Targeting RNA Helicase DDX3X with a Small Molecule Inhibitor for Breast Cancer Bone Metastasis Treatment.

Patients who present with breast cancer bone metastasis only have limited palliative treatment strategies and efficacious drug treatments are needed. In breast cancer patient data, high levels of the RNA helicase DDX3 are associated with poor overall survival and bone metastasis. Consequently, our objective was to target DDX3 in a mouse breast cancer bone metastasis model using a small molecule inhibitor of DDX3, RK-33. Histologically confirmed live imaging indicated no bone metastases in the RK-33 treated cohort, as opposed to placebo-treated mice. We generated a cell line from a bone metastatic lesion in mouse and found that it along with a patient-derived bone metastasis cell line gained resistance to conventional chemotherapeutics but not to RK-33. Finally, differential levels of DDX3 were observed in breast cancer patient metastatic bone samples. Overall, this study indicates that DDX3 is a relevant clinical target in breast cancer bone metastasis and that RK-33 can be a safe and effective treatment for these patients.

Inhibition of DDX3X ameliorated CD4+ T cells pyroptosis and improves survival in septic mice.

Over-expression of DDX3X mRNA is associated with T cell loss in septic patients. This study aimed to investigate the molecular mechanism of DDX3X on T cell reduction in sepsis. The sepsis model was established using lipopolysaccharide stimulation in vitro and cecal ligation and puncture (CLP) surgery in vivo. Results showed that the expression of DDX3X was significantly upregulated in CD4+ T cells in sepsis. RK-33, the inhibitor of DDX3X, was found to dramatically increase CD4+ T cell counts and prolong the survival rate of mice with sepsis. The results also showed that the expression of caspase-1/GSDMD in CD4+ T cells was significantly increased in vitro and in vivo, and RK-33 can substantially reduce CD4+ T cell pyroptosis through inhibiting NLRP3/caspase-1/GSDMD. Globally, our results suggest that DDX3X is involved in the loss of CD4+ T cells partly through activating the pyroptotic pathway during sepsis, which may provide potential targets for therapeutic interventions in this highly lethal disease.

RNA Helicase DDX3 Regulates RAD51 Localization and DNA Damage Repair in Ewing Sarcoma.

We previously demonstrated that RNA helicase DDX3X (DDX3) can be a therapeutic target in Ewing sarcoma (EWS), but its role in EWS biology remains unclear. The present work demonstrates that DDX3 plays a unique role in DNA damage repair (DDR). We show that DDX3 interacts with several proteins involved in homologous recombination, including RAD51, RECQL1, RPA32, and XRCC2. In particular, DDX3 colocalizes with RAD51 and RNA:DNA hybrid structures in the cytoplasm of EWS cells. Inhibition of DDX3 RNA helicase activity increases cytoplasmic RNA:DNA hybrids, sequestering RAD51 in the cytoplasm, which impairs nuclear translocation of RAD51 to sites of double-stranded DNA breaks thus increasing sensitivity of EWS to radiation treatment, both in vitro and in vivo. This discovery lays the foundation for exploring new therapeutic approaches directed at manipulating DDR protein localization in solid tumors.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy toward them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3X (DDX3), a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented support the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

RK-33 Is a Broad-Spectrum Antiviral Agent That Targets DEAD-Box RNA Helicase DDX3X.

Viral disease is one of the greatest burdens for human health worldwide, with an urgent need for efficacious antiviral strategies. While antiviral drugs are available, in many cases, they are prone to the development of drug resistance. A way to overcome drug resistance associated with common antiviral therapies is to develop antivirals targeting host cellular co-factors critical to viral replication, such as DEAD-box helicase 3 X-linked (DDX3X), which plays key roles in RNA metabolism and the antiviral response. Here, we use biochemical/biophysical approaches and infectious assays to show for the first time that the small molecule RK-33 has broad-spectrum antiviral action by inhibiting the enzymatic activities of DDX3X. Importantly, we show that RK-33 is efficacious at low micromolar concentrations in limiting infection by human parainfluenza virus type 3 (hPIV-3), respiratory syncytial virus (RSV), dengue virus (DENV), Zika virus (ZIKV) or West Nile virus (WNV)-for all of which, no Food and Drug Administration (FDA)-approved therapeutic is widely available. These findings establish for the first time that RK-33 is a broad-spectrum antiviral agent that blocks DDX3X's catalytic activities in vitro and limits viral replication in cells.

Pharmacological inhibition of DEAD-Box RNA Helicase 3 attenuates stress granule assembly.

