First demonstration of cerebrospinal fluid and plasma A beta lowering with oral administration of a beta-site amyloid precursor protein-cleaving enzyme 1 inhibitor in nonhuman primates.

beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Macrocyclic inhibitors of beta-secretase: functional activity in an animal model.

A macrocyclic inhibitor of beta-secretase was designed by covalently cross-linking the P1 and P3 side chains of an isophthalamide-based inhibitor. Macrocyclization resulted in significantly improved potency and physical properties when compared to the initial lead structures. More importantly, these macrocyclic inhibitors also displayed in vivo amyloid lowering when dosed in a murine model.

Novel mutations introduced at the beta-site of amyloid beta protein precursor enhance the production of amyloid beta peptide by BACE1 in vitro and in cells.

Abnormal production and accumulation of amyloid-beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at thebeta-site in amyloid beta protein precursor (AbetaPP/APP) to generate the N-terminus of Abeta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEV" (APP_NFEV) in vitro and in cells. In vitro, the APP_NFEV exhibits 100-fold enhanced cleavage rate relative to the "wild-type" substrate (APPwt) and 10-fold increase relative to the Swedish-type mutation variant (APPsw). In cells, it was preferably cleaved among 24 APP beta-site mutations tested. More importantly, the APP_NFEV mutant failed to generate any detectable Abeta peptides in BACE1-KO mouse fibroblast cells. The production of Abeta peptides was restored by co-transfecting human BACE1, demonstrating that BACE1 is the only enzyme responsible for the processing of APP_NFEV in these cells. Analysis of APP_NFEV cleavage products secreted in the media revealed that in cells BACE1 cleaves APP_NFEV at the position between NF and EV, identical to that observed in vitro. A BACE inhibitor blocked the processing of the APP_NFEV beta-site in vitro and in cells. Our data indicates that the "NFEV" mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.

Structure-based design of potent and selective cell-permeable inhibitors of human beta-secretase (BACE-1).

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.

CCR5 antagonists: 3-(pyrrolidin-1-yl)propionic acid analogues with potent anti-HIV activity.

[reaction: see text] A novel approach to alpha,alpha-disubstituted-beta-amino acids (beta(2,2)-amino acids) was employed in the synthesis of a series of 3-(pyrrolidin-1-yl)propionic acids possessing high affinity for the CCR5 receptor and potent anti-HIV activity. The rat pharmacokinetics for these new analogues featured higher bioavailabilities and lower rates of clearance as compared to cyclopentane 1.

Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents.

Myostatin is a highly conserved member of the transforming growth factor-ß ligand family known to regulate muscle growth via activation of activin receptors. A fusion protein consisting of the extracellular ligand-binding domain of activin type IIB receptor with the Fc portion of human immunoglobulin G (ActRIIB-Fc) was used to inhibit signaling through this pathway. Here, we study the effects of this fusion protein in adult, 18-month-old, and orchidectomized mice. Significant muscle growth and enhanced muscle function were observed in adult mice treated for 3 days with ActRIIB-Fc. The ActRIIB-Fc-treated mice had enhanced fast fatigable muscle function, with only minor enhancement of fatigue-resistant fiber function. The ActRIIB-Fc-treated 18-month-old mice and orchidectomized mice showed significantly improved muscle function. Treatment with ActRIIB-Fc also increased bone mineral density and serum levels of a marker of bone formation. These observations highlight the potential of targeting ActRIIB receptor to treat age-related and hypogonadism-associated musculoskeletal degeneration.

Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

BACKGROUND: Notch 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 FINDINGS: Here 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 EC(50) 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/SIGNIFICANCE: Antibodies 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.

BACE-1 inhibition by a series of psi[CH2NH] reduced amide isosteres.

A series of beta-site amyloid precursor protein cleaving enzyme (BACE-1) inhibitors containing a psi(CH2NH) reduced amide bond were synthesized. Incorporation of this reduced amide isostere as a non-cleavable peptide surrogate afforded inhibitors possessing low nanomolar potencies in both an enzymatic and cell-based assay.

Synthesis and evaluation of CCR5 antagonists containing modified 4-piperidinyl-2-phenyl-1-(phenylsulfonylamino)-butane.

