Chemical probes targeting lysosomal surface proteins for cellular visualization and tracking

Overview

This project develops chemical probes that selectively bind to proteins residing on the lysosomal surface, providing researchers with a direct tool for visualizing and tracking lysosomes in cells and organisms. Unlike conventional lysosomal markers such as LAMPs that localize to the lumen, the targeted proteins are integral to the lysosomal membrane and fully accessible from the cytosolic side. This means probes do not need to cross multiple membranes to reach their targets, enabling more efficient and specific labeling.

The research focuses on three well-characterized lysosomal surface proteins: the Ragulator complex, SLC38A9, and SLC38A7. These proteins are strict lysosome residents with known structural features and potential small-molecule binding pockets. Expression patterns vary, with Ragulator and SLC38A9 expressed ubiquitously, while SLC38A7 is highly expressed in the brain and liver. The resulting probes could support research in cell biology, neuroscience, metabolic disease, and drug discovery targeting lysosomal function.

Technical specifications

Target proteins and rationale:

• Ragulator complex — A multi-protein complex whose p18 subunit enables highly efficient lysosomal targeting via gene fusion. Multiple cryo-EM structures have been solved, revealing potential small-molecule binding pockets on its surface.
• SLC38A9 — A 12-transmembrane helix transporter strictly localized to lysosomes, harboring dedicated drug-binding pockets.
• SLC38A7 — Another 12-transmembrane transporter with strict lysosomal localization and high expression in brain and liver tissues.

Discovery and validation approach:

• Screening a 6-million DNA-encoded library of small molecules against purified, crystal-quality target proteins
• Hit validation using SPR (surface plasmon resonance) and in-cell binding assays
• Analog development and structure-activity relationship studies to optimize binders
• Cryo-EM structure determination of probe-protein complexes to guide design

Key advantages:

• Cytosolic accessibility eliminates the need for probes to penetrate membranes
• Strict lysosomal localization of targets reduces off-target binding
• Multiple targets with different expression profiles enable tissue-selective applications

Technology readiness level

This project begins at an advanced starting point. The team has already purified crystal-quality samples of Ragulator, SLC38A9, and SLC38A7, and has initiated screening against a 6-million-member DNA-encoded library. Multiple cryo-EM structures of Ragulator have been solved, and binding pockets have been identified.

Over the next two years, the project will progress through hit identification, analog optimization, and structural validation of probe-protein interactions. The current stage corresponds to early-to-mid lead discovery. Successful completion will yield validated chemical probes ready for use as research tools, with potential downstream applications in lysosome-related disease research and therapeutic target validation.