In Situ Proteome Profiling and Bioimaging Applications of Small-Molecule Affinity-Based Probes Derived From DOT1L...

Area of Science:

• Biochemistry
• Chemical Biology
• Cancer Research

Background:

• DOT1L methylates histone H3 lysine 79 (H3K79), crucial for cancer development.
• Small-molecule DOT1L inhibitors like FED1 show promise but have limited cellular efficacy.
• Investigating DOT1L's biological roles requires understanding inhibitor behavior in cells.

Purpose of the Study:

• To design and synthesize cell-permeable, photo-reactive affinity-based probes (AfBPs) P1 and P2 as FED1 structural mimics.
• To evaluate the in vitro and in situ binding and inhibitory effects of P1 and P2 against DOT1L.
• To identify potential cellular off-targets of FED1 using proteome profiling.

Main Methods:

• Synthesis of photo-reactive and "clickable" AfBPs (P1, P2) based on FED1 structure.
• In vitro and live-cell assays to assess DOT1L inhibition and probe localization.
• Quantitative LC-MS/MS proteome profiling to identify FED1 off-targets.
• Validation of off-targets using pull-down/Western blotting (PD/WB) and CETSA.

Main Results:

• P1 and P2 demonstrated cell-permeability but predominantly localized outside the cell nucleus.
• This sub-cellular mislocalization explains the poor cellular activity of FED1.
• Proteome profiling identified over 100 potential FED1 off-targets, including NOP2.
• NOP2 was validated as a likely off-target of FED1.

Conclusions:

• The sub-cellular localization of FED1-mimicking probes is critical for its therapeutic efficacy.
• The study elucidates a key reason for the limited cellular activity of FED1.
• NOP2 emerges as a potential off-target, offering new avenues for cancer therapy research.