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Summary Metabotropic glutamate receptor 5 (mGluR5) has been implicated in Alzheimer’s disease (AD) pathology. We sought to understand whether mGluR5’s role in AD requires glutamate signaling. We used a potent mGluR5 silent allosteric modulator (SAM, BMS-984923) to separate its well-known physiological role in glutamate signaling from a pathological role in mediating amyloid-β oligomer (Aβo) action. Binding of the SAM to mGluR5 does not change glutamate signaling but strongly reduces mGluR5 interaction with cellular prion protein (PrP C ) bound to Aβo. The SAM compound prevents Aβo-induced signal transduction in brain slices and in an AD transgenic mouse model, the APPswe/PS1ΔE9 strain. Critically, 4 weeks of SAM treatment rescues memory deficits and synaptic depletion in the APPswe/PS1ΔE9 transgenic mouse brain. Our data show that mGluR5’s role in Aβo-dependent AD phenotypes is separate from its role in glutamate signaling and silent allosteric modulation of mGluR5 has promise as a disease-modifying AD intervention with a broad therapeutic window. [ABSTRACT FROM AUTHOR]

Abstract: To selectively target doxorubicin (Dox) to tumor tissue and thereby improve the therapeutic index and/or efficacy of Dox, matrix metalloproteinases (MMP) activated peptide–Dox prodrugs were designed and synthesized by coupling MMP-cleavable peptides to Dox. Preferred conjugates were good substrates for MMPs, poor substrates for neprilysin, an off-target proteinase, and stable in blood ex vivo. When administered to mice with HT1080 xenografts, conjugates, such as 19, preferentially released Dox in tumor relative to heart tissue and prevented tumor growth with less marrow toxicity than Dox. [Copyright &y& Elsevier]

Alzheimer's disease is associated with the accumulation of amyloid-β (Aβ) in the brain. In particular, the 42-amino acid form, Aβ1-42, is thought to play a key role in the disease. It is therefore of interest that diverse compounds, known as γ-secretase modulators (GSM), can selectively decrease Aβ1-42 production without inhibiting the production of other forms of Aβ. Here we describe the novel discovery of synergistic inhibition of Aβ by certain combinations of GSMs. Cell cultures were treated with pairwise combinations of GSMs to determine how Aβ peptide production was affected. Analysis of isobolograms and calculation of the combination index showed that BMS-869780 and GSM-2 were highly synergistic. Additional combinations of GSMs revealed that inhibition of Aβ occurred only when one GSM was of the “acid GSM” structural class and the other was of the “non-acid GSM” class. A total of 15 representative acid/non-acid GSM combinations were shown to inhibit Aβ production, whereas 10 pairwise combinations containing two acid GSMs or containing two non-acid GSMs did not inhibit Aβ. We also discovered that lasalocid, a natural product, is a potent GSM. Lasalocid is unique in that it did not synergize with other GSMs. Synergism did not translate in vivo perhaps because of biochemical differences between the cell culture model and brain. These findings reinforce the pharmacological differences between different structural classes of GSMs, and may help to exploit the potential of γ-secretase as a drug target. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This Letter describes the synthesis and structure–activity relationships of a series of furo[2,3- d ][1,3]thiazinamine BACE1 inhibitors. The co-crystal structure of a representative thiazinamine 2e bound with the BACE1 active site displayed a binding mode driven by interactions with the catalytic aspartate dyad and engagement of the biaryl amide toward the S1 and S3 pockets. This work indicates that furo[2,3- d ]thiazine can serve as a viable bioisostere of the known furo[3,4- d ]thiazine. [ABSTRACT FROM AUTHOR]

