title: Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors.
abstract: Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC50against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC50shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned.
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.|Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: Joel R
authors.identifier: 0000-0001-6855-2969
authors.initials: JR
authors.lastname: Walker
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.|Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: Mary P
authors.initials: MP
authors.lastname: Hall
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.|Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: Chad A
authors.initials: CA
authors.lastname: Zimprich
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.|Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Robers
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.|Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Duellman
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.|Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Machleidt
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.|Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: Jacquelynn
authors.initials: J
authors.lastname: Rodriguez
authors.affiliation: Promega Biosciences LLC , 277 Granada Drive, San Luis Obispo, California 93401, United States.|Promega Corporation , 2800 Woods Hollow Road, Madison, Wisconsin 53711-5399, United States.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhou
mesh_terms: D002463:Cell Membrane Permeability; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D020128:Inhibitory Concentration 50; D008156:Luciferases / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D008156:Luciferases