Human MX-1 Research Articles

Induction and antiviral activity of ferret myxovirus resistance (Mx) protein 1 against influenza A viruses

Myxovirus resistance (Mx) proteins are products of interferon stimulated genes (ISGs) and Mx proteins of different species have been reported to mediate antiviral activity against a number of viruses, including influenza A viruses (IAV). Ferrets are widely considered to represent the ‘gold standard’ small animal model for studying pathogenesis and immunity to human IAV infections, however little is known regarding the antiviral activity of ferret Mx proteins. Herein, we report induction of ferret (f)Mx1/2 in a ferret lung cell line and in airway tissues from IAV-infected ferrets, noting that fMx1 was induced to higher levels that fMx2 both in vitro and in vivo. Overexpression confirmed cytoplasmic expression of fMx1 as well as its ability to inhibit infection and replication of IAV, noting that this antiviral effect of fMx1was modest when compared to cells overexpressing either human MxA or mouse Mx1. Together, these studies provide the first insights regarding the role of fMx1 in cell innate antiviral immunity to influenza viruses. Understanding similarities and differences in the antiviral activities of human and ferret ISGs provides critical context for evaluating results when studying human IAV infections in the ferret model.

The Antiviral Activity of Equine Mx1 against Thogoto Virus Is Determined by the Molecular Structure of Its Viral Specificity Region.

Mammalian myxovirus resistance (Mx) proteins are interferon-induced, large dynamin-like GTPases with a broad antiviral spectrum. Here, we analyzed the antiviral activity of selected mammalian Mx1 proteins against Thogoto virus (THOV). Of those, equine Mx1 (eqMx1) showed antiviral activity comparable to that of the human MX1 gene product, designated huMxA, whereas most Mx1 proteins were antivirally inactive. We previously demonstrated that the flexible loop L4 protruding from the stalk domain of huMxA, and especially the phenylalanine at position 561 (F561), determines its antiviral specificity against THOV (P. S. Mitchell, C. Patzina, M. Emerman, O. Haller, et al., Cell Host Microbe 12:598-604, 2012, https://doi.org/10.1016/j.chom.2012.09.005). However, despite the similar antiviral activity against THOV, the loop L4 sequence of eqMx1 substantially differs from the one of huMxA. Mutational analysis of eqMx1 L4 identified a tryptophan (W562) and the adjacent glycine (G563) as critical antiviral determinants against THOV, whereas the neighboring residues could be exchanged for nonpolar alanines without affecting the antiviral activity. Further mutational analyses revealed that a single bulky residue at position 562 and the adjacent tiny residue G563 were sufficient for antiviral activity. Moreover, this minimal set of L4 amino acids transferred anti-THOV activity to the otherwise inactive bovine Mx1 (boMx1) protein. Taken together, our data suggest a fairly simple architecture of the antiviral loop L4 that could serve as a mutational hot spot in an evolutionary arms race between Mx-escaping viral variants and their hosts. IMPORTANCE Most mammals encode two paralogs of the interferon-induced Mx proteins: Mx1, with antiviral activity largely against RNA viruses, like orthomyxoviruses and bunyaviruses; and Mx2, which is antivirally active against HIV-1 and herpesviruses. The human Mx1 protein, also called huMxA, is the best-characterized example of mammalian Mx1 proteins and was recently shown to prevent zoonotic virus transmissions. To evaluate the antiviral activity of other mammalian Mx1 proteins, we used Thogoto virus, a tick-transmitted orthomyxovirus, which is efficiently blocked by huMxA. Interestingly, we detected antiviral activity only with equine Mx1 (eqMx1) but not with other nonprimate Mx1 proteins. Detailed functional analysis of eqMx1 identified amino acid residues in the unstructured loop L4 of the stalk domain critical for antiviral activity. The structural insights of the present study explain the unique position of eqMx1 antiviral activity within the collection of nonhuman mammalian Mx1 proteins.

