Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors.

Area of Science:

• Biochemistry
• Molecular Biology
• Assay Development

Background:

• NanoLuc (Nluc) luciferase is a smaller, brighter, and superior alternative to traditional firefly (Fluc) and Renilla (Rluc) luciferases.
• The superior properties of Nluc present opportunities for biological, biomedical, and safety applications.
• There is an urgent need for Nluc inhibitors to enable selective bioluminescent suppression and multiplexing compatibility.

Purpose of the Study:

• To discover and develop potent Nluc inhibitors.
• To create both cell-permeable and cell-impermeable Nluc inhibitors.
• To explore Nluc inhibitor structural motifs for future assay development.

Main Methods:

• Screening a PubChem compound library to identify initial Nluc inhibitors.
• Derivatizing structural fragments of a hit compound to optimize potency and cell permeability.
• Evaluating compound permeability by comparing EC50 shifts in live and lysed cells expressing Nluc.
• Utilizing luminescence imaging to confirm inhibitor activity on intracellular and extracellular Nluc.

Main Results:

• Identified a family of potent, single-digit nanomolar Nluc inhibitors.
• Developed both cell-permeable inhibitors targeting intracellular Nluc and cell-impermeable inhibitors targeting extracellular Nluc.
• Demonstrated high specificity, with no observed activity against firefly luciferase or NanoBit.
• Confirmed low cellular toxicity of the developed inhibitors.

Conclusions:

• Novel NanoLuc inhibitors with varying permeability have been successfully developed.
• These inhibitors exhibit high specificity and low toxicity, making them suitable for diverse applications.
• The identified structural motifs provide a foundation for developing novel assays based on relief-of-inhibition mechanisms.