Jere E. Meredith

Integrins, adhesion and apoptosis.

Integrin-mediated adhesion to extracellular matrix proteins is required for survival of many cell types. This phenomenon appears to be a mechanism of tumour suppression and to participate in embryogenesis. Here, our current understanding of how integrin-dependent signals prevent apoptosis and implications of anchorage-dependent survival for development, physiology and pathology are discussed.

Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable γ-Secretase Inhibitor

During the course of our research efforts to develop a potent and selective γ-secretase inhibitor for the treatment of Alzheimers disease, we investigated a series of carboxamide-substituted sulfonamides. Optimization based on potency, Notch/amyloid-β precursor protein selectivity, and brain efficacy after oral dosing led to the discovery of 4 (BMS-708163). Compound 4 is a potent inhibitor of γ-secretase (Aβ40 IC50 = 0.30 nM), demonstrating a 193-fold selectivity against Notch. Oral administration of 4 significantly reduced Aβ40 levels for sustained periods in brain, plasma, and cerebrospinal fluid in rats and dogs.

Changing ligand specificities of αvβ1 and αvβ3 integrins by swapping a short diverse sequence of the β subunit

Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin αvβ3 recognizes fibrinogen, von Willebrand factor, and vitronectin, while αvβ1 does not. We studied the mechanisms for defining ligand specificity of these integrins by swapping the highly diverse sequences in the I domain-like structure of the β1 and β3 subunits. When the sequence CTSEQNC (residues 187–193) of β1 is replaced with the corresponding CYDMKTTC sequence of β3, the ligand specificity of αvβ1 is altered. The mutant (αvβ1–3-1), like αvβ3, recognizes fibrinogen, von Willebrand factor, and vitronectin (a gain-of-function effect). The αvβ1–3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin, suggesting that the mutant transduces intracellular signals on adhesion. The reciprocal β3–1-3 mutation blocks binding of αvβ3 to these multiple ligands and to LM609, a function-blocking anti-αvβ3 antibody. These results suggest that the highly divergent sequence is a key determinant of integrin ligand specificity. Also, the data support a recent hypothetical model of the I domain of β, in which the sequence is located in the ligand binding site.

Hyperdynamic Microtubules, Cognitive Deficits, and Pathology Are Improved in Tau Transgenic Mice with Low Doses of the Microtubule-Stabilizing Agent BMS-241027

Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimers disease (AD) and other tauopathies. It is hypothesized that the hyperphosphorylated, conformationally altered, and multimeric forms of tau lead to a disruption of MT stability; however, direct evidence is lacking in vivo. In this study, an in vivo stable isotope-mass spectrometric technique was used to measure the turnover, or dynamicity, of MTs in brains of living animals. We demonstrated an age-dependent increase in MT dynamics in two different tau transgenic mouse models, 3xTg and rTg4510. MT hyperdynamicity was dependent on tau expression, since a reduction of transgene expression with doxycycline reversed the MT changes. Treatment of rTg4510 mice with the epothilone, BMS-241027, also restored MT dynamics to baseline levels. In addition, MT stabilization with BMS-241027 had beneficial effects on Morris water maze deficits, tau pathology, and neurodegeneration. Interestingly, pathological and functional benefits of BMS-241027 were observed at doses that only partially reversed MT hyperdynamicity. Together, these data suggest that tau-mediated loss of MT stability may contribute to disease progression and that very low doses of BMS-241027 may be useful in the treatment of AD and other tauopathies.

The Amyloid-β Rise and γ-Secretase Inhibitor Potency Depend on the Level of Substrate Expression

The amyloid-β (Aβ) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-β precursor protein (APP) through consecutive proteolytic cleavages by β-site APP-cleaving enzyme and γ-secretase. Unexpectedly γ-secretase inhibitors can increase the secretion of Aβ peptides under some circumstances. This “Aβ rise” phenomenon, the same inhibitor causing an increase in Aβ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Aβ rise depends on the β-secretase-derived C-terminal fragment of APP (βCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Aβ, known as “p3,” formed by α-secretase cleavage, did not exhibit a rise. In addition to the Aβ rise, low βCTF or C99 expression decreased γ-secretase inhibitor potency. This “potency shift” may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, γ-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Aβ under conditions of low substrate expression. The Aβ rise was also observed in rat brain after dosing with the γ-secretase inhibitor BMS-299897. The Aβ rise and potency shift are therefore relevant factors in the development of γ-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Aβ rise, including the “incomplete processing” and endocytic models, are discussed.