Stress granules (SGs) are non-membranous cytosolic protein-RNA aggregates that process mRNAs through stalled translation initiation in response to cellular stressors and in disease. DEAD-Box RNA helicase 3 (DDX3) is an active target of drug development for the treatment of viral infections, cancers, and neurodegenerative diseases. DDX3 plays a critical role in RNA metabolism, including SGs, but the role of DDX3 enzymatic activity in SG dynamics is not well understood. Here, we address this question by determining the effects of DDX3 inhibition on the dynamics of SG assembly and disassembly. We use two small molecule inhibitors of DDX3, RK33 and 16D, with distinct inhibitory mechanisms that target DDX3's ATPase activity and RNA helicase site, respectively. We find that both DDX3 inhibitors reduce the assembly of SGs, with a more pronounced reduction from RK-33. In contrast, both compounds only marginally affect the disassembly of SGs. RNA-mediated knockdown of DDX3 caused a similar reduction in SG assembly and minimal effect on SG disassembly. Collectively, these results reveal that the enzymatic activity of DDX3 is required for the assembly of SGs and pharmacological inhibition of DDX3 could be relevant for the treatment of SG-dependent pathologies.

Targeting RNA helicase DDX3 in stem cell maintenance and teratoma formation.

DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. Besides the role of DDX3 in transformed cells, there is evidence to indicate that DDX3 expression is at its highest levels during early embryonic development and is also expressed in germ cells of adults. Even though there is a distinct pattern of DDX3 expression during embryonic development and in adults, very little is known regarding its role in embryonic stem cells and pluripotency. In this work, we examined the relationship between DDX3 and human embryonic stem cells and its differentiated lineages. DDX3 expression was analyzed by immunohistochemistry in human embryonic stem cells and embryonal carcinoma cells. From the data obtained, it was evident that DDX3 was overexpressed in undifferentiated stem cells compared to differentiated cells. Moreover, when DDX3 expression was abrogated in multiple stem cells, proliferation was decreased, but differentiation was facilitated. Importantly, this resulted in reduced potency to induce teratoma formation. Taken together, these findings indicate a distinct role for DDX3 in stem cell maintenance.

PLGA nanoparticle formulation of RK-33: an RNA helicase inhibitor against DDX3.

BACKGROUND: The DDX3 helicase inhibitor RK-33 is a newly developed anticancer agent that showed promising results in preclinical research (Bol et al. EMBO Mol Med, 7(5):648-649, 2015). However, due to the physicochemical and pharmacological characteristics of RK-33, we initiated development of alternative formulations of RK-33 by preparing sustained release nanoparticles that can be administered intravenously. METHODS: In this study, RK-33 was encapsulated in poly(lactic-co-glycolic acid) (PLGA), one of the most well-developed biodegradable polymers, using the emulsion solvent evaporation method. RESULTS: Hydrodynamic diameter of RK-33-PLGA nanoparticles was about 245 nm with a negative charge, and RK-33-PLGA nanoparticles had a payload of 1.4 % RK-33. RK-33 was released from the PLGA nanoparticles over 7 days (90 ± 5.7 % released by day 7) and exhibited cytotoxicity to human breast carcinoma MCF-7 cells in a time-dependent manner. Moreover, RK-33-PLGA nanoparticles were well tolerated, and systemic retention of RK-33 was markedly improved in normal mice. CONCLUSIONS: PLGA nanoparticles have a potential as a parenteral formulation of RK-33.

Osthole induces apoptosis, suppresses cell-cycle progression and proliferation of cancer cells.

BACKGROUND: The aim of the present study was to determine the effects of osthole on cell proliferation and viability, cell-cycle progression and induction of apoptosis in human laryngeal cancer RK33 and human medulloblastoma TE671 cell lines. MATERIALS AND METHODS: Cell viability was measured by means of the MTT method and cell proliferation by the 5-bromo-2-deoxyuridine (BrdU) incorporation assay. Cell-cycle progression was determined by flow cytometry, and induction of apoptosis by release of oligonucleosomes to the cytosol. The gene expression was estimated by a quantitative polymerase chain reaction (qPCR) method. High-performance counter-current chromatography (HPCCC) was applied for isolation of osthole from fruits of Mutellina purpurea. RESULTS: Osthole decreased proliferation and cell viability of cancer cells in a dose-dependent manner. The tested compound induced apoptosis, increased the cell numbers in G1 and decreased cell number in S/G2 phases of the cell cycle, differentially regulating CDKN1A and TP53 gene expression depending on cancer cell type. CONCLUSION: Osthole could be considered as a potential compound for cancer therapy and chemoprevention.