Mechanism and Structure-Guided Optimization of SLC1A1/EAAT3-Selective Inhibitors in Kidney Cancer

Area of Science:

• Biochemistry
• Structural Biology
• Oncology

Background:

• Renal Cell Carcinomas (RCCs) exhibit metabolic dependence on the SLC1A1/EAAT3 transporter.
• Pharmacological targeting of SLC1A1/EAAT3 presents significant challenges.
• Understanding the structural basis of SLC1A1/EAAT3 inhibition is crucial for therapeutic development.

Purpose of the Study:

• To determine the cryo-EM structure of human SLC1A1/EAAT3 bound to the inhibitor 3e.
• To elucidate the binding mechanism and allosteric inhibition of SLC1A1/EAAT3 by bicyclic imidazo[1,2-α]pyridine-3-amine (BIA) derivatives.
• To guide the development of novel, more potent SLC1A1/EAAT3 inhibitors for RCC treatment.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of human SLC1A1/EAAT3.
• Biochemical assays to assess inhibitor binding and transport activity.
• Site-directed mutagenesis to investigate the role of the allosteric binding pocket.
• Medicinal chemistry to synthesize novel BIA derivatives.

Main Results:

• The cryo-EM structure revealed that compound 3e binds to a membrane-embedded allosteric pocket in the apo state of SLC1A1/EAAT3.
• Inhibition involves blocking substrate and sodium binding, and preventing essential transport movements.
• Mutations in the allosteric pocket abolished 3e binding and its cytotoxicity in RCC cells.
• Newly developed BIA derivatives, PBJ1 and PBJ2, demonstrated enhanced cytotoxicity via SLC1A1/EAAT3 inhibition.

Conclusions:

• Compound 3e effectively inhibits SLC1A1/EAAT3 by binding to a unique allosteric site.
• The identified binding site and mechanism provide a basis for designing selective SLC1A1/EAAT3 inhibitors.
• Structure-guided development of BIA derivatives offers a promising therapeutic strategy against RCC by targeting metabolic vulnerabilities.