An evolutionarily conserved N-terminal leucine is essential for MX1 GTPase antiviral activity against different families of RNA viruses

Myxovirus resistance protein 1 (MX1) and MX2 are homologous, dynamin-like large GTPases, induced upon interferon exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus, by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with a GTPase domain and a carboxy-terminal stalk domain, connected by a bundle signaling element. These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging, and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against influenza A virus, Vesicular Stomatitis Virus, and La Crosse virus. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse, and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities but does modify MX1 protein subcellular localization. The discovery of this essential and highly conserved residue defines this region as key for antiviral activity and may reveal insights as to the mechanism(s) of action of MX1 proteins.

Metastable biomolecular condensates of interferon-inducible antiviral Mx-family GTPases: A paradigm shift in the last three years

Membraneless organelles (MLOs) in the cytoplasm and nucleus in the form of phase-separated biomolecular condensates are increasingly viewed as critical in regulating diverse cellular functions. We summarize a paradigm shift over the last 3 years in the field of interferon (IFN)-inducible antiviral Mx-family GTPases. Expression of the ‘myxovirus resistance proteins’ MxA in human cells and its ortholog Mx1 in murine cells is increased 50- to 100-fold by Type I (IFN-α and -β) and III IFNs (IFN-λ). Human MxA forms cytoplasmic structures, while murine Mx1 forms nuclear bodies. Since 2002, it has been widely thought that human (Hu) MxA is associated with the membraneous smooth endoplasmic reticulum (ER). In a paradigm shift, our recent data showed that HuMxA formed membraneless phase-separated biomolecular condensates in the cytoplasm. Some of the HuMxA condensates adhered to intermediate filaments generating a reticular pattern. Murine (Mu) Mx1, which was predominantly nuclear, was also confirmed to be in phase-separated nuclear biomolecular condensates. A subset of Huh7 cells showed association of GFP-MuMx1 with intermediate filaments in the cytoplasm. While cells with cytoplasmic GFP-HuMxA condensates and cytoplasmic GFP-MuMx1 filaments showed an antiviral phenotype towards vesicular stomatitis virus (VSV), those with only nuclear GFP-MuMx1 bodies did not. The new data bring forward the paradigm that both human MxA and murine Mx1 give rise to phase-separated biomolecular condensates, albeit in different subcellular compartments, and that differences in the subcellular localization of condensates of different Mx proteins determines the spectrum of their antiviral activity.

From the Editor’s Desk…

From the Editor’s Desk…

Interferon-inducible MX2 is a host restriction factor of hepatitis B virus replication

Non-cytolytic cure of HBV-infected hepatocytes by cytokines, including type I interferons (IFNs), is of importance for resolving acute and chronic infection. However, as IFNs stimulate hundreds of genes, those most relevant for HBV suppression remain largely unknown. Amongst them are the large myxovirus resistance (Mx) GTPases. Human MX1 (or MxA) is active against many RNA viruses, while MX2 (or MxB) was recently found to restrict HIV-1, HCV, and herpesviruses. Herein, we investigated the anti-HBV activity of MX2. The potential anti-HBV activity of MX2 and functional variants were assessed in transfected and HBV-infected hepatoma cells and primary human hepatocytes, employing multiple assays to analyze the synthesis and decay of HBV nucleic acids. The specific roles of MX2 in IFN-α-driven inhibition of HBV transcription and replication were assessed by MX2-specific shRNA interference (RNAi). Both MX2 alone and IFN-α substantially inhibited HBV replication, due to significant deceleration of the synthesis and slight acceleration of the turnover of viral RNA. RNAi knockdown of MX2 significantly reduced the inhibitory effects of IFN-α. Strikingly, MX2 inhibited HBV infection by reducing covalently closed circular DNA (cccDNA), most likely by indirectly impairing the conversion of relaxed circular DNA to cccDNA rather than by destabilizing existing cccDNA. Various mutations affecting the GTPase activity and oligomerization status reduced MX2's anti-HBV activity. MX2 is an important IFN-α inducible effector that decreases HBV RNA levels but can also potently inhibit HBV infection by indirectly impairing cccDNA formation. MX2 likely has the potential for therapeutic applications aimed at curing HBV infection by eliminating cccDNA. This study shows that the protein MX2, which is induced by interferon-α, has important anti-hepatitis B virus (HBV) effector functions. MX2 can reduce the amount of covalently closed circular DNA, which is the form of DNA that HBV uses to maintain viral persistence within hepatocytes. MX2 also reduces HBV RNA levels by downregulating synthesis of viral RNA. MX2 likely represents a novel intrinsic HBV inhibitor that could have therapeutic potential, as well as being useful for improving our understanding of the complex biology of HBV and the antiviral mechanisms of interferon-α.

