The α₂δ‐1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α₂δ‐1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α₂δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α₂δ-1 (α₂δ-1_S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α₂δ‐1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α₂δ‐1 and suggest that gabapentin does not directly target TSP/α₂δ-1 interaction to alleviate neuropathic pain.

The human phosphatidylinositol 4‐phosphate 5‐kinase type I α (hPIP5K1α) participates in the phosphoinositide‐3‐kinase/protein kinase B/mammalian target of rapamycin signaling pathway. Despite the evidence that hPIP5K1α plays a role in the development of prostate cancer (PCa), only one inhibitor is known to date. With the aim of identifying new inhibitors, a nonradiometric assay for measurement of the hPIP5K1α enzyme activity was developed. The assay is based on the separation of the fluorescently labeled substrate phosphatidylinositol‐4‐phosphate (PI(4)P) and the resulting product phosphatidylinositol‐4,5‐bisphosphate (PIP₂) by capillary electrophoresis (CE). Furthermore, an inactive mutant K261A of hPIP5K1α was generated by site‐directed mutagenesis and used as a control. Michaelis–Menten analysis revealed a K_m value of 21.6 µm and V_max of 0.65 pmol·min⁻¹ for the cosubstrate ATP. The average Z’ value was determined to be 0.86, indicating a high reliability of the assay. An in silico screening of an in‐house compound library was performed employing the crystal structure of zebrafish PIP5K1α. By applying this strategy, three compounds with a 2‐amino‐3‐cyano‐4H‐pyranobenzoquinone scaffold were identified and tested using the CE‐based assay. These compounds inhibited hPIP5K1α to > 90% at a concentration of 50 µm. Subsequently, the inhibitory activity of all compounds with a pyranobenzoquinone scaffold (29) was tested on hPIP5K1α. Compound 4‐(2‐amino‐3‐cyano‐6‐hydroxy‐5,8‐dioxo‐7‐undecyl‐5,8‐dihydro‐4H‐chromen‐4‐yl)benzoic acid appeared to be the most potent inhibitor of hPIP5K1α identified so far with an IC₅₀ value of 1.55 µm, exhibiting a substrate‐competitive mode of action. The effects of this compound on cell viability and the induction of apoptosis were investigated in LNCaP, DU145, and PC3 PCa cells.

Protein kinase CK2 is involved in regulating cellular processes, such as cell cycle, proliferation, migration, and apoptosis, making it an attractive anticancer target. We previously described a prenyloxy-substituted indeno[1,2-b]indole (5-isopropyl-4-(3-methylbut-2-enyloxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p)) as a very potent inhibitor of CK2 holoenzyme (IC₅₀ = 25 nM). Here, we report the broad-spectrum anticancer activity of 4p and provide substantial progress on its pharmacokinetic properties. Using a cell-based CK2 activity assay and live-cell imaging of cultured A431, A549, and LNCaP cancer cell lines, cellular CK2 target engagement was shown as well as strong antiproliferative, anti-migratory and apoptosis-inducing effects of 4p. Furthermore, evidence was found for the ability of 4p to disrupt A549 spheroid cohesion. A series of LC-MS/MS experiments revealed high and rapid cellular uptake (intracellular concentration is approximately 5 µM after 1 h incubation) and low metabolic stability of 4p. These results point to the value of 4p as a potent CK2 inhibitor with promising anticancer activities and should trigger future medicinal chemistry efforts to improve the drug-like properties of this compound.

Highly pleiotropic and constitutively active protein kinase CK2 is a key target in cancer therapy, but only one small‐molecule inhibitor has reached clinical trials—CX‐4945. In this study, we present the indeno[1,2‐b]indole derivative 5‐isopropyl‐4‐methoxy‐7‐methyl‐5,6,7,8‐tetrahydroindeno[1,2‐b]indole‐9,10‐dione (5a‐2) that decreased the intracellular CK2 activity in A431, A549, and LNCaP tumor cell lines analogous to CX‐4945 (> 75% inhibition at 20 µm) and similarly blocked CK2‐specific Akt phosphorylation in LNCaP cells. Cellular uptake analysis demonstrated higher intracellular concentrations of 5a‐2 (408.3 nm) compared with CX‐4945 (119.3 nm). This finding clarifies the comparable effects of both compounds on the intracellular CK2 activity despite their different inhibitory potency in vitro [IC₅₀ = 25 nm (5a‐2) and 3.7 nm (CX‐4945)]. Examination of the effects of both CK2 inhibitors on cancer cells using live‐cell imaging revealed notable differences. Whereas CX‐4945 showed a stronger pro‐apoptotic effect on tumor cells, 5a‐2 was more effective in inhibiting tumor cell migration. Our results showed that 49% of intracellular CX‐4945 was localized in the nuclear fraction, whereas 71% of 5a‐2 was detectable in the cytoplasm. The different subcellular distribution, and thus the site of CK2 inhibition, provides a possible explanation for the different cellular effects. Our study indicates that investigating CK2 inhibition‐mediated cellular effects in relation to the subcellular sites of CK2 inhibition may help to improve our understanding of the preferential roles of CK2 within different cancer cell compartments.

The fall armyworm, Spodoptera frugiperda, is a highly invasive pest that poses severe threats to food crop production worldwide. Several methods have been applied to control fall armyworm infestations, including egg parasitoids, chemical insecticides, and transgenic crops expressing Bacillus thuringiensis (Bt) toxins. However, the currently available control methods are either poorly effective or facing challenges such as resistance development and environmental concerns. The rapidly evolving molecular tools of genetic engineering, particularly clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas), have been increasingly utilized in recent years in attempts to develop novel pest management strategies and genetically resistant crops. CRISPR/Cas‐based genome editing offers a precise and efficient molecular tool that has been used successfully in several functional genomic studies of the fall armyworm over the last few years. In this review, we summarize recent progress in using CRISPR/Cas for precise genome editing of the fall armyworm, enabling the targeted modification of key genes essential for the survival and propagation of the pest. The potential of the CRISPR/Cas system to develop insect‐resistant crops and pest management approaches that can be integrated into the current management strategies of the pest is discussed.