Biomedical subjects

Amy S Espeseth

Publications and source records attributed to Amy S Espeseth.

9 recordsLinked to original sources

PubMed2006

Discovery of oxadiazoyl tertiary carbinamine inhibitors of beta-secretase (BACE-1).

We describe the discovery and optimization of tertiary carbinamine derived inhibitors of the enzyme beta-secretase (BACE-1). These novel non-transition-state-derived ligands incorporate a single primary amine to interact with the catalytic aspartates of the target enzyme. Optimization of this series provided inhibitors with intrinsic and functional potency comparable to evolved transition state isostere derived inhibitors of BACE-1.

Amyloid Precursor Protein Secretases↗

PubMed2006

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.

Amides↗

PubMed2006

A genome wide analysis of ubiquitin ligases in APP processing identifies a novel regulator of BACE1 mRNA levels.

Proteolysis of beta-amyloid precursor protein (APP) into amyloid beta peptide (Abeta) by beta- and gamma-secretases is a critical step in the pathogenesis of Alzheimer's Disease (AD), but the pathways regulating secretases are not fully characterized. Ubiquitinylation, which is dysregulated in AD, may affect APP processing. Here, we describe a screen for APP processing modulators using an siRNA library targeting 532 predicted ubiquitin ligases. Seven siRNA pools diminished Abeta production. Of these, siRNAs targeting PPIL2 (hCyp-60) suppressed beta-site cleavage. Knockdown of PPIL2 mRNA decreased BACE1 mRNA, while overexpression of PPIL2 cDNA enhanced BACE1 mRNA levels. Microarray analysis of PPIL2 or BACE1 knockdown indicated that genes affected by BACE1 knockdown are a subset of those dependent upon PPIL2; suggesting that BACE1 expression is downstream of PPIL2. The association of PPIL2 with BACE expression and its requirement for Abeta production suggests new approaches to discover disease modifying agents for AD.

Amyloid Precursor Protein Secretases↗

PubMed2006

Robust statistical methods for hit selection in RNA interference high-throughput screening experiments.

RNA interference (RNAi) high-throughput screening (HTS) experiments carried out using large (>5000 short interfering [si]RNA) libraries generate a huge amount of data. In order to use these data to identify the most effective siRNAs tested, it is critical to adopt and develop appropriate statistical methods. To address the questions in hit selection of RNAi HTS, we proposed a quartile-based method which is robust to outliers, true hits and nonsymmetrical data. We compared it with the more traditional tests, mean +/- k standard deviation (SD) and median +/- 3 median of absolute deviation (MAD). The results suggested that the quartile-based method selected more hits than mean +/- k SD under the same preset error rate. The number of hits selected by median +/- k MAD was close to that by the quartile-based method. Further analysis suggested that the quartile-based method had the greatest power in detecting true hits, especially weak or moderate true hits. Our investigation also suggested that platewise analysis (determining effective siRNAs on a plate-by-plate basis) can adjust for systematic errors in different plates, while an experimentwise analysis, in which effective siRNAs are identified in an analysis of the entire experiment, cannot. However, experimentwise analysis may detect a cluster of true positive hits placed together in one or several plates, while platewise analysis may not. To display hit selection results, we designed a specific figure called a plate-well series plot. We thus suggest the following strategy for hit selection in RNAi HTS experiments. First, choose the quartile-based method, or median +/- k MAD, for identifying effective siRNAs. Second, perform the chosen method experimentwise on transformed/normalized data, such as percentage inhibition, to check the possibility of hit clusters. If a cluster of selected hits are observed, repeat the analysis based on untransformed data to determine whether the cluster is due to an artifact in the data. If no clusters of hits are observed, select hits by performing platewise analysis on transformed data. Third, adopt the plate-well series plot to visualize both the data and the hit selection results, as well as to check for artifacts.

Cluster Analysis↗

PubMed2005

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.

Amyloid Precursor Protein Secretases↗

PubMed2005

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.

Alzheimer Disease↗

PubMed2004

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.

Amyloid Precursor Protein Secretases↗

PubMed2004

A naphthyridine carboxamide provides evidence for discordant resistance between mechanistically identical inhibitors of HIV-1 integrase.

The increasing incidence of resistance to current HIV-1 therapy underscores the need to develop antiretroviral agents with new mechanisms of action. Integrase, one of three viral enzymes essential for HIV-1 replication, presents an important yet unexploited opportunity for drug development. We describe here the identification and characterization of L-870,810, a small-molecule inhibitor of HIV-1 integrase with potent antiviral activity in cell culture and good pharmacokinetic properties. L-870,810 is an inhibitor with an 8-hydroxy-(1,6)-naphthyridine-7-carboxamide pharmacophore. The compound inhibits HIV-1 integrase-mediated strand transfer, and its antiviral activity in vitro is a direct consequence of this ascribed effect on integration. L-870,810 is mechanistically identical to previously described inhibitors from the diketo acid series; however, viruses selected for resistance to L-870,810 contain mutations (integrase residues 72, 121, and 125) that uniquely confer resistance to the naphthyridine. Conversely, mutations associated with resistance to the diketo acid do not engender naphthyridine resistance. Importantly, the mutations associated with resistance to each of these inhibitors map to distinct regions within the integrase active site. Therefore, we propose a model of the two inhibitors that is consistent with this observation and suggests specific interactions with discrete binding sites for each ligand. These studies provide a structural basis and rationale for developing integrase inhibitors with the potential for unique and nonoverlapping resistance profiles.

Animals↗

PubMed2002

Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes.

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3' end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg(2+). The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.

Anti-HIV Agents↗