Investigation of biaryl heterocycles as inhibitors of Wee1 kinase

In continuation of our previous research towards the discovery of potent, selective and drug-like Wee1 inhibitors, 2 novel series of biaryl heterocycles were designed, synthesized and evaluated. The new biaryl cores were designed to enable structure−activity exploration of substituents at C-8 or N-8 which were used for tuning compound properties and to improve compound profiles. The lead molecule 33 demonstrated a desirable pharmacokinetic profile and potentiated the anti-proliferative activity of irinotecan in vivo when dosed orally in the human breast MX-1 xenograft model.

In vivo evasion of MxA by avian influenza viruses requires human signature in the viral nucleoprotein.

Zoonotic transmission of influenza A viruses can give rise to devastating pandemics, but currently it is impossible to predict the pandemic potential of circulating avian influenza viruses. Here, we describe a new mouse model suitable for such risk assessment, based on the observation that the innate restriction factor MxA represents an effective species barrier that must be overcome by zoonotic viruses. Our mouse lacks functional endogenous Mx genes but instead carries the human MX1 locus as a transgene. Such transgenic mice were largely resistant to highly pathogenic avian H5 and H7 influenza A viruses, but were almost as susceptible to infection with influenza viruses of human origin as nontransgenic littermates. Influenza A viruses that successfully established stable lineages in humans have acquired adaptive mutations which allow partial MxA escape. Accordingly, an engineered avian H7N7 influenza virus carrying a nucleoprotein with signature mutations typically found in human virus isolates was more virulent in transgenic mice than parental virus, demonstrating that a few amino acid changes in the viral target protein can mediate escape from MxA restriction in vivo. Similar mutations probably need to be acquired by emerging influenza A viruses before they can spread in the human population.

174: Transgenic mice expressing interferon-regulated human influenza virus restriction factor MxA

In mice, influenza virus resistance is strongly dependent on the interferon (IFN)-regulated Mx1 gene which encodes a potent restriction factor that inhibits a poorly defined early step of the viral life cycle. Humans possess two IFN-regulated Mx genes with antiviral activity. The human Mx1 gene (encoding MxA protein) confers resistance to a broad range of RNA viruses in cell culture, including influenza A viruses, whereas the human Mx2 gene (encoding MxB protein) has been shown to inhibit HIV-1. To determine whether human MxA plays a decisive role in defending the intact organism against influenza A viruses, we developed a mouse strain that lacks functional endogenous Mx genes but carries the complete human Mx locus as a transgene. MxA-transgenic mice exhibited solid resistance to infection with highly pathogenic H5N1 and H7N7 avian influenza viruses, as well as influenza-like Thogoto virus. However, transgenic mice differed only slightly from non-transgenic littermates with regard to resistance to H1N1 and H3N2 influenza viruses of human origin, suggesting that seasonal human influenza viruses have acquired adaptive mutations which permit MxA evasion. To identify adaptive mutations which confer Mx resistance, we passaged mouse-adapted H7N7 avian influenza virus SC35M in MxA-transgenic mice. Here we will describe an escape variant of SC35M resulting from this screen that induces fatal disease in MxA-transgenic mice and exhibits enhanced virulence for mice with intact endogenous Mx1 genes.

