Small-molecule allosteric inhibitors of GPX4.

Encouraged by the dependence of drug-resistant, metastatic cancers on GPX4, we examined biophysical mechanisms of GPX4 inhibition, which revealed an unexpected allosteric site. We found that this site was involved in native regeneration of GPX4 under low glutathione conditions. Covalent binding of inhibitors to this allosteric site caused a conformational change, inhibition of activity, and subsequent cellular GPX4 protein degradation. To verify this site in an unbiased manner, we screened a library of compounds and identified and validated that an additional compound can covalently bind in this allosteric site, inhibiting and degrading GPX4. We determined co-crystal structures of six different inhibitors bound in this site. We have thus identified an allosteric mechanism for small molecules targeting aggressive cancers dependent on GPX4.

Interrogation of Eukaryotic Stop Codon Readthrough Signals by in Vitro RNA Selection.

RNA signals located downstream of stop codons in eukaryotic mRNAs can stimulate high levels of translational readthrough by the ribosome, thereby giving rise to functionally distinct C-terminally extended protein products. Although many readthrough events have been previously discovered in Nature, a broader description of the stimulatory RNA signals would help to identify new reprogramming events in eukaryotic genes and provide insights into the molecular mechanisms of readthrough. Here, we explore the RNA reprogramming landscape by performing in vitro translation selections to enrich RNA readthrough signals de novo from a starting randomized library comprising >1013 unique sequence variants. Selection products were characterized using high-throughput sequencing, from which we identified primary sequence and secondary structure readthrough features. The activities of readthrough signals, including three novel sequence motifs, were confirmed in cellular reporter assays. Then, we used machine learning and our HTS data to predict readthrough activity from human 3'-untranslated region sequences. This led to the discovery of >1.5% readthrough in four human genes (CDKN2B, LEPROTL1, PVRL3, and SFTA2). Together, our results provide valuable insights into RNA-mediated translation reprogramming, offer tools for readthrough discovery in eukaryotic genes, and present new opportunities to explore the biological consequences of stop codon readthrough in humans.

FINO2 initiates ferroptosis through GPX4 inactivation and iron oxidation.

Ferroptosis is a non-apoptotic form of regulated cell death caused by the failure of the glutathione-dependent lipid-peroxide-scavenging network. FINO2 is an endoperoxide-containing 1,2-dioxolane that can initiate ferroptosis selectively in engineered cancer cells. We investigated the mechanism and structural features necessary for ferroptosis initiation by FINO2. We found that FINO2 requires both an endoperoxide moiety and a nearby hydroxyl head group to initiate ferroptosis. In contrast to previously described ferroptosis inducers, FINO2 does not inhibit system xc- or directly target the reducing enzyme GPX4, as do erastin and RSL3, respectively, nor does it deplete GPX4 protein, as does FIN56. Instead, FINO2 both indirectly inhibits GPX4 enzymatic function and directly oxidizes iron, ultimately causing widespread lipid peroxidation. These findings suggest that endoperoxides such as FINO2 can initiate a multipronged mechanism of ferroptosis.

Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches.

Protein synthesis in eukaryotes is regulated by diverse reprogramming mechanisms that expand the coding capacity of individual genes. Here, we exploit one such mechanism, termed -1 programmed ribosomal frameshifting (-1 PRF), to engineer ligand-responsive RNA switches that regulate protein expression. First, efficient -1 PRF stimulatory RNA elements were discovered by in vitro selection; then, ligand-responsive switches were constructed by coupling -1 PRF stimulatory elements to RNA aptamers using rational design and directed evolution in Saccharomyces cerevisiae. We demonstrate that -1 PRF switches tightly control the relative stoichiometry of two distinct protein outputs from a single mRNA, exhibiting consistent ligand response across whole populations of cells. Furthermore, -1 PRF switches were applied to build single-mRNA logic gates and an apoptosis module in yeast. Together, these results showcase the potential for harnessing translation-reprogramming mechanisms for synthetic biology, and they establish -1 PRF switches as powerful RNA tools for controlling protein synthesis in eukaryotes.