Abstract: Background: γ-Secretase inhibitors (GSIs) are being investigated for their potential to modify the progression of Alzheimer disease based on their ability to regulate amyloid-β (Aβ) accumulation. BMS-708163 (avagacestat) is an oral GSI designed for selective inhibition of Aβ synthesis currently in development for the treatment of mild to moderate and predementia AD. In addition to the desired effect on Aβ synthesis, GSIs affect Notch processing, which is thought to mediate some toxic adverse effects reported with this drug class. Avagacestat produced up to 190-fold greater selectivity for Aβ synthesis than Notch processing in preclinical studies and may therefore produce less toxic adverse events than other less selective compounds. Presented here are the results of the first in-human study for this new GSI compound. Objective: The goal of this study was to assess the tolerability profile, pharmacokinetic properties, and effects on pharmacodynamic markers (Aβ, trefoil factor family 3 protein, dual specificity phosphatase 6, and hairy and enhancer of split-1) of single, oral doses of avagacestat in healthy, young, male volunteers. Methods: This was a multicenter, randomized, double-blind, placebo-controlled, single-ascending dose study in 8 healthy young men (age, 18–45 years) per dosing panel. Each study participant was randomized to receive a single dose of placebo (n = 2) or avagacestat (n = 6 for each dose) as an oral solution in 1 of 9 sequential dose panels (0.3, 1.5, 5, 15, 50, 100, 200, 400, and 800 mg). For determination of avagacestat, blood samples were obtained before dosing and for up to 144 hours after dosing. For participants in the 800-mg avagacestat dose panel, additional samples were obtained at 216, 312, and 648 hours. For 40–amino acid isoform of Aβ (Aβ1–40) assessment, plasma samples were collected before avagacestat administration and up to 72 hours after dosing. Results: Avagacestat concentrations peaked quickly after oral administration and then had a biphasic decrease in concentrations with a prolonged terminal phase. Exposures were proportional with doses up to 200 mg. Avagacestat was well tolerated at single oral doses up to 800 mg, with a biphasic effect on plasma Aβ1–40. Adverse events were predominately mild to moderate in severity with no evidence of dose dependence up to 200 mg. Conclusions: Results from this single-ascending dose study suggest that avagacestat was tolerated at a single-dose range of 0.3 to 800 mg and suitable for further clinical development. ClinicalTrials.gov identifier: NCT01454115. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: A series of N-((2S,3R)-1-(3,5-difluorophenyl)-3-hydroxy-4-(3-methoxybenzylamino)-butan-2-yl)benzamides has been synthesized as BACE inhibitors. A variety of P2 and P3 substituents has been explored, and these efforts have culminated in the identification of several 1,3,5-trisubstituted phenylcarboxyamides with potent BACE inhibitory activity. [Copyright &y& Elsevier]

Abstract: In this report we describe a novel radioligand, [3H](S)-2-((S)-3-Acetylamino-3-sec-butyl-2-oxo-pyrrolidin-1-yl)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-methoxy-benzylamino)-propyl]-4-phenyl-butyramide ([3H]BMS-599240), that exhibits robust specific binding in homogenates from cell cultures overexpressing β-site amyloid precursor protein cleaving enzyme-1 (BACE1). Radioligand binding exhibited high affinity, K d =2 nM, commensurate with its inhibitory potency against BACE1. Inhibition of radioligand binding in the presence of a range of different BACE1 inhibitors exhibited the same rank order of potency as for inhibition of BACE1 enzymatic activity. BACE1-dependent binding of the radioligand was also demonstrated in mouse brain homogenates, where genetic ablation of BACE1 eliminated high affinity binding. Thus, the radioligand [3H]BMS-599240 is a novel tool potentially useful for evaluation of BACE1 enzyme in biological samples, and for evaluation of inhibitor binding to BACE1. [Copyright &y& Elsevier]

The enzyme γ-secretase has long been considered a potential pharmaceutical target for Alzheimer disease. Presenilin (the catalytic subunit of γ-secretase) and signal peptide peptidase (SPP) are related transmembrane aspartyl proteases that cleave transmembrane substrates. SPP and γ-secretase are pharmacologically similar in that they are targeted by many of the same small molecules, including transition state analogs, non-transition state inhibitors, and amyloid β-peptide modulators. One difference between presenilin and SPP is that the proteolytic activity of presenilin functions only within a multisubunit complex, whereas SPP requires no additional protein cofactors for activity. In this study, γ-secretase inhibitor radioligands were used to evaluate SPP and γ-secretase inhibitor binding pharmacology. We found that the SPP enzyme exhibited distinct binding sites for transition state analogs, non-transition state inhibitors, and the nonsteroidal anti-inflammatory drug sulindac sulfide, analogous to those reported previously for γ-secretase. In the course of this study, cultured cells were found to contain an abundance of SPP binding activity, most likely contributed by several of the SPP family proteins. The number of SPP binding sites was in excess of γ-secretase binding sites, making it essential to use selective radioligands for evaluation of γ-secretase binding under these conditions. This study provides further support for the idea that SPP is a useful model of inhibitory mechanisms and structure in the SPP/presenilin protein family. [ABSTRACT FROM AUTHOR]

The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) ↓ (L/Y)(T/V/M/R)(R/K), where ↓ denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E ↓ LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest k[sub cat]/K[sub m] values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower k[sub cat]/K[sub m] values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer. [ABSTRACT FROM AUTHOR]

In an effort to improve physical properties by introducing polar functionality into the bicyclic pyrimidine gamma-secretase modulator (GSM) clinical candidate BMS-932481, we prepared several oxidative products of BMS-932481. Among the analogs that were prepared, the C-5 alcohol 3 was identified as the predominant metabolite of BMS-932481 found in rat and human liver microsomes. Alcohol 3 was determined to be chemically unstable, leading to the hypothesis that 3 may lead to the production of reactive species both in vitro and in vivo. [ABSTRACT FROM AUTHOR]