A series of novel benzimidazole derivatives were designed and synthesised based on the structures of reported oral available ALK inhibitor and HDAC inhibitor, pracinostat. In enzymatic assays, compound 3b, containing a 2-acyliminobenzimidazole moiety and hydroxamic acid side chain, could inhibit both ALK and HDAC6 (IC₅₀ = 16 nM and 1.03 µM, respectively). Compound 3b also inhibited various ALK mutants known to be involved in crizotinib resistance, including mutant L1196M (IC₅₀, 4.9 nM). Moreover, 3b inhibited the proliferation of several cancer cell lines, including ALK-addicted H2228 cells. To evaluate its potential for treating cancers in vivo, 3b was used in a human A549 xenograft model with BALB/c nude mice. At 20 mg/kg, 3b inhibited tumour growth by 85% yet had a negligible effect on mean body weight. These results suggest a attracting route for the further research and optimisation of dual ALK/HDAC inhibitors.

Secondary mutation, T790M, conferring tyrosine kinase inhibitors (TKIs) resistance beyond oncogenic epidermal growth factor receptor (EGFR) mutations presents a challenging unmet need. Although TKI-resistant mechanisms are intensively investigated, the underlying responses of cancer cells adapting drug perturbation are largely unknown. To illuminate the molecular basis linking acquired mutation to TKI resistance, affinity purification coupled mass spectrometry was adopted to dissect EGFR interactome in TKI-sensitive and TKI-resistant non-small cell lung cancer cells. The analysis revealed TKI-resistant EGFR-mutant interactome allocated in diverse subcellular distribution and enriched in endocytic trafficking, in which gefitinib intervention activated autophagy-mediated EGFR degradation and thus autophagy inhibition elevated gefitinib susceptibility. Alternatively, gefitinib prompted TKI-sensitive EGFR translocating toward cell periphery through Rab7 ubiquitination which may favor efficacy to TKIs suppression. This study revealed that T790M mutation rewired EGFR interactome that guided EGFR to autophagy-mediated degradation to escape treatment, suggesting that combination therapy with TKI and autophagy inhibitor may overcome acquired resistance in non-small cell lung cancer.

Protein post-translational modifications (PTMs) are crucial in plant cellular processes, particularly in protein folding and signal transduction. N-glycosylation and phosphorylation are notably significant PTMs, playing essential roles in regulating plant responses to environmental stimuli. However, current sequential enrichment methods for simultaneous analysis of phosphoproteome and N-glycoproteome are labor-intensive and time-consuming, limiting their throughput. Addressing this challenge, this study introduces a novel tandem S-Trap-IMAC-HILIC (S-Trap: suspension trapping; IMAC: immobilized metal ion affinity chromatography; HILIC: hydrophilic interaction chromatography) strategy, termed TIMAHAC, for simultaneous analysis of plant phosphoproteomics and N-glycoproteomics. This approach integrates IMAC and HILIC into a tandem tip format, streamlining the enrichment process of phosphopeptides and N-glycopeptides. The key innovation lies in the use of a unified buffer system and an optimized enrichment sequence to enhance efficiency and reproducibility. The applicability of TIMAHAC was demonstrated by analyzing the Arabidopsis phosphoproteome and N-glycoproteome in response to abscisic acid (ABA) treatment. Up to 1954 N-glycopeptides and 11,255 phosphopeptides were identified from Arabidopsis, indicating its scalability for plant tissues. Notably, distinct perturbation patterns were observed in the phosphoproteome and N-glycoproteome, suggesting their unique contributions to ABA response. Our results reveal that TIMAHAC offers a comprehensive approach to studying complex regulatory mechanisms and PTM interplay in plant biology, paving the way for in-depth investigations into plant signaling networks.

The promising role of cold atmospheric plasma jet (CAPJ) treatment in promoting wound healing has been widely documented in therapeutic implications. However, the fact that not all subjects respond equally to CAPJ necessitates the investigation of the underlying cellular mechanisms, which have been rarely understood so far. Given that wound healing is a complex and prolonged process, post-plasma-activated medium (PAM) treated keratinocytes were collected at two time points, 2 hours (receiving) and 24 hours (recovery), for (phospho)proteomic analysis to systematically dissect the molecular basis of CAPJ-promoted wound healing. The receiving (phospho)proteomics datasets, referred to the time point of 2 hours, revealed an apparent increase in the phosphorylation of CK2 and its-mediated PI3K/AKT and MAPK signaling pathways, accompanied by a prompted downstream physiological response of cell migration. Additionally, incorporating the network analysis of predicted kinases and their direct interactors, we reiterated that CAPJ influenced cell growth and migration, thereby paving the way for its role in subsequent wound healing processes. Further determining the proteome profiles at recovery phase, which is the time point of 24 hours, displayed a totally different view from the receiving proteome which had almost no change. The up-regulation of ROBOs/SLITs expression and vesicle trafficking and fusion-related proteins, along with the abundant presence of 14-3-3 family proteins, indicated that the persistent effect of PAM on the wound healing process could potentially promote keratinocyte-fibroblast crosstalk and stimulate extracellular matrix (ECM) synthesis upon epithelialization. Consistent with proteome patterns, CAPJ-treated wound tissues indeed showed a denser and well-organized ECM architecture, implying hastened epithelialization during wound healing. Collectively, we delineated the molecular basis of CAPJ-accelerated wound healing at early and late responses, providing valuable insights for treatment selection and the development of therapeutic strategies to achieve better outcomes.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m ⁵ C), is found on the RNA of HIV-1, where m ⁵ C enhances the translation of HIV-1 RNA. However, whether m ⁵ C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m ⁵ C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m ⁵ C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m ⁵ C mainly deposited by the cellular methyltransferase NSUN2. Loss of m ⁵ C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m ⁵ C deposition led to a significant decrease in HBV replication. Thus, our data indicate m ⁵ C methylations as a critical mediator of the epsilon elements’ function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m ⁵ C methyltransfer process on HBV epsilon as an antiviral strategy.

Protein phosphorylation can induce signal transduction to change sperm motility patterns during sperm capacitation. However, changes in the phosphorylation of sperm proteins in mice are still incompletely understood. Here, capacitation-related phosphorylation in mouse sperms were firstly investigated by label-free quantitative (LFQ) phosphoproteomics coupled with bioinformatics analysis using ingenuity pathway analysis (IPA) methods such as canonical pathway, upstream regulator, and network analysis. Among 1632 phosphopeptides identified at serine, threonine, and tyrosine residues, 1050 novel phosphosites, corresponding to 402 proteins, were reported. Gene heatmaps for IPA canonical pathways showed a novel role for GSK-3 in GP6 signaling pathways associated with capacitation for 60 min. At the same time, the reduction of the abundant isoform-specific GSK-3α expression was shown by western blot (WB) while the LFQ pY of this isoform slightly decreased and then increased. The combined results from WB and LFQ methods explain the less inhibitory phosphorylation of GSK-3α during capacitation and also support the predicted increases in its activity. In addition, pAKAP4 increased at the Y156 site but decreased at the Y811 site in a capacitated state, even though IPA network analysis and WB analysis for overall pAKAP revealed upregulated trends. The potential roles of GSK-3 and AKAP4 in fertility are discussed.