New antibacterial compounds are urgently needed, especially for infections caused by the top-priority Gram-negative bacteria that are increasingly difficult to treat. Lipid A is a key component of the Gram-negative outer membrane and the LpxH enzyme plays an important role in its biosynthesis, making it a promising antibacterial target. Inspired by previously reported ortho-N-methyl-sulfonamidobenzamide-based LpxH inhibitors, novel benzamide substitutions were explored in this work to assess their in vitro activity. Our findings reveal that maintaining wild-type antibacterial activity necessitates removal of the N-methyl group when shifting the ortho-N-methyl-sulfonamide to the meta-position. This discovery led to the synthesis of meta-sulfonamidobenzamide analogs with potent antibacterial activity and enzyme inhibition. Moreover, we demonstrate that modifying the benzamide scaffold can alter blocking of the cardiac voltage-gated potassium ion channel hERG. Furthermore, two LpxH-bound X-ray structures show how the enzyme-ligand interactions of the meta-sulfonamidobenzamide analogs differ from those of the previously reported ortho analogs. Overall, our study has identified meta-sulfonamidobenzamide derivatives as promising LpxH inhibitors with the potential for optimization in future antibacterial hit-to-lead programs.

Chemical risk assessments typically focus on single substances, often overlooking real-world co-exposures to chemical mixtures. Mixture toxicology studies using representative mixtures can reveal potential chemical interactions, but these do not account for the unique chemical profiles that occur in the blood of diverse individuals. Here we used the H295R steroidogenesis assay to screen personalized mixtures of 24 persistent organic pollutants (POPs) for cytotoxicity and endocrine disruption. Each mixture was reconstructed at a human exposure relevant concentration (1×), as well as at 10- and 100-fold higher concentration (10×, 100×) by acoustic liquid handling based on measured blood concentrations in a Swedish cohort. Among the twelve mixtures tested, nine mixtures decreased the cell viability by 4–18%, primarily at the highest concentration. While the median and maximum mixtures based on the whole study population induced no measurable effects on steroidogenesis at any concentration, the personalized mixture from an individual with the lowest total POPs concentration was the only mixture that affected estradiol synthesis (35% increase at the 100× concentration). Mixtures reconstructed from blood levels of three different individuals stimulated testosterone synthesis at the 1× (11–15%) and 10× concentrations (12–16%), but not at the 100× concentration. This proof-of-principle personalized toxicity study illustrates that population-based representative chemical mixtures may not adequately account for the toxicological risks posed to individuals. It highlights the importance of testing a range of real-world mixtures at relevant concentrations to explore potential interactions and non-monotonic effects. Further toxicological studies of personalized contaminant mixtures could improve chemical risk assessment and advance the understanding of human health, as chemical exposome data become increasingly available.

Many persistent organic pollutants (POPs) are suspected endocrine disruptors and it is important to investigate their effects at low concentrations relevant to human exposure. Here, the OECD test guideline #456 steroidogenesis assay was downscaled to a 96-well microplate format to screen 24 POPs for their effects on viability, and testosterone and estradiol synthesis using the human adrenocortical cell line H295R. The compounds (six polyfluoroalkyl substances, five organochlorine pesticides, ten polychlorinated biphenyls and three polybrominated diphenyl ethers) were tested at human-relevant levels (1 nM to 10 µM). Increased estradiol synthesis, above the OECD guideline threshold of 1.5-fold solvent control, was shown after exposure to 10 µM PCB-156 (153%) and PCB-180 (196%). Interestingly, the base hormone synthesis varied depending on the cell batch. An alternative data analysis using a linear mixed-effects model that include multiple independent experiments and considers batch-dependent variation was therefore applied. This approach revealed small but statistically significant effects on estradiol or testosterone synthesis for 17 compounds. Increased testosterone levels were demonstrated even at 1 nM for PCB-74 (18%), PCB-99 (29%), PCB-118 (16%), PCB-138 (19%), PCB-180 (22%), and PBDE-153 (21%). The MTT assay revealed significant effects on cell viability after exposure to 1 nM of perfluoroundecanoic acid (12%), 3 nM PBDE-153 (9%), and 10 µM of PCB-156 (6%). This shows that some POPs can interfere with endocrine signaling at concentrations found in human blood, highlighting the need for further investigation into the toxicological mechanisms of POPs and their mixtures at low concentrations relevant to human exposure.

Glioblastoma (GBM) is a malignant brain tumor with few therapeutic options. The disease presents with a complex spectrum of genomic aberrations, but the pharmacological consequences of these aberrations are partly unknown. Here, we report an integrated pharmacogenomic analysis of 100 patient-derived GBM cell cultures from the human glioma cell culture (HGCC) cohort. Exploring 1,544 drugs, we find that GBM has two main pharmacological subgroups, marked by differential response to proteasome inhibitors and mutually exclusive aberrations in TP53 and CDKN2A/B. We confirm this trend in cell and in xenotransplantation models, and identify both Bcl-2 family inhibitors and p53 activators as potentiators of proteasome inhibitors in GBM cells, We can further predict the responses of individual cell cultures to several existing drug classes, presenting opportunities for drug repurposing and design of stratified trials. Our functionally profiled biobank provides a valuable resource for the discovery of new treatments for GBM.

