UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1’s ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.

Hyperpolarized (HP) carbon-13 [¹³C] enables the specific investigation of dynamic metabolic and physiologic processes via in vivo MRI-based molecular imaging. As the leading HP metabolic agent, [1-¹³C]pyruvate plays a pivotal role due to its rapid tissue uptake and central role in cellular energetics. Dissolution dynamic nuclear polarization (d-DNP) is considered the gold standard method for the production of HP metabolic probes; however, development of a faster, less expensive technique could accelerate the translation of metabolic imaging via HP MRI to routine clinical use. Signal Amplification by Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) achieves rapid hyperpolarization by using parahydrogen (p-H₂) as the source of nuclear spin order. Currently, SABRE is clinically limited due to the toxicity of the iridium catalyst, which is crucial to the SABRE process. To mitigate Ir contamination, we introduce a novel iteration of the SABRE catalyst, incorporating bis(polyfluoroalkylated) imidazolium salts. This novel perfluorinated SABRE catalyst retained polarization properties while exhibiting an enhanced hydrophobicity. This modification allows the easy removal of the perfluorinated SABRE catalyst from HP [1-¹³C]-pyruvate after polarization in an aqueous solution, using the ReD-SABRE protocol. The residual Ir content after removal was measured via ICP-MS at 177 ppb, which is the lowest reported to date for pyruvate and is sufficiently safe for use in clinical investigations. Further improvement is anticipated once automated processes for delivery and recovery are initiated. SABRE-SHEATH using the perfluorinated SABRE catalyst can become an attractive low-cost alternative to d-DNP to prepare biocompatible HP [1-¹³C]-pyruvate formulations for in vivo applications in next-generation molecular imaging modalities.

The analytical quality of compounds subjected to high-throughput screening (HTS) impacts accurate interpretation of assay results, with poor quality samples potentially leading to false negatives or positives. The Tox21 "10K" library consists of over 8900 unique compounds, spanning a diverse landscape of environmental and pharmaceutical chemicals, posing opportunities and challenges for analytical quality control (QC) determinations. Tox21 sample plates stored in DMSO at ambient conditions for 0 (T0) and/or 4 months (T4), totaling more than 13K unique sample identifiers (Tox21 IDs), were subjected to various analyses, including liquid and gas chromatography mass spectrometry (LC-MS, GC-MS) and nuclear magnetic resonance (NMR). Results for each sample at T0 or T4 underwent expert review and, where possible, a QC grade conveying purity, identity, and concentration was assigned. Herein, we relate details of the methods applied and report on the original (v0) Tox21 ID level results. Thirteen QC grades were condensed to 5 quality scores to aid global analysis, resulting in reinterpretation and improvement of >700 sample grades. Of the 92% T0 samples successfully graded, 76% exceeded 90% purity. For 76% of samples that were also tested at T4, 89% showed no evidence of sample loss or degradation. Prioritized quality bins were used to summarize thousands of replicate sample-level QC results to a compound-level QC score to support structure-based analyses. ToxPrint chemotype analysis identified structural features enriched in unstable compounds, as well as in high and low quality T0 subsets. Predicted vapor pressure was weakly correlated with low-concentration QC indicators, reflecting likely entanglement with method amenability and quality issues. Finally, an ongoing EPA effort to re-evaluate the original QC spectra is generating insights that will further modify QC grades. Tox21 QC spectra and results will be made available in a new public QC browser, facilitating further evaluation to support HTS interpretation and modeling applications.