Transfer of the amino-terminal nuclear envelope targeting domain of human MX2 converts MX1 into an HIV-1 resistance factor.

The myxovirus resistance 2 (MX2) protein of humans has been identified recently as an interferon (IFN)-inducible inhibitor of human immunodeficiency virus type 1 (HIV-1) that acts at a late postentry step of infection to prevent the nuclear accumulation of viral cDNA (C. Goujon et al., Nature 502:559-562, 2013, http://dx.doi.org/10.1038/nature12542; M. Kane et al., Nature 502:563-566, 2013, http://dx.doi.org/10.1038/nature12653; Z. Liu et al., Cell Host Microbe 14:398-410, 2013, http://dx.doi.org/10.1016/j.chom.2013.08.015). In contrast, the closely related human MX1 protein, which suppresses infection by a range of RNA and DNA viruses (such as influenza A virus [FluAV]), is ineffective against HIV-1. Using a panel of engineered chimeric MX1/2 proteins, we demonstrate that the amino-terminal 91-amino-acid domain of MX2 confers full anti-HIV-1 function when transferred to the amino terminus of MX1, and that this fusion protein retains full anti-FluAV activity. Confocal microscopy experiments further show that this MX1/2 fusion, similar to MX2 but not MX1, can localize to the nuclear envelope (NE), linking HIV-1 inhibition with MX accumulation at the NE. MX proteins are dynamin-like GTPases, and while MX1 antiviral function requires GTPase activity, neither MX2 nor MX1/2 chimeras require this attribute to inhibit HIV-1. This key discrepancy between the characteristics of MX1- and MX2-mediated viral resistance, together with previous observations showing that the L4 loop of the stalk domain of MX1 is a critical determinant of viral substrate specificity, presumably reflect fundamental differences in the mechanisms of antiviral suppression. Accordingly, we propose that further comparative studies of MX proteins will help illuminate the molecular basis and subcellular localization requirements for implementing the noted diversity of virus inhibition by MX proteins. Interferon (IFN) elicits an antiviral state in cells through the induction of hundreds of IFN-stimulated genes (ISGs). The human MX2 protein has been identified as a key effector in the suppression of HIV-1 infection by IFN. Here, we describe a molecular genetic approach, using a collection of chimeric MX proteins, to identify protein domains of MX2 that specify HIV-1 inhibition. The amino-terminal 91-amino-acid domain of human MX2 confers HIV-1 suppressor capabilities upon human and mouse MX proteins and also promotes protein accumulation at the nuclear envelope. Therefore, these studies correlate the cellular location of MX proteins with anti-HIV-1 function and help establish a framework for future mechanistic analyses of MX-mediated virus control.

Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection

Animal cells harbour multiple innate effector mechanisms that inhibit virus replication. For the pathogenic retrovirus human immunodeficiency virus type-1 (HIV-1), these include widely expressed restriction factors1 such as APOBEC3 proteins2, TRIM5α3, tetherin/BST24,5 and SAMHD16,7, as well as additional factors that are stimulated by type-1 interferon (IFN)8,9,10,11,12,13,14. Here, we employ both ectopic expression and gene silencing experiments to define the human dynamin-like, IFN-induced guanosine triphosphatase (GTPase), myxovirus resistance 2 (MX2 or MxB) protein, as a potent inhibitor of HIV-1 infection and as a major effector of IFNα-mediated resistance to HIV-1 infection. MX2 suppresses infection by all HIV-1 strains tested, has similar to modest effects on divergent simian immunodeficiency viruses (SIVs), and does not inhibit other retroviruses such as murine leukaemia virus (MLV). The capsid (CA) region of the viral Gag protein dictates susceptibility to MX2, and the block to infection occurs at a late post-entry step with the nuclear accumulation and chromosomal integration of nascent viral cDNA both being suppressed. Finally, human MX1 (or MxA), a closely related protein that has long been recognised as a broadly acting inhibitor of RNA/DNA viruses, including the orthomyxovirus influenza A virus15,16, does not affect HIV-1,whereas MX2 is ineffective against influenza virus. MX2 is therefore a cell-autonomous, anti-HIV-1 resistance factor whose purposeful mobilisation may represent a new therapeutic approach for the treatment of HIV/AIDS.

