The presenilin containing gamma-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. gamma-Secretase catalyzes the final step in the generation of Abeta(40) and Abeta(42) peptides from APP. Amyloid beta-peptides (Abeta peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Abeta peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide gamma-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Abeta production with low nanomolar potency in cellular and cell-free assays of gamma-secretase function, and displaces a tritiated analog of GSI-953 from enriched gamma-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Abeta levels, and a reversal of contextual fear-conditioning deficits that are correlated with Abeta load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Abeta levels, confirming pharmacodynamic activity of GSI-953 in humans.

Development of a large-scale enantioselective synthesis of a lead compound containing a 3-aryl-3-trifluoromethyl-2-aminopropanol core is described. A single isomer of 3,3-disubstituted acrylic acid derivative was prepared via Perkin condensation or Horner-Wadsworth-Emmons olefination, followed by hydrolysis. The acid was converted to a chiral acryloxazolidinone derivative. Hydrogenation of the latter on Pd/C in the presence of MgBr(2) proceeded via a chelation-controlled conformation to yield the desired isomer with high selectivity. Subsequent Evans azidation, hydrogenation, reductive cleavage of the chiral auxiliary, and sulfonylation afforded the target compound as a single isomer in high overall yield.

gamma-Secretase inhibitors have been shown to reduce the production of beta-amyloid, a component of the plaques that are found in brains of patients with Alzheimer's disease. A novel series of heterocyclic sulfonamide gamma-secretase inhibitors that reduce beta-amyloid levels in cells is reported. Several examples of compounds within this series demonstrate a higher propensity to inhibit the processing of amyloid precursor protein compared to Notch, an alternative gamma-secretase substrate.

Gamma secretase is responsible for the intramembraneous cleavage of the Alzheimer's Precursor Protein (APP), the Notch receptor, and several other substrates. While inhibition of this protease results in potentially therapeutic reductions in the neurotoxic Abeta peptide, severe side effects might result from inhibiting Notch processing. We report a novel thiophene sulfonamide gamma-secretase inhibitor, GSI-953, that selectively inhibits cleavage of APP while sparing Notch processing. In vitro assays of Abeta production and Notch function, measurements of Abeta levels in plasma and brain of Tg2576 mouse, and assessments of cognitive function using the contextual fear conditioning model are described, as well as human plasma Abeta levels and initial biomarker data. This compound inhibits Abeta production with low nM potency in vitro in cellular and cell-free assays. Cellular assays of Notch cleavage reveal that this compound is >15-fold selective for the inhibition of APP cleavage. In the Tg2576 transgenic mouse, this compound causes a robust reduction in brain and plasma Abeta levels and reverses memory deficits that are correlated with Abeta load. A lowering of plasma Abeta levels in human demonstrates target engagement. These data demonstrate that GSI-953 is a potent and selective gamma-secretase inhibitor with potential for therapeutic utility in Alzheimer's Disease. For these reasons, GSI-953 has been advanced into human clinical trials.

ADVERTISEMENT RETURN TO ISSUEPerspectiveNEXTRecent Advances in the Identification of γ-Secretase Inhibitors To Clinically Test the Aβ Oligomer Hypothesis of Alzheimer's DiseaseAnthony F. Kreft, Robert Martone, and Alexander Porte*View Author Information Wyeth Research, CN 8000, Princeton, New Jersey 08543*To whom correspondence should be addressed. Telephone: 732-274-4559. Fax: 732-274-4505. E-mail: [email protected]Cite this: J. Med. Chem. 2009, 52, 20, 6169–6188Publication Date (Web):August 20, 2009Publication History Received17 February 2009Published online20 August 2009Published inissue 22 October 2009https://pubs.acs.org/doi/10.1021/jm900188zhttps://doi.org/10.1021/jm900188zreview-articleACS PublicationsCopyright © 2009 American Chemical SocietyRequest reuse permissionsArticle Views3972Altmetric-Citations89LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Central nervous system,Peptides and proteins,Redox reactions,Rodent models,Screening assays Get e-Alerts

Blocking the synthesis of the AD pathogen beta amyloid peptide (Αβ) through inhibition of APP processing by gamma secretase (GS) is a promising therapeutic strategy that may provide the first disease-modifying anti-Alzheimer's drug (DMAAD). In animal models, GSI-953 is a Notch-sparing gamma secretase inhibitor (GSI) and this compound has subsequently entered clinical trials. Our objective was to characterize the relative affinity of GSI-953 for binding to GS and compare it to the potency of GS inhibition. A cell-free GS binding assay was developed employing membranes isolated from human neuroblastoma SY5Y cells and the tritiated GSI-953 analog TGSI (5-chloro-N-[(1S,2R)–4,4,4-trifluoro-1-(hydroxyl[3H2]methyl)–2-methylbutyl]thiophene-2-sulfonamide). GSI-953 and benchmark GSIs (DAPT, LY411575, LY450139, DuPont E, L-685458, a BMS sulfonamide and an Amgen sulfonamide) were profiled in this assay for their ability to displace the radiolabeled GSI-953 analog. For comparison to GS binding affinity, the GSIs were also profiled for inhibition of Αβ synthesis in a cellular assay (hAPPCHO cells). GSI-953 competitively displaced TGSI from the GS complex in the cell-free binding assay (IC50=8 nM) and its affinity for GS was comparable to its GSI potency in the cellular assay (EC50Αβ42=15 nM). The stereospecific nature of both the binding to and inhibition of GS by GSI-953 was confirmed by the low affinity (IC50>10,000 nM) and GSI activity (EC50Αβ42>30,000 nM) observed for the enantiomer of GSI-953. Benchmark GSIs DAPT (IC50=29 nM), LY411575 (IC50=3 nM), LY450139 (IC50=26 nM), DuPont E (IC50<10 nM), a BMS sulfonamide (IC50=57 nM) and an Amgen sulfonamide (IC50=986 nM) were able to competitively displace TGSI from GS at concentrations comparable to their EC50s for inhibition of Αβ synthesis. The transition state inhibitor L-685458 was only able to partially displace TGSI. GSI-953 and benchmark GSIs (except L-685458) can competitively displace TGSI from GS suggesting that these GSIs may bind to the same site. Among the GSIs profiled, a good correlation was observed between GS binding affinity and GSI potency.

