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Cerebrospinal fluid (CSF) is a complex fluid filling the ventricular system and surrounding the brain and spinal cord. Although the bulk of CSF is created by the choroid plexus, a significant fraction derives from the interstitial fluid in the brain and spinal cord parenchyma. For this reason, CSF can often be used as a source of pharmacodynamic and prognostic biomarkers to reflect biochemical changes occurring within the brain. For instance, CSF biomarkers can be used to diagnose and track progression of disease as well as understand pharmacokinetic and pharmacodynamic relationships in clinical trials. To facilitate the use of these biomarkers in humans, studies in preclinical species are often valuable. This review summarizes methods for preclinical CSF collection for biomarkers from mice, rats, and nonhuman primates. In addition, dosing directly into CSF is increasingly being used to improve drug levels in the brain. Therefore, this review also summarizes the state of the art in CSF dosing in these preclinical species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In Alzheimer's disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.

Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer's disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid β-peptide (Aβ), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aβ and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aβ and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aβ levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aβ40 and Aβ42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aβ40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aβ levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

A series of (N-benzyl-N-phenylsulfonamido)alkyl amides were developed from classic and parallel synthesis strategies. Compounds with good in vitro and in vivo γ-secretase activity were identified and described., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimer's 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 synthesis, evaluation, and structure-activity relationships of a class of γ-lactam 1,3-diaminopropan-2-ol transition-state isostere inhibitors of BACE are discussed. Two strategies for optimizing lead compound 1a are presented. Reducing the overall size of the inhibitors resulted in the identification of γ-lactam 1i, whereas the introduction of conformational constraint on the prime-side of the inhibitor generated compounds such as the 3-hydroxypyrrolidine inhibitor 28n. The full in vivo profile of 1i in rats and 28n in Tg 2576 mice is presented., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The synthesis, evaluation, and structure-activity relationships of a set of related constrained diaminopropane inhibitors of BACE-1 are described. The full in vivo profile of an optimized inhibitor in both normal and P-gp deficient mice is compared with data generated in normal rats., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Heterocyclic replacement of the isophthalamide phenyl ring in hydroxyethylamine (HEA) BACE-1 inhibitors was explored. A variety of indole-1,3-dicarboxamide HEAs exhibited potent BACE-1 enzyme inhibition, but displayed poor cellular activity. Improvements in cellular activity and aspartic protease selectivity were observed for 7-azaindole-1,3-dicarboxamide HEAs. A methylprolinol-bearing derivative (10n) demonstrated robust reductions in rat plasma Aβ levels, but did not lower rat brain Aβ due to poor central exposure. The same analog exhibited a high efflux ratio in a bidirectional Caco-2 assay and was likely a substrate of the efflux transporter P-glycoprotein. X-ray crystal structures are reported for two indole HEAs in complex with BACE-1., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Levels of tau in cerebrospinal fluid (CSF) are elevated in Alzheimer's disease (AD) patients. It is believed this elevation is related to the tau pathology and neurodegeneration observed in AD, but not all tauopathies have increased CSF tau. There has been little pre-clinical work to investigate mechanisms of increased CSF tau due to the difficulty in collecting CSF samples from mice, the most commonly used pre-clinical models. We developed methods to collect CSF from mice without contamination from tau in brain tissue, which is approximately 50,000 fold more abundant in brain than CSF. Using these methods, we measured CSF tau from 3xTg, Tg4510, and Tau Alone transgenic mice. All three lines of mice showed age-dependent increases in CSF tau. They varied in phenotype from undetectable to severe tau pathology and neurodegeneration, suggesting that degenerating neurons are unlikely to be the only source of pathologic CSF tau. Overall, CSF tau levels mirrored expression levels and changes of tau in the brain, but they did not always correlate exactly. CSF tau was often more sensitive to changes in brain transgene expression and pathology. In addition, we also developed ELISA assays specific to different regions of the tau protein. We used these assays to provide evidence that CSF tau exists as fragments, with little intact C-terminus and partial loss of the N-terminus. Taken together, these assays and mouse models may be used to facilitate a deeper understanding of CSF tau in neurodegenerative disease.

During the course of our research efforts to develop a potent and selective γ-secretase inhibitor for the treatment of Alzheimer's 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.

