Chromosome Region Maintenance Research Articles

Discovery of SZJK-0421: A Novel Potent, Low Toxicity, Selective Second Generation of CRM1 Inhibitor for the Treatment of Both Hematological and Solid Tumors.

Nuclear export factor chromosome region maintenance 1 (CRM1) mediated the transport of various growth-regulatory proteins and was frequently overexpressed in many hematologic and solid tumors. Selinexor (KPT-330) was the only approved CRM1 inhibitor, but the severe gastrointestinal and central nervous system toxicities limited its clinical application. In this manuscript, a series of novel second-generation CRM1 inhibitors were designed, in which SZJK-0421 was a more reversible inhibitor than KPT-330. The treatment of various tumor cells with SZJK-0421 significantly inhibited the function of CRM1. SZJK-0421 displayed good liver microsome stabilities and pharmacokinetic properties. Most importantly, SZJK-0421 reduced the direct damage to the gastrointestinal mucosa, and the brain plasma distribution ratio of SZJK-0421 was very low in Sprague-Dawley (SD) rats (3%), which avoided gastrointestinal reactions such as central nausea and vomiting caused by large permeability of blood-brain barrier. In addition, SZJK-0421 exhibited strong anticancer efficacy in xenograft models of both solid and hematological tumors.

Discovery of Potent Covalent CRM1 Inhibitors Via a Customized Structure-Based Virtual Screening Pipeline and Bioassays.

CRM1 (chromosomal region maintenance 1, also referred to as exportin 1 or XPO1) plays a crucial role in maintaining the appropriate nuclear levels of tumor suppressor proteins (TSPs), growth regulatory proteins (GRPs), and antiapoptotic proteins, thereby contributing significantly to their anticancer effects. Dysregulation of CRM1-mediated nuclear transport, observed in a range of cancers such as colon cancer as well as autoimmune diseases, highlights its significance in various disease processes. In this paper, we employed a customized structure-based virtual screening campaign to search for novel covalent CRM1 inhibitors and purchased 50 potentially active compounds for in vitro bioassays. Among these candidates, AN-988 displayed a notably higher binding affinity (KD = 615 nM) toward CRM1, as determined by the biolayer interferometry (BLI) assay. Furthermore, AN-988 exhibited a strong suppression of colorectal cancer cell proliferation and remarkable anti-inflammatory effects. Notably, AN-988 induced cell apoptosis and cell cycle arrest in a time- and dose-dependent manner by effectively inhibiting the translocation of FOXO3a from the nucleus to the cytosol, thereby preserving the activity of FOXO3a. Collectively, our study identified AN-988 as a promising CRM1 inhibitor, underscoring its potential as a preclinical colon cancer therapy candidate.

Cellular RNA-binding proteins LARP4 and PABPC1 synergistically facilitate viral translation of coronavirus PEDV

Coronaviruses are causing epizootic diseases and thus are a substantial threat for both domestic and wild animals. These viruses depend on the host translation machinery to complete their life cycle. The current paper identified cellular RNA-binding proteins (RBPs), La-related protein 4 (LARP4) and polyadenylate-binding protein cytoplasmic 1 (PABPC1), as critical regulators of efficient translation of the coronavirus porcine epidemic diarrhea virus (PEDV) mRNA. In Vero cells, PEDV infection caused LARP4 to migrate from the nucleus to the cytoplasm in a chromosome region maintenance1 (CRM1)-independent pathway. In the absence of the nuclear export signal of LARP4, viral translation was not promoted by LARP4. A further study unveiled that the cytoplasmic LARP4 binds to the 3′-terminal untranslated region (3′UTR) of PEDV mRNA with the assistance of PABPC1 to facilitate viral translation. LARP4 knockdown reduced the promotion of the PABPC1-induced 3′UTR translation activity. Moreover, the rabbit reticulocyte lysate (RRL) system revealed that the prokaryotic expressed protein LARP4 and PABPC1 enhance PEDV mRNA translation. To our knowledge, this is the first study demonstrating that PEDV induces nucleo-cytoplasmic shuttling of LARP4 to enhance its own replication, which broadens our insights into how viruses use host's RBPs for the efficient translation of viral mRNA.