Abstract 4502: Eribulin alters vascular function in human triple-negative (TN) breast MX-1 and MDA-MB-231 tumor xenograft models as measured by DCE-MRI.

Abstract Objective: Eribulin mesylate (ERI) is a simplified synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. Literature reports have increasingly shown tubulin binding agents have vascular disruptive activity. In this study, we evaluated the effect of eribulin on tumor vasculature using DCE-MRI of TN human breast MX-1 xenografts in nude rats. Methods: The effects of ERI on human TN breast MX-1 and MDA-MB-231 tumor are examined in nude rats and nude mice. ERI was administered at MTD dose (0.3 mg/kg at Q4D4 in nude rats and 3.0 mg/kg at D1 in nude mice). In nude rats, DCE-MRI was conducted on a Bruker 4.7 T scanner with Magnevist contrast. Imaging was conducted on day -1, 6 hrs, day 2, day 5-6 and day 10. In nude mice, morphology of tumor vasculature was analyzed with immunohistochemistry (IHC) staining with anti-mouse endothelial marker CD31 antibody. Microvessel density (MVD) and vessel perimeter were analyzed using Aperio Image Scope. Results and discussion: ERI inhibited tumor growth of MX-1 (day 5; 290 +/- 169 vs 1195 +/- 36 mm3, N=5, p<0.01) and MDA-MB-231 (day 6; 295 +/- 113 vs 570 +/- 56 mm3, N=5, p<0.05) in nude rats compared to vehicle (VEH). DCE-MRI analysis demonstrated that, at 6 hrs, Ktrans (1/s) in the tumor rim was decreased in the ERI group (MX-1; 0.471 +/- 0.061 vs 0.611 +/- 0.082, p<0.05 vs VEH and MDA-MB-231; 0.290 +/- 0.130 vs 0.537 +/- 0.132, p<0.05 vs VEH). Interestingly, Ktrans in the tumor core was increased in the ERI group at later time points (MX-1 day 5; 0.723 +/- 0.155 vs 0.097 +/- 0.073, p<0.05 vs VEH and MDA-MB-231 day 10; 0.612 +/- 0.187 vs 0.103 +/- 0.090, p<0.01 vs VEH). The effects of ERI on tumor vasculature function were reproduced in a mouse MX-1 xenograft model. The trend towards a decrease in Ktrans at 6hrs (confirmed by Hoechst dye perfusion of mouse tumor samples taken at 6 hrs showing a 70% reduction compared to VEH. N=5, p<0.05) suggests ERI may cause an acute shutdown of vascular function. In endothelial cells, lack of significant change by IHC at 6hrs and no increased apoptosis at 24hrs in vitro indicate the acute activity may be different from vascular disrupting agents. IHC analysis of tumor vasculature morphology was performed to investigate the increase in the tumor core Ktrans at day 5. Results demonstrated that ERI treated tumors had higher MVD compared to VEH along with decreased vessel perimeter in mice, indicating ERI affected tumor vascular morphology, suggestive of vascular normalization. Conclusions: ERI altered tumor vasculature acutely in the tumor rim and increase vascular perfusion in the tumor core 5 days after treatment. In addition to regulation of mitosis, ERI may have induced normalization of tumor vasculature in preclinical TN breast cancer cell models. Further analysis of the mechanism of ERI on vascular normalization will be warranted. Citation Format: Paul J. McCracken, Ken Ito, Mamoru Yanagimachi, Xavier Tizon, Peggy Provent, Shanqin Xu, Namita Kumar, Denice Welsh, Tyler J. Teceno, Galina Kuznetsov, Yasuhiro Funahashi. Eribulin alters vascular function in human triple-negative (TN) breast MX-1 and MDA-MB-231 tumor xenograft models as measured by DCE-MRI. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4502. doi:10.1158/1538-7445.AM2013-4502

Abstract 1413: Eribulin caused re-modeling of tumor vasculature altering gene expression profiling in angiogenesis and Epithelial Mesenchymal Transition (EMT) signaling pathway of host cells within human breast cancer cell (BCC) xenografts in nude mice.