BMS-299897 is a gamma-secretase inhibitor that was effective in reducing amyloid beta-peptide (A beta) in transgenic mice and guinea pigs. Therefore, pharmacokinetic and drug metabolism studies were conducted in animals to support its clinical development. The compound appeared to have low to intermediate total body clearance and was orally bioavailable (24-100%). The oral absorption of BMS-299897 from solid dosage forms appeared to be dissolution rate-limited. BMS-299897 was distributed into extravascular space (V(ss) >or= 1.3 l kg(-1)), including brain (brain-to-plasma ratio = 0.13-0.50). BMS-299897 appeared to be a P-glycoprotein (P-gp) substrate as the brain-to-plasma ratio was two-fold higher in the mdr1a knockout mouse as compared with the wild-type. Apparent autoinduction by BMS-299897 was observed in murine and rat efficacy and toxicity studies. In vitro, BMS-299897 was a weaker inducer of cytochrome P450 3A4 (CYP3A4) and a weaker transactivator of human pregnane X receptor (hPXR) as compared with rifampicin. Induction of human UGT1A and UGT2B was evaluated in primary human hepatocytes, but the results were inconclusive. A low potential for autoinduction in humans was predicted at a clinical dose of 250 mg and the prediction was consistent with the findings from a clinical multiple-dose study with BMS-299897 in probable Alzheimer's patients.

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

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Amyloid beta (Abeta) 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, beta-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 Abeta. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Abeta formation in cultured cells with IC(50) values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Abeta peptides by mass spectrometry showed that these inhibitors decreased Abeta by inhibiting BACE1. An assay for Abeta1-40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Abeta1-40, but not brain Abeta1-40, 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 Abeta1-40 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 Abeta1-40 and brain Abeta1-40 dose responses for these three compounds revealed differences in relative ED(50) values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

Therapeutic approaches for Alzheimer's disease (AD) are guided by four disease characteristics: amyloid plaques, neurofibrillar tangles (NFT), neurodegeneration, and dementia. Amyloid plaques are composed largely of 4 kDa beta-amyloid (Abeta) peptides, with the more amyloidogenic, 42 amino acid form (Abeta42) as the primary species. Because multiple, rare mutations that cause early-onset, familial AD lead to increased production or aggregation of Abeta42, amyloid therapeutics aim to reduce the amount of toxic Abeta42 aggregates. Amyloid-based therapies include gamma-secretase inhibitors and modulators, BACE inhibitors, aggregation blockers, catabolism inducers, and anti-Abeta biologics. Tangles are composed of paired helical filaments of hyperphosphorylated tau protein. Tau-based therapeutics include kinase inhibitors, microtubule stabilizers, and catabolism inducers. Therapeutic strategies for neurodegeneration target multiple mechanisms, including excitotoxicity, mitochondrial dysfunction, oxidative damage, and inflammation or stimulation of neuronal viability. Although not disease modifying, cognition enhancers are important to treat the symptom of dementia. Strategies for cognition enhancement include cholinesterase inhibitors, and other approaches to enhance the signaling of cholinergic and glutamatergic neurons. In summary, plaques, tangles, neurodegeneration and dementia guide the development of multiple therapeutic approaches for AD and are the subject of this review.

The synthesis and gamma-secretase inhibition data for a series of carbamate-appended N-alkylsulfonamides are described. Carbamate 54 was found to significantly reduce brain Abeta in transgenic mice. 54 was also found to possess markedly improved brain levels in transgenic mice compared to previously disclosed 1 and 2.

The synthesis and gamma-secretase inhibition data for a series of nitrogen-appended N-alkylsulfonamides (11-47) are described. Inhibition of brain Abeta in transgenic mice was demonstrated by two of these compounds (23 and 44).

A series of amino-caprolactam sulfonamides were developed from a screening hit. Compounds with good in vitro and in vivo gamma-secretase activity are reported.

