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The chemical diversification of natural products provides a robust and general method for the creation of stereochemically rich and structurally diverse small molecules. The resulting compounds have physicochemical traits different from those in most screening collections, and as such are an excellent source for biological discovery. Herein, we subject the diterpene natural product pleuromutilin to reaction sequences focused on creating ring system diversity in few synthetic steps. This effort resulted in a collection of compounds with previously unreported ring systems, providing a novel set of structurally diverse and highly complex compounds suitable for screening in a variety of different settings. Biological evaluation identified the novel compound ferroptocide, a small molecule that rapidly and robustly induces ferroptotic death of cancer cells. Target identification efforts and CRISPR knockout studies reveal that ferroptocide is an inhibitor of thioredoxin, a key component of the antioxidant system in the cell. Ferroptocide positively modulates the immune system in a murine model of breast cancer and will be a useful tool to study the utility of pro-ferroptotic agents for treatment of cancer. A set of stereochemically complex and structurally diverse compounds were created from the diterpene natural product pleuromutilin using the complexity-to-diversity strategy. Phenotypic screening identified a compound that induces rapid ferroptotic death of cancer cells. Experiments to probe the mechanism revealed the compound to be an inhibitor of thioredoxin.

The mammalian thioredoxins are a family of small (approximately 12 kDa) redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase and in turn reduce oxidized cysteine groups on proteins. The two main thioredoxins are thioredoxin-1, a cytosolic and nuclear form, and thioredoxin-2, a mitochondrial form. Thioredoxin-1 has been studied more. It performs many biological actions including the supply of reducing equivalents to thioredoxin peroxidases and ribonucleotide reductase, the regulation of transcription factor activity, and the regulation of enzyme activity by heterodimer formation. Thioredoxin-1 stimulates cell growth and is an inhibitor of apoptosis. Thioredoxins may play a role in a variety of human diseases including cancer. An increased level of thioredoxin-1 is found in many human tumors, where it is associated with aggressive tumor growth. Drugs are being developed that inhibit thioredoxin and that have antitumor activity.

Thioredoxin reductase 1 (TrxR1) is a homodimeric flavoprotein crucially involved in the regulation of cellular redox homeostasis, growth, and differentiation. Its importance in various diseases makes TrxR1 a highly interesting drug target. Here we present the first crystal structures of human TrxR1 in complex with its substrate thioredoxin (Trx). The carboxy-terminal redox centre is found about 20 Å apart from the amino-terminal redox centre, with no major conformational changes in TrxR or Trx. Thus, our structure confirms that the enzyme uses a flexible C-terminal arm for electron transport to its substrates, which is stabilized by a guiding bar for controlled transfer. This notion is supported by mutational analyses. Furthermore, essential residues of the interface region were characterized both structurally and functionally. The structure provides templates for future drug design, and contributes to our understanding of redox regulatory processes in mammals. Thioredoxin reductase 1 is a flavoprotein that is involved in the regulation of redox homeostasis. Fritz-Wolf et al . report the first crystal structures of thioredoxin reductase 1 in complex with its substrate, thioredoxin, and confirm that the enzyme uses a flexible carboxy-terminal arm for electron transport to its substrates.

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis 1 . NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer 2 – 6 . However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated 4 , 7 , 8 . Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system. Structural and mutational studies and cellular assays show that the inflammasome sensor NLRP1 forms a complex with thioredoxin, which acts as a negative regulator of inflammasome activity.

β-Lactoglobulin (BLG), a member of lipocalin family, is one of the major bovine milk allergens. This protein exists as a dimer of two identical subunits and contains two intramolecular disulfide bonds that are responsible for its resistance to trypsin digestion and allergenicity. This study aimed to evaluate the effect of reduction of disulfide bonds of BLG with different rice thioredoxins (Trxs) on its digestibility and allergenicity. Therefore, the active recombinant forms of three rice Trx isoforms (OsTrx1, OsTrx20, and OsTrx23) and one rice NADPH-dependent Trx reductase isoform (OsNTRB) were expressed in Escherichia coli. Based on SDS-PAGE, HPLC analysis, and competitive ELISA, the reduction of disulfide bonds of BLG with OsNTRB/OsTrx23, OsNTRB/OsTrx1, GSH/OsTrx1, or GSH/OsTrx20 increased its trypsin digestibility and reduced its immunoreactivity. The finding of this study opens new insights for application of plant Trxs in the improvement of food protein digestibility. Especially, the use of OsTrx20 and OsTrx1 are more cost-effective than E. coli and animal Trxs due to their reduction by GSH and no need to NADPH and Trx reductase as mediator enzyme.

Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms.

Cysteine thiols of many cancer-associated proteins are attractive targets of anticancer agents. Herein, we unequivocally demonstrate a distinct thiol-targeting property of gold(III) mesoporphyrin IX dimethyl ester (AuMesoIX) and its anticancer activities. While the binding of cysteine thiols with metal complexes usually occurs via M–S bond formation, AuMesoIX is unique in that the meso-carbon atom of the porphyrin ring is activated by the gold(III) ion to undergo nucleophilic aromatic substitution with thiols. AuMesoIX was shown to modify reactive cysteine residues and inhibit the activities of anticancer protein targets including thioredoxin, peroxiredoxin, and deubiquitinases. Treatment of cancer cells with AuMesoIX resulted in the formation of gold-bound sulfur-rich protein aggregates, oxidative stress-mediated cytotoxicity, and accumulation of ubiquitinated proteins. Importantly, AuMesoIX exhibited effective antitumor activity in mice. Our study has uncovered a gold(III)-induced ligand scaffold reactivity for thiol targeting that can be exploited for anticancer applications.

Since many biomarkers of both the tumor and its microenvironment are expected to involve differential expression of divalent proteins capable of protein or peptide ligand interaction, we are developing multivalent nanodevices for the identification of biomarkers in prostate cancer. We compared a multivalent thioredoxin-targeted nanodevice with monovalent thioredoxin in binding to human prostate cell line(s) and freshly frozen tissue specimens obtained after resection from patients with biopsy-proven prostate cancer. The nanodevice binds specifically with enhanced avidity to tumor microenvironment-associated stromal cells in prostate cancer tissue specimens. Cells that bind the nanodevice also reacted with antibodies to dimeric thioredoxin reductases 1 and 2, suggesting the utility of the nanodevice as a potentially specific and functional marker of tumor stromal cells.

Hydrogen peroxide (H2 O2 ) acts as a signaling messenger by triggering the reversible oxidation of redox-regulated proteins. It remains unclear how proteins can be oxidized by signaling levels of H2 O2 in the presence of peroxiredoxins, which are highly efficient peroxide scavengers. Here we show that the rapid formation of disulfide bonds in cytosolic proteins is enabled, rather than competed, by cytosolic 2-Cys peroxiredoxins. Under the conditions tested, the combined deletion or depletion of cytosolic peroxiredoxins broadly frustrated H2 O2 -dependent protein thiol oxidation, which is the exact opposite of what would be predicted based on the assumption that H2 O2 oxidizes proteins directly. We find that peroxiredoxins enable rapid and sensitive protein thiol oxidation by relaying H2 O2 -derived oxidizing equivalents to other proteins. Although these findings do not rule out the existence of Prx-independent H2 O2 signaling mechanisms, they suggest a broader role for peroxiredoxins as sensors and transmitters of H2 O2 signals than hitherto recognized.

Latent bioreactive unnatural amino acids (Uaas) have been widely used in the development of covalent drugs and identification of protein interactors, such as proteins, DNA, RNA and carbohydrates. However, it is challenging to perform high-throughput identification of Uaa cross-linking products due to the complexities of protein samples and the data analysis processes. Enrichable Uaas can effectively reduce the complexities of protein samples and simplify data analysis, but few cross-linked peptides were identified from mammalian cell samples with these Uaas. Here we develop an enrichable and multiple amino acids reactive Uaa, eFSY, and demonstrate that eFSY is MS cleavable when eFSY-Lys and eFSY-His are the cross-linking products. An identification software, AixUaa is developed to decipher eFSY mass cleavable data. We systematically identify direct interactomes of Thioredoxin 1 (Trx1) and Selenoprotein M (SELM) with eFSY and AixUaa. The high-throughput identification of cross-linking peptides is challenging. Here, the authors developed an enrichable and cleavable latent bioreactive unnatural amino acid eFSY, and a software AixUaa to improve the cross-linking peptides indentification in live cells.