In the framework of REMEDi4ALL's mission to strengthen reproducibility in drug repurposing, we conducted a multicenter ring study to evaluate the reproducibility and translational value of combining histone deacetylase inhibitors (HDACi) with simvastatin (SIM) in pancreatic ductal adenocarcinoma (PDAC) cells. Initial inconsistencies due to misaligned protocols were resolved through harmonized SOPs across three European laboratories (INT-NA, ITMP, KI), enabling robust and comparable results. Under standardized procedures, valproic acid (VPA) and SIM emerged as the most synergistic combination, confirming our previous findings. The reproducible enhancement of antitumor activity when VPA/SIM was added to the standard chemotherapy (gemcitabine+nab-paclitaxel) further supports its translational relevance. Beyond validating this drug combination, the resulting open dataset enables systematic evaluation of technical confounders affecting drug sensitivity and provides a reference for data harmonization across heterogeneous assay platforms. All centers independently delivered high-quality data, demonstrating their readiness for future REMEDi4ALL repurposing efforts with enhanced rigor, robustness and interoperability.

In the rapidly advancing landscape of drug discovery and repurposing, efficient access and integration of chemical and bioactivity data from public repositories have become essential. To address this need, we developed two complementary annotation pipelines (KNIME- and Python-based) that automate the extraction and integration of curated chemical and bioactivity data from public repositories. These pipelines support any user-provided compound library, enabling reproducible workflows that integrate data from heterogeneous sources such as ChEMBL and PubChem. As part of the REMEDi4ALL project, with the aim of establishing a European platform for drug repurposing, we validated our framework using a harmonized subset of the Specs repurposing collection, which includes >5000 compounds available at the partner institutes. We also developed two interactive dashboards that support multilayered analyses and visualization by integrating chemical properties, bioactivity profiles, and relational data. Our results demonstrate that this framework streamlines the collection of harmonized data and facilitates analyses that are critical for drug repurposing efforts, while remaining versatile for broader applications in drug discovery. Moreover, the analysis of the annotations reveals that the Specs subset includes chemical scaffolds representative of a significant portion of approved drugs and compounds undergoing clinical evaluation, underscoring its potential as a rich source of drug repurposing candidates. Both pipeline protocols are publicly available online, and the dashboards are open access.

Inhibition of insulin-regulated aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign of approximately 10,000 compounds, identifying novel small-molecule-based compounds acting as inhibitors of the enzymatic activity of IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of 48 imidazo [1,5-α]pyridine-based inhibitors, including delineation of their mode of action as non-competitive inhibitors with a small L-leucine-based IRAP substrate. The best compound displays an IC50 value of 1.0 µM. We elucidate the importance of two chiral sites in these molecules and find they have little impact on the compound’s metabolic stability or physicochemical properties. The carbonyl group of a central urea moiety was initially believed to mimic substrate binding to a catalytically important Zn2+ ion in the active site, although the plausibility of this binding hypothesis is challenged by observation of excellent selectivity versus the closely related aminopeptidase N (APN). Taken together with the non-competitive inhibition pattern, we also consider an alternative model of allosteric binding.

Proteomics and phosphoproteomics data analyses II

Supplementary drug screen data

RNA sequencing data A23187 vs control (DMSO)

Abstract Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741–52. ©2017 AACR.

RNA sequencing data niguldipine vs control (DMSO)

VGCC channel identification

VGCC channel identification

Abstract Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.

Drug resistance in Mycobacterium tuberculosis is an emerging threat that makes the discovery of new candidate drugs a priority. In particular, drugs with high sterilizing activity within host cells are needed to improve efficacy and reduce treatment duration. We aimed to develope and validate a High Content Screening assay based on Mycobacterium tuberculosis-infected primary human monocyte-derived macrophages as its natural reservoir. Infected primary human monocyte-derived macrophages were exposed to control antibiotics or tested compounds on 384 well plates. Intracellular bacterial growth and macrophage numbers were evaluated using an ImageXpress High Content Screening system and Z'-factor was calculated to assess the reproducibility. The combination of isoniazid and rifampicin as a positive control rendered a Z'-factor above 0.4, demonstrating suitability of the assay for screening and compound profiling purposes. In a validation experiment, isoniazid, rifampicin, moxifloxacin and levofloxacin all effectively inhibited intracellular growth as expected. Finally, a pilot screening campaign including 5700 compounds from diverse libraries resulted in the identification of three compounds with confirmed antimycobacterial activity in the low micromolar range and low host cell toxicity. The assay represents an attractive screening platform for both academic research on host-pathogen mechanisms in tuberculosis and for the identification and characterization of novel antimycobacterial compounds.

Insulin-regulated aminopeptidase (IRAP) is a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV) improves the memory and learning in rats. The majority of the known IRAP inhibitors are peptidic in character and suffer from poor pharmacokinetic properties. Herein, we present a series of small non-peptide IRAP inhibitors derived from a spiro-oxindole dihydroquinazolinone screening hit (pIC(50) 5.8). The compounds were synthesized either by a simple microwave (MW)-promoted three-component reaction, or by a two-step one-pot procedure. For decoration of the oxindole ring system, rapid MW-assisted Suzuki-Miyaura cross-couplings (1 min) were performed. A small improvement of potency (pIC(50) 6.6 for the most potent compound) and an increased solubility could be achieved. As deduced from computational modelling and MD simulations it is proposed that the S-configuration of the spiro-oxindole dihydroquinazolinones accounts for the inhibition of IRAP.

Due to a polar or even charged binding interface, DNA-binding proteins are considered extraordinarily difficult targets for development of small-molecule ligands and only a handful of proteins have been targeted successfully to date. Recently, however, it has been shown that development of selective and efficient inhibitors of 8-oxoguanine DNA glycosylase is possible. Here, we describe the initial druggability assessment of DNA glycosylases in a computational setting and experimentally investigate several methods to target endonuclease VIII-like 1 (NEIL1) with small-molecule inhibitors. We find that DNA glycosylases exhibit good predicted druggability in both DNA-bound and -unbound states. Furthermore, we find catalytic sites to be highly flexible, allowing for a range of interactions and binding partners. One flexible catalytic site was rationalized for NEIL1 and further investigated experimentally using both a biochemical assay in the presence of DNA and a thermal shift assay in the absence of DNA.

DNA binding as an anti-inflammatory Mice that lack the gene encoding 8-oxoguanine DNA glycosylase 1 (OGG1) show resistance to inflammation. This enzyme binds to sites of oxidative DNA damage and initiates DNA base excision repair. Visnes et al. developed a small-molecule drug that acts as a potent and selective active-site inhibitor that stops OGG1 from recognizing its DNA substrate (see the Perspective by Samson). The drug inhibited DNA repair and modified OGG1 chromatin dynamics, which resulted in the inhibition of proinflammatory pathway genes. The drug was well tolerated by mice and suppressed lipopolysaccharide- and tumor necrosis factor–α–mediated neutrophilic inflammation in the lungs. Science , this issue p. 834 ; see also p. 748

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).

The dCTP pyrophosphatase 1 (dCTPase) is involved in the regulation of the cellular dNTP pool and has been linked to cancer progression. Here we report on the discovery of a series of 3,6-disubstituted triazolothiadiazoles as potent dCTPase inhibitors. Compounds 16 and 18 display good correlation between enzymatic inhibition and target engagement, together with efficacy in a cellular synergy model, deeming them as a promising starting point for hit -to-lead development.