Synthesis of analogs containing more rigid bicyclic piperidine replacements for the 4-benzyloxycarbonyl-(ethyl)amino-piperidine moiety of the CCR5 antagonist structure, 1, is described. Although similar binding affinity to the lead was achieved with some analogs they were overall less potent anti-HIV agents suggesting that other features besides CCR5 binding are required for good anti-viral activity.

Potent 1,3,4-trisubstituted pyrrolidine CCR5 receptor antagonists: effects of fused heterocycles on antiviral activity and pharmacokinetic properties.

A series of 1,3,4-trisubstituted pyrrolidine CCR5 receptor antagonists containing a variety of fused heterocycles at the 4-position of the piperidine side chain has been discovered, which are orally bioavailable with potent anti-HIV activity.

Biochemical and cell-based assays for characterization of BACE-1 inhibitors.

The deposition of beta-amyloid peptides (A beta42 and A beta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). A beta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. BACE-1 has been shown to be the major beta-secretase and is a primary therapeutic target for AD. In this article, two novel assays for the characterization of BACE-1 inhibitors are reported. The first is a sensitive 96-well HPLC biochemical assay that uses a unique substrate containing an optimized peptide cleavage sequence, NFEV, spanning from the P2-P2' positions This substrate was processed by BACE-1 approximately 10 times more efficiently than was the widely used substrate containing the Swedish (NLDA) sequence. As a result, the concentration of the enzyme required for the assay can be as low as 100 pM, permitting the evaluation of inhibitors with subnanomolar potency. The assay has also been applied to related aspartyl proteases such as cathepsin D (Cat D) and BACE-2. The second assay is a homogeneous electrochemiluminescence assay for the evaluation of BACE-1 inhibition in cultured cells that assesses the level of secreted amyloid EV40_NF from HEK293T cells stably transfected with APP containing the novel NFEV sequence. To illustrate the use of these assays, the properties of a potent, cell-active BACE-1 inhibitor are described.

Compounds that bind APP and inhibit Abeta processing in vitro suggest a novel approach to Alzheimer disease therapeutics.

Extracellular deposits of aggregated amyloid-beta (Abeta) peptides are a hallmark of Alzheimer disease; thus, inhibition of Abeta production and/or aggregation is an appealing strategy to thwart the onset and progression of this disease. The release of Abeta requires processing of the amyloid precursor protein (APP) by both beta- and gamma-secretase. Using an assay that incorporates full-length recombinant APP as a substrate for beta-secretase (BACE), we have identified a series of compounds that inhibit APP processing, but do not affect the cleavage of peptide substrates by BACE1. These molecules also inhibit the processing of APP and Abeta by BACE2 and selectively inhibit the production of Abeta(42) species by gamma-secretase in assays using CTF99. The compounds bind directly to APP, likely within the Abeta domain, and therefore, unlike previously described inhibitors of the secretase enzymes, their mechanism of action is mediated through APP. These studies demonstrate that APP binding agents can affect its processing through multiple pathways, providing proof of concept for novel strategies aimed at selectively modulating Abeta production.

Altering expression levels of human immunodeficiency virus type 1 gp120-gp41 affects efficiency but not kinetics of cell-cell fusion.

Human immunodeficiency virus (HIV) entry into a host cell requires the fusion of virus and cellular membranes that is driven by interaction of the viral envelope glycoproteins gp120 and gp41 (gp120/gp41) with CD4 and a coreceptor, typically either CXCR4 or CCR5. The stoichiometry of gp120/gp41:CD4:CCR5 necessary to initiate membrane fusion is not known. To allow an examination of early events in gp120/gp41-driven membrane fusion, we developed a novel real-time cell-cell fusion assay. Using this assay to study fusion kinetics, we found that altering the cell surface density of gp120/gp41 affected the maximal extent of fusion without dramatically altering fusion kinetics. Collectively, these observations are consistent with the view that gp120/gp41-driven membrane fusion requires the formation of a threshold number of fusion-active intercellular gp120/gp41:CD4:CCR5 complexes. Furthermore, the probability of reaching this threshold is governed, in part, by the surface density of gp120/gp41.

