A Chemical Proteomic Analysis of Illudin-Interacting Proteins

Abstract

The illudin natural product family are fungal secondary metabolites with a characteristic spirocyclopropyl-substituted fused 6,5-bicyclic ring system. They have been extensively studied for their cytotoxicity in various tumor cell types, and semisynthetic derivatives with improved therapeutic characteristics have progressed to clinical trials. Although it is believed that this potent alkylating compound class acts mainly through DNA modification, little is known about its binding to protein sites in a cellular context. To reveal putative protein targets of the illudin family in live cancer cells, we employed a semisynthetic strategy to access a series of illudin-based probes for activity-based protein profiling (ABPP). While the probes largely retained potent cytotoxicity, proteomic profiling studies unraveled multiple protein hits, suggesting that illudins exert their mode of action not from addressing a specific protein target but rather from DNA modification and unselective protein binding.

Keywords: antitumor agents; drug discovery; natural products; protein binding; proteomics.

Figure 1

Structures of illudin M and S and semisynthetic analogue irofulven (HMAF).

Scheme 1

Proposed mechanism for DNA alkylation involving conjugate addition to the α,β‐unsaturated enone to form an electrophilic intermediate either through a cellular nucleophile (1) or thorough reductive bioactivation using NADPH for hydride delivery (4) followed by nucleophilic opening of the spirocyclopropyl ring to yield a stable aromatic product (2, 3, 5, or 6).

Figure 2

Semisynthetic strategy to access illudin‐based ABPP probes: Isolation of natural products ILM‐0 and ILS‐0 from O. olearius, followed by introduction of terminal alkyne handles via synthetic chemistry to access illudin‐based probes for proteomic profiling. Reaction conditions: a) 5‐hexynoic acid, DIPC, CH₂Cl₂, RT, 24 h, 61 %; b) 5‐hexynoic acid, DIPC, DMAP, CH₂Cl₂, RT, 24 h, 55 %; c) 2‐(3‐(but‐3‐yn‐1‐yl)‐3H‐diazirin‐3‐yl)acetic acid,35 DIPC, DMAP, CH₂Cl₂, RT, 24 h, 65 %; d) 3‐ethynylphenol, PPh₃, DIAD, THF, RT, 24 h, 6 %; e) PyrSO₃, DIPEA, DMSO, CH₂Cl₂, −20 °C, 1 h, 87 %; f) i. propargylamine, Na₂SO₄, RT, 24 h; ii. NaBH(OAc)₃, RT, 16 h, 28 %.

Figure 3

A) The concept of ABPP: Live cells are treated with probes, followed by optional UV irradiation. Cells are lysed, terminal alkynes functionalized with a reporter group by CuAAC and probe‐labeled proteins visualized via fluorescence SDS‐PAGE or identified via mass spectrometry‐based proteomics after downstream protein enrichment. B) Analysis of protein binding: In‐gel fluorescence analysis after labeling with ILS‐1, ILS‐2, ILS‐3, ILM‐1, and ILS‐1 PP (10 μm; 1 h) in A549 cells. For Coomassie stained gel, see Figure S1.

Figure 4

A) Protein labeling profiles of illudin‐based probes ILS‐1, ILS‐2, and ILS‐1 PP (3 μm; incubation for 1 h 37 °C; in case of ILS‐1 PP, UV irradiation for 30 min, 4 °C) in A549 cells. Volcano plots illustrate the log₂‐fold enrichment of proteins compared to DMSO in the soluble fraction. Identified proteins were aligned with diazirine‐associated off‐target protein binding published by Kleiner et al.35 and classified accordingly as low‐confidence (red), medium‐confidence (yellow), or high‐confidence (black) hits. Data represent average values; n=6 (two independent experiments performed in triplicate). For protein labeling profiles of insoluble proteins, see Figure S8. For details, see the Supporting Table in the Supporting Information. B) Overlap of proteins identified in the ILS‐1, ILS‐2, and ILS‐1 PP enrichment (log₂ protein ratio≥1, p≤0.05). C) List of proteins enriched (log₂ protein ratio≥1, p≤0.05) and shared by ILS‐1, ILS‐2, or ILS‐1 PP.