Nanobodies targeting the hepatocyte growth factor: potential new drugs for molecular cancer therapy.

Hepatocyte growth factor (HGF) and its receptor c-Met are associated with increased aggressiveness of tumors and poor prognostic outcome of patients with cancer. Here, we report the development and characterization of therapeutic anti-HGF (αHGF)-Nanobodies and their potential for positron emission tomographic (PET) imaging to assess HGF expression in vivo. Two αHGF-Nanobodies designated 1E2 and 6E10 were identified, characterized, and molecularly fused to an albumin-binding Nanobody unit (Alb8) to obtain serum half-life extension. The resulting Nanobody formats were radiolabeled with the positron emitter zirconium-89 ((89)Zr, t(1/2;) = 78 hours), administered to nude mice bearing U87 MG glioblastoma xenografts, and their biodistribution was assessed. In addition, their therapeutic effect was evaluated in the same animal model at doses of 10, 30, or 100 µg per mouse. The (89)Zr-Nanobodies showed similar biodistribution with selective tumor targeting. For example, 1E2-Alb8 showed decreased blood levels of 12.6%ID/g ± 0.6%ID/g, 7.2%ID/g ± 1.0%ID/g, 3.4%ID/g ± 0.3%ID/g, and 0.3%ID/g ± 0.1%ID/g at 1, 2, 3, and 7 days after injection, whereas tumor uptake levels remained relatively stable at these time points: 7.8%ID/g ± 1.1%ID/g, 8.9%ID/g ± 1.0%ID/g, 8.7%ID/g ± 1.5%ID/g, and 7.2%ID/g ±1.6%ID/g. Uptake in normal tissues was lower than in tumor, except for kidneys. In a therapy study, all Nanobody-treated mice showed tumor growth delay compared with the control saline group. In the 100-µg group, four of six mice were cured after treatment with 1E2-Alb8 and 73 days follow-up, and three of six mice when treated with 6E10-Alb8. These results provide evidence that Nanobodies 1E2-Alb8 and 6E10-Alb8 have potential for therapy and PET imaging of HGF-expressing tumors.

A fast and sensitive method for measuring the integrity of siRNA-carrier complexes in full human serum.

SiRNA based therapeutics are currently under investigation for treatment of cancer and viral infections. Upon intravenous administration, the nanoscopic delivery systems which carry the siRNA need to be stable in serum, an aspect which is often overlooked in numerous publications on siRNA delivery systems. Techniques currently available for studying the dissociation of siRNA-liposome complexes are time consuming and incompatible with full serum. We therefore developed a fluorescence fluctuation spectroscopy (FFS) based method which allows to monitor the integrity of siRNA-carrier complexes. The method can very rapidly provide quantitative information on the complex integrity in biological media, like full human serum, and at very low siRNA concentrations (approximately 20 nM siRNA). Information on the integrity of intravenously injected siRNA nanoparticles in serum is crucial. Consequently, the FFS method reported in this work may find broad applicability in the field of siRNA-carrier design.

Novel virtual screening protocol based on the combined use of molecular modeling and electron-ion interaction potential techniques to design HIV-1 integrase inhibitors.

HIV-1 integrase (IN) is an essential enzyme for viral replication and represents an intriguing target for the development of new drugs. Although a large number of compounds have been reported to inhibit IN in biochemical assays, no drug active against this enzyme has been approved by the FDA so far. In this study, we report, for the first time, the use of the electron-ion interaction potential (EIIP) technique in combination with molecular modeling approaches for the identification of new IN inhibitors. An innovative virtual screening approach, based on the determination of both short- and long-range interactions between interacting molecules, was employed with the aim of identifying molecules able to inhibit the binding of IN to viral DNA. Moreover, results from a database screening on the commercial Asinex Gold Collection led to the selection of several compounds. One of them showed a significant inhibitory potency toward IN in the overall integration assay. Biological investigations also showed, in agreement with modeling studies, that these compounds prevent recognition of DNA by IN in a fluorescence fluctuation assay, probably by interacting with the DNA binding domain of IN.