Novel-and Not So Novel-Inhibitors of the Multifunctional CRM1 Protein.

Chromosome Region Maintenance 1 (CRM1), also known as Exportin 1 (XPO1), is a protein that is critical for transport of proteins and RNA to the cytoplasm through the nuclear pore complex. CRM1 inhibition with small molecule inhibitors is currently being studied in many cancers, including leukemias, solid organ malignancies and brain tumors. We review the structure of CRM1, its role in nuclear export, the current availability of CRM1 inhibitors, and the role of CRM1 in a number of distinct cellular processes. A deeper understanding of how CRM1 functions in nuclear export as well as other cellular processes may allow for the development of additional novel CRM1 inhibitors.

Cadmium targeting transcription factor EB to inhibit autophagy-lysosome function contributes to acute kidney injury

IntroductionEnvironmental and occupational exposure to cadmium (Cd) has been shown to cause acute kidney injury (AKI). Previous studies have demonstrated that autophagy inhibition and lysosomal dysfunction are important mechanisms of Cd-induced AKI. ObjectivesTranscription factor EB (TFEB) is a critical transcription regulator that modulates autophagy-lysosome function, but its role in Cd-induced AKI is yet to be elucidated. Thus, in vivo and in vitro studies were conducted to clarify this issue. Methods and resultsData firstly showed that reduced TFEB expression and nuclear translocation were evident in Cd-induced AKI models, accompanied by autophagy-lysosome dysfunction. Pharmacological and genetic activation of TFEB improved Cd-induced AKI via alleviating autophagy inhibition and lysosomal dysfunction, whereas Tfeb knockdown further aggravated this phenomenon, suggesting the key role of TFEB in Cd-induced AKI by regulating autophagy. Mechanistically, Cd activated mechanistic target of rapamycin complex 1 (mTORC1) to enhance TFEB phosphorylation and thereby inhibiting TFEB nuclear translocation. Cd also activated chromosome region maintenance 1 (CRM1) to promote TFEB nuclear export. Meanwhile, Cd activated general control non-repressed protein 5 (GCN5) to enhance nuclear TFEB acetylation, resulting in the decreased TFEB transcriptional activity. Moreover, inhibition of CRM1 or GCN5 alleviated Cd-induced AKI by enhancing TFEB activity, respectively. ConclusionIn summary, these findings reveal that TFEB phosphorylation, nuclear export and acetylation independently suppress TFEB activity to cause Cd-induced AKI via regulating autophagy-lysosome function, suggesting that TFEB activation might be a promising treatment strategy for Cd-induced AKI.

Decreased Ubiquitination and Acetylation of Histones 3 and 4 Are Associated with Obesity-Induced Disorders of Spermatogenesis in Mice.

Obesity, a chronic metabolic disorder, is related to cardiovascular diseases, diabetes, cancer, and reproductive disorders. The relationship between obesity and male infertility is now well recognized, but the mechanisms involved are unclear. We aimed to observe the effect of obesity on spermatogenesis and to investigate the role of histone ubiquitination and acetylation modifications in obesity-induced spermatogenesis disorders. Thirty male C57BL/6J mice were randomly divided into two groups. The control group was fed with a general maintenance diet (12% fat), while a high-fat diet (HFD) group was fed with 40% fat for 10 weeks; then, they were mated with normal females. The fertility of male mice was calculated, testicular and sperm morphology were observed, and the expression levels of key genes and the levels of histone acetylation and ubiquitination modification during spermatogenesis were detected. The number of sperm was decreased, as well as the sperm motility, while the number of sperm with malformations was increased. In the testes, the mRNA and protein expression levels of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), chromosome region maintenance-1 protein (CRM1), high-mobility group B2 (HMGB2), phosphoglycerate kinase 2 (PGK2), and testicular angiotensin-converting enzyme (tACE) were decreased. Furthermore, obesity led to a decrease in ubiquitinated H2A (ubH2A) and reduced levels of histone H3 acetylation K18 (H3AcK18) and histone H4 acetylation K5, K8, K12, and K16 (H4tetraAck), which disrupted protamine 1 (Prm1) deposition in testis tissue. These results suggest that low levels of histone ubiquitination and acetylation are linked with obesity-induced disorders during spermatogenesis, contributing to a better understanding of obesity-induced damage to male reproduction.

