{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:18:17Z","timestamp":1785421097148,"version":"3.56.0"},"posted":{"date-parts":[[2025,12,8]]},"group-title":"Biochemistry","reference-count":56,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T00:00:00Z","timestamp":1765152000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["P30 GM124169"],"award-info":[{"award-number":["P30 GM124169"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"award":["S10OD021832"],"award-info":[{"award-number":["S10OD021832"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"award":["S10OD026941"],"award-info":[{"award-number":["S10OD026941"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35 GM134945"],"award-info":[{"award-number":["R35 GM134945"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T32GM127216 (AMB)"],"award-info":[{"award-number":["T32GM127216 (AMB)"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000015","name":"Department of Energy","doi-asserted-by":"crossref","award":["DE-AC02-05CH11231"],"award-info":[{"award-number":["DE-AC02-05CH11231"]}],"id":[{"id":"10.13039\/100000015","id-type":"DOI","asserted-by":"crossref"}]},{"award":["I-1561"],"award-info":[{"award-number":["I-1561"]}],"id":[{"id":"https:\/\/ror.org\/00np6vq88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["10070558 \/ 51006865"],"award-info":[{"award-number":["10070558 \/ 51006865"]}],"id":[{"id":"https:\/\/ror.org\/02qenvm24","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  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\n                  <jats:underline>M<\/jats:underline>\n                  ass spectrometry Integrated with equilibrium\n                  <jats:underline>D<\/jats:underline>\n                  ialysis for the discovery of\n                  <jats:underline>A<\/jats:underline>\n                  llostery\n                  <jats:underline>S<\/jats:underline>\n                  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\n                  <jats:sup>R374H<\/jats:sup>\n                  and FicD\n                  <jats:sup>R374C<\/jats:sup>\n                  variants implicated in causing hereditary spastic paraplegia, but not the FicD\n                  <jats:sup>R371S<\/jats:sup>\n                  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.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>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.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2025.12.08.693056","type":"posted-content","created":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T04:50:11Z","timestamp":1765255811000},"source":"Crossref","is-referenced-by-count":0,"title":["Inhibition of FicD-mediated AMPylation and deAMPylation by Isoprenoid Diphosphates"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4546-7267","authenticated-orcid":false,"given":"Aubrie M.","family":"Blevins","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Peng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3041-2615","authenticated-orcid":false,"given":"Lisa N.","family":"Kinch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zihan","family":"Monshad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea G.","family":"Paredes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christina","family":"Volz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2710-9765","authenticated-orcid":false,"given":"Jared","family":"Rutter","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5829-748X","authenticated-orcid":false,"given":"Amanda K.","family":"Casey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5067-7608","authenticated-orcid":false,"given":"Kevin G.","family":"Hicks","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0678-7620","authenticated-orcid":false,"given":"Kim","family":"Orth","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025121010250856000_2025.12.08.693056v1.1","doi-asserted-by":"crossref","first-page":"168418","DOI":"10.1016\/j.jmb.2023.168418","article-title":"The Essential Functions of Molecular Chaperones and Folding Enzymes in Maintaining Endoplasmic Reticulum Homeostasis","volume":"436","year":"2024","journal-title":"J Mol Biol"},{"key":"2025121010250856000_2025.12.08.693056v1.2","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a013201"},{"key":"2025121010250856000_2025.12.08.693056v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.tips.2019.07.003"},{"key":"2025121010250856000_2025.12.08.693056v1.4","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1038\/s41392-023-01570-w","article-title":"Endoplasmic reticulum stress: molecular mechanism and therapeutic targets","volume":"8","year":"2023","journal-title":"Signal