Overexpression of the lens epithelium-derived growth factor/p75 integrase binding domain inhibits human immunodeficiency virus replication.

We initially identified lens epithelium-derived growth factor/p75 (LEDGF/p75) as a binding partner of human immunodeficiency virus type 1 (HIV-1) integrase. To investigate the role of LEDGF/p75 in HIV replication and its potential as a new antiviral target, we stably overexpressed two different fragments containing the integrase binding domain (IBD) of LEDGF/p75 fused to enhanced green fluorescent protein (eGFP). HIV-1 replication was severely inhibited by overexpression of the eGFP-IBD fusion proteins, while no inhibition was observed in cell lines overexpressing the interaction-deficient D366A mutant. Quantitative PCR pinpointed the block to the integration step, whereas nuclear import was not affected. Competition of the IBD fusion proteins with endogenous LEDGF/p75 for binding to integrase led to a potent defect in HIV-1 replication in both HeLaP4- and MT-4-derived cell lines. A previously described diketo acid-resistant HIV-1 strain remained fully susceptible to inhibition, suggesting that this strategy will also work in patients who harbor strains resistant to the current experimental integrase inhibitors. These data support LEDGF/p75 as an important cofactor for HIV replication and provide proof of concept for the LEDGF/p75-integrase interaction as a novel target for treating HIV-1 infection.

Probing diffusion laws within cellular membranes by Z-scan fluorescence correlation spectroscopy.

The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F. Lenne. 2005. Biophys. J. 89:4029-4042) described a method to probe the nature of the lateral microheterogeneities of the membrane by varying the beam size in the FCS instrument. The dependence of the width of the autocorrelation function at half-maximum, i.e., the diffusion time, on the transverse area of the confocal volume gives information on the nature of the imposed confinement. We describe an alternative approach that yields essentially the same information, and can readily be applied on commercial FCS instruments by measuring the diffusion time and the particle number at various relative positions of the cell membrane with respect to the waist of the laser beam, i.e., by performing a Z-scan.

Diffusion of sphingomyelin and myelin oligodendrocyte glycoprotein in the membrane of OLN-93 oligodendroglial cells studied by fluorescence correlation spectroscopy.

Evidence has been accumulated that the plasma membrane of various mammalian cell types is heterogeneous in structure and may contain lipid microdomains (lipid rafts). This study focuses on the membrane organization of living oligodendrocytes, which are the myelin-producing cells of the central nervous system. Fluorescence correlation spectroscopy (FCS) was used to monitor the lateral diffusion of a lipid and of a protein in the oligodendroglial cell line OLN-93. The lipid was fluorescently labelled sphingomyelin (Bodipy FL-C5 SM). The protein was the myelin oligodendrocyte glycoprotein (MOG). In order to monitor the lateral diffusion of MOG, OLN-93 cells were transfected with a MOG-EGFP (enhanced green fluorescent protein) fusion plasmid. The measurements were performed at room temperature. FCS data were analyzed for two-dimensional (2D) diffusion according to three models which all included a triplet fraction: (a) 2D 1 component (2D1C), (b) 2D anomalous diffusion (2D1Calpha), and (c) 2D 2 components (2D2C). Preliminary results indicate that for the lipid case, the best fits are obtained with 2D2C. In the case of MOG-EGFP, 2D2C and 2D1Calpha give fits of similar quality. The parameter estimates obtained with 2D1Calpha, however, have a lower standard deviation. The anomaly parameter for MOG-EGFP is 0.59+/-0.01.

Measuring protein-protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type 1 integrase and LEDGF/p75.

Recently we described the interaction of human immunodeficiency virus type 1 (HIV-1)1 integrase (IN) with a cellular protein, lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75). We now present the study of the diffusion behavior of the three independent domains of IN and LEDGF/p75 using fluorescence correlation microscopy (FCM). We show that diffusion in the cell of the different enhanced green fluorescent protein (EGFP) fusion proteins is described by two components with different fractions and that the average parameters in the nucleus are comparable with those in the cytoplasm. In addition, we demonstrate that specific interaction between EGFP-fused HIV-1 IN and LEDGF/p75 results in a shift in diffusion coefficient (D). The opposite shift was observed in an IN-deletion mutant that does not exhibit LEDGF/p75 binding or in a LEDGF/p75 knock-down experiment using siRNA. We thus demonstrate that protein-protein interactions can be studied in living cells, using single-color FCM (scFCM).