Reduction of brain beta-amyloid peptide (Abeta) synthesis by gamma-secretase inhibitors is a promising approach for the treatment of Alzheimer's disease. However, measurement of central pharmacodynamic effects in the Alzheimer's disease patient will be a challenge. Determination of drug occupancy may facilitate the analysis of efficacy of gamma-secretase inhibitors in a clinical setting. In this study, the relationship of gamma-secretase site occupancy and brain Abeta40 reduction by gamma-secretase inhibitors was examined in Tg2576 mice. [3H](2R,3S)-2-Isobutyl-N1-((S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-3-propylsuccinamide (IN973) was used as a gamma-secretase radioligand, since it has been shown to bind to gamma-secretase in rat, rhesus, and human brains with high affinity and specificity. We extended these findings by showing that [3H]IN973 bound to gamma-secretase in Tg2576 brains with an affinity, specificity, and regional localization very similar to the other species. To quantify gamma-secretase occupancy by gamma-secretase inhibitors, an ex vivo binding assay was developed using [3H]IN973 and frozen brain sections from drug-treated mice. Gamma-secretase occupancy and brain Abeta40 reduction were found to be highly correlated in animals dosed with either 2-[(1R)-1-[[4-chlorophenyl)-sulfonyl](2,5-difluorophenyl)amino] ethyl]-5-fluoro-benzenepropanoic acid (BMS-299897) or (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) over a wide range of doses and times postdose, with the exception of the earliest times postdose. This lag in Abeta40 response to gamma-secretase inhibition is probably related to the delayed clearance of previously produced Abeta40. The excellent correlation between brain Abeta40 and gamma-secretase occupancy suggests that a positron emission tomography ligand for gamma-secretase could be a valuable biomarker to determine whether gamma-secretase inhibitors bind to their target in humans.

A series of N-alkylbenzenesulfonamides were developed from a high throughput screening hit. Classic and parallel synthesis strategies were employed to produce compounds with good in vitro and in vivo gamma-secretase activity.

We report on the design of benzodiazepinones as peptidomimetics at the carboxy terminus of hydroxyamides. Structure-activity relationships of diazepinones were investigated and orally active gamma-secretase inhibitors were synthesized. Active metabolites contributing to Abeta reduction were identified by analysis of plasma samples from Tg2576 mice. In particular, (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) was identified with an acceptable pharmacodynamic and pharmacokinetic profile. Chronic dosing of BMS-433796 in Tg2576 mice suggested a narrow therapeutic window and Notch-mediated toxicity at higher doses.

Recent findings suggest that Alzheimer's dementia may be mediated by soluble beta amyloid (Abeta) more than the deposits of aggregated, insoluble Abeta, and vulnerability to cognitive deficits after scopolamine challenge may help identify AD even in patients that are still pre-symptomatic. The objectives of the present experiments were to determine if vulnerability to cognitive deficits after scopolamine challenge is related to levels of soluble Abeta, and if levels of soluble Abeta are more closely related to cognitive deficits than levels of insoluble Abeta, even in aged, transgenic mice, after they have developed very high levels of insoluble Abeta. Aged F-344 rats and young mice over-expressing the Swedish mutation in the human amyloid precursor protein (APPsw; Tg2576+) had elevated levels of soluble Abeta, and were more vulnerable to scopolamine challenge in the Morris water maze (MWM), relative to young rats and Tg2576- mice; but, among individual animals, higher levels of soluble Abeta were not correlated with vulnerability to scopolamine. On the other hand, in aged Tg2576+ mice, cognitive deficits were related to levels of soluble Abeta, not insoluble Abeta, despite the fact that the levels of insoluble Abeta were thousands of times higher than the levels of soluble Abeta. The results of the present experiments suggest that vulnerability to cognitive deficits after scopolamine challenge is not related to elevated levels of soluble Abeta, but that high levels of soluble Abeta are more closely correlated with cognitive deficits than the amount insoluble Abeta, even after large amounts of aggregated, insoluble Abeta have been deposited.

The amyloid hypothesis, which states that beta-amyloid (Abeta) aggregates cause the onset and progression of Alzheimer's disease (AD), is a leading proposal to explain AD aetiology. Based on this hypothesis, compounds that inhibit gamma-secretase, one of the enzymes responsible for forming Abeta, are potential therapeutics for AD. Preclinical studies clearly establish that gamma-secretase inhibitors can reduce brain Abeta in rodent models. The initial investigation of the effects of a gamma-secretase inhibitor on Abeta-induced cognitive deficits in transgenic mice showed that modest Abeta reductions (15-30%) are sufficient to reverse Abeta-induced cognitive deficits in Tg2576 mice. Extending these studies to other gamma-secretase inhibitors and other models with Abeta-induced cognitive deficits will be important. Unfortunately, gamma-secretase inhibitors also cause abnormalities in the gastrointestinal tract, thymus and spleen in rodents. These changes likely result from inhibition of Notch cleavage, a transmembrane receptor involved in regulating cell-fate decisions. Two recent studies in rodents suggest that Abeta reduction using gamma-secretase inhibitors can be partially separated from Notch inhibition. Given the uncertain Abeta reduction target and the potential for mechanism-based toxicity, biomarkers for efficacy and toxicity would be helpful in clinical trials. The first report of gamma-secretase inhibitors in clinical trials was recently published. In this study, LY-450139 reduced plasma Abeta, but not cerebrospinal fluid Abeta. Taken together, the results of studies to date suggest that gamma-secretase inhibitors have the potential to address a large unmet medical need if the technical challenges can be overcome.

