Chad L. Moore

Transition-state analogue inhibitors of γ-secretase bind directly to presenilin-1

The β-amyloid precursor protein (β-APP), which is involved in the pathogenesis of Alzheimer’s disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.

Transition-state analogue inhibitors of γ-secretase bind directlyto presenilin-1

The β-amyloid precursor protein (β-APP), which is involved in the pathogenesis of Alzheimer’s disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.

γ-Secretase inhibitors repress thymocyte development

A major therapeutic target in the search for a cure to the devastating Alzheimers disease is γ-secretase. This activity resides in a multiprotein enzyme complex responsible for the generation of Aβ42 peptides, precipitates of which are thought to cause the disease. γ-Secretase is also a critical component of the Notch signal transduction pathway; Notch signals regulate development and differentiation of adult self-renewing cells. This has led to the hypothesis that therapeutic inhibition of γ-secretase may interfere with Notch-related processes in adults, most alarmingly in hematopoiesis. Here, we show that application of γ-secretase inhibitors to fetal thymus organ cultures interferes with T cell development in a manner consistent with loss or reduction of Notch1 function. Progression from an immature CD4−/CD8− state to an intermediate CD4+/CD8+ double-positive state was repressed. Furthermore, treatment beginning later at the double-positive stage specifically inhibited CD8+ single-positive maturation but did not affect CD4+ single-positive cells. These results demonstrate that pharmacological γ-secretase inhibition recapitulates Notch1 loss in a vertebrate tissue and present a system in which rapid evaluation of γ-secretase-targeted pharmaceuticals for their ability to inhibit Notch activity can be performed in a relevant context.

A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis.

Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent γ-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimers disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog γ-secretase inhibitor. We propose that γ-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

Experimental Evaluation of the FluChip Diagnostic Microarray for Influenza Virus Surveillance

ABSTRACT Global surveillance of influenza is critical for improvements in disease management and is especially important for early detection, rapid intervention, and a possible reduction of the impact of an influenza pandemic. Enhanced surveillance requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of influenza virus strains. Low-density oligonucleotide microarrays with highly multiplexed “signatures” for influenza viruses offer many of the desired characteristics. However, the high mutability of the influenza virus represents a design challenge. In order for an influenza virus microarray to be of utility, it must provide information for a wide range of viral strains and lineages. The design and characterization of an influenza microarray, the FluChip-55 microarray, for the relatively rapid identification of influenza A virus subtypes H1N1, H3N2, and H5N1 are described here. In this work, a small set of sequences was carefully selected to exhibit broad coverage for the influenza A and B viruses currently circulating in