Cytoplasmic localization of SETDB1‑induced Warburg effect via c‑MYC‑LDHA axis enhances migration and invasion in breast carcinoma.

SET domain bifurcated 1 (SETDB1), a pivotal histone lysine methyltransferase, is transported to the cytoplasm via a chromosome region maintenance 1 (CMR1)‑dependent pathway, contributing to non‑histone methylation. However, the function and underlying mechanism of cytoplasmic SETDB1 in breast cancer remain elusive. In the present study, immunohistochemistry revealed that elevated cytoplasmic SETDB1 was correlated with lymph node metastasis and more aggressive breast cancer subtypes. Functionally, wound healing and Transwell assays showed that cytoplasmic SETDB1 is key for cell migration and invasion, as well as induction of epithelial‑mesenchymal transition (EMT), which was reversed by leptomycin B (LMB, a CMR1 inhibitor) treatment. Furthermore, RNA‑seq and metabolite detection revealed that cytoplasmic SETDB1 was associated with metabolism pathway and elevated levels of metabolites involved in the Warburg effect, including glucose, pyruvate, lactate and ATP. Immunoblotting and reverse transcription‑quantitative PCR verified that elevation of cytoplasmic SETDB1 contributed to elevation of c‑MYC expression and subsequent upregulation of lactate dehydrogenase A (LDHA) expression. Notably, gain‑ and loss‑of‑function approaches revealed that LDHA overexpression in T47D cells enhanced migration and invasion by inducing EMT, while its depletion in SETDB1‑overexpressing MCF7 cells reversed SETDB1‑induced migration and invasion, as well as the Warburg effect and EMT. In conclusion, subcellular localization of cytoplasmic SETDB1 may be a pivotal factor in breast cancer progression. The present study offers valuable insight into the novel functions and mechanisms of cytoplasmic SETDB1.

Statement of Retraction: The abnormal expression of chromosomal region maintenance 1 (CRM1)-survivin axis in ovarian cancer and its related mechanisms regulating proliferation and apoptosis of ovarian cancer cells

Statement of Retraction: The abnormal expression of chromosomal region maintenance 1 (CRM1)-survivin axis in ovarian cancer and its related mechanisms regulating proliferation and apoptosis of ovarian cancer cells

Nucleocytoplasmic shuttling of BEFV M protein-modulated by lamin A/C and chromosome maintenance region 1 through a transcription-, carrier- and energy-dependent pathway

This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.

CRM1/XPO1-FOXO1 Pathway Is Associated with Clinical Outcome and Represents a Promising Target in B-Cell Acute Lymphocytic Leukemia