Nef does not affect the efficiency of human immunodeficiency virus type 1 fusion with target cells.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef stimulates viral infectivity by an unknown mechanism. Recent studies have suggested that Nef may act by regulating the efficiency of virus entry into cells. Here we provide evidence to the contrary. Using a quantitative assay of HIV-1 virus-cell fusion, we observed equivalent rates and extents of fusion of wild-type and Nef-defective HIV-1 particles with MT-4 cells and CD4-expressing HeLa cells. In studies using soluble CD4 (sCD4) to inhibit infection, wild-type and Nef-defective HIV-1 escaped the sCD4 block with similar kinetics. We conclude that Nef acts at a postentry step in infection, probably by facilitating intracellular transport of the HIV-1 ribonucleoprotein complex.

Coupling of human immunodeficiency virus type 1 fusion to virion maturation: a novel role of the gp41 cytoplasmic tail.

Retrovirus particles are not infectious until they undergo proteolytic maturation to form a functional core. Here we report a link between human immunodeficiency virus type 1 (HIV-1) core maturation and the ability of the virus to fuse with target cells. Using a recently developed reporter assay of HIV-1 virus-cell fusion, we show that immature HIV-1 particles are 5- to 10-fold less active for fusion with target cells than are mature virions. The fusion of mature and immature virions was rendered equivalent by truncating the gp41 cytoplasmic domain or by pseudotyping viruses with the glycoprotein of vesicular stomatitis virus. An analysis of a panel of mutants containing mutated cleavage sites indicated that HIV-1 fusion competence is activated by the cleavage of Gag at any site between the MA and NC segments and not as an indirect consequence of an altered core structure. These results suggest a mechanism by which binding of the gp41 cytoplasmic tail to Gag within immature HIV-1 particles inhibits Env conformational changes on the surface of the virion that are required for membrane fusion. This "inside-out" regulation of HIV-1 fusion could play an important role in the virus life cycle by preventing the entry of immature, noninfectious particles.

CCR5 antagonists: bicyclic isoxazolidines as conformationally constrained N-1-substituted pyrrolidines.

A series of CCR5 antagonists containing bicyclic isoxazolidines was generated through a nitrone mediated cycloaddition with olefins bearing the preferred pharmacophores previously described. Potent antagonists (3 and 16) were generated with enhanced affinity for the CCR5 receptor while maintaining antiviral activity against HIV.

1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists. Part 3: polar functionality and its effect on anti-HIV-1 activity.

Incorporation of acidic functional groups into a lead CCR5 antagonist identified from a targeted combinatorial library resulted in compounds with enhanced anti-HIV-1 activity and attenuated L-type calcium channel affinity.

Syntheses and SAR studies of 4-(heteroarylpiperdin-1-yl-methyl)-pyrrolidin-1-yl-acetic acid antagonists of the human CCR5 chemokine receptor.

Efforts toward the exploration of the title compounds as CCR5 antagonists are disclosed. The basis for such work stems from the fact that cellular proliferation of HIV-1 requires the cooperative assistance of both CCR5 and CD4 receptors. The synthesis and SAR of pyrrolidineacetic acid derivatives as CCR5 antagonists displaying potent binding and antiviral properties in a HeLa cell-based HIV-1 infectivity assay are discussed.

Antagonists of human CCR5 receptor containing 4-(pyrazolyl)piperidine side chains. Part 1: Discovery and SAR study of 4-pyrazolylpiperidine side chains.

Replacement of the flexible connecting chains between the piperidine moiety and an aromatic group in previous CCR5 antagonists with heterocycles, such as pyrazole and isoxazole, provided potent CCR5 antagonists with excellent anti-HIV-1 activity in vitro. SAR studies revealed optimal placement of an unsubstituted nitrogen atom in the heterocycle to be meta to the bond connected to the 4-position of piperidine. Truncation of a benzyl group to a phenyl group afforded compounds with dramatically improved oral bioavailability, albeit with reduced activity.

1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists. Part 4: synthesis of N-1 acidic functionality affording analogues with enhanced antiviral activity against HIV.

A series of alpha-(pyrrolidin-1-yl)acetic acids is presented as selective and potent antivirals against HIV. Several of the pyrrolidine zwitterions demonstrated reasonable in vitro properties, enhanced antiviral activities and improved pharmacokinetic profiles over pyrrolidine 1.