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The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor\'s opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.

Acute myeloid leukemia (AML) is a genetically diverse and challenging malignancy, with mutations in the FLT3 gene being particularly common and deleterious. Gilteritinib, a potent FLT3 inhibitor, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsed/refractory AML with FLT3 mutations. Although gilteritinib was developed based on its inhibitory activity against FLT3 kinase, it is important to understand the precise mechanisms of its antileukemic activity in managing drug resistance and discovering biomarkers. This study was designed to elucidate the effect of gilteritinib on the FLT3 expression level. The results showed that gilteritinib induced a dose-dependent decrease in both FLT3 phosphorylation and expression. This reduction was particularly pronounced after 48 h of treatment. The decrease in FLT3 expression was found to be independent of changes in FLT3 mRNA transcription, suggesting post-transcriptional regulatory mechanisms. Further studies were performed in various AML cell lines and cells with both FLT3 wild-type and FLT3 mutant exhibited FLT3 reduction by gilteritinib treatment. In addition, other FLT3 inhibitors were evaluated for their ability to reduce FLT3 expression. Other FLT3 inhibitors, midostaurin, crenolanib, and quizartinib, also reduced FLT3 expression, consistent with the effect of gilteritinib. These findings hold great promise for optimizing gilteritinib treatment in AML patients. However, it is important to recognize that further research is warranted to gain a full understanding of these mechanisms and their clinical implications in the context of FLT3 reduction.

Kinases, a class of enzymes controlling various substrates phosphorylation, are pivotal in both physiological and pathological processes. Although their conserved ATP binding pockets pose challenges for achieving selectivity, this feature offers opportunities for drug repositioning of kinase inhibitors (KIs). This study presents a cost-effective in silico prediction of KIs drug repositioning via analyzing cross-docking results. We established the KIs database (278 unique KIs, 1834 bioactivity data points) and kinases database (357 kinase structures categorized by the DFG motif) for carrying out cross-docking. Comparative analysis of the docking scores and reported experimental bioactivity revealed that the Atypical, TK, and TKL superfamilies are suitable for drug repositioning. Among these kinase superfamilies, Olverematinib, Lapatinib, and Abemaciclib displayed enzymatic activity in our focused AKT-PI3K-mTOR pathway with IC50 values of 3.3, 3.2 and 5.8 μM. Further cell assays showed IC50 values of 0.2, 1.2 and 0.6 μM in tumor cells. The consistent result between prediction and validation demonstrated that repositioning KIs via in silico method is feasible.

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that has been associated with congenital neurological defects in fetuses born to infected mothers. At present, no vaccine or antiviral therapy is available to combat this devastating disease. Repurposing drugs that target essential host factors exploited by viruses is an attractive therapeutic approach. Here, we screened a panel of clinically approved small-molecule kinase inhibitors for their antiviral effects against a clinical isolate of ZIKV and thoroughly characterized their mechanisms of action. We found that the Raf kinase inhibitors Dabrafenib and Regorafenib potently impair the replication of ZIKV, but not that of its close relative dengue virus. Time-of-addition experiments showed that both inhibitors target ZIKV infection at post-entry steps. We found that Dabrafenib, but not Regorafenib, interfered with ZIKV genome replication by impairing both negative- and positive-strand RNA synthesis. Regorafenib, on the other hand, altered steady-state viral protein levels, viral egress, and blocked NS1 secretion. We also observed Regorafenib-induced ER fragmentation in ZIKV-infected cells, which might contribute to its antiviral effects. Because these inhibitors target different steps of the ZIKV infection cycle, their use in combination therapy may amplify their antiviral effects which could be further explored for future therapeutic strategies against ZIKV and possibly other flaviviruses.
OBJECTIVE: There is an urgent need to develop effective therapeutics against re-emerging arboviruses associated with neurological disorders like Zika virus (ZIKV). We identified two FDA-approved kinase inhibitors, Dabrafenib and Regorafenib, as potent inhibitors of contemporary ZIKV strains at distinct stages of infection despite overlapping host targets. Both inhibitors reduced viral titers by ~1 to 2 log10 (~10-fold to 100-fold) with minimal cytotoxicity. Furthermore, we show that Dabrafenib inhibits ZIKV RNA replication whereas Regorafenib inhibits ZIKV translation and egress. Regorafenib has the added benefit of limiting NS1 secretion, which contributes to the pathogenesis and disease progression of several flaviviruses. Because these inhibitors affect distinct post-entry steps of ZIKV infection, their therapeutic potential may be amplified by combination therapy and likely does not require prophylactic administration. This study provides further insight into ZIKV-host interactions and has implications for the development of novel antivirals against ZIKV and possibly other flaviviruses.