Background: Currently, a proportion of B-Cell Acute Lymphocytic Leukemia (B-ALL) patients still show no response to existing therapeutic drugs and existing therapeutic drugs cannot further improve the prognosis of refractory and relapsed B-ALL. Thus, the development of a new therapeutic target to battle this deadly disease is imperative. Exportin-1 (XPO1), also known as chromosomal region maintenance 1(CRM1), plays an important role in the progression of tumorigenesis. However, the prognostic and therapeutic values of XPO1 in B-ALL have not been reported. Methods: The difference in XPO1 and Foxo1 expression between normal B cells and leukemia cells was using the Human Protein Atlas (HPA) dataset and RT-qPCR results. Correlations between XPO1 expression level and clinical outcome were analyzed using the RNA-seq data of 110 newly diagnosed B−ALL patients in Nanfang Hospital. Cell proliferation assays, apoptosis assays, and cell cycle analysis were performed to analyze the anti-tumor effects of selinexor (KPT-330) in vitro. Bioinformatics analysis, western blot, and RT-qPCR were performed to explore the underlying mechanism. Results ： Higher XPO1 levels or Foxo1 levels were associated with poor prognosis in newly diagnosed B-ALL patients. Using bone marrow (BM)， peripheral blood (PB) cells from B-ALL and normal people as well as B-ALL cell lines, we found that XPO1 and Foxo1 expression was significantly increased in B-ALL patients and B-ALL cell lines. And notably, there was a positive correlation between the expression of Foxo1 and XPO1, which was verified by the data in the HPA database, and the results were consistent. Foxo1 is an important transcription factor in B-ALL, mainly localized in the nucleus, and is important for leukemia cell survival. KPT-330 (Selinexor), a selective Inhibitor of XPO1, mechanistically inhibited the expression of Foxo1, a leukemia transcription factor (reduced in both the nuclear and cytoplasmic compartments), and led to a strong repression of Foxo1 target genes associated with cell cycle, DNA replication, and transcriptional regulation. This induced growth inhibition, apoptosis, and cycle arrest in B-ALL cells. Moreover, in combination with Foxo1 Inhibitor (AS1842856), the proliferation inhibition and apoptosis-inducing effects of KPT-330 on B-ALL could be significantly enhanced. Furthermore, the KPT-330 strongly triggered apoptosis in leukemic but with minimal effects on normal cells implying that drugs of this class show promise for the targeted therapy of B-ALL leukemia cells. Conclusions: Our findings demonstrate that XPO1 is a promising prognostic indicator and a valid target for antitumor treatment with selective inhibitor KPT-330. This research was supported by the National Natural Science Foundation of China(NFSC82170163, 81970147), Clinical Study of Nanfang Hospital(LC2016ZD009/2019CR012).

Viral Subversion of the Chromosome Region Maintenance 1 Export Pathway and Its Consequences for the Cell Host.

In eukaryotic cells, the spatial distribution between cytoplasm and nucleus is essential for cell homeostasis. This dynamic distribution is selectively regulated by the nuclear pore complex (NPC), which allows the passive or energy-dependent transport of proteins between these two compartments. Viruses possess many strategies to hijack nucleocytoplasmic shuttling for the benefit of their viral replication. Here, we review how viruses interfere with the karyopherin CRM1 that controls the nuclear export of protein cargoes. We analyze the fact that the viral hijacking of CRM1 provokes are-localization of numerous cellular factors in a suitable place for specific steps of viral replication. While CRM1 emerges as a critical partner for viruses, it also takes part in antiviral and inflammatory response regulation. This review also addresses how CRM1 hijacking affects it and the benefits of CRM1 inhibitors as antiviral treatments.

Discovery of Aminoratjadone Derivatives as Potent Noncovalent CRM1 Inhibitors.

Cancer cells frequently utilize elevated nuclear export to escape tumor suppression and gain proliferative advantage. Chromosome Region Maintenance 1 (CRM1/XPO1) mediates macromolecule nuclear export and plays an important role in tumorigenesis and progression. The clinical approval of its covalent inhibitor KPT-330 (Selinexor) validates the feasibility of targeting CRM1 to treat cancers. Here, we synthesized four aminoratjadone derivatives and found that two of them, KL1 and KL2, are noncovalent CRM1 inhibitors. The two compounds underwent spontaneous hydrolysis in aqueous buffers, and the resulting products were more active against CRM1. High-resolution crystal structures revealed the CRM1-binding mode of these compounds and explained the observed structure-activity relationships. In cells, KL1 and KL2 localized CRM1 in the nuclear periphery and led to depletion of nuclear CRM1, thereby inhibiting the nuclear export and growth of colorectal cancer cells at submicromolar concentrations. This work lays the foundation for further development of aminoratjadone-based noncovalent CRM1 inhibitors.

