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Despite a strong rationale for why cancer cells are susceptible to redox-targeting drugs, such drugs often face tumor resistance or dose-limiting toxicity in preclinical and clinical studies. An important reason is the lack of specific biomarkers to better select susceptible cancer entities and stratify patients. Using a large panel of lung cancer cell lines, we identified a set of “antioxidant-capacity” biomarkers (ACB), which were tightly repressed, partly by STAT3 and STAT5A/B in sensitive cells, rendering them susceptible to multiple redox-targeting and ferroptosis-inducing drugs. Contrary to expectation, constitutively low ACB expression was not associated with an increased steady state level of reactive oxygen species (ROS) but a high level of nitric oxide, which is required to sustain high replication rates. Using ACBs, we identified cancer entities with a high percentage of patients with favorable ACB expression pattern, making it likely that more responders to ROS-inducing drugs could be stratified for clinical trials.

Plasmoredoxin is a 22 ​kDa thiol–disulfide oxidoreductase involved in cellular redox regulatory processes and antioxidant defense. The 1.6 ​Å structure of the protein, solved via X-ray crystallography, adopts a modified thioredoxin fold. The structure reveals that plasmoredoxin, unique for malarial parasites, forms a new subgroup of thioredoxin-like proteins together with tryparedoxin, unique for kinetoplastids. Unlike most members of this superfamily, Plrx does not have a proline residue within the CxxC redox motif. In addition, the Plrx structure has a distinct C-terminal domain. Similar to human thioredoxin, plasmoredoxin forms monomers and dimers, which are also structurally similar to the human thioredoxin dimer, and, as in humans, plasmoredoxin is inactive as a dimer. Monomer–dimer equilibrium depends on the surrounding redox conditions, which could support the parasite in reacting to oxidative challenges. Based on structural considerations, the residues of the dimer interface are likely to interact with target proteins. In contrast to human and Plasmodium falciparum thioredoxin, however, there is a cluster of positively charged residues at the dimer interface of plasmoredoxin. These intersubunit (lysine) residues might allow binding of the protein to cellular membranes or to plasminogen. Malaria parasites lack catalase and glutathione peroxidase and therefore depend on their other glutathione and thioredoxin-dependent redox relays. Plasmoredoxin could be part of a so far unknown electron transfer system that only occurs in these parasites. Since the surface charge of plasmoredoxin differs significantly from other members of the thioredoxin superfamily, its three-dimensional structure can provide a model for designing selective redox-modulatory inhibitors.

Background: The heterologous expression of parasitic proteins is challenging because the sequence composition often differs significantly from host preferences. However, the production of such proteins is important because they are potential drug targets and can be screened for interactions with new lead compounds. Here we compared two expression systems for the production of an active recombinant aldehyde dehydrogenase (SmALDH_312) from Schistosoma mansoni, which causes the neglected tropical disease schistosomiasis. Results: We produced SmALDH_312 successfully in the bacterium Escherichia coli and in the baculovirus expression vector system (BEVS). Both versions of the recombinant protein were found to be active in vitro, but the BEVS-derived enzyme showed 3.7-fold higher specific activity and was selected for further characterization. We investigated the influence of Mg2+, Ca2+ and Mn2+, and found out that the specific activity of the enzyme increased 1.5-fold in the presence of 0.5 mM Mg2+. Finally, we characterized the kinetic properties of the enzyme using a design-of-experiment approach, revealing optimal activity at pH 7.6 and 41°C. Conclusions: Although, E. coli has many advantages, such as rapid expression, high yields and low costs, this system was outperformed by BEVS for the production of a schistosome ALDH. BEVS therefore provides an opportunity for the expression and subsequent evaluation of schistosome enzymes as drug targets.How to cite: Harnischfeger J, Beutler M, Salzig D, et al. Biochemical characterization of the recombinant schistosome tegumental protein SmALDH_312 produced in E. coli and baculovirus expression vector system. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2021.08.002

Summary: Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.

Background: The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). Methods: A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. Findings: EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. Interpretation: This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.