Makiko Hamatake

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)

The novel CXCR4 antagonist KRH-3955 is an orally bioavailable and extremely potent inhibitor of human immunodeficiency virus type 1 infection: comparative studies with AMD3100.

ABSTRACT The previously reported CXCR4 antagonist KRH-1636 was a potent and selective inhibitor of CXCR4-using (X4) human immunodeficiency virus type 1 (HIV-1) but could not be further developed as an anti-HIV-1 agent because of its poor oral bioavailability. Newly developed KRH-3955 is a KRH-1636 derivative that is bioavailable when administered orally with much more potent anti-HIV-1 activity than AMD3100 and KRH-1636. The compound very potently inhibits the replication of X4 HIV-1, including clinical isolates in activated peripheral blood mononuclear cells from different donors. It is also active against recombinant X4 HIV-1 containing resistance mutations in reverse transcriptase and protease and envelope with enfuvirtide resistance mutations. KRH-3955 inhibits both SDF-1α binding to CXCR4 and Ca2+ signaling through the receptor. KRH-3955 inhibits the binding of anti-CXCR4 monoclonal antibodies that recognize the first, second, or third extracellular loop of CXCR4. The compound shows an oral bioavailability of 25.6% in rats, and its oral administration blocks X4 HIV-1 replication in the human peripheral blood lymphocyte-severe combined immunodeficiency mouse system. Thus, KRH-3955 is a new promising agent for HIV-1 infection and AIDS.

Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant Mycobacterium bovis bacille Calmette-Guerin expressing human immunodeficiency virus type 1 Gag.

ABSTRACT Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.

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.

Identification of the P-TEFb complex-interacting domain of Brd4 as an inhibitor of HIV-1 replication by functional cDNA library screening in MT-4 cells

We conducted a phenotypic cDNA screening using a T cell line‐based assay to identify human genes that render cells resistant to human immunodeficiency virus type 1 (HIV‐1). We isolated potential HIV‐1 resistance genes, including the carboxy terminal domain (CTD) of bromodomain‐containing protein 4 (Brd4). Expression of GFP‐Brd4‐CTD was tolerated in MT‐4 and Jurkat cells in which HIV‐1 replication was markedly inhibited. We provide direct experimental data demonstrating that Brd4‐CTD serves as a specific inhibitor of HIV‐1 replication in T cells. Our method is a powerful tool for the identification of host factors that regulate HIV‐1 replication in T cells.

Cyclin K/CPR4 inhibits primate lentiviral replication by inactivating Tat/positive transcription elongation factor b-dependent long terminal repeat transcription.

The positive transcription elongation factor b complexes comprise CDK9 and a C-type cyclin, required for the efficient expression of both eukaryotic and primate lentivirus-encoded genes. Cyclin K/CPR4 is the least studied of the positive transcription elongation factor b-forming cyclins. Here, we demonstrate that cyclin K/CPR4-containing positive transcription elongation factor b complexes are unresponsive to Tat and HEXIM1-mediated inactivation. Enhancing expression of cyclin K/CPR4 inhibited the human and simian immunodeficiency viral replication. These data indicate that cyclin K/CPR4 functions as a natural inhibitor of primate lentiviruses.

Inhibition of HIV replication by a CD4‐reactive Fab of an IgM clone isolated from a healthy HIV‐seronegative individual

HIV replication is restricted by some anti‐CD4 mouse mAb in vitro and in vivo. However, a human monoclonal anti‐CD4 Ab has not been isolated. We screened EBV‐transformed peripheral B cells from 12 adult donors for CD4‐reactive Ab production followed by functional reconstitution of Fab genes. Three independent IgM Fab clones reactive specifically to CD4 were isolated from a healthy HIV‐seronegative adult (∼0.0013% of the peripheral B cells). The germ line combinations for the VH and VL genes were VH3‐33/L6, VH3‐33/L12, and VH4‐4/L12, respectively, accompanied by somatic hypermutations. Genetic analysis revealed a preference for V‐gene usage to develop CD4‐reactive Ab. Notably, one of the CD4‐reactive clones, HO538‐213, with an 1×10−8 M dissociation constant (Kd) to recombinant human CD4, limited the replication of R5‐tropic and X4‐tropic HIV‐1 strains at 1–2.5 μg/mL in primary mononuclear cells. This is the first clonal genetic analysis of human monoclonal CD4‐reactive Ab. A mAb against CD4 isolated from a healthy individual could be useful in the intervention of HIV/AIDS.

A Triazinone Derivative Inhibits HIV-1 Replication by Interfering with Reverse Transcriptase Activity.

A novel HIV‐1 inhibitor, 6‐(tert‐butyl)‐4‐phenyl‐4‐(trifluoromethyl)‐1H,3H‐1,3,5‐triazin‐2‐one (compound 1), was identified from a compound library screened for the ability to inhibit HIV‐1 replication. EC50 values of compound 1 were found to range from 107.9 to 145.4 nm against primary HIV‐1 clinical isolates. In in vitro assays, HIV‐1 reverse transcriptase (RT) activity was inhibited by compound 1 with an EC50 of 4.3 μm. An assay for resistance to compound 1 selected a variant of HIV‐1 with a RT mutation (RTL100I); this frequently identified mutation confers mild resistance to non‐nucleoside RT inhibitors (NNRTIs). A recombinant HIV‐1 bearing RTL100I exhibited a 41‐fold greater resistance to compound 1 than the wild‐type virus. Compound 1 was also effective against HIV‐1 with RTK103N, one of the major mutations that confers substantial resistance to NNRTIs. Computer‐assisted docking simulations indicated that compound 1 binds to the RT NNRTI binding pocket in a manner similar to that of efavirenz; however, the putative compound 1 binding site is located further from RTK103 than that of efavirenz. Compound 1 is a novel NNRTI with a unique drug‐resistance profile.

Dominant‐negative derivative of EBNA1 represses EBNA1‐mediated transforming gene expression during the acute phase of Epstein–Barr virus infection independent of rapid loss of viral genome

The oncogenic human herpes virus, the Epstein–Barr virus (EBV), expresses EBNA1 in almost all forms of viral latency. EBNA1 plays a major role in the maintenance of the viral genome and in the transactivation of viral transforming genes, including EBNA2 and latent membrane protein (LMP‐1). However, it is unknown whether inhibition of EBNA1 from the onset of EBV infection disrupts the establishment of EBV’s latency and transactivation of the viral oncogenes. To address this, we measured EBV infection kinetics in the B cell lines BALL‐1 and BJAB, which stably express a dominant‐negative EBNA1 (dnE1) fused to green fluorescent protein (GFP). The EBV genome was surprisingly unstable 1 week post‐infection: the average loss rate of EBV DNA from GFP‐ and GFP‐dnE1‐expressing cells was 53.4% and 41.0% per cell generation, respectively, which was substantially higher than that of an ‘established’oriP replicon (2–4%). GFP‐dnE1 did not accelerate loss of the EBV genome, suggesting that EBNA1‐dependent licensing of the EBV genome occurs infrequently during the acute phase of EBV infection. In the subacute phase, establishment of EBV latency was completely blocked in GFP‐dnE1‐expressing cells. In contrast, C/W promoter‐driven transcription was strongly restricted in GFP‐dnE1‐expressing cells at 2 days post‐infection. These data suggest that inhibition of EBNA1 from the onset of EBV infection is effective in blocking the positive feedback loop in the transactivation of viral transforming genes, and in eradicating the EBV genome during the subacute phase. Our results suggest that gene transduction of GFP‐dnE1 could be a promising therapeutic and prophylactic approach toward EBV‐associated malignancies.