Ifigeneia Akrani

The tandem Tudor domain (TTD) of UHRF1 is a compelling epigenetic target for novel cancer therapeutics. Here, we integrate theoretical simulations, sophisticated biophysical evaluations and cellular assays to characterize potent TTD binders. Screening of small drug‐ and fragment‐like collections identifies several TTD ligands, with the most promising hit being the local anesthetic hydroxyprocaine. The ligand is characterized in terms of its binding requisites by calorimetry, affording a K _d of 1.46 μM and a well‐balanced thermodynamic profile. Molecular dynamics simulations combined with heat capacity measurements and osmotic stress titrations confirm that hydroxyprocaine binds stably to the TTD by displacing approximately 26 interfacial water molecules upon complexation. A targeted follow‐up screen focusing on sodium channel blockers yields two additional, although less promising hits, mexiletine and triamterene. In the DU145 prostate cancer cell line, hydroxyprocaine treatment significantly up‐regulates key downstream targets including the tumor‐suppressor p53 and, to a lesser degree, the stress and inflammation regulators p38 and p65, respectively, while exhibiting very low cytotoxicity. Finally, a previously undocumented interdomain interaction between TTD and its N‐terminal adjacent Ubiquitin‐like domain is reported, introducing a novel, potentially druggable UHRF1 regulatory feature. Together, these findings establish hydroxyprocaine as a highly viable chemical scaffold for TTD‐targeted drug development.

Regardless of the massive global efforts to combat the virus causing SARS‐CoV‐2 syndrome, the infection remains a substantial health challenge worldwide in the years following 2019. To this direction, targeting the main viral protease M ^pro has been proposed as a tractable and particularly promising approach toward developing effective and safe COVID‐19 antivirals. By applying an integrated workflow combining a previously developed in silico consensus ranking protocol with two orthogonal in vitro methods, the NCI/DTP repository is screened and the discovery of an original naphthol scaffold with M ^pro inhibitory properties is reported. The hit is characterized in terms of structure and binding thermodynamics by combining X‐ray crystallography and isothermal titration calorimetry where a binding affinity constant of 1.55 μM is determined. The compound is further evaluated against virus‐infected cells, where an EC ₅₀ value of 7.23 μM and comparable toxicity with nirmatrelvir are measured. Chemical synthesis is additionally employed to facilitate optimal exploration of the structure–activity relationship landscape regarding the new hit. By integrating computational, biophysical, and enzymatic methods, the suggested approach allows the combination of a structural hypothesis with functional evidence and shows its capacity toward identifying and rationally optimizing structurally original noncovalent M ^pro inhibitors.

Members of the nucleobase ascorbate transporter (NAT) family (SLC23) are elevator-type transporters that are responsible for the uptake of nucleobases and ascorbate. In fungi, NAT members are also responsible for the specific uptake of antifungal nucleobase analogues, such as oxypurinol, allopurinol, or 8-azaguanine. Here, we report nearly full-length cryo-EM structures of UapA, a high-affinity purine transporter from the model fungus Aspergillus nidulans , in inward-facing apo- and substrate-loaded conformations at 2.06 to 3.5 Å in detergent and lipid nanodiscs. The high-resolution structures reveal the role of water molecules and lipids in substrate binding, specificity, transporter dimerization, and activity. Notably, the N-tail of UapA is found to be structured, interacting with both the core and scaffold domains, which in combination with functional data suggests a dual role in trafficking and transport dynamics. Overall, our study provides unprecedented structural and functional insights into an elevator-type fungal transporter, which may well contribute to the exploitation of NAT transporters as specific gateways for targeted pharmacological antifungal approaches.

Insecticide resistance in mosquitoes, largely mediated by cytochrome P450 monooxygenases (CYPs), compromises the efficacy of vector control tools. In this study, chemically-wise selected natural extracts and compounds were screened for their CYP inhibition potential. Among 37 tested plant extracts and fractions, a decarboxylated acidic fraction of industrial hemp ( Cannabis sativa Linnaeus var. ‘Futura 75’) emerged as a promising hit, and phytochemical profiling identified cannabidiol (CBD) as its major component (IC₅₀ = 18.37 μM for CYP9K1). CBD was used as a scaffold to generate semisynthetic analogues; of which a piperazinyl analogue outperformed the natural scaffold demonstrating significantly greater potency (IC₅₀ = 2.50 μM for CYP9K1). Docking studies using homology-derived CYP9K1 models also supported a stronger binding affinity of the piperazinyl analogue relative to CBD. Toxicity assays using pyrethroid-resistant Anopheles gambiae Giles adults confirmed that neither CBD nor the piperazinyl analogue had intrinsic toxicity, yet the semisynthetic analogue significantly enhanced deltamethrin efficacy, showing a threefold synergistic effect. The safety profile of the cannabis compounds for non-target organisms was evaluated through human cell line cytotoxicity tests and bee toxicity assays, suggesting low non-target organism toxicity. Our study describes the identification of a plant-derived synergist lead with strong potential as an insecticide additive to combat metabolic resistance in malaria-transmitting mosquitoes.