Emiko Urano

Ligand‐independent higher‐order multimerization of CXCR4, a G‐protein‐coupled chemokine receptor involved in targeted metastasis

CXCR4, a G‐protein‐coupled receptor of CXCL12/stromal cell‐derived factor‐1α, mediates a wide range of physiological and pathological processes, including the targeted metastasis of cancer cells. CXCR4 has been shown to homo‐oligomerize in several experimental systems. However, it remains unclear with which domains CXCR4 interacts homotypically, and whether it dimerizes or forms a higher‐order complex. To address these issues, we used bioluminescent resonance energy transfer and bimolecular fluorescence complementation analyses to measure the homotypic interactions of CXCR4 in living cells. Both assays indicated that CXCR4 interacts homotypically, which is consistent with previous studies. By studying CXCR4 mutants lacking various domains, we found that multiple transmembrane domains probably serve as potential molecular interaction surfaces for oligomerization. The relative contribution of the amino‐ or carboxy‐termini to oligomerization was small. To differentiate between a dimer and a multimer consisting of more than two molecules, bioluminescent resonance energy transfer–bimolecular fluorescence complementation analysis was conducted. It revealed that CXCR4 engages in higher‐order oligomerization in a ligand‐independent fashion. This is the first report providing direct experimental evidence for the higher‐order multimerization of CXCR4 in vivo. We hypothesize that CXCR4 distributes to the cell surface as a multimer, in order to effectively sense, with increased avidity, the chemotaxis‐inducing ligand in the microenvironment. Studying the structure and function of the oligomeric state of CXCR4 may lead us to develop novel CXCR4 inhibitors that disassemble the molecular cluster of CXCR4. (Cancer Sci 2009; 100: 95–102)

Cell-Permeable Stapled Peptides Based on HIV-1 Integrase Inhibitors Derived from HIV-1 Gene Products

HIV-1 integrase (IN) is an enzyme which is indispensable for the stable infection of host cells because it catalyzes the insertion of viral DNA into the genome and thus is an attractive target for the development of anti-HIV agents. Earlier, we found Vpr-derived peptides with inhibitory activity against HIV-1 IN. These Vpr-derived peptides are originally located in an α-helical region of the parent Vpr protein. Addition of an octa-arginyl group to the inhibitory peptides caused significant inhibition against HIV replication associated with an increase in cell permeability but also relatively high cytotoxicity. In the current study, stapled peptides, a new class of stabilized α-helical peptidomimetics were adopted to enhance the cell permeability of the above lead peptides. A series of stapled peptides, which have a hydrocarbon link formed by a ruthenium-catalyzed ring-closing metathesis reaction between successive turns of α-helix, were designed, synthesized, and evaluated for biological activity. In cell-based assays some of the stapled peptides showed potent anti-HIV activity comparable with that of the original octa-arginine-containing peptide (2) but with lower cytotoxicity. Fluorescent imaging experiments revealed that these stapled peptides are significantly cell permeable, and CD analysis showed they form α-helical structures, whereas the unstapled congeners form β-sheet structures. The application of this stapling strategy to Vpr-derived IN inhibitory peptides led to a remarkable increase in their potency in cells and a significant reduction of their cytotoxicity.

Cytokine signatures of transformed B cells with distinct Epstein―Barr virus latencies as a potential diagnostic tool for B cell lymphoma

Immunocompromised individuals, including those infected with human immunodeficiency virus (HIV), are at increased risk of Epstein–Barr virus (EBV)‐associated aggressive B cell malignancies such as Burkitt’s lymphoma (BL) or diffuse large B cell lymphoma (DLBCL). Differential diagnosis of these lymphomas requires histopathological, immunohistochemical and cytogenetic assessments. Rapid, less invasive approaches to the diagnosis of EBV‐associated B cell lymphomas are needed. Here, high‐throughput cytokine profiling of BL cell lines and EBV‐transformed B lymphoblastoid cell lines (B‐LCL), representing DLBCL, was carried out. By monitoring the production of 42 different cytokines, unique cytokine signatures were identified for BL and B‐LCL/DLBCL. The BL cells produced interleukin (IL)‐10, 10 kDa interferon gamma‐induced protein (IP‐10)/CXCL10, macrophage‐derived chemokine (MDC)/CCL22, macrophage inflammatory protein (MIP)‐1α/CCL3 and MIP‐1β/CCL4. In addition to these five cytokines, the cytokine signature of B‐LCL/DLBCL cells included IL‐8/CXCL8, IL‐13, platelet‐derived growth factor (PDGF)‐AA, and regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5. Epstein–Barr virus latency was responsible for the increased production of IL‐10, MDC/CCL22 and MIP‐1α/CCL3 in BL cells, suggesting that EBV‐mediated BL‐genesis involves these three cytokines. These results suggest that high‐throughput cytokine profiling might be a valuable tool for the differential diagnosis and might deepen our understanding of the pathogenesis of EBV‐associated B cell malignancies. (Cancer Sci 2011; 102: 1236–1241)