Transduct Target Ther"},{"key":"2025121010250856000_2025.12.08.693056v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-020-0250-z"},{"key":"2025121010250856000_2025.12.08.693056v1.6","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1007\/978-3-030-40204-4_8","article-title":"The Impact of the ER Unfolded Protein Response on Cancer Initiation and Progression: Therapeutic Implications","volume":"1243","year":"2020","journal-title":"Adv Exp Med Biol"},{"key":"2025121010250856000_2025.12.08.693056v1.7","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a013169"},{"key":"2025121010250856000_2025.12.08.693056v1.8","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.1000415"},{"key":"2025121010250856000_2025.12.08.693056v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/35014014"},{"key":"2025121010250856000_2025.12.08.693056v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/S1534-5807(02)00203-4"},{"key":"2025121010250856000_2025.12.08.693056v1.11","doi-asserted-by":"crossref","unstructured":"A. Bakunts et al., Ratiometric sensing of BiP-client versus BiP levels by the unfolded protein response determines its signaling amplitude. Elife 6 (2017).","DOI":"10.7554\/eLife.27518"},{"key":"2025121010250856000_2025.12.08.693056v1.12","doi-asserted-by":"crossref","unstructured":"M. Vitale et al., Inadequate BiP availability defines endoplasmic reticulum stress. Elife 8 (2019).","DOI":"10.7554\/eLife.41168"},{"key":"2025121010250856000_2025.12.08.693056v1.13","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1038\/s41580-024-00794-0","article-title":"Homeostasis control in health and disease by the unfolded protein response","volume":"26","year":"2025","journal-title":"Nat Rev Mol Cell Biol"},{"key":"2025121010250856000_2025.12.08.693056v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.tcb.2017.05.006"},{"key":"2025121010250856000_2025.12.08.693056v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/s41573-021-00320-3"},{"key":"2025121010250856000_2025.12.08.693056v1.16","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-019-0326-2"},{"key":"2025121010250856000_2025.12.08.693056v1.17","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M114.612515"},{"key":"2025121010250856000_2025.12.08.693056v1.18","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M114.618348"},{"key":"2025121010250856000_2025.12.08.693056v1.19","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.12621"},{"key":"2025121010250856000_2025.12.08.693056v1.20","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0005818"},{"key":"2025121010250856000_2025.12.08.693056v1.21","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2208317119"},{"key":"2025121010250856000_2025.12.08.693056v1.22","doi-asserted-by":"crossref","unstructured":"S. Preissler et al., AMPylation targets the rate-limiting step of BiP\u2019s ATPase cycle for its functional inactivation. Elife 6 (2017).","DOI":"10.7554\/eLife.29428"},{"key":"2025121010250856000_2025.12.08.693056v1.23","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.3337"},{"key":"2025121010250856000_2025.12.08.693056v1.24","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M117.799296"},{"key":"2025121010250856000_2025.12.08.693056v1.25","doi-asserted-by":"publisher","DOI":"10.1126\/science.1166382"},{"key":"2025121010250856000_2025.12.08.693056v1.26","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2014.10.007"},{"key":"2025121010250856000_2025.12.08.693056v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.tim.2021.08.003"},{"key":"2025121010250856000_2025.12.08.693056v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/nature10729"},{"key":"2025121010250856000_2025.12.08.693056v1.29","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0064901"},{"key":"2025121010250856000_2025.12.08.693056v1.30","doi-asserted-by":"crossref","first-page":"eadd5108","DOI":"10.1126\/sciadv.add5108","article-title":"Pseudomonas effector AvrB is a glycosyltransferase that rhamnosylates plant guardee protein RIN4","volume":"10","year":"2024","journal-title":"Sci Adv"},{"key":"2025121010250856000_2025.12.08.693056v1.31","doi-asserted-by":"publisher","DOI":"10.15252\/embj.2019102177"},{"key":"2025121010250856000_2025.12.08.693056v1.32","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1516930113"},{"key":"2025121010250856000_2025.12.08.693056v1.33","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-09023-1"},{"key":"2025121010250856000_2025.12.08.693056v1.34","doi-asserted-by":"publisher","DOI":"10.1038\/nn.3102"},{"key":"2025121010250856000_2025.12.08.693056v1.35","doi-asserted-by":"crossref","first-page":"167348","DOI":"10.1016\/j.bbadis.2024.167348","article-title":"Loss