Abstract Objective: Eribulin mesylate (ERI) is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. The objective of this study was to examine the effect of ERI on tumor vasculature with immunohistchemical (IHC) analysis and gene expression profiling (GEP) in normal host cells, such as endothelial cells and vascular mural cells within tumor microenvironments in human BCC xenograft models Methods: Anti-tumor activity of ERI was examined at doses of 1.5 and 3.0 mg/kg, i.v. at day 1, in human BCC MX-1, MDA-MB-231 and MDA-MB-453 sc xenografts in nude mice. For IHC and GEP analysis, tumor tissues were collected at day 4 and day 8. IHC analysis was performed using mouse CD31 antibody to stain endothelial cells. Microvessel density (MVD) and vessel perimeter were determined by using Aperio Image Scope. GEP analysis for mouse host and human tumor cells within tumor tissues was done by using mouse and human TaqMan Low Density Arrays (TLDAs) consisting of a set of 92 genes related to angiogenesis, metastasis/EMT and cell differentiation signal pathways. Results shows % of non-treatment group (NT). Results: ERI showed significant anti-tumor activity against all three human BCC xenografts in a dose dependent manner. IHC analysis showed that ERI altered morphology of tumor vasculature day 8 after treatments and increased number of vessels with small size of perimeter (<300um), but decreased large size of vessels (>300um) in both MX-1 and MDA-MB-231 xenograft models (p<0.05 vs NT). ERI altered morphology of tumor vascular, which resembled normalized vasculature in two of triple negative (TN) breast cancer xenograft models. Next, GEP analysis revealed that in three human BCCs xenograft models, expression of endothelial (CD31, CD105) and pericyte markers (αSMA, NG2) were decreased in host cells 4 days after treatment of ERI (1.5mg/kg). ERI also decreased the expression of angiogenesis regulating genes (VEGF; 22.6%, Dll4; 30.4%, Notch4; 42.6%, Tie2; 63.4%) and genes in the EMT/metastasis pathway (TGFB1, ZEB1 and TWIST) in two of TN BCC (65.1%, 65.0%, 56.5% in MX-1 and 41.6%, 55.8%, 46.1% in MDA-MB-231, respectively) models compared to NT (p<0.05), suggesting anti-EMT activity in host tissues within tumors. Conclusions: ERI induced re-modeling of tumor vasculature in human BCC xenograft models. GEP related to angiogenesis and EMT/metastasis pathway was significantly affected with ERI treatment in host cells under tumor microenvironments. ERI might cause remodeling of tumor vasculature by regulating GEP in host cells. Further investigation may be warranted to examine if the activity of ERI against host cells in tumor tissues contributed to anti-tumor activity of ERI. Citation Format: Junji Matsui, Osamu Toyama, Mitsuhiro Ino, Taro Semba, Mai Uesugi, Hiroki Muto, Judith L. Oestreicher, Kentaro Takahashi, Kentaro Matsuura, Yoshiaki Sato, Taisuke Uehara, Takayuki Kimura, Hideki Watanabe, Yoichi Ozawa, Makoto Asano, Yusuke Adachi, Ken Aoshima, Yasuhiro Funahashi. Eribulin caused re-modeling of tumor vasculature altering gene expression profiling in angiogenesis and Epithelial Mesenchymal Transition (EMT) signaling pathway of host cells within human breast cancer cell (BCC) xenografts in nude mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2013-1413

Antagonists of growth hormone-releasing hormone suppress in vivo tumor growth and gene expression in triple negative breast cancers