Separate elements are required for ligand-dependent and -independent internalization of metastatic potentiator CXCR4

The C‐terminal cytoplasmic domain of the metastatic potentiator CXCR4 regulates its function and spatiotemporal expression. However, little is known about the mechanism underlying constitutive internalization of CXCR4 compared to internalization mediated by its ligand, stromal cell‐derived factor‐1 alpha (SDF‐1α)/CXCL12. We established a system to analyze the role of the CXCR4 cytoplasmic tail in steady‐state internalization using the NP2 cell line, which lacks endogenous CXCR4 and SDF‐1α. Deleting more than six amino acids from the C‐terminus dramatically reduced constitutive internalization of CXCR4. Alanine substitution mutations revealed that three of those amino acids Ser344 Glu345 Ser346 are essential for efficient steady‐state internalization of CXCR4. Mutating Glu345 to Asp did not disrupt internalization, suggesting that the steady‐state internalization motif is S(E/D)S. When responses to SDF‐1α were tested, cells expressing CXCR4 mutants lacking the C‐terminal 10, 14, 22, 31 or 44 amino acids did not show downregulation of cell surface CXCR4 or the cell migration induced by SDF‐1α. Interestingly, however, we identified two mutants, one with E344A mutation and the other lacking the C‐terminal 17 amino acids, that were defective in constitutive internalization but competent in ligand‐promoted internalization and cell migration. These data demonstrate that ligand‐dependent and ‐independent internalization is genetically separable and that, between amino acids 336 and 342, there is a negative regulatory element for ligand‐promoted internalization. Potential involvement of this novel motif in cancer metastasis and other CXCR4‐associated disorders such as warts, hypogammaglobulinemia, infections and myelokathexis (WHIM) syndrome is discussed. (Cancer Sci 2007; 98: 373–379)

Derivatives of 5-nitro-furan-2-carboxylic acid carbamoylmethyl ester inhibit RNase H activity associated with HIV-1 reverse transcriptase.

The RNase H activity associated with human immunodeficiency virus type 1 (HIV-1) is an attractive target for an antiretroviral drug development. We screened 20000 small-molecular-weight compounds for RNase H inhibitors and identified a novel RNase H-inhibiting structure characterized by a 5-nitro-furan-2-carboxylic acid carbamoylmethyl ester (NACME) moiety. Two NACME derivatives, 5-nitro-furan-2-carboxylic acid adamantan-1-carbamoylmethyl ester (compound 1) and 5-nitro-furan-2-carboxylic acid [[4-(4-bromo-phenyl)-thiazol-2-yl]-(tetrahydro-furan-2-ylmethyl)-carbamoyl]-methyl ester (compound 2), effectively blocked HIV-1 and MLV RT-associated RNase H activities with IC(50)s of 3-30 microM but had little effect on bacterial RNase H activity in vitro. Additionally, 20-25 microM compound 2 effectively inhibited HIV-1 replication. An in silico docking simulation indicated that the conserved His539 residue, and two metal ions in the RNase H catalytic center are involved in RNase H inhibition by NACME derivatives. Taken together, these data suggest that NACME derivatives may be potent lead compounds for development of a novel class of antiretroviral drugs.

A novel therapeutic molecule against HTLV-1 infection targeting provirus.

Human T-cell leukemia virus type 1 (HTLV-1), which causes adult T-cell leukemia (ATL) in humans, establishes a life-long latent infection. Current therapies are not very effective against HTLV-1-associated disorders. A novel therapeutic approach may help to combat HTLV-1 infection. A molecular therapy that targets the proviral genome is favorable because the therapeutic effect occurs specifically in HTLV-1-infected cells, regardless of whether they express viral genes. In this proof-of-concept study, we developed a therapeutic molecule based on zinc finger nuclease (ZFN) to achieve this goal. We designed a ZFN that specifically recognized conserved region of HTLV-1 long terminal repeat (LTR) and introduced it into various HTLV-1-positive human T-cell lines, including HTLV-1-transformed and ATL-derived cell lines. The ZFN disrupted the promoter function of HTLV-1 LTR and specifically killed HTLV-1-infected cells. We also showed a potential approach of this therapeutic molecule to remove the proviral genome from HTLV-1-infected cells, something that has not been possible before. The therapeutic effect of ZFN was confirmed in an in vivo model of ATL. This strategy may form the basis of a therapy that can eradicate HTLV-1 infection. Similar approaches can be used to target other malignancy-associated viruses.

Inhibiting lentiviral replication by HEXIM1, a cellular negative regulator of the CDK9/cyclin T complex.

