Abstract
Genetic mutations cause a wide spectrum of human disease by disrupting protein folding, both during and after synthesis. Transient de-novo folding intermediates therefore represent potential drug targets for pharmacological correction of protein folding disorders. Here we develop a FRET-based high-throughput screening (HTS) assay in 1,536-well format capable of identifying small molecules that interact with nascent polypeptides and correct genetic, cotranslational folding defects. Ribosome nascent chain complexes (RNCs) containing donor and acceptor fluorophores were isolated from cell free translation reactions, immobilized on Nickel-NTA/IDA beads, and imaged by high-content microscopy. Quantitative FRET measurements obtained from as little as 0.4 attomole of protein/bead enabled rapid assessment of conformational changes with a high degree of reproducibility. Using this assay, we performed a pilot screen of ~ 50,000 small molecules to identify compounds that interact with RNCs containing the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) harboring a disease-causing mutation (A455E). Screen results yielded 133 primary hits and 1 validated hit that normalized FRET values of the mutant nascent peptide. This system provides a scalable, tractable, structure-based discovery platform for screening small molecules that bind to or impact the folding of protein substrates that are not amenable to traditional biochemical analyses.
Highlights
Genetic mutations cause a wide spectrum of human disease by disrupting protein folding, both during and after synthesis
We previously showed that: (i) NBD1 acquires its structure cotranslationally through a distinct series of carefully choreographed folding events, (ii) Cystic fibrosis (CF)-causing mutations located within NBD1 can disrupt the nascent polypeptide folding landscape, and (iii) genetic suppressor mutations that restore cotranslational folding can partially restore trafficking of full-length CFTR1,2,24
Bead binding characteristics were optimized using His10-tagged and non-His-tagged Cyan Fluorescent Protein (CFP)-NBD1 Ribosome nascent chain complexes (RNCs) incubated with 2 × 1 05 17 μm beads, 5 × 1 04 34 μm beads, or 6 × 103 100 μm beads which provide similar aggregate binding surface area
Summary
Genetic mutations cause a wide spectrum of human disease by disrupting protein folding, both during and after synthesis. Quantitative FRET measurements obtained from as little as 0.4 attomole of protein/bead enabled rapid assessment of conformational changes with a high degree of reproducibility Using this assay, we performed a pilot screen of ~ 50,000 small molecules to identify compounds that interact with RNCs containing the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) harboring a disease-causing mutation (A455E). HTS efforts have largely focused on F508del and have identified numerous corrector molecules[34] These extensive efforts to correct F508del CFTR folding in cells, using small molecules, have led to recent FDA approval of three combination drugs: Lumacaftor (VX-809) + Ivacaftor in 2015, Tezacaftor (VX-661) + Ivacaftor in 2018, and Elexacaftor (VX-445) + Tezacaftor + Ivacaftor in 201935–38. This has suggested that some CFTR correctors may act on one or more transient biosynthetic intermediates[42] and that understanding cotranslational folding pathways may provide a potential novel approach for developing new treatment s trategies[24]
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