TRPV4 cation channel activation by cytochrome P450-mediated derivatives of arachidonic acid (AA), epoxyeicosatrienoic acids (EETs), constitute a major mechanisms of endothelium-derived vasodilatation. Besides, TRPV4 mechano/osmosensitivity depends on phospholipase A₂(PLA₂) activation and subsequent production of AA and EETs. However, the lack of evidence for a direct interaction of EETs with TRPV4 together with claims of EET-independent mechanical activation of TRPV4 has cast doubts on the validity of this mechanism. We now report: 1) The identification of an EET-binding pocket that specifically mediates TRPV4 activation by 5′,6′-EET, AA and hypotonic cell swelling, thereby suggesting that all these stimuli shared a common structural target within the TRPV4 channel; and 2) A structural insight into the gating of TRPV4 by a natural agonist (5′,6′-EET) in which K535 plays a crucial role, as mutant TRPV4-K535A losses binding of and gating by EET, without affecting GSK1016790A, 4α-phorbol 12,13-didecanoate and heat mediated channel activation. Together, our data demonstrates that the mechano- and osmotransducing messenger EET gates TRPV4 by a direct action on a site formed by residues from the S2-S3 linker, S4 and S4-S5 linker.

The accessory β1 subunit modulates the Ca²⁺- and voltage-activated K⁺ (BK) channel gating properties mainly by increasing its apparent Ca²⁺ sensitivity. β1 plays an important role in the modulation of arterial tone and blood pressure by vascular smooth muscle cells (SMCs). 17β-estradiol (E2) increases the BK channel open probability (P_o) in SMCs, through a β1 subunit-dependent modulatory effect. Here, using molecular modeling, bioinformatics, mutagenesis, and electrophysiology, we identify a cluster of hydrophobic residues in the second transmembrane domain of the β1 subunit, including the residues W163 and F166, as the binding site for E2. We further show that the increase in P_o induced by E2 is associated with a stabilization of the voltage sensor in its active configuration and an increase in the coupling between the voltage sensor activation and pore opening. Since β1 is a key molecular player in vasoregulation, the findings reported here are of importance in the design of novel drugs able to modulate BK channels.

Helicobacter pylori (Hp) infections pose a global health challenge demanding innovative therapeutic strategies by which to eradicate them. Urease, a key Hp virulence factor hydrolyzes urea, facilitating bacterial survival in the acidic gastric environment. In this study, a multi-methodological approach combining pharmacophore- and structure-based virtual screening, molecular dynamics simulations, and MM-GBSA calculations was employed to identify novel inhibitors for Hp urease (HpU). A refined dataset of 8,271,505 small molecules from the ZINC15 database underwent pharmacokinetic and physicochemical filtering, resulting in 16% of compounds for pharmacophore-based virtual screening. Molecular docking simulations were performed in successive stages, utilizing HTVS, SP, and XP algorithms. Subsequent energetic re-scoring with MM-GBSA identified promising candidates interacting with distinct urease variants. Lys219, a residue critical for urea catalysis at the urease binding site, can manifest in two forms, neutral (LYN) or carbamylated (KCX). Notably, the evaluated molecules demonstrated different interaction and energetic patterns in both protein variants. Further evaluation through ADMET predictions highlighted compounds with favorable pharmacological profiles, leading to the identification of 15 candidates. Molecular dynamics simulations revealed comparable structural stability to the control DJM, with candidates 5, 8 and 12 (CA5, CA8, and CA12, respectively) exhibiting the lowest binding free energies. These inhibitors suggest a chelating capacity that is crucial for urease inhibition. The analysis underscores the potential of CA5, CA8, and CA12 as novel HpU inhibitors. Finally, we compare our candidates with the chemical space of urease inhibitors finding physicochemical similarities with potent agents such as thiourea.

Targeted therapy indications for actionable variants in non-small-cell lung cancer (NSCLC) have primarily been studied in Caucasian populations, with limited data on Latin American patients. This study utilized a 52-genes next-generation sequencing (NGS) panel to analyze 1560 tumor biopsies from NSCLC patients in Chile, Brazil, and Peru. The RNA sequencing reads and DNA coverage were correlated to improve the detection of the actionable MET exon 14 skipping variant (METex14). The pathogenicity of MET variants of uncertain significance (VUSs) was assessed using bioinformatic methods, based on their predicted driver potential. The effects of the predicted drivers VUS T992I and H1094Y on c-MET signaling activation, proliferation, and migration were evaluated in HEK293T, BEAS-2B, and H1993 cell lines. Subsequently, c-Met inhibitors were tested in 2D and 3D cell cultures, and drug affinity was determined using 3D structure simulations. The prevalence of MET variants in the South American cohort was 8%, and RNA-based diagnosis detected 27% more cases of METex14 than DNA-based methods. Notably, 20% of METex14 cases with RNA reads below the detection threshold were confirmed using DNA analysis. The novel actionable T992I and H1094Y variants induced proliferation and migration through c-Met/Akt signaling. Both variants showed sensitivity to crizotinib and savolitinib, but the H1094Y variant exhibited reduced sensitivity to capmatinib. These findings highlight the importance of RNA-based METex14 diagnosis and reveal the drug sensitivity profiles of novel actionable MET variants from an understudied patient population.

Bacterial outer membrane vesicles (OMVs) are nanoscale extracellular structures produced by Gram-negative bacteria that are critical for microbial biology and host-pathogen interactions and have great potential in biotechnological applications. Despite the availability of fluorescent dyes for OMV studies, many are repurposed from eukaryotic extracellular vesicle research and are not explicitly optimized for OMVs, leading to challenges in achieving consistent labeling, minimizing background noise, and preserving vesicle integrity during analyses. This study evaluates B2, a benzimidazole-derived fluorophore, for OMV labeling in advanced techniques like flow cytometry and confocal microscopy. OMVs were isolated from Escherichia coli strains BL21 and O157, and their integrity was confirmed using transmission electron microscopy (TEM). B2 staining protocols were optimized for OMVs, and fluorescence analyses revealed specific interactions with the vesicle membranes, reducing aggregation and enhancing signal uniformity. Flow cytometry indicated near-complete labeling efficiency (98–100%) with minimal background interference. Confocal microscopy further validated B2’s effectiveness, showing evident OMV internalization into epithelial HT-29 cells and compatibility with other fluorophores. Density functional theory (DFT) calculations, including Fukui function analysis, identified key electrophilic and nucleophilic regions in B2 that facilitate specific hydrogen bonding and polar interactions with membrane components. Non-covalent interaction (NCI) analysis revealed pronounced intramolecular hydrogen bonding along with discrete regions of weak van der Waals interactions. Molecular dynamics simulations suggest that B2 exhibits an affinity for both the hydrophobic core of the lipid bilayer and the core oligosaccharide region of the LPS layer, which collectively ensures sustained retention of the dye. The findings presented in this study position B2 as a valuable fluorophore for OMV research.