Objective:Tat-dependent transcriptional elongation is crucial for the replication of HIV-1 and depends on positive transcription elongation factor b complex (P-TEFb), composed of cyclin dependent kinase 9 (CDK9) and cyclin T. Hexamethylene bisacetamide-induced protein 1 (HEXIM1) inhibits P-TEFb in cooperation with 7SK RNA, but direct evidence that this inhibition limits the replication of HIV-1 has been lacking. In the present study we examined whether the expression of FLAG-tagged HEXIM1 (HEXIM1-f) affected lentiviral replication in human T cell lines. Methods:HEXIM1-f was introduced to five human T cell lines, relevant host for HIV-1, by murine leukemia virus vector and cells expressing HEXIM1-f were collected by fluorescence activated cell sorter. The lentiviral replication kinetics in HEXIM1-f-expressing cells was compared with that in green fluorescent protein (GFP)-expressing cells. Results:HIV-1 and simian immunodeficiency virus replicated less efficiently in HEXIM1-f-expressing cells than in GFP-expressing cells of the five T cell lines tested. The viral revertants were not immediately selected in culture. In contrast, the replication of vaccinia virus, adenovirus, and herpes simplex virus type 1 was not limited. The quantitative PCR analyses revealed that the early phase of viral life cycle was not blocked by HEXIM1. On the other hand, Tat-dependent transcription in HEXIM1-f-expressing cells was substantially repressed as compared with that in GFP-expressing cells. Conclusion:These data indicate that HEXIM1 is a host factor that negatively regulates lentiviral replication specifically. Elucidating the regulatory mechanism of HEXIM1 might lead to ways to control lentiviral replication.

Substitution of the myristoylation signal of human immunodeficiency virus type 1 Pr55Gag with the phospholipase C-δ1 pleckstrin homology domain results in infectious pseudovirion production

The matrix domain (MA) of human immunodeficiency virus type 1 Pr55Gag is covalently modified with a myristoyl group that mediates efficient viral production. However, the role of myristoylation, particularly in the viral entry process, remains uninvestigated. This study replaced the myristoylation signal of MA with a well-studied phosphatidylinositol 4,5-biphosphate-binding plasma membrane (PM) targeting motif, the phospholipase C-δ1 pleckstrin homology (PH) domain. PH–Gag–Pol PM targeting and viral production efficiencies were improved compared with Gag–Pol, consistent with the estimated increases in Gag–PM affinity. Both virions were recovered in similar sucrose density-gradient fractions and had similar mature virion morphologies. Importantly, PH–Gag–Pol and Gag–Pol pseudovirions had almost identical infectivity, suggesting a dispensable role for myristoylation in the virus life cycle. PH–Gag–Pol might be useful in separating the myristoylation-dependent processes from the myristoylation-independent processes. This the first report demonstrating infectious pseudovirion production without myristoylated Pr55Gag.

Alkyl Amine Bevirimat Derivatives are Potent and Broadly Active HIV-1 Maturation Inhibitors

ABSTRACT Concomitant with the release of human immunodeficiency virus type 1 (HIV-1) particles from the infected cell, the viral protease cleaves the Gag polyprotein precursor at a number of sites to trigger virus maturation. We previously reported that a betulinic acid-derived compound, bevirimat (BVM), blocks HIV-1 maturation by disrupting a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown in multiple clinical trials to be safe and effective in reducing viral loads in HIV-1-infected patients. However, naturally occurring polymorphisms in the SP1 region of Gag (e.g., SP1-V7A) led to a variable response in some BVM-treated patients. The reduced susceptibility of SP1-polymorphic HIV-1 to BVM resulted in the discontinuation of its clinical development. To overcome the loss of BVM activity induced by polymorphisms in SP1, we carried out an extensive medicinal chemistry campaign to develop novel maturation inhibitors. In this study, we focused on alkyl amine derivatives modified at the C-28 position of the BVM scaffold. We identified a set of derivatives that are markedly more potent than BVM against an HIV-1 clade B clone (NL4-3) and show robust antiviral activity against a variant of NL4-3 containing the V7A polymorphism in SP1. One of the most potent of these compounds also strongly inhibited a multiclade panel of primary HIV-1 isolates. These data demonstrate that C-28 alkyl amine derivatives of BVM can, to a large extent, overcome the loss of susceptibility imposed by polymorphisms in SP1.

