an HSP70 chaperone and master regulator of the UPR. FicD activity is regulated by ER-stress, catalyzing BiP AMPylation under low stress conditions to hold inactive chaperone in reserve. In stressed cells, FicD deAMPylates BiP, acutely increasing its active pool to assist in protein folding. Variants in UPR machinery, including those in the FicD gene, are linked to hereditary diseases. Despite the known role of FicD in UPR, in-vivo regulation of its activity remains elusive, and identifying metabolites that alter FicD activity could prove useful pharmaceutically. We applied an unbiased high-throughput screening platform, known as M ass spectrometry Integrated with equilibrium D ialysis for the discovery of A llostery S ystematically (MIDAS), to identify novel small molecule metabolites that might regulate FicD activity. MIDAS revealed interactions between FicD and two mavelonate pathway intermediates : geranyl-pyrophosphate and farnesyl-pyrophosphate. Biochemical characterization indicates that both potently inhibit FicD-mediated AMPylation and deAMPylation. The crystal structure of FicD bound to farnesyl-pyrophosphate demonstrates a competitive inhibition mechanism, with the pyrophosphate adopting the alpha and beta phosphate positions of ATP and the hydrocarbon chain filling the nucleoside pocket. FicD variants previously appeared as biochemically indistinguishable, yet lead to different human pathologies. We demonstrate farnesyl-pyrophosphate inhibits FicD ^R374H and FicD ^R374C variants implicated in causing hereditary spastic paraplegia, but not the FicD ^R371S variant associated with neonatal diabetes. This study furthers our understanding of FicD inhibitors and distinguishes disease causing variants, providing insight into pharmacological targeting of UPR activity.

FicD regulates UPR signaling in metazoans by fine-tuning BiP chaperone capacity. Therefore, targeting FicD activity may be a tractable method of altering UPR signaling for therapeutic benefit. We identify geranyl- and farnesyl-pyrophosphate as specific FicD inhibitors. Notably, these small molecules differentially inhibit disease-causing variants of FicD. A structure of farnesyl-pyrophosphate bound to the FicD active site helps explain the differential inhibition of pathogenic variants and provides insight into interactions that can be differentially exploited for modifying FicD activity. Their composition provides a novel chemical foundation for future drug development efforts targeting FicD activity.