Abstract B067: CRM1 inhibitor selinexor induces DNA damage repair-related vulnerability in prostate cancer cells

Abstract Background: Advanced metastatic prostate cancer is the second leading cause of death in men in the United States. Androgen deprivation therapy has been used as a front-line therapy; however, despite initial success, it fails subsequently and results in the development of castration-resistant prostate cancer. Due to these unmet therapeutic needs, there is an urgency to identify novel targets to develop next-generation therapeutics. Chromosome region maintenance 1 (CRM1) is one of the seven members of mammalian export proteins that function to export the proteins using nuclear export signal (NES). Several tumors, including prostate cancer, overexpress CRM1 to efflux tumor suppressor proteins to the cytoplasm and exhibit dysregulated nuclear export. CRM1-mediated export can lead to functional inactivation of p53, cyclinD1, p16, p21, and p27. Essential DNA repair proteins such as Rad51 and BRCA1 exhibit leucine-containing hydrophobic residues rich in NES and are known to be shuttled by CRM1. Based on this, we hypothesize that CRM1 inhibition may affect the expression, kinetics, localization, and stability of DNA repair network proteins. Methods: Androgen receptor-positive and negative prostate cancer cells were treated with Selinexor. The dose of the drug and duration was dependent on the endpoint measurement. Cell death was monitored using MTT assay and western blot measurement of cleaved PARP. Total RNA and cell lysate were collected to measure the relative levels of BRCA-1, RAD51, p53, and other members of the DNA repair pathway (HR and NHEJ). DNA damage was estimated using the number of micronuclei and immunofluorescence staining for γ-H2AX and COMET assay. Results: Selinexor treatment in prostate cancer cells affects the functioning of the DNA repair network and can be combined with inhibitors of DNA repair proteins. Citation Format: Rajendra Kumar, Janet Mendonca, Yuhan Yang, Abhishek Shetty, Lillian Wilson, Kavya Boyapati, Deven Topiwala, Keerti Soundappan, Eleni Panagopoulos, Michael Carducci, Sushant Kachhap. CRM1 inhibitor selinexor induces DNA damage repair-related vulnerability in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B067.

The CRM1‐mediated nuclear export effectuates the role conversion of tumour suppressor gene BATF2 in colorectal cancer

The CRM1‐mediated nuclear export effectuates the role conversion of tumour suppressor gene BATF2 in colorectal cancer

Subcellular Translocation of Yorkie through the PRP4K-CRM1 Axis Regulates Antimicrobial Peptides Transcription and Defense against Bacterial Infection in Crab.

The Hippo signaling pathway plays important roles in innate immunity. In the current study, we found that bacterial infection did not influence mRNA and protein levels of yorkie (Yki), which is an important terminal molecule of the Hippo signaling pathway. However, bacterial infection promoted the translocation of Yki from the nucleus to the cytoplasm in Chinese mitten crab (Eriocheir sinensis), thus attenuating Yki-suppressed transcription of antimicrobial peptides through Cactus. Chromosome region maintenance 1 (CRM1)-silenced crab hemocytes significantly suppressed Yki translocation from the nucleus to the cytoplasm upon bacterial infection, resulting in significantly increased expression of Cactus, decreased expression of antimicrobial peptides, and higher bacterial susceptibility, which demonstrated the regulatory role of CRM1 in subcellular localization of Yki. However, RNA interference of Scalloped (Sd) exhibited no effect on the subcellular localization of Yki and its regulation of Cactus/antimicrobial peptides. Moreover, we elucidated that both CRM1 and Sd could interact with Yki and that the PRP4K-mediated phosphorylation of a conserved serine amino acid residue in the nuclear export signal of Yki is essential for interaction between Yki and CRM1; however, the phosphorylation did not affect the binding of Yki with Sd. We also found that bacterial infection significantly promoted the expression of PRP4K in hemocytes; RNA interference of PRP4K and phosphatase inhibitor suppressed Yki translocation from the nucleus to the cytoplasm, promoting Cactus expression and inhibiting antimicrobial peptide expression. Thus, subcellular localization of Yki regulates antibacterial infection through both PRP4K and CRM1 in crabs.