Sphingosine-1-phosphate (S1P) formed via catalytic actions of sphingosine kinase 1 (SphK1) behaves as a pro-survival substance and activates downstream target molecules associated with various pathologies, including initiation, inflammation, and progression of cancer. Here, we aimed to investigate the SphK1 inhibitory potentials of thymoquinone (TQ), Artemisinin (AR), and Thymol (TM) for the therapeutic management of lung cancer. We implemented docking, molecular dynamics (MD) simulations, enzyme inhibition assay, and fluorescence measurement studies to estimate binding affinity and SphK1 inhibitory potential of TQ, AR, and TM. We further investigated the anti-cancer potential of these compounds on non-small cell lung cancer (NSCLC) cell lines (H1299 and A549), followed by estimation of mitochondrial ROS, mitochondrial membrane potential depolarization, and cleavage of DNA by comet assay. Enzyme activity and fluorescence binding studies suggest that TQ, AR, and TM significantly inhibit the activity of SphK1 with IC50 values of 35.52 µM, 42.81 µM, and 53.68 µM, respectively, and have an excellent binding affinity. TQ shows cytotoxic effect and anti-proliferative potentials on H1299 and A549 with an IC50 value of 27.96 µM and 54.43 µM, respectively. Detection of mitochondrial ROS and mitochondrial membrane potential depolarization shows promising TQ-induced oxidative stress on H1299 and A549 cell lines. Comet assay shows promising TQ-induced oxidative DNA damage. In conclusion, TQ, AR, and TM act as potential inhibitors for SphK1, with a strong binding affinity. In addition, the cytotoxicity of TQ is linked to oxidative stress due to mitochondrial ROS generation. Overall, our study suggests that TQ is a promising inhibitor of SphK1 targeting lung cancer therapy.

Serine/threonine protein kinases (CK2, PIM-1, RIO1) are constitutively active, highly conserved, pleiotropic, and multifunctional kinases, which control several signaling pathways and regulate many cellular functions, such as cell activity, survival, proliferation, and apoptosis. Over the past decades, they have gained increasing attention as potential therapeutic targets, ranging from various cancers and neurological, inflammation, and autoimmune disorders to viral diseases, including COVID-19. Despite the accumulation of a vast amount of experimental data, there is still no \"recipe\" that would facilitate the search for new effective kinase inhibitors. The aim of our study was to develop an effective screening method that would be useful for this purpose. A combination of Density Functional Theory calculations and molecular docking, supplemented with newly developed quantitative methods for the comparison of the binding modes, provided deep insight into the set of desirable properties responsible for their inhibition. The mathematical metrics helped assess the distance between the binding modes, while heatmaps revealed the locations in the ligand that should be modified according to binding site requirements. The Structure-Binding Affinity Index and Structural-Binding Affinity Landscape proposed in this paper helped to measure the extent to which binding affinity is gained or lost in response to a relatively small change in the ligand\'s structure. The combination of the physico-chemical profile with the aforementioned factors enabled the identification of both \"dead\" and \"promising\" search directions. Tests carried out on experimental data have validated and demonstrated the high efficiency of the proposed innovative approach. Our method for quantifying differences between the ligands and their binding capabilities holds promise for guiding future research on new anti-cancer agents.

