Selective inhibition of human translation termination by a drug-like compound

Abstract

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

“…S2). This reconstruction at 2.2 Å resolution allowed us to build the most accurate structure of a mammalian 80S ribosome so far and directly visualize many protein and virtually all rRNA modifications identified for the human ribosome based on quantitative mass spectrometry and as interpreted in a recent human ribosome structure (20,21), consistent with the complete conservation of all modified residues between rabbit and human rRNAs (figs. S4 and S5; and tables S1 to S3).…”

Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

“…Only a handful of other ligand-dependent arrest peptides have been structurally characterized to date 26,[39][40][41][42] , including only one other bacterial amino acid-sensing peptide 26 . The bestcharacterized group comprises drug-dependent arrest peptides belonging to the Erm family, which undergo translational arrest in response to macrolide antibiotics to regulate the expression of macrolide-resistance genes.…”

Context-specific action of macrolide antibiotics on the eukaryotic ribosome

“…10 ), but its specificity and mode of action are significantly different. In contrast to TEL, which arrests translation at distinct sites, PF846 slows down the progression of the ribosome over several consecutive mRNA codons at the site of arrest 5 – 7 . At each of these codons the ribosome operates with a different combination of donor and acceptor ligands and therefore, in contrast to TEL, PF846 specificity is less dependent on the nature of the PTC substrates, but rather on the unusual trajectory of the nascent chain in the NPET 6 , 7 .…”

“…In contrast to TEL, which arrests translation at distinct sites, PF846 slows down the progression of the ribosome over several consecutive mRNA codons at the site of arrest 5 – 7 . At each of these codons the ribosome operates with a different combination of donor and acceptor ligands and therefore, in contrast to TEL, PF846 specificity is less dependent on the nature of the PTC substrates, but rather on the unusual trajectory of the nascent chain in the NPET 6 , 7 . Consistently, while macrolides inhibit peptide bond formation 24 – 27 , PF846 interferes with ribosome translocation and with translation termination 6 , 7 .…”

“…Therefore, the discovery of PF846, a small molecule that binds to eukaryotic ribosomes and selectively inhibits translation of only a subset of polypeptides, including the therapeutically-significant protein PCSK9 involved in cholesterol homeostasis, came as a big and welcomed surprise 5 , 6 . Illuminating biochemical and structural studies have shown that PF846 binds in the nascent peptide exit tunnel (NPET) of the large ribosomal subunit and interferes with translation of several specific nascent polypeptides, that assume an idiosyncratic conformation in the tunnel 6 , 7 . However, because many details of the mechanism of PF846 action remain unknown, predicting the proteins whose synthesis would be inhibited by this compound would be a difficult task.…”