Background/Objectives: Indolo[2,3-a]pyrrolo[3,4-c]carbazole scaffold is successfully used as an efficient structural motif for the design and development of different antitumor agents. In this study, we investigated the anti-glioblastoma therapeutic potential of glycosylated indolocarbazole analog LCS1269 utilizing in vitro, in vivo, and in silico approaches. Methods: Cell viability was estimated by an MTT assay. The distribution of cell cycle phases was monitored using flow cytometry. Mitotic figures were visualized by fluorescence microscopy. Quantitative RT-PCR was used to evaluate the gene expression. The protein expression was assessed by Western blotting. Molecular docking and computational ADMET were approved for the probable protein target simulations and predicted pharmacological assessments, respectively. Results: Our findings clearly suggest that LCS1269 displayed a significant cytotoxic effect against diverse glioblastoma cell lines and patient-derived glioblastoma cultures as well as strongly suppressed xenograft growth in nude mice. LCS1269 exhibited more potent anti-proliferative activity toward glioblastoma cell lines and patient-derived glioblastoma cultures compared to conventional drug temozolomide. We further demonstrated that LCS1269 treatment caused the severe G2 phase arrest of cell cycle in a dose-dependent manner. Mechanistically, we proposed that LCS1269 could affect the CDK1 activity both by targeting active site of this enzyme and indirectly, in particular through the modulation of the Wee1/Myt1 and FOXM1/Plk1 signaling pathways, and via p21 up-regulation. LCS1269 also showed favorable pharmacological characteristics in in silico ADME prediction in comparison with staurosporine, rebeccamycin, and becatecarin as reference drugs. Conclusions: Further investigations of LCS1269 as an anti-glioblastoma medicinal agent could be very promising.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. The success of modern multimodal standards approved in anti-glioblastoma therapy remains limited. Consequently, new therapeutics are urgently needed. In this study, utilizing ex vivo, in silico, and in vitro approaches, we investigated the LCS1269 effects on two potential targets, DNA and Top I. We also elucidated the influence of LCS1269 on signaling pathways and GBM cell viability. Based on our docking data and competition studies results, we demonstrated that LCS1269 may bind to DNA, demonstrating selectivity toward AT-rich regions. We also showed that LCS1269 could dock both Top I/DNA binary complex and Top I active sites. LCS1269 caused Top I dysfunction and downregulated the expression of Top I. Moreover, the LCS1269 treatment of GBM cells facilitated DNA damage and the activation of the ATM/Chk1/BRCA1/Rad51 pathway. Meanwhile, DNA damage response induction and ATM/Chk1/BRCA1/Rad51 pathway activation were insufficient to prevent GBM cell death triggered by LCS1269 treatment. Our work shows that DNA and Top I are promising molecular targets of LCS1269, thus providing insight on several novel mechanisms of its anti-tumor activity. Nonetheless, we did not perform a biophysical validation of the LCS1269–DNA interaction, which is a limitation of our study.

Carfilzomib (CFZ) is a cornerstone in the treatment of relapsed multiple myeloma (MM). However, its efficacy is limited by resistance mediated by the overexpression of the ABC-transporter P-glycoprotein (P-gp). The signaling pathways driving the emergence of P-gp in MM remain unclear. To investigate this, we generated CFZ-resistant AMO-1/CFZ cells with P-gp overexpression by long-term selection. RNA sequencing of control AMO-1 and AMO-1/CFZ, sorted into two subpopulations, P-gp HIGH and P-gp LOW, implicated the Ras/MEK/ERK pathway as the most likely signaling cascade involved in P-gp upregulation. We therefore evaluated two clinically used MAPK pathway inhibitors, cobimetinib and ulixertinib, for their ability to re-sensitize AMO-1/CFZ cells to CFZ. Co-administration at non-toxic concentrations enhanced sensitivity 5-fold with cobimetinib and 17-fold with ulixertinib. Analysis of the combined MTT assay results, rhodamine efflux experiments, molecular docking, and Western blotting revealed distinct actions. Ulixertinib primarily functions as a potent direct P-gp inhibitor. Conversely, non-toxic concentrations of cobimetinib sensitizes cells by suppressing MAPK signaling, though it also exhibits P-gp inhibition at higher concentrations. At the IC₅₀ concentration, both inhibitors reduced P-gp expression. In conclusion, combining CFZ with MAPK pathway inhibitors like cobimetinib or ulixertinib represents a promising strategy to overcome P-gp-mediated resistance in MM.