Nuclear export of BATF2 enhances colorectal cancer proliferation through binding to CRM1.

During the tumourigenesis and development of colorectal cancer (CRC), the inactivation of tumour suppressor genes is closely involved, although detailed molecular mechanisms remain elusive. Accumulating studies, including ours, have demonstrated that basic leucine zipper transcription factor ATF (activating transcription factor)-like 2 (BATF2) is a capable tumour suppressor that localises in the nucleus. However, its different subcellular localisation, potential functions and underlying mechanisms are unclear. The translocation of BATF2 and its clinical relevance were detected using CRC samples, cell lines and xenograft nude mice. Candidate BATF2-binding proteins were screened using co-immunoprecipitation, quantitative label-free liquid chromatography-tandem mass spectrometry proteomic analysis, Western blotting and immunofluorescence. Recombinant plasmids, point mutations and siRNAs were applied to clarify the binding sites between BATF2 and chromosome region maintenance 1 (CRM1). The present study found that BATF2 was mainly localised in the cytoplasm, rather than nucleus, of CRC cells in vitro and in vivo, while cytoplasmic BATF2 expression was inversely correlated with the prognosis of CRC patients. Furthermore, we identified the nuclear export and subsequent ubiquitin-mediated degradation of BATF2 in CRC cells. Mechanistically, a functional nuclear export sequence (any amino acid) was characterised in BATF2 protein, through which BATF2 bound to CRM1 and translocated out of nucleus, ultimately enhancing CRC growth via inducing activator protein 1 (AP-1)/cyclin D1/phosphorylated retinoblastoma protein (pRb) signalling pathway. Additionally, nuclear export of BATF2 can be retarded by the mutation of NES in BATF2 or the knockdown of CRM1, whereas CRM1 expression was negatively associated with nuclear BATF2 expression and the prognosis of CRC patients. These findings revealed the biological effects and underlying mechanisms of cytoplasmic localisation of BATF2. Furthermore, suppressing nuclear export of BATF2 via mutating its NES region or inhibiting CRM1 expression may serve as a promising therapeutic strategy against CRC.

High Throughput Fluorescence-Based In Vitro Experimental Platform for the Identification of Effective Therapies to Overcome Tumour Microenvironment-Mediated Drug Resistance in AML.

The interactions between Acute Myeloid Leukaemia (AML) leukemic stem cells and the bone marrow (BM) microenvironment play a critical role during AML progression and resistance to drug treatments. Therefore, the identification of novel therapies requires drug-screening methods using in vitro co-culture models that closely recreate the cytoprotective BM setting. We have developed a new fluorescence-based in vitro co-culture system scalable to high throughput for measuring the concomitant effect of drugs on AML cells and the cytoprotective BM microenvironment. eGFP-expressing AML cells are co-cultured in direct contact with mCherry-expressing BM stromal cells for the accurate assessment of proliferation, viability, and signaling in both cell types. This model identified several efficacious compounds that overcome BM stroma-mediated drug resistance against daunorubicin, including the chromosome region maintenance 1 (CRM1/XPO1) inhibitor KPT-330. In silico analysis of genes co-expressed with CRM1, combined with in vitro experiments using our new methodology, also indicates that the combination of KPT-330 with the AURKA pharmacological inhibitor alisertib circumvents the cytoprotection of AML cells mediated by the BM stroma. This new experimental model and analysis provide a more precise screening method for developing improved therapeutics targeting AML cells within the cytoprotective BM microenvironment.

Direct interaction between mortalin and HIF-1α at the mitochondria inhibits apoptosis by blocking recruitment of Bax.

