We describe the development of cell-permeable beta-secretase inhibitors that demonstratively inhibit the production of the secreted amino terminal fragment of an artificial amyloid precursor protein in cell culture. In addition to potent inhibition in a cell-based assay (IC50 < 100 nM), these inhibitors display impressive selectivity against other biologically relevant aspartyl proteases.

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BackgroundNotch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.Principal FindingsHere we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC50 values as low as 5±3 nM and 0.13±0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR “class I” point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare “class II” or “class III” mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.Conclusions/SignificanceAntibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.

The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.Human immunodeficiency virus (HIV) therapies have been highly successful in slowing disease progression, increasing health and well-being, and prolonging life. However, viral resistance is now becoming common, and since most existing drugs target only two viral proteins, reverse transcriptase and protease, cross-resistance is a significant problem. One solution to the issue of resistance is development of new complementary therapies based on novel mechanisms of action. The discovery that the chemokine receptors CCR5 and CXCR4, in addition to CD4, are required for viral entry not only furthered understanding of the fusion and infection process but provided two new targets for therapeutic intervention (3,12,14,17,18,22,44).The entry mechanism as currently understood is an ordered process in which the viral envelope protein, gp120, following interaction with CD4, undergoes a conformational change allowing binding to the appropriate chemokine receptor, CCR5 for macrophagetropic or R5 strains, and CXCR4 for T-celltropic or X4 s...

Like the CCR5 chemokine receptors of humans and rhesus macaques, the very homologous (ϳ98 -99% identical) CCR5 of African green monkeys (AGMs) avidly binds ␤-chemokines and functions as a coreceptor for simian immunodeficiency viruses. However, AGM CCR5 is a weak coreceptor for tested macrophage-tropic (R5) isolates of human immunodeficiency virus type 1 (HIV-1). Correspondingly, gp120 envelope glycoproteins derived from R5 isolates of HIV-1 bind poorly to AGM CCR5. We focused on a unique extracellular amino acid substitution at the juncture of transmembrane helix 4 (TM4) and extracellular loop 2 (ECL2) (Arg for Gly at amino acid 163 (G163R)) as the likely source of the weak R5 gp120 binding and HIV-1 coreceptor properties of AGM CCR5. Accordingly, a G163R mutant of human CCR5 was severely attenuated in its ability to bind R5 gp120s and to mediate infection by R5 HIV-1 isolates. Conversely, the R163G mutant of AGM CCR5 was substantially strengthened as a coreceptor for HIV-1 and had improved R5 gp120 binding affinity relative to the wild-type AGM CCR5. These substitutions at amino acid position 163 had no effect on chemokine binding or signal transduction, suggesting the absence of structural alterations. The 2D7 monoclonal antibody has been reported to bind to ECL2 and to block HIV-1 binding and infection. Whereas 2D7 antibody binding to CCR5 was unaffected by the G163R mutation, it was prevented by a conservative ECL2 substitution (K171R), shared between rhesus and AGM CCR5s. Thus, it appears that the 2D7 antibody binds to an epitope that includes Lys-171 and may block HIV-1 infection mediated by CCR5 by occluding an HIV-1-binding site in the vicinity of Gly-163. In summary, our results identify a site for gp120 interaction that is critical for R5 isolates of HIV-1 in the central core of human CCR5, and we propose that this site collaborates with a previously identified region in the CCR5 amino terminus to enable gp120 binding and HIV-1 infections.Infection by human immunodeficiency virus type 1 (HIV-1) 1 involves adsorption onto cell-surface CD4, followed by interaction of the viral gp120⅐gp41 envelope glycoprotein complexes with a coreceptor (1-6). Association of gp120 with CD4 induces a conformational change that exposes previously buried epitopes in gp120 and gp41 (7-9), including a site for gp120 interaction with the coreceptor (10 -13). The latter interaction is thought to cause an additional conformational change that facilitates fusion of the viral and cellular membranes, resulting in transfer of the viral cores into the cytosol (14). The known coreceptors are all G protein-coupled receptors with seven transmembrane domains (TM) that normally signal in response to cognate chemokine ligands (15). The major coreceptor for macrophage-tropic (R5) isolates of HIV-1 is CCR5, a receptor for the ␤-chemokines MIP1␣, MIP1␤, and RANTES (1-5, 16). The coreceptor for T cell-tropic HIV-1 isolates is CXCR4, a receptor for the ␣-chemokine stromal cell-derived factor (6, 17, 18). R5 isolates are generally responsible f...