Cleavage of the Cytoplasmic Domain of the Integrin β3 Subunit during Endothelial Cell Apoptosis

In this study, we report that the cytoplasmic domain of the integrin β3 subunit is a target for limited proteolysis during apoptosis of human umbilical vein endothelial cells. Calpain inhibitors inhibited the cleavage of the β3 cytoplasmic domain, indicating that calpain is required. Calpain-mediated proteolysis of fodrin was also detected, indicating that calpain is activated during endothelial cell apoptosis. A phosphatase inhibitor, sodium orthovanadate, inhibited endothelial cell apoptosis and cleavage β3, suggesting that protein dephosphorylation preceded integrin cleavage in the apoptosis signaling pathway. β3 cleavage was observed in cells that were viable, suggesting that it is an early event and not the consequence of post-death proteolysis. The extent of β3 cleavage correlated with a loss in the capacity of cells to reattach to matrix proteins. Loss of reattachment capacity during apoptosis was significantly retarded by a calpain inhibitor. As the β3cytoplasmic domain is required for integrin signaling and interaction with the cytoskeleton, our results suggest that cleavage in the β3 cytoplasmic domain by calpain or a calpain-like protease negatively regulates integrin-mediated adhesion, signaling, and cytoskeleton association.

P-Glycoprotein Efflux and Other Factors Limit Brain Amyloid β Reduction by β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitors in Mice

Alzheimers disease (AD) is a progressive neurodegenerative disease. Amyloid β (Aβ) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Aβ. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Aβ formation in cultured cells with IC50 values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Aβ peptides by mass spectrometry showed that these inhibitors decreased Aβ by inhibiting BACE1. An assay for Aβ1–40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Aβ1–40, but not brain Aβ1–40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Aβ1–40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Aβ1–40 and brain Aβ1–40 dose responses for these three compounds revealed differences in relative ED50 values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

Acyl guanidine inhibitors of β-secretase (BACE-1): optimization of a micromolar hit to a nanomolar lead via iterative solid- and solution-phase library synthesis.

This report describes the discovery and optimization of a BACE-1 inhibitor series containing an unusual acyl guanidine chemotype that was originally synthesized as part of a 6041-membered solid-phase library. The synthesis of multiple follow-up solid- and solution-phase libraries facilitated the optimization of the original micromolar hit into a single-digit nanomolar BACE-1 inhibitor in both radioligand binding and cell-based functional assay formats. The X-ray structure of representative inhibitors bound to BACE-1 revealed a number of key ligand:protein interactions, including a hydrogen bond between the side chain amide of flap residue Gln73 and the acyl guanidine carbonyl group, and a cation-π interaction between Arg235 and the isothiazole 4-methoxyphenyl substituent. Following subcutaneous administration in rats, an acyl guanidine inhibitor with single-digit nanomolar activity in cells afforded good plasma exposures and a dose-dependent reduction in plasma Aβ levels, but poor brain exposure was observed (likely due to Pgp-mediated efflux), and significant reductions in brain Aβ levels were not obtained.

Mutational analysis of cell cycle inhibition by integrin β(1C)

Integrin β1C is an alternatively spliced cytoplasmic variant of the β1 subunit that potently inhibits cell cycle progression. In this study, we analyzed the requirements for growth suppression by β1C. A chimera containing the extracellular/transmembrane domain of the Tac subunit of the human interleukin 2 receptor (gp55) fused to the cytoplasmic domain of β1C (residues 732–805) strongly inhibited growth in mouse 10T1/2 cells even at low expression levels, whereas chimeras containing the β1A, β1B, β1D, β3, and β5 cytoplasmic domains had weak and variable effects. The β1C cytoplasmic domain is composed of a membrane proximal region (732–757) common to all β1variants and a COOH-terminal 48-amino acid domain (758–805) unique to β1C. The β1C-specific domain (758–805) was sufficient to block cell growth even when expressed as a soluble cytoplasmic green fluorescent protein fusion protein. These results indicate that growth inhibition by β1C does not require the intact receptor and can function in the absence of membrane targeting. Analysis of deletions within the β1C-specific domain showed that the 18-amino acid sequence 775–792 is both necessary and sufficient for maximal growth inhibition, although the 13 COOH-terminal residues (793–805) also had weak activity. Finally, β1C is known to be induced in endothelial cells in response to tumor necrosis factor and is down-regulated in prostate epithelial cells after transformation. The green fluorescent protein/β1C (758–805) chimera blocked growth in the human endothelial cell line EV304 and in the transformed prostate epithelial cell line DU145, consistent with a role for β1C as a growth inhibitor in vivo.

An activated rac mutant functions as a dominant negative for membrane ruffling

Previous studies have shown that point mutations in the effector domain of Rac1 block specific downstream pathways such as PAK, JNK/SAPK kinases and membrane ruffling. Specifically, the F37A mutation, made in a constitutively activated Q61L background, activates PAK but fails to induce membrane ruffles. We now show that Q61L/F37A Rac not only fails to induce ruffling but potently blocks membrane ruffling induced by serum or PDGF. In the presence of serum, cells do extend filopodia, suggesting that this mutant only blocks a subset of the effectors that induce cytoskeletal reorganization. At later times, this rac mutant induces membrane blebbing, but not apoptosis. These results show that Q61L/F37A Rac, is constitutively activated with respect to PAK activation but functions as a dominant negative for another pathway, membrane ruffling. That an effector domain point mutant can simultaneously function as a dominant negative and dominant positive for different pathways implies that effects of these variants on cell functions must be interpreted with caution.