gamma-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer's disease (AD). gamma-Secretase inhibitors reduce brain beta-amyloid peptide (Abeta), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human beta-amyloid precursor protein (APP) are valuable models to examine the dynamics of Abeta changes with gamma-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a gamma-secretase inhibitor, showed dose- and time dependent reductions of Abeta in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF Abeta levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of Abeta in plaques showed reductions in CSF Abeta in the absence of measurable changes in plaque Abeta in the brain after up to 2 weeks of treatment. Hence, CSF Abeta levels were a valuable measure of gamma-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8(+) thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for gamma-secretase inhibitors to reduce brain Abeta without causing Notch-mediated toxicity.

2,3-Benzodiazepin-1,4-diones were designed as peptidomimetics at the carboxy terminus of hydroxyamides. Inhibition of brain Abeta production was improved by one of the compounds containing constrained modification.

Using a cell-based assay, we have identified optimal residues and key recognition elements necessary for inhibition of gamma-secretase. An (S)-hydroxy group or 3,5-difluorophenylacetyl group at the amino terminus and N-methyltertiary amide moiety at the carboxy terminus provided potent gamma-secretase inhibitors with an IC(50) <10 nM.

Antagonists of serotonin 6 (5-HT6) receptors have been reported to enhance cognition in animal models of learning, although this finding has not been universal. We have assessed the therapeutic potential of the specific 5-HT6 receptor antagonists 4-amino-N-(2,6-bis-methylamino-pyrimidin-4-yl)-benzenesulfonamide (Ro 04-6790) and 5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide (SB-271046) in rodent models of cognitive function. Although mice express the 5-HT6 receptor and the function of this receptor has been investigated in mice, all reports of activity with 5-HT6 receptor antagonists have used rat models. In the present study, receptor binding revealed that the pharmacological properties of the mouse receptor are different from the rat and human receptor: Ro 04-6790 does not bind to the mouse 5-HT6 receptor, so all in vivo testing included in the present report was conducted in rats. We replicated previous reports that 5-HT6 receptor antagonists produce a stretching syndrome previously shown to be mediated through cholinergic mechanisms, but Ro 04-6790 and SB-271046 failed to attenuate scopolamine-induced deficits in a test of contextual fear conditioning. We also failed to replicate the significant effects reported previously in both an autoshaping task and in a version of the Morris water maze. The results of our experiments are not consistent with previous reports that suggested that 5-HT6 antagonists might have therapeutic potential for cognitive disorders.

The phenotypic and functional characteristics of activated T cells and recruited unactivated T cells at an inflammatory site were examined using a V beta 4+ myelin basic protein-specific T cell clone in a passively transferred model of experimental allergic encephalomyelitis. A high percentage of the T cells isolated from the central nervous system (CNS) were V beta 4+. This population exhibited the characteristics of activated T cells based on the proportion of cells in the blast state, their ability to proliferate in response to IL-2 or CNS Ag, and their expression of activation/memory cell markers. Activated V beta 4+ T cells were also observed in the periphery. Large numbers of V beta 4- T cells, which are entirely host-recruited, were also found in the CNS, where they demonstrated the properties of memory cells. There were differences in adhesion molecule expression between CNS V beta 4+ T cells and peripheral V beta 4+ T cells, although both populations were in activated state. V beta 4+ T cells at the site of Ag expression (the spinal cord) demonstrated higher levels of LFA-1 and CD44, but lower levels of VLA-4 and intercellular adhesion molecule-1, than did V beta 4+ T cells in the spleen. In contrast, the levels of all of these adhesion molecules on recruited V beta 4- T cells were higher in the CNS than in the periphery. This experimental model allows the detailed characterization of different T cell populations isolated from the same inflammatory site.