BACKGROUND: Atypical Teratoid Rhabdoid Tumor (ATRT) is a rare, devastating, and largely incurable pediatric brain tumor. Although recent studies have uncovered three molecular subgroups of ATRTs with distinct disease patterns, and signaling features, the therapeutic profiles of ATRT subgroups remain incompletely elucidated.
METHODS: We examined the effect of 465 kinase inhibitors on a panel of ATRT subgroup-specific cell lines. We then applied multi-omics analyses to investigate the underlying molecular mechanism of kinase inhibitor efficacy in ATRT subgroups.
RESULTS: We observed that ATRT cell lines are broadly sensitive to inhibitors of the PI3K and MAPK signaling pathways, as well as CDKs, AURKA/B kinases, and PLK1. We identified two classes of multi-kinase inhibitors (MKIs) predominantly targeting receptors tyrosine kinase (RTKs) including PDGFR and EGFR/ERBB2 in MYC/TYR ATRT cells. The PDGFRB inhibitor, Dasatinib, synergistically affected MYC/TYR ATRT cell growth when combined with broad-acting PI3K and MAPK pathway inhibitors, including Rapamycin and Trametinib. We observed that MYC/TYR ATRT cells were also distinctly sensitive to various inhibitors of ERBB2 signaling. Transcriptional, H3K27Ac ChIPSeq, ATACSeq, and HiChIP analyses of primary MYC/TYR ATRTs revealed ERBB2 expression which correlated with differential methylation and activation of a distinct enhancer element by DNA looping. Significantly, we show the brain penetrant EGFR/ERBB2 inhibitor, Afatinib, specifically inhibited in vitro and in vivo growth of MYC/TYR ATRT cells.
CONCLUSIONS: Taken together our studies suggest combined treatments with PDGFR and ERBB2-directed TKIs with inhibitors of the PI3K and MAPK pathways as an important new therapeutic strategy for the MYC/TYR subgroup of ATRTs.

Protein kinase CK2 is a holoenzyme composed of two regulatory subunits (CK2β) and two catalytic subunits (CK2α and CK2α\'). CK2 controls several cellular processes, including proliferation, inflammation, and cell death. However, CK2α and CK2α\' possess different expression patterns and substrates and therefore impact each of these processes differently. Elevated CK2α participates in the development of cancer, while increased CK2α\' has been associated with neurodegeneration, especially Huntington\'s disease (HD). HD is a fatal disease for which no effective therapies are available. Genetic deletion of CK2α\' in HD mouse models has ameliorated neurodegeneration. Therefore, pharmacological inhibition of CK2α\' presents a promising therapeutic strategy for treating HD. However, current CK2 inhibitors are unable to discriminate between CK2α and CK2α\' due to their high structural homology, especially in the targeted ATP-binding site. Using computational analyses, we found a potential type IV (\"D\" pocket) allosteric site that contained different residues between CK2α and CK2α\' and was distal from the ATP-binding pocket featured in both kinases. We decided to look for allosteric modulators that might interact in a biased fashion with the type IV pocket on both CK2α and CK2α\'. We screened a commercial library containing ∼29,000 allosteric-kinase-inhibitor-like compounds using a CK2α\' activity-dependent ADP-Glo Kinase assay. Obtained hits were counter-screened against CK2α using the ADP-Glo Kinase assay, revealing two CK2α\'-biased compounds. These two compounds might serve as the basis for further medicinal chemistry optimization for the potential treatment of HD.

mTORC1 is aberrantly activated in renal cell carcinoma (RCC) and is targeted by rapalogs. As for other targeted therapies, rapalogs clinical utility is limited by the development of resistance. Resistance often results from target mutation, but mTOR mutations are rarely found in RCC. As in humans, prolonged rapalog treatment of RCC tumorgrafts (TGs) led to resistance. Unexpectedly, explants from resistant tumors became sensitive both in culture and in subsequent transplants in mice. Notably, resistance developed despite persistent mTORC1 inhibition in tumor cells. In contrast, mTORC1 became reactivated in the tumor microenvironment (TME). To test the role of the TME, we engineered immunocompromised recipient mice with a resistance mTOR mutation (S2035T). Interestingly, TGs became resistant to rapalogs in mTORS2035T mice. Resistance occurred despite mTORC1 inhibition in tumor cells and could be induced by coculturing tumor cells with mutant fibroblasts. Thus, enforced mTORC1 activation in the TME is sufficient to confer resistance to rapalogs. These studies highlight the importance of mTORC1 inhibition in nontumor cells for rapalog antitumor activity and provide an explanation for the lack of mTOR resistance mutations in RCC patients.