The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding.

We have previously shown that the p75 isoform of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF) interacts tightly with human immunodeficiency virus (HIV)-1 integrase (IN) and is essential for nuclear targeting of this protein in human cells (Cherepanov, P., Maertens, G., Proost, P., Devreese, B., Van Beeumen, J., Engelborghs, Y., De Clercq, E., and Debyser, Z. (2003) J. Biol. Chem. 278, 372-381; Maertens, G., Cherepanov, P., Pluymers, W., Busschots, K., De Clercq, E., Debyser, Z., and Engelborghs, Y. (2003) J. Biol. Chem. 278, 33528-33539). Here the interaction between recombinant LEDGF/p75 and HIV-1 IN was examined in a pull-down binding test. LEDGF/p75 was shown to increase the solubility of HIV-1 IN. Next, fluorescent correlation spectroscopy was used to measure the interaction of LEDGF/p75 or the complex of HIV-1 IN and LEDGF/p75 with a specific double-stranded DNA oligonucleotide. Whereas LEDGF/p75 displayed only a moderate affinity for DNA, it strongly promoted the binding of HIV-1 IN to DNA. This effect was specific for the p75 isoform of LEDGF and was not seen with p52. In the pull-down assay LEDGF/p75 interacted with HIV-1, HIV-2, and feline immunodeficiency virus IN, but not with the IN of human T-cell lymphotropic virus type 2, Moloney murine leukemia virus, or Rous sarcoma virus. These results strongly suggest that the interaction of LEDGF/p75 with IN is specific to lentiviridae. LEDGF/p75 stimulated the binding of HIV-1 and HIV-2 IN, but not Moloney murine leukemia virus or Rous sarcoma virus IN, to an aspecific DNA. These results provide supporting evidence for our hypothesis that LEDGF/p75 plays a role in the tethering of lentiviral IN to the chromosomal DNA.

Correct diffusion coefficients of proteins in fluorescence correlation spectroscopy. Application to tubulin oligomers induced by Mg2+ and Paclitaxel.

In view of recent warnings for artifacts in fluorescence correlation spectroscopy, the diffusion coefficient of a series of labeled proteins in a wide range of molecular mass (43-670 kD) was determined and shown to be correct with respect to published values and the theory. Fluorescence correlation spectroscopy was then applied to the study of fluorescently labeled tubulin and its oligomerization in vitro induced by Mg2+ ions, paclitaxel, and a fluorescent derivative of paclitaxel (Flutax2). By applying relations derived from the theory of Oosawa, we were able to determine the association constant of the oligomers induced by Mg2+. With Flutax2 our experiments show that at nanomolar concentration, the fluorescent derivative is able to recruit tubulin dimers and to form oligomers of defined size. Flutax2 does not bind to microtubules preformed with paclitaxel, but it becomes preferentially incorporated into microtubules when Flutax2 oligomers are preformed, and microtubule formation is induced by paclitaxel addition. This shows that their incorporation into microtubules is faster than the displacement of the prebound drug. Experiments using fluorescently labeled tubulin and (unlabeled) paclitaxel confirm the induction of tubulin oligomers at limiting paclitaxel concentrations.

The Saccharomyces cerevisiae alcohol acetyl transferase Atf1p is localized in lipid particles.

