Living Cell-Target Responsive Accessibility Profiling Reveals Silibinin Targeting ACSL4 for Combating Ferroptosis

Abstract

We report a living cell-target responsive accessibility profiling (LC-TRAP) approach to identify the targetome of silibinin (SIL), a well-established hepatoprotective natural product (NP), in HepG2 cells. Proteins showing accessibility changes, probed by covalent lysine labeling reagents and leveraged by multiplexed quantitative proteomics, following the administration of SIL to the living cells were assigned as potential targets. Among the assigned targetome, ACSL4, an enzyme essential for ferroptosis induction, might be involved in the hepatoprotective effects of SIL and hence was intensively validated. We first demonstrated that SIL protected HepG2 cells from ferroptosis dependent on ACSL4. Then, we used biophysical assays and a SIL-derivatized chemical probe to corroborate that SIL can bind to ACSL4. The ensuing enzymatic assays showed that SIL inhibited ACSL4 enzymatic activity, thereby mitigating the ACSL4-mediated ferroptosis. As such, we revealed that ACSL4 inhibition, using SIL as a model compound, represents a promising hepatoprotective strategy. Further, since TRAP probes the accessibility changes of reactive proteinaceous lysines, it can pinpoint the proximal regions where the ligand engagement may occur. Thus, the LC-TRAP analysis of SIL, the newly discovered ligand of ACSL4, and arachidonic acid (AA), the substrate, intriguingly showed that SIL and AA both affected the conformation of the K536-proximal region of ACSL4, albeit through distinct binding patterns. Collectively, we describe a straightforward LC-TRAP workflow that does not involve ligand-derived probe synthesis and is widely applicable to target discovery of NPs.

Natural Flavonoids and Ferroptosis: Potential Therapeutic Opportunities for Human Diseases

“…128 Specifically, biochanin A not only alleviated intracellular iron overload by upregulating the expression of SLC40A1 and down-regulating the expression of TFR but also reduced lipid peroxidation by regulating the NRF2/SLC7A11/GPX4 axis. These findings suggest that most isoflavones can simultaneously target 132 Further studies confirmed that silibinin could suppress the enzymatic activity of ACSL4 by directly binding to it, which inhibited ferroptosis in hepatocytes and thus exerted hepatoprotective effects. Dihydroquercetin, a plant flavanonol isolated from yew, protected against SiO 2 -induced lung fibrosis in C57BL/6 mice.…”

“…In addition, other types of flavonoids, including flavanonols, flavanols, and biflavonoids, can also act as potent regulators of ferroptosis. − Yan et al discovered that ACSL4 was the target of silibinin in HepG2 cells by developing a living cell-target responsive accessibility profiling . Further studies confirmed that silibinin could suppress the enzymatic activity of ACSL4 by directly binding to it, which inhibited ferroptosis in hepatocytes and thus exerted hepatoprotective effects.…”

Cellular protein painting for structural and binding sites analysis via lysine reactivity profiling with o-phthalaldehyde

“…TEPP-46 binds and activate PKM2 (pyruvate kinase M2) by promoting the tetramerization of the protein (30). A recent work treated cells with TEPP-46 followed chemical labelling after cell lysis with isotope-coded formaldehyde (CD2O) to identify structural conformation changes induced in PMK2 (31). To further validate the use of RAPID-OPA to study conformational changes in protein structures directly in cells, HEK293T cells were treated with TEPP-46 for 20 min, followed by RAPID-OPA where proteins were labelled with OPA before cell lysis and analyzed with MS in PRM mode (Fig.…”

Studying Protein–Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry

“…The integration of protein-denaturation principles with lysine-reactive qXL-MS provides a protein–ligand assay. Utilizing additional strategies such as Activity-Based Protein Profiling and Target-Responsive Accessibility Profiling could allow greater orthogonal access in evaluating ligand–target interactions. , The strategy presented has the potential to complement other protein-denaturation MS-based approaches in studying drug–target interactions on the cellular scale.…”