A Synthetic C34 Trimer of HIV-1 gp41 Shows Significant Increase in Inhibition Potency

The development of new anti-HIV-1 drugs such as inhibitors of protease and integrase has been contributed to highly active anti-retroviral therapy (HAART) for the treatment of AIDS. The entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope glycoprotein (Env), a type I transmembrane protein that consists of surface subunit gp120 and noncovalently associated transmembrane subunit gp41. Sequential binding of HIV-1 gp120 to its cell receptor CD4 and a co-receptor (CCR5 or CXCR4) can trigger a series of conformational rearrangements in gp41 to mediate fusion between viral and cellular membranes. The protein gp41 is hidden beneath gp120, and its ectodomain contains helical Nand C-terminal leucine/isoleucine heptad repeat domains, NHR and C-HR. Particular regions of N-HR and C-HR are involved in membrane fusion, and 36-mer and 34-mer peptides, which are derived from N-HR and C-HR, have been designated as the N-terminal helix (N36) and C-terminal helix (C34), respectively. In the membrane fusion of HIV-1, these helices assemble to form a six-helical bundle (6-HB) consisting of a central parallel trimer of N36 surrounded by C34 in an antiparallel hairpin fashion. Synthetic peptides derived from these helices have potent antiviral activity against both laboratory-adapted strains and primary isolates of HIV-1. They inhibit the membrane fusion stage of HIV-1 infection in a dominant-negative manner by binding to the counterpart regions of gp41 (N-HR or C-HR), blocking formation of the viral gp41 core. Several potent anti-HIV-1 peptides based on the C-HR region have been discovered, 8] and T20 was subsequently developed as the clinical anti-HIV-1 drug enfuvirtide (Roche/Trimeris). 10–13] It is a 36-mer peptide derived from the gp41 C-HR sequence and can bind to the N-HR to prevent formation of the 6-HB in a dominant-negative fashion. T20 therapy has brought safety, potent antiretroviral activity, and immunological benefit to patients, but its clinical application is limited by the development of resistance. The C-terminal helix C34 is also a C-HR-derived peptide, and contains the amino acid residues required for docking into the hydrophobic pocket, termed the “deep pocket”, of the trimer of the N-HR region. This peptide potently inhibits HIV-1 fusion in vitro. To date, several gp41 mimetics, especially those of N36 regions, which assemble these helical peptides with branched peptide linkers, have been synthesized as antigens. Recently, by using a novel template with C3-symmetric linkers of equal length, we synthesized a three-helix bundle mimetic that corresponds to the trimeric form of N36. The antisera obtained from mice immunized by the peptide antigen showed strong recognition against the N36 trimer peptide with structural preference. At the same time, the trimer peptide was also investigated as a fusion inhibitor. However, the trimer N36 showed only a threefold increase in inhibition of HIV-1 fusion relative to the N36 monomer. In terms of N36 content, the trimer and monomer have nearly the same inhibitory potency. This phenomenon is consistent with the results from other studies. The multimerization of the functional unit, such as synthetic ligands against receptors, show synergistic binding and strong binding activity. Thus, we hypothesized that our strategy using C3-symmetric linkers in the design of trimer mimics of gp41 could be applied to the C34 peptide, which shows significant inhibition potency in the monomeric form. In the present study, we designed and synthesized a novel three-helical bundle structure of the trimeric form of C34. This equivalent mimic of the trimeric form of C34 was evaluated as a novel form of fusion inhibitor. The C-terminal region of gp41 is known to be an assembly site involving a trimeric coiled-coil conformation. In the design of the C34-derived peptides C34REG-thioester (Figure 1 A) and C34REG (Figure 1 B), the triplet repeat of arginine and glutamic acid (RERERE) was added to the C-terminal end of the C34 sequence (residues 628–661) to increase aqueous solubility, and for C34REG-thioester, a glycine thioester was fused to the C terminus. To form a triple helix corresponding precisely to the gp41 pre-fusion form, we designed the novel C3-symmetric template depicted in Figure 1 C. This designed template linker has three branches of equal length, a hydrophilic structure, and a ligation site for coupling with C34REG-thioester. The template was synthesized as shown in Scheme 1. This approach uses native chemical ligation for chemoselective coupling of unprotected C34REG-thioester with a three-armed cysteine scaffold to produce triC34e (Figure 2). 25] Circular dichroism (CD) spectra of C34REG and triC34e are shown in Figure 3 A. The peptides were dissolved in 50 mm sodium phosphate buffer with 150 mm NaCl, pH 7.2. Both spectra display minima at ~200 nm, indicating that these peptides form random structures. We previously reported that the [a] Dr. W. Nomura, C. Hashimoto, A. Ohya, Dr. T. Tanaka, Dr. T. Narumi, T. Nakahara, Prof. Dr. H. Tamamura Institute of Biomaterials and Bioengineering Tokyo Medical and Dental University 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062 (Japan) E-mail : tamamura.mr@tmd.ac.jp [b] Dr. K. Miyauchi, Dr. E. Urano, Dr. J. A. Komano AIDS Research Center, National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640 (Japan) [c] Prof. Dr. N. Yamamoto Department of Microbiology, Yong Loo Lin School of Medicine National University of Singapore 5 Science Drive 2, Singapore 117597 (Singapore) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100542.