Hypoxia-inducible factor-1, a heterodimer of alpha (HIF-1α) and beta (HIF-1β or ARNT) subunits, is a major regulator of the transcriptional response to hypoxia. However, HIF-1α, the oxygen-regulated subunit, also exerts nontranscriptional functions through interaction with proteins other than ARNT. We have previously shown that the subcellular localization and protein interactions of HIF-1α are controlled by ERK-mediated phosphorylation at Ser641/643. When HIF-1α is modified at these sites, it is nuclear, binds to ARNT, interacts with nucleophosmin 1 (NPM1) and activates transcription of hypoxia-target genes. On the contrary, unmodified HIF-1α is bound by chromosomal region maintenance 1 (CRM1), exits the nucleus and, via its association with mortalin, is targeted to the mitochondria to form an antiapoptotic complex. To further characterize the latter function, recombinant fragments of HIF-1α and mortalin were used for in vitro binding assays and immunoprecipitation experiments to map the respective binding sites and show that their interaction is direct and functional. We could also show that embelin, a natural product and known inhibitor of the mortalin-p53 interaction, also disrupts the mortalin-HIF-1α association and, furthermore, removes unmodified HIF-1α from mitochondria. Mitochondrial dissociation of HIF-1α, either by embelin or overexpression of a HIF-1α peptide harbouring the mortalin binding site, under stress conditions leads to mitochondrial localization of the pro-apoptotic protein B-cell lymphoma 2-associated X protein (Bax) and induction of apoptosis. We suggest that when ERK activity is low under hypoxia, binding of HIF-1α to mortalin inhibits mitochondrial recruitment of Bax and protects cells from apoptotic cell death.

Inhibition of influenza A virus replication by a marine derived quinolone alkaloid targeting virus nucleoprotein.

Owing to the emergence of drug resistance and high morbidity and mortality, the need for novel anti-influenza A virus (IAV) drugs with divergent targets is highly sought after. Herein, a novel quinolone alkaloid (QLA) derived from marine fungus was discovered with broad-spectrum anti-IAV activities with low toxicity. Distinct from current anti-IAV drugs, QLA may block virus replication and viral RNA (vRNA) export from the nucleus by targeting virus nucleoprotein (NP). QLA can block the binding of chromosome region maintenance 1 (CRM1) to nuclear export signal 3 (NES3) of NP to inhibit the nuclear export of NP and vRNP. QLA may also affect vRNP assembly by interfering with the binding of NP to RNA rather than NP oligomerization. Arg305 and Phe488-Gly490 may be required for the interaction between QLA and NP, and the binding pocket around these amino acids may be a promising target for anti-IAV drugs. Importantly, oral administration of QLA can protect the mice against IAV-induced death and weight loss, superior to the effects of the clinical drug oseltamivir. In summary, the marine derived compound QLA has the potential to be developed into a novel anti-IAV agent targeting virus NP protein in the future. This article is protected by copyright. All rights reserved.

Searching for Novel Noncovalent Nuclear Export Inhibitors through a Drug Repurposing Approach.

Chromosomal region maintenance protein 1 (CRM1) is a validated anticancer drug target, and its covalent inhibitor KPT-330 has been approved for marketing. However, the development of CRM1 inhibitors, especially the noncovalent ones, is still very limited. Drug repurposing is an effective strategy to develop drug leads for new targets. In this work, we virtually screened a library of marketed drugs and identified zafirlukast as a new CRM1 inhibitor. Biochemical and structural analysis revealed that zafirlukast was a noncovalent CRM1 inhibitor that bound to four subpockets in the nuclear-export-signal (NES) groove. Methylation of the sulfonamide group rendered zafirlukast completely inactive against CRM1. Zafirlukast inhibited the growth of a variety of cancer cells and worked synergistically with the drug doxorubicin. Taken together, these works laid a solid foundation for reshaping zafirlukast as a valuable lead compound for further design of noncovalent, specific, and potent CRM1 inhibitors toward the treatment of various cancers.