of Fic causes progressive neurodegeneration in a Drosophila model of hereditary spastic paraplegia","volume":"1870","year":"2024","journal-title":"Biochim Biophys Acta Mol Basis Dis"},{"key":"2025121010250856000_2025.12.08.693056v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.biochi.2024.05.012"},{"key":"2025121010250856000_2025.12.08.693056v1.37","doi-asserted-by":"crossref","first-page":"e040192","DOI":"10.1161\/JAHA.124.040192","article-title":"FICD (FIC Domain Protein Adenylyl Transferase) Deficiency Protects Mice From Hypertrophy-Induced Heart Failure and Promotes BiP (Binding Immunoglobulin Protein) -Mediated Activation of the Unfolded Protein Response and Endoplasmic Reticulum-Selective Autophagy in Cardiomyocytes","volume":"14","year":"2025","journal-title":"J Am Heart Assoc"},{"key":"2025121010250856000_2025.12.08.693056v1.38","doi-asserted-by":"crossref","first-page":"e1011723","DOI":"10.1371\/journal.pgen.1011723","article-title":"Loss of FIC-1-mediated AMPylation activates the UPRER and upregulates cytosolic HSP70 chaperones to suppress polyglutamine toxicity","volume":"21","year":"2025","journal-title":"PLoS Genet"},{"key":"2025121010250856000_2025.12.08.693056v1.39","doi-asserted-by":"publisher","DOI":"10.15252\/emmm.202216491"},{"key":"2025121010250856000_2025.12.08.693056v1.40","doi-asserted-by":"crossref","first-page":"102120","DOI":"10.1016\/j.molmet.2025.102120","article-title":"Pre-clinical model of dysregulated FicD AMPylation causes diabetes by disrupting pancreatic endocrine homeostasis","volume":"95","year":"2025","journal-title":"Mol Metab"},{"key":"2025121010250856000_2025.12.08.693056v1.41","doi-asserted-by":"publisher","DOI":"10.1016\/j.gim.2022.08.019"},{"key":"2025121010250856000_2025.12.08.693056v1.42","doi-asserted-by":"crossref","first-page":"1193","DOI":"10.1002\/mdc3.70033","article-title":"FIC Domain Protein Adenylyltransferase (FICD)-Related Complex Hereditary Spastic Paraplegia with Diabetes Mellitus","volume":"12","year":"2025","journal-title":"Mov Disord Clin Pract"},{"key":"2025121010250856000_2025.12.08.693056v1.43","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2021.100991"},{"key":"2025121010250856000_2025.12.08.693056v1.44","doi-asserted-by":"publisher","DOI":"10.1126\/science.abm3452"},{"key":"2025121010250856000_2025.12.08.693056v1.45","doi-asserted-by":"crossref","first-page":"880","DOI":"10.1021\/acschembio.4c00847","article-title":"Small-Molecule FICD Inhibitors Suppress Endogenous and Pathologic FICD-Mediated Protein AMPylation","volume":"20","year":"2025","journal-title":"ACS Chem Biol"},{"key":"2025121010250856000_2025.12.08.693056v1.46","doi-asserted-by":"publisher","DOI":"10.1007\/s12551-024-01188-4"},{"key":"2025121010250856000_2025.12.08.693056v1.47","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv1070%J"},{"key":"2025121010250856000_2025.12.08.693056v1.48","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbagen.2009.03.030"},{"key":"2025121010250856000_2025.12.08.693056v1.49","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-25076-7"},{"key":"2025121010250856000_2025.12.08.693056v1.50","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.7b00145"},{"key":"2025121010250856000_2025.12.08.693056v1.51","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-22596-0"},{"key":"2025121010250856000_2025.12.08.693056v1.52","first-page":"159515","article-title":"The lipid side of unfolded protein response","volume":"1869","year":"2024","journal-title":"Biochim Biophys Acta Mol Cell Biol Lipids"},{"key":"2025121010250856000_2025.12.08.693056v1.53","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2015.07.056"},{"key":"2025121010250856000_2025.12.08.693056v1.54","doi-asserted-by":"publisher","DOI":"10.1126\/scisignal.aad0446"},{"key":"2025121010250856000_2025.12.08.693056v1.55","doi-asserted-by":"crossref","first-page":"2889","DOI":"10.3762\/bjoc.15.283","article-title":"Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering","volume":"15","year":"2019","journal-title":"Beilstein J Org Chem"},{"key":"2025121010250856000_2025.12.08.693056v1.56","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20121899"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2025.12.08.693056","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:08:25Z","timestamp":1782277705000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2025.12.08.693056"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,8]]},"references-count":56,"URL":"https:\/\/doi.org\/10.64898\/2025.12.08.693056","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1073\/pnas.2533457123","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,12,8]]},"subtype":"preprint"}}