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Identification of 5-Carboxyfluorescein as a Probe Substrate of SLC46A3 and Its Application in a Fluorescence-Based In
Ryuto Tomabechi1, Miki Miyasato1, Taeka Sato1
1Department of Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji, Tokyo192-0392, Japan.
Abstract:
The therapeutic modalities that involve the endocytosis pathway, including antibody-drug conjugates (ADCs), have recently been developed. Since the drug escape from endosomes/lysosomes is a determinant of their efficacy, it is important to optimize the escape, and the cellular evaluation system is needed. SLC46A3, a lysosomal membrane protein, has been implicated in the pharmacological efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC used for the treatment of breast cancer, and the cellular uptake efficacy of lipid-based nanoparticles. Recently, we identified the SLC46A3 function as a proton-coupled steroid conjugate and bile acid transporter, which can directly transport active catabolites of T-DM1. Thus, the rapid and convenient assay systems for evaluating the SLC46A3 function may help to facilitate ADC development and to clarify the physiological roles in endocytosis. Here, we show that SLC46A3 dC, which localizes to the plasma membrane owing to lacking a lysosomal-sorting motif, has a great ability to transport 5-carboxyfluorescein (5-CF), a fluorescent probe, in a pH-dependent manner. 5-CF uptake mediated by SLC46A3 was significantly inhibited by compounds reported to be SLC46A3 substrates/inhibitors and competitively inhibited by estrone 3-sulfate, a typical SLC46A3 substrate. The inhibition assays followed by uptake studies revealed that SG3199, a pyrrolobenzodiazepine dimer, which has been used as an ADC payload, is a substrate of SLC46A3. Accordingly, the fluorescence-based assay system for the SLC46A3 function using 5-CF can provide a valuable tool to evaluate the interaction of drugs/drug candidates with SLC46A3.
Insights
Researchers developed a new assay to evaluate SLC46A3 transporter function, crucial for antibody-drug conjugate (ADC) efficacy. This system aids in optimizing drug delivery and understanding endocytosis for cancer therapies.
Area of Science:
- Cell Biology
- Pharmacology
- Drug Delivery Systems
Background:
- Therapeutic modalities utilizing the endocytosis pathway, such as antibody-drug conjugates (ADCs), are emerging.
- Optimizing drug escape from endosomes/lysosomes is critical for ADC efficacy, necessitating robust cellular evaluation systems.
- SLC46A3, a lysosomal membrane protein, influences the efficacy of trastuzumab emtansine (T-DM1) and nanoparticle uptake.
Purpose of the Study:
- To develop rapid and convenient assay systems for evaluating SLC46A3 function.
- To facilitate antibody-drug conjugate (ADC) development and clarify SLC46A3's physiological roles in endocytosis.
- To identify novel substrates and inhibitors of SLC46A3 for improved therapeutic strategies.
Main Methods:
- Utilized a modified SLC46A3 (SLC46A3 dC) localized to the plasma membrane.
- Employed 5-carboxyfluorescein (5-CF) as a fluorescent probe to measure pH-dependent transport activity.
- Conducted inhibition assays using known SLC46A3 substrates/inhibitors and performed competitive inhibition studies with estrone 3-sulfate.
Main Results:
- SLC46A3 dC demonstrated significant pH-dependent transport of 5-CF.
- Transport activity was inhibited by known SLC46A3 substrates/inhibitors and competitively by estrone 3-sulfate.
- Identified SG3199, an ADC payload, as a novel substrate of SLC46A3.
Conclusions:
- A fluorescence-based assay system using 5-CF effectively evaluates SLC46A3 function.
- This assay system provides a valuable tool for assessing drug and drug candidate interactions with SLC46A3.
- Findings support the optimization of ADCs and enhance understanding of SLC46A3's role in endocytosis and drug efficacy.
