Tihomir Tomašič

Voltage-gated sodium channel Na _V 1.7 is a key mediator of electrical excitability and signal transmission in peripheral nociceptors and has emerged as a highly attractive therapeutic target for the development of novel analgesic agents. However, the development of selective Na _V 1.7 inhibitors has been characterized by significant challenges, with repeated failures in clinical trials despite encouraging preclinical data. In this study, we developed and validated a series of ligand-based pharmacophore models (LBPMs) that can be useful for the discovery of novel Na _V 1.7 inhibitors with improved selectivity profiles. Using the VGSC database as our primary data source, we focused on sulfonamide-based inhibitors targeting the voltage-sensing domain IV (VSD-IV). Validation against active compounds and decoys demonstrated that most models achieved good discrimination performance with high areas under the curve (AUC) and strong enrichment factors. External validation using 22 inhibitors extracted from recent literature confirmed the models’ capability to identify novel Na _V 1.7 inhibitors. Virtual screening of 3 million commercially available compounds retrieved promising hits and known inhibitors, with molecular docking studies revealing binding modes consistent with established sulfonamide-based inhibitors. Experimental validation identified one compound with measurable selectivity for Na _V 1.7 over Na _V 1.5, providing preliminary support for the utility of the developed virtual screening workflow. In parallel, we developed Na _V 1.5 LBPMs to assess selectivity profiles and minimize potential cardiotoxic effects. Overall, our findings provide valuable computational tools and structural insights for the rational design of selective Na _V 1.7 inhibitors, offering important starting points for developing analgesics with reduced off-target effects.

Heat shock protein 90 (Hsp90) is an established and clinically validated anticancer target. By regulating the folding and maturation of diverse client proteins, it is indirectly involved in carcinogenesis. Key breast cancer oncogenic drivers, including estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, are established Hsp90 clients, making breast cancer models widely used for Hsp90 inhibitor evaluation. However, N‐terminal inhibitors are well known to induce a heat shock response (HSR), representing a major limitation in cancer therapy. This has driven the development of alternative modalities, such as C‐terminal inhibitors, which in several cases avoid HSR induction. To evaluate Hsp90 inhibitors and characterize their proteomic effects, most studies employ western blotting (WB) as the primary method. On WB, different inhibitors are tested across various concentrations, time points, and breast cancer cell lines for their effects on client, heat shock, and other proteins. This generates extensive data sets that outline the expected WB performance of Hsp90 inhibitors. In this review, we focus on 10 protein families that are commonly used to assess Hsp90 inhibition in in vitro breast cancer models and represent all 10 cancer hallmarks. We provide basic structural and functional information on these families and comprehensively describe the effects of N‐ and C‐terminal inhibitors on their levels. We summarize WB‐based observations for more than 100 Hsp90 inhibitors, outlining the characteristic effects of N‐ and C‐terminal inhibition, highlighting opportunities for future research, and ending with a suggestion for the WB method design based on best practices observed in literature.

A recent publication by our research team has introduced a novel group of hHv1 inhibitors, the 5-phenyl-2-aminoimidazoles. To guide rational drug design of selective derivatives, our goal was to map the molecular mechanism of compound 13 (i.e., NZ-58), a potent member of the series. For this, we utilized site-directed mutagenesis and conventional patch-clamp technique. Applying NZ-58 at various membrane potentials, and using different pulse frequencies, we could unveil the binding mode, which showed closed-state inhibition and no use dependence. The changes in inhibition of mutated hHv1 constructs confirmed the binding pocket suggested by molecular docking comprising countercharges D112 and D185. Combining the results of ionic and gating current measurements revealed an interesting molecular mechanism that traps the S4 helix. Knowing the interacting partners of NZ-58, this mechanism aligned well with previously found functional models of hHv1. By integrating pharmacological and biophysical perspectives, this work lays the groundwork for future advancements in rational drug design.