This study evaluated the effects of a modern antagonistic analog of GHRH on tumor growth and on expression of inflammatory cytokine genes in two models of human triple negative breast cancers (TNBC). The TNBC subtype is refractory to the treatment options available for other hormone-independent breast cancers. Inflammatory cytokines play a major role in the cellular signaling associated with breast cancer pathogenesis and enhance epithelial-mesenchymal transitions (EMT), drug resistance, and metastatic potential. Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide which regulates the synthesis and release of growth hormone by the pituitary and is an autocrine/paracrine growth factor for multiple human cancers. The effects of analogs of GHRH on tumoral cytokine expression have not been previously investigated. Animals bearing xenografts of the human TNBC cell lines, HCC1806 and MX-1, were treated with MIA-602, an antagonistic analog of GHRH. Treatment with MIA-602 significantly reduced tumor growth. We quantified transcript levels of the genes for several inflammatory cytokines. Expression of INFγ, IL-1α, IL-4, IL-6, IL-8, IL-10, and TNFα, was significantly reduced by treatment with MIA-602. We conclude that treatment of TNBC with GHRH antagonists reduces tumor growth through an action mediated by tumoral GHRH receptors and produces a suppression of inflammatory cytokine signaling. Silencing of GHRH receptors in vitro with siRNA inhibited the expression of GHRH-R genes and inflammatory cytokine genes in HCC1806 and MX-1 cells. Further studies on GHRH antagonists may facilitate the development of new strategies for the treatment of resistant cancers.

Interferon-induced Antiviral Protein MxA Interacts with the Cellular RNA Helicases UAP56 and URH49

Mx proteins are a family of large GTPases that are induced exclusively by interferon-α/β and have a broad antiviral activity against several viruses, including influenza A virus (IAV). Although the antiviral activities of mouse Mx1 and human MxA have been studied extensively, the molecular mechanism of action remains largely unsolved. Because no direct interaction between Mx proteins and IAV proteins or RNA had been demonstrated so far, we addressed the question of whether Mx protein would interact with cellular proteins required for efficient replication of IAV. Immunoprecipitation of MxA revealed its association with two closely related RNA helicases, UAP56 and URH49. UAP56 and its paralog URH49 play an important role in IAV replication and are involved in nuclear export of IAV mRNAs and prevention of dsRNA accumulation in infected cells. In vitro binding assays with purified recombinant proteins revealed that MxA formed a direct complex with the RNA helicases. In addition, recombinant mouse Mx1 was also able to bind to UAP56 or URH49. Furthermore, the complex formation between cytoplasmic MxA and UAP56 or URH49 occurred in the perinuclear region, whereas nuclear Mx1 interacted with UAP56 or URH49 in distinct dots in the nucleus. Taken together, our data reveal that Mx proteins exerting antiviral activity can directly bind to the two cellular DExD/H box RNA helicases UAP56 and URH49. Moreover, the observed subcellular localization of the Mx-RNA helicase complexes coincides with the subcellular localization, where human MxA and mouse Mx1 proteins act antivirally. On the basis of these data, we propose that Mx proteins exert their antiviral activity against IAV by interfering with the function of the RNA helicases UAP56 and URH49.