SAR on HTS hits 1 and 2 led to the potent, Notch-1-sparing GSI 9, which lowered brain Abeta in Tg2576 mice at 100 mg/kg po. Converting the metabolically labile methyl groups in 9 to trifluoromethyl groups afforded the more stable analogue 10, which had improved in vivo potency. Further side chain modification afforded the potent Notch-1-sparing GSI begacestat (5), which was selected for development for the treatment of Alzheimer's disease.

Using a cell-based assay, we have identified a new series of Notch-sparing gamma-secretase inhibitors from HTS screening leads 2a and 2e. Lead optimization studies led to the discovery of analog 8e with improved gamma-secretase inhibitory potency and Notch-sparing selectivity.

Accumulation of beta-amyloid (Abeta), produced by the proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase, is widely believed to be associated with Alzheimer's disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) gamma-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-beta,beta-diethylalaninol 7.b.2 (Abeta(40/42) EC(50)=28 nM), which is efficacious in reduction of Abeta production in vivo.

Gamma secretase is responsible for the intramembraneous cleavage of the Alzheimer's Precursor Protein (APP), the Notch receptor, and several other substrates. While inhibition of this protease results in potentially therapeutic reductions in the neurotoxic Abeta peptide, severe side effects might result from inhibiting Notch processing. We report a novel thiophene sulfonamide gamma-secretase inhibitor, GSI-953 thatselectively inhibits cleavage of APP while sparing Notch processing. In vitro assays of Abeta production and Notch function: measurements of Abeta levels in plasma and brain of Tg2576 mouse, and assessments of cognitive function using the contextual fear conditioning model. This compound inhibits Abeta production with low nM potency in vitro in cellular and cell-free assays. Cellular assays of Notch cleavage reveal that this compound is >15-fold selective for the inhibition of APP cleavage. In the Tg2576 transgenic mouse, this compound causes a robust reduction in brain and plasma Abeta levels and reverses memory deficits that are correlated with Abeta load. These data demonstrate that GSI-953 is a potent and selective gamma-secretase inhibitor with potential for therapeutic utility in Alzheimer's disease. For these reasons, GSI-953 has been advanced into human clinical trials.

A promising Alzheimer's disease (AD) therapeutic strategy is the reduction of the pathogenic beta amyloid peptide (Aβ) by inhibiting gamma secretase (GS). Aβ peptides can serve as biomarkers of pharmacodynamic activity of GS inhibitors (GSI). However, a complex and dynamic relationship exists for production and clearance of Aβ peptides in brain, CSF and plasma compartments. Here, we report the PK/PD relationship of our Notch-selective GSI-953 on Aβ biomarkers in different compartments in both rodent and human subjects. Following single oral administration of GSI-953, plasma, CSF and brain tissue from transgenic mice expressing the human APP transgene, Tg2576, and wild-type mice were collected for the measurement of Aβ peptides and GSI-953 levels.Plasma and CSF samples were obtained in clinical studies following single oral administration of GSI-953 in healthy young and AD subjects. Aβ peptides were measured using validated immunoassays and GSI-953 levels were determined using a validated LC/MS/MS assay. High doses of GSI-953 significantly reduced Aβ40 levels in all three compartments: brain, CSF and plasma in Tg2576 mice. In contrast, a lower dose of GSI-953 significantly reduced Aβ40 only in brain and plasma but not in CSF. In the human study, plasma but not CSF Aβ40 levels were dose-dependently reduced following single oral doses of GSI-953 in healthy young and AD subjects. A GSI-953 plasma:CSF exposure ratio of approximately 10 was observed in both mice and humans. These biomarker data demonstrate target engagement by GSI-953. Here we show that a single dose of GSI-953 produces dose-dependent reductions of plasma but not CSF Aβ40 in humans. These data are consistent with Ab40 results obtained at a lower dose of GSI-953 in the rodent model. A similar GSI-953 exposure relationship in these biological compartments in mice and humans implies that brain concentrations of GSI-953 that lowered brain Aβ in the mouse model are likely achieved at the doses tested in humans. This translational work illustrates the utility of parallel assessment of biomarkers and PK/PD relationships in preclinical and clinical settings.