Ubiquitin Rhodamine 110: A Fluorescent Probe Illuminates the Activity of deubiquitinase

I. Introduction

II. Molecular Design and Detection Principle of Ubiquitin-Rhodamine 110

(I) Molecular Structural Characteristics

(II) Fluorescence Detection Mechanism

III. Core Applications in USP1 Activity Detection

(I) In vitro Enzymatic Property Research

Kinetic analysis shows that the cleavage efficiency (kcat/Km) of the USP1-UAF1 complex on the probe is significantly higher than that of USP1 alone, confirming the activating effect of UAF1;

In point mutation experiments, the fluorescence signal almost completely disappears after mutation of C90 (a key residue in the catalytic triad), directly verifying the necessity of the catalytic core.

(II) Intracellular Activity Visualization

In HeLa cells, the fluorescent signal is mainly enriched in the nucleus (consistent with the nuclear localization of USP1);

After ultraviolet-induced DNA damage, the intracellular fluorescent intensity in the nucleus increases by 2.3 times within 1 hour, indicating that USP1 activity is activated to participate in DNA repair;

The fluorescent signal in USP1 knockout cells decreases by 72%, confirming the specificity of the probe.

IV. Technical Advantages and Limitations

(I) Significant Advantages

High Sensitivity and Specificity: The detection limit reaches the nanomolar level, and the ubiquitin moiety ensures response only to DUBs without cross-reaction with other proteases;

Real-time Dynamic Monitoring: The fluorescent signal changes in real-time with the enzymatic cleavage reaction, enabling the acquisition of kinetic parameters, which is superior to endpoint detection methods such as Western blot;

High-throughput Compatibility: Suitable for 96/384-well plate systems, no need for radioactive labeling, and can quickly screen inhibitors.

(II) Existing Limitations

Substrate Simulation Limitations: The monoubiquitin structure cannot fully simulate the natural conformation of polyubiquitin chains or ubiquitinated target proteins, which may affect the recognition efficiency of some DUBs;

Cell Delivery Challenges: Unmodified probes have poor membrane penetration, and chemical modifications (such as PEGylation) may reduce their binding activity with enzymes;

Complex System Interference: Components in serum or cell lysates may quench fluorescence, requiring optimization of buffer systems to reduce background.

V. Application Value in Drug R&D

In in vitro screening, the inhibitory activity of compounds can be quickly evaluated by monitoring changes in fluorescence intensity. For example, the IC50 determination of KSQ-4279 (a USP1 allosteric inhibitor) shows that the half-inhibitory concentration deviates from the cell experiment results by less than 15%;

Combined with fluorescence polarization technology, it can distinguish the mode of action of inhibitors (competitive/non-competitive), providing a basis for structure-activity relationship analysis;

In cell-level experiments, modified probes can verify the intracellular activity of inhibitors. For example, after treatment with candidate compounds, the reduction range of intracellular fluorescent signals in tumor cells is linearly correlated with the inhibitor concentration, providing preliminary data for in vivo experiments.

VI. Summary and Outlook