Anti-tumor necrosis factor (TNF) antibodies inhibit passively transferred experimental allergic encephalomyelitis (EAE) in SJL mice. The possibility that this occurs through interference in TNF's upregulation of endothelial cell adhesion molecules was investigated. Expression of both vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on spinal cord vessels increased during EAE. The upregulation of VCAM-1 was markedly reduced or prevented by anti-TNF treatment. Leukocytic infiltration was 15-fold lower in anti-TNF-treated than diseased animals. Spinal cord endothelial expression of VCAM-1, though not ICAM-1 or fibronectin, positively correlated with the extent of T cell, B cell or monocyte infiltration in each animal.

Protein carboxylmethyltransferase (PCM) is an enzyme whose function in eucaryotic cells remains controversial. Early studies suggested that protein carboxylmethylation subserved a regulatory, post-translational role in such diverse processes as secretion, neuronal receptor function, chemotaxis, and cellular differentiation. Later work strongly supported a totally unrelated role for this enzyme, i.e., the repair of spontaneously altered aspartate residues in cellular proteins. More recent evidence, however, suggests that a distinct, membrane-associated PCM catalyzes the methylation of alpha-carboxyl groups of C-terminal cysteines on discrete proteins. In view of these recent investigations, the data supporting a regulatory role for PCM are critically discussed and re-evaluated. There now appears to be compelling evidence that PCM(s) subserves both repair and regulatory functions in eucaryotic cells, catalyzing post-translational modifications of proteins involved in cell division, hormonal secretion, calmodulin-associated events and the interaction of guanyl nucleotide-linked proteins with the cell membrane.

Protein carboxylmethyltransferase has been proposed to play a role in the regulation of neuroblastoma differentiation (Kloog et al., 1983). When we investigated this hypothesis further, different results for methyl ester formation were obtained when measured in acid-precipitated proteins and in proteins separated by acidic polyacrylamide gel electrophoresis, following the incubation of intact neuroblastoma cells with [3H]methionine. These unexpected findings led to the development of a modified assay using S-[3H]-adenosylmethionine as the radiolabeled precursor for quantitating carboxyl methylation in intact cells. Data obtained from either acid-precipitated proteins or those separated on an electrophoresis gel following S-[3H]adenosylmethionine incubation directly correlated with data obtained from proteins separated by electrophoresis following [3H]methionine incubation. Using each of the three methods, an approximately twofold increase in the carboxyl methylation of cellular proteins was detected in neuroblastoma cells differentiated by reducing the serum concentration from 10 to 0.5%, but not in those cells differentiated with either 5 mM hexamethylene bisacetamide or 2% dimethyl sulfoxide. The finding that all detectable methyl acceptor proteins are increasingly methylated following 0.5% serum treatment and that this modification is substoichiometric suggests that protein carboxyl methylation is not an essential component of the differentiation process in neuroblastoma cells.

The noncompetitive S-adenosylhomocysteine (AdoHcy) hydrolase antagonist adenosine dialdehyde (AD) has been shown to suppress the growth of cultured C-1300 murine neuroblastoma (MNB) cells. The enzymatic sites at which AD and other nucleoside analogues exert their cytotoxic effects have been postulated to include protein carboxylmethyltransferase (PCM), AdoHcy hydrolase, and ribonucleotide reductase. AD (10(-5) M) increased PCM activity 350% in suspensions prepared from disrupted cells after 72 h of drug exposure; in contrast, 3-deazaadenosine (10(-4) M) increased PCM activity 57%, whereas AdoHcy and sinefungin had no effect. When intact MNB cells were incubated with AD for varying time periods up to 72 h and then pulse labeled with the S-adenosylmethionine precursor L-[3H]-methionine, AD (10(-8) to 5 X 10(-6) M) produced a concentration-dependent inhibition of protein carboxylmethylation which persisted for up to 6 h. Following extended periods of AD treatment (48 to 72 h), AD (10(-6) to 10(-5) M) produced a 250% increment in protein carboxylmethylation, similar in magnitude to that observed in disrupted cell preparations. This increase in carboxylmethylation was observed at timepoints when AdoHcy hydrolase activity remained suppressed. The inhibitory effect of AD on AdoHcy hydrolase activity was maximal within 4 h and still apparent after 72 h of incubation. In contrast, AD treatment had no effect on the ribonucleotide reductase activity of MNB cells. These data suggest that the cytotoxic effect of AD on MNB cells results directly from its inhibition of AdoHcy hydrolase activity and indirectly through its suppression of methyltransferase enzyme systems. The potential linkage between the observed long-term elevations in PCM activity and AD-induced cytotoxicity remains to be defined.