A LOPAC1280 screen using this assay protocol performed well (Z' = 0.63, S:B 7:1). Compounds with apparent toxicity within the assay time were excluded by a separate viability counter-assay. Moderate cut-off criteria (IC50 <20 µM and curve form; see Supporting Datafile) gave a <1.5% hit rate (18 of 1278 compounds, plus TRi-1 and TRi-2; Fig. , Fig. ). None of the LOPAC1280 compounds were expected to have truly TrxR-selective inhibitory activity in cells: the panel serves to demonstrate HTS assay performance and likely hit rates in larger-scale screening, and to identify trends among hit classes. Pleasingly, the hit rate was manageably low, and the hits are indeed likely selenol-reactive species: 3 heavy metal complexes, 13 organic electrophiles or redox-active species, and a known TrxR-inhibiting porphyrin which may act via redox; encouragingly, only one non-obvious hit was present, the glutamate receptor ligand AIDA (Fig. ). pIC50 values also followed likely cellular reactivity: no change despite ca. 1.1 LogP difference between lipophilic vinylsulfones BAY 11-7085 (methyl) and BAY 11-7082 (tert-butyl); but 10-fold lower for permeability-limited drug aurothioglucose than its ester prodrug auranofin; etc (Fig. , Fig. ). Matching expectations, with the exception of auranofin, none of these species approached the potency of TRi-1. One opportunity afforded by RX1-enabled cellular screening is to compare, for the first time, cell-free to cellular TrxR inhibition by species, to assess trends for cellularly-useful or -less-useful inhibitors. 1047 LOPAC compounds were assessed across both the screens , of which 1011 generated robust enough data quality for comparisons (Table ; Supporting Information section 4.2). Of these, 993 compounds were inactive in both assays, and 7 were active in both. This 99% overlap of conclusive results between RX1 and the purified enzyme screen speaks strongly to the precision of the cellular RX1 assay. Only 2 compounds, IPA-3 and chloro-APB, were active in the cellular screen despite inactivity in the enzymatic assay: and these are catechol-like species with plausible redox activity in cells. To us, this indicates that the cellular RX1 assay has excellent robustness against false positives. Given that hits were classed up to 20 µM, while RX1 relies on precipitation of >1 µM of a flat aryl fluorophore for signal generation, we had feared false positive inhibition from the many LOPAC PAINS (pan-assay interference compounds) that have aggregation effects, or could alkylate the reduced intermediate C (Fig. ). However, not even powerful cellular PAINS rottlerin, rhodanine and myrcetin gave apparent potencies above 3% of that of the reference inhibitor TRi-1 (Fig. ). Finally, 9 compounds were inactive in the RX1 assay despite activity in the enzymatic screen. These were also redox-actives and electrophiles (catechols, α-haloketone, nitrosyl donor, Michael acceptors, wortmannin), which may indicate filtering of low-quality hits by the more stringent cellular assay (Table and Supporting Datafile).