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Summary γ-Secretase, a four-subunit transmembrane aspartic proteinase, is a highly valued drug target in Alzheimer's disease and cancer. Despite significant progress in structural studies, the respective molecular mechanisms and binding modes of γ-secretase inhibitors (GSIs) and modulators (GSMs) remain uncertain. Here, we developed biotinylated cleavable-linker photoprobes based on the BMS-708163 GSI to study its interaction with γ-secretase. Comparison of four cleavable linkers indicated that the hydrazine-labile N -1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker was cleaved most efficiently to release photolabeled and affinity-captured presenilin-1 (PS1), the catalytic subunit of γ-secretase. Peptide mapping showed that the BMS-708163-based probe photoinserted at L282 of PS1. This insertion site was consistent with the results of molecular dynamics simulations of the γ-secretase complex with inhibitor. Taken together, this work reveals the binding site of a GSI and offers insights into the mechanism of action of this class of inhibitors. [ABSTRACT FROM AUTHOR]

Utilizing a phenotypic screen, we identified chemical matter that increased astrocytic apoE secretion in vitro. We designed a clickable photoaffinity probe based on a pyrrolidine lead compound and carried out probe-based quantitative chemical proteomics in human astrocytoma CCF-STTG1 cells to identify liver x receptor β (LXRβ) as the target. Binding of the small molecule ligand stabilized LXRβ, as shown by cellular thermal shift assay (CETSA). In addition, we identified a probe-modified peptide by mass spectrometry and proposed a model where the photoaffinity probe is bound in the ligand-binding pocket of LXRβ. Taken together, our findings demonstrated that the lead chemical matter bound directly to LXRβ, and our results highlight the power of chemical proteomic approaches to identify the target of a phenotypic screening hit. Additionally, the LXR photoaffinity probe and lead compound described herein may serve as valuable tools to further evaluate the LXR pathway. • A phenotypic screen identifies novel chemical matter regulating apoE secretion in vitro • Chemical biology strategy identifies LXRβ as the functional target of phenotypic hit • Study highlight the critical nature of counter-screening strategy as part of hit triage • Photoaffinity probe and potent lead may serve as valuable tools to probe LXR biology Phenotypic screens can potentially deliver first-in-class drugs. Seneviratne et al. describe a screen and hit triage strategy to identify small molecule activators of apoE secretion in human astrocytes. The team applied comprehensive chemical biology tactics for target deconvolution to identify LXRβ as the target of the lead chemical matter disclosed. [ABSTRACT FROM AUTHOR]

Sterol regulatory element-binding proteins (SREBPs) are master transcriptional regulators of the mevalonate pathway and lipid metabolism and represent an attractive therapeutic target for lipid metabolic disorders. SREBPs are maintained in the endoplasmic reticulum (ER) in a tripartite complex with SREBP cleavage-activating protein (SCAP) and insulin-induced gene protein (INSIG). When new lipid synthesis is required, the SCAP-SREBP complex dissociates from INSIG and undergoes ER-to-Golgi transport where the N-terminal transcription factor domain is released by proteolysis. The mature transcription factor translocates to the nucleus and stimulates expression of the SREBP gene program. Previous studies showed that dipyridamole, a clinically prescribed phosphodiesterase (PDE) inhibitor, potentiated statin-induced tumor growth inhibition. Dipyridamole limited nuclear accumulation of SREBP, but the mechanism was not well resolved. In this study, we show that dipyridamole selectively blocks ER-to-Golgi movement of the SCAP-SREBP complex and that this is independent of its PDE inhibitory activity. • Dipyridamole, a PDE inhibitor, decreases lipogenesis by inhibiting SREBP maturation • Dipyridamole blocks SREBP independent of its PDE inhibitory action • A clickable photoprobe dipyridamole derivative binds to INSIG and SCAP • Dipyridamole derivatives are potentially therapeutic for lipid disorders Esquejo et al. report that dipyridamole, an FDA-approved anti-thrombotic phosphodiesterase (PDE) inhibitor, impedes endoplasmic reticulum to Golgi trafficking of sterol regulatory element-binding proteins (SREBPs) preventing their activation. A chemically modified version of dipyridamole that has no effect on PDE remains effective at SREBP blockage. [ABSTRACT FROM AUTHOR]

Here, we describe a protocol for a photoaffinity labeling probe strategy for target deconvolution in live cells. We made a chemical probe by incorporation of a photoreactive group to covalently cross-link with adjacent amino acid residues upon UV irradiation. Click chemistry-based enrichment captures labeled proteins for proteomic analysis. Here, we detail specifics for finding targets of LXRβ, but the protocol has potential for application to other targets. For complete details on the use and execution of this protocol, please refer to Seneviratne et al. (2020)., Competing Interests: All authors are or were Pfizer employees at the time of assembling the manuscript. W.L. is an employee at Dewpoint Therapeutics., (© 2021 The Author(s).)