The yeast alcohol acetyl transferase I, Atf1p, is responsible for the major part of volatile acetate ester production in fermenting Saccharomyces cerevisiae cells. Some of these esters, such as ethyl acetate and isoamyl acetate, are important for the fruity flavours of wine, beer and other fermented beverages. In order to reveal the subcellular localization of Atf1p and further unravel the possible physiological role of this protein, ATF1::GFP fusion constructs were overexpressed in brewer's yeast. The transformant strain showed a significant increase in acetate ester formation, similar to that of an ATF1 overexpression strain, indicating that the Atf1p-GFP fusion protein was active. UV fluorescence microscopy revealed that the fusion protein was localized in small, sphere-like organelles. These organelles could be selectively stained by the fluorescent dye Nile red, indicating that they contained high amounts of neutral lipids and/or sterols, a specific characteristic of yeast lipid particles. Purification of lipid particles from wild type and ATF1 deletion cells confirmed that the Atf1p-GFP fusion protein was located in these organelles. Furthermore, a clear alcohol acetyl transferase activity could be measured in the purified lipid particles of both wild type and transformed cells. The localization of Atf1p in lipid particles may indicate that Atf1p has a specific role in the lipid and/or sterol metabolism that takes place in these particles.

Development of resistance against diketo derivatives of human immunodeficiency virus type 1 by progressive accumulation of integrase mutations.

The diketo acid L-708,906 has been reported to be a selective inhibitor of the strand transfer step of the human immunodeficiency virus type 1 (HIV-1) integration process (D. Hazuda, P. Felock, M. Witmer, A. Wolfe, K. Stillmock, J. A. Grobler, A. Espeseth, L. Gabryelski, W. Schleif, C. Blau, and M. D. Miller, Science 287:646-650, 2000). We have now studied the development of antiviral resistance to L-708,906 by growing HIV-1 strains in the presence of increasing concentrations of the compound. The mutations T66I, L74M, and S230R emerged successively in the integrase gene. The virus with three mutations (T66I L74M S230R) was 10-fold less susceptible to L-708,906, while displaying the sensitivity of the wild-type virus to inhibitors of the RT or PRO or viral entry process. Chimeric HIV-1 strains containing the mutant integrase genes displayed the same resistance profile as the in vitro-selected strains, corroborating the impact of the reported mutations on the resistance phenotype. Phenotypic cross-resistance to S-1360, a diketo analogue in clinical trials, was observed for all strains. Interestingly, the diketo acid-resistant strain remained fully sensitive to V-165, a novel integrase inhibitor (C. Pannecouque, W. Pluymers, B. Van Maele, V. Tetz, P. Cherepanov, E. De Clercq, M. Witvrouw, and Z. Debyser, Curr. Biol. 12:1169-1177, 2002). Antiviral resistance was also studied at the level of recombinant integrase. Single mutations did not appear to impair specific enzymatic activity. However, 3' processing and strand transfer activities of the recombinant integrases with two (T66I L74M) and three (T66I L74M S230R) mutations were notably lower than those of the wild-type integrase. Although the virus with three mutations was resistant to inhibition by diketo acids, the sensitivity of the corresponding enzyme to L-708,906 or S-1360 was reduced only two- to threefold. As to the replication kinetics of the selected strains, the replication fitness for all strains was lower than that of the wild-type HIV-1 strain.

DNA-induced polymerization of HIV-1 integrase analyzed with fluorescence fluctuation spectroscopy.

Human immunodeficiency virus type 1 (HIV-1) integrase is essential for viral replication. Integrase inserts the viral DNA into the host DNA. We studied the association of integrase to fluorescently labeled oligonucleotides using fluorescence correlation spectroscopy. The binding of integrase to the fluorescent oligonucleotides resulted in the appearance of bright spikes during fluorescence correlation spectroscopy measurements. These spikes arise from the formation of high molecular mass protein-DNA complexes. The fluorescence of the free DNA was separated from the spikes with a statistical method. From the decrease of the concentration of free oligonucleotides, a site association constant was determined. The DNA-protein complexes were formed rapidly in a salt-dependent manner with site association constants ranging between 5 and 40 microm(-1) under different conditions. We also analyzed the kinetics of the DNA-protein complex assembly and the effect of different buffer components. The formation of the fluorescent protein-DNA complex was inhibited by guanosine quartets, and the inhibition constant was determined at 1.8 +/- 0.6 x 10(8) m(-1). Displacement of bound DNA with G-quartets allowed the determination of the dissociation rate constant and proves the reversibility of the association process.