The Viral Nucleoprotein Determines Mx Sensitivity of Influenza A Viruses

Host restriction factors play a crucial role in preventing trans-species transmission of viral pathogens. In mammals, the interferon-induced Mx GTPases are powerful antiviral proteins restricting orthomyxoviruses. Hence, the human MxA GTPase may function as an efficient barrier against zoonotic introduction of influenza A viruses into the human population. Successful viruses are likely to acquire adaptive mutations allowing them to evade MxA restriction. We compared the 2009 pandemic influenza A virus [strain A/Hamburg/4/09 (pH1N1)] with a highly pathogenic avian H5N1 isolate [strain A/Thailand/1(KAN-1)/04] for their relative sensitivities to human MxA and murine Mx1. The H5N1 virus was highly sensitive to both Mx GTPases, whereas the pandemic H1N1 virus was almost insensitive. Substitutions of the viral polymerase subunits or the nucleoprotein (NP) in a polymerase reconstitution assay demonstrated that NP was the main determinant of Mx sensitivity. The NP of H5N1 conferred Mx sensitivity to the pandemic H1N1 polymerase, whereas the NP of pandemic H1N1 rendered the H5N1 polymerase insensitive. Reassortant viruses which expressed the NP of H5N1 in a pH1N1 genetic background and vice versa were generated. Congenic Mx1-positive mice survived intranasal infection with these reassortants if the challenge virus contained the avian NP. In contrast, they succumbed to infection if the NP of pH1N1 origin was present. These findings clearly indicate that the origin of NP determines Mx sensitivity and that human influenza viruses acquired adaptive mutations to evade MxA restriction. This also explains our previous observations that human and avian influenza A viruses differ in their sensitivities to Mx.

Synthesis and Biological Activities of Topoisomerase I Inhibitors, 6-Arylmethylamino Analogues of Edotecarin

The replacement of 1,3-dihydroxy-2-propylamino moiety at the N6-position of edotecarin (1) by arylmethylamino groups yielded a number of more potent topoisomerase I inhibitors with better cytotoxic (CTX) activities in vitro than edotecarin. Among them, the three most potent pyridylmethyl analogues, compounds 22g, 22m, and 23c, showed better antitumor activities against MKN-45 human stomach cancer or MX-1 human breast cancer xenografted mice than those of edotecarin. Furthermore, compounds 22m and 23c exhibited complete response against MX-1 cells implanted in mice.

Discovery of N-(4-Methoxyphenyl)-N,2-dimethylquinazolin-4-amine, a Potent Apoptosis Inducer and Efficacious Anticancer Agent with High Blood Brain Barrier Penetration

As a continuation of our structure-activity relationship (SAR) studies on 4-anilinoquinazolines as potent apoptosis inducers and to identify anticancer development candidates, we explored the replacement of the 2-Cl group in our lead compound 2-chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine (6b, EP128265, MPI-0441138) by other functional groups. This SAR study and lead optimization resulted in the identification of N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine (6h, EP128495, MPC-6827) as an anticancer clinical candidate. Compound 6h was found to be a potent apoptosis inducer with EC(50) of 2 nM in our cell-based apoptosis induction assay. It also has excellent blood brain barrier penetration, and is highly efficacious in human MX-1 breast and other mouse xenograft cancer models.

Carbonate and carbamate derivatives of 4-demethylpenclomedine as novel anticancer agents.

PurposeThe purpose of this investigation was to synthesize a series of carbonate and carbamate derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma non-toxic metabolite of penclomedine (PEN) seen in patients. DM-PEN has been observed to be an active antitumor agent in mouse human xenograft tumor models and non-neurotoxic in a rat model, however, activity in intracranially implanted human glioma xenograft models have not been reported. The major goal was to identify derivatives that are active in brain tumors.MethodsDerivatives were prepared from DM-PEN and evaluated in vivo against human U251 glioblastoma, D54 glioblastoma and MX-1 breast tumor xenografts and mammary tumor 16/C that were implanted in the mammary fat pad or intracranially (IC).ResultsCarbonate and carbamate derivatives were found to be superior to DM-PEN against IC growing human glioblastoma xenografts.ConclusionThe activity of the carbonates and carbamates against human tumor xenografts in vivo suggests consideration of these two series of derivatives of DM-PEN for clinical development.

Nested clade analysis: an extensively validated method for strong phylogeographic inference

Petit (2007) speculates that three papers (Knowles, Maddison, 2002; Panchal, Beaumont, 2007; Petit, Grivet, 2002) have delivered the coup de grâce for nested clade phylogeographic analysis (NCPA). As Petit (2007) notes, NCPA is one of the most frequently used analytical tools in the area of phylogeography, so this speculation must be evaluated carefully.