Mitochondria‐targeting moieties (MTMs) are molecular fragments designed to deliver covalently tethered functional cargo to mitochondria, providing a modular strategy for chemical biology tools, imaging agents, and mitochondria‐targeted therapies. Phosphonium‐ or nitrogen cation‐based MTMs are not inert vectors and exhibit intrinsic bioactivity on mitochondrial and cellular levels to various extents. Here, we systematically evaluated a panel of N ⁺ ‐based cations to determine how structural features influence subcellular distribution and inherent bioactivity. Live‐cell imaging of fluorescent dye conjugates revealed that 3,5‐diphenylpyridinium (DPPy ⁺ ) exhibits cellular uptake and mitochondrial targeting comparable to the benchmark triphenylphosphonium (TPP ⁺ ), whereas conjugates with unsubstituted pyridinium preferentially accumulate in lysosomes. Profiling of inert cargo derivatives showed that DPPy ⁺ has lower intrinsic activity on mitochondrial membrane potential and oxidative phosphorylation, as well as on cellular respiration and viability than TPP ⁺ . The combination of efficient mitochondrial delivery and low intrinsic bioactivity translated to bioactive cargo: a Kv1.3 inhibitor conjugate with DPPy ⁺ induced apoptosis in cancer cell lines and demonstrated improved cancer selectivity relative to the TPP ⁺ conjugate in pancreatic organoid models. These results position lipophilic pyridinium cations as effective TPP ⁺ surrogates with enhanced biocompatibility for mitochondria‐targeted therapeutic and diagnostic agents, while revealing the structure‐dependent competing lysosomal accumulation of permanent nitrogen cations.

Mitochondria‐targeting moieties (MTMs) are molecular fragments designed to deliver covalently tethered functional cargo to mitochondria, providing a modular strategy for chemical biology tools, imaging agents, and mitochondria‐targeted therapies. Phosphonium‐ or nitrogen cation‐based MTMs are not inert vectors and exhibit intrinsic bioactivity on mitochondrial and cellular levels to various extents. Here, we systematically evaluated a panel of N ⁺ ‐based cations to determine how structural features influence subcellular distribution and inherent bioactivity. Live‐cell imaging of fluorescent dye conjugates revealed that 3,5‐diphenylpyridinium (DPPy ⁺ ) exhibits cellular uptake and mitochondrial targeting comparable to the benchmark triphenylphosphonium (TPP ⁺ ), whereas conjugates with unsubstituted pyridinium preferentially accumulate in lysosomes. Profiling of inert cargo derivatives showed that DPPy ⁺ has lower intrinsic activity on mitochondrial membrane potential and oxidative phosphorylation, as well as on cellular respiration and viability than TPP ⁺ . The combination of efficient mitochondrial delivery and low intrinsic bioactivity translated to bioactive cargo: a Kv1.3 inhibitor conjugate with DPPy ⁺ induced apoptosis in cancer cell lines and demonstrated improved cancer selectivity relative to the TPP ⁺ conjugate in pancreatic organoid models. These results position lipophilic pyridinium cations as effective TPP ⁺ surrogates with enhanced biocompatibility for mitochondria‐targeted therapeutic and diagnostic agents, while revealing the structure‐dependent competing lysosomal accumulation of permanent nitrogen cations.

Lysosomal and endoplasmic reticulum (ER) Ca²⁺ release mutually influence each other’s functions. Recent work revealed that ER-located ryanodine receptor(s) (RyR(s)) Ca²⁺ release channels suppress autophagosome turnover by the lysosomes. In familial Alzheimer’s disease, inhibiting RyR hyperactivity restored autophagic flux by normalizing lysosomal vacuolar H⁺-ATPase (vATPase) levels. However, the mechanisms by which RyRs control lysosomal function and how this involves the vATPase remain unknown. Here, we show that RyRs interact with the ATP6v0a1 subunit of the vATPase, contributing to ER-lysosomal contact site formation. This interaction suppresses RyR-mediated Ca²⁺ release, leading to reduced lysosomal exocytosis. Pharmacological inhibition of RyR activity mimics these effects on lysosomal exocytosis. Retaining lysosomes inside cells via RyR inhibition increases ER-lysosomal contact site formation, rendering lysosomes more available for autophagic flux. In summary, these findings establish RyR/ATP6v0a1 complexes as ER-lysosomal tethers that dynamically and Ca²⁺ dependently regulate the intracellular availability of lysosomes to participate in autophagic flux.