Chemical proteomics encompasses novel drug target deconvolution methods in which compound modification is not required. Herein we use Thermal Proteome Profiling, Functional Identification of Target by Expression Proteomics and multiplexed redox proteomics for deconvolution of auranofin targets to aid elucidation of its mechanisms of action. Auranofin (Ridaura (R)) was approved for treatment of rheumatoid arthritis in 1985. Because several clinical trials are currently ongoing to repurpose auranofin for cancer therapy, comprehensive characterization of its targets and effects in cancer cells is important. Together, our chemical proteomics tools confirmed thioredoxin reductase 1 (TXNRD1, EC:1.8.1.9) as a main auranofin target, with perturbation of oxidoreductase pathways as the top mechanism of drug action. Additional indirect targets included NFKB2 and CHORDC1. Our comprehensive data can be used as a proteomic signature resource for further analyses of the effects of auranofin. Here we also assessed the orthogonality and complementarity of different chemical proteomics methods that can furnish invaluable mechanistic information and thus the approach can facilitate drug discovery efforts in general.

Background: Fanconi anemia is a rare disease clinically characterized by malformations, bone marrow failure and an increased risk of solid tumors and hematologic malignancies. The only therapies available are hematopoietic stem cell transplantation for bone marrow failure or leukemia, and surgical resection for solid tumors. Therefore, there is still an urgent need for new therapeutic options. With this aim, we developed a novel high-content cell-based screening assay to identify drugs with therapeutic potential in FA.

Results: A TALEN-mediated FANCA-deficient U2OS cell line was stably transfected with YFP-FANCD2 fusion protein. These cells were unable to form fluorescent foci or to monoubiquitinate endogenous or exogenous FANCD2 upon DNA damage and were more sensitive to mitomycin C when compared to the parental wild type counterpart. FANCA correction by retroviral infection restored the cell line's ability to form FANCD2 foci and ubiquitinate FANCD2. The feasibility of this cell-based system was interrogated in a high content screening of 3802 compounds, including a Prestwick library of 1200 FDA-approved drugs. The potential hits identified were then individually tested for their ability to rescue FANCD2 foci and monoubiquitination, and chromosomal stability in the absence ofFANCA.

Conclusions: While, unfortunately, none of the compounds tested were able to restore cellular FANCA-deficiency, our study shows the potential capacity to screen large compound libraries in the context of Fanconi anemia therapeutics in an optimized and cost-effective platform.

Objective: The peroxisome proliferator-activated receptor-gamma coactivator-1 alpha 1 (PGC-1 alpha 1) regulates genes involved in energy metabolism. Increasing adipose tissue energy expenditure through PGC-1 alpha 1 activation is potentially beneficial for systemic metabolism. Pharmacological PGC-1 alpha 1 activators could be valuable tools in the fight against obesity and metabolic disease. Finding such compounds has been challenging partly because PGC-1 alpha 1 is a transcriptional coactivator with no known ligand-binding properties. While, PGC-1 alpha 1 activation is regulated by several mechanisms, protein stabilization is a crucial limiting step due to its short half-life under unstimulated conditions.

Methods: We designed a cell-based high-throughput screening system to identify PGC-1 alpha 1 protein stabilizers. Positive hits were tested for their ability to induce endogenous PGC-1 alpha 1 protein accumulation and activate target gene expression in brown adipocytes. Select compounds were analyzed for their effects on global gene expression and cellular respiration in adipocytes.

Results: Among 7,040 compounds screened, we highlight four small molecules with high activity as measured by: PGC-1 alpha 1 protein accumulation, target gene expression, and uncoupled mitochondrial respiration in brown adipocytes.

Conclusions: We identify compounds that induce PGC-1 alpha 1 protein accumulation and show that this increases uncoupled respiration in brown adipocytes. This screening platform establishes the foundation for a new class of therapeutics with potential use in obesity and associated disorders.

The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.

A high-throughput screening campaign using a commercial compound library (ChemBridge DiverSET) revealed diverse chemotypes as inhibitors of the human dCTP pyrophosphatase 1 (dCTPase). Triazole, triazolopyrimidine, triazinoindole, quinoline hydrazone and arylpiperazine hits were clustered, confirmed by IC50 determinations, and their preliminary structure-activity-relationships (SAR) and ligand efficiency scores are discussed in this letter.

Two screening campaigns using commercial (Chembridge DiverSET) and proprietary (Chemical Biology Consortium Sweden, CBCS) compound libraries, revealed a number of pyridone- and pyrimidinone-derived systems as inhibitors of the human dCTP pyrophosphatase 1 (dCTPase). In this letter, we present their preliminary structure-activity-relationships (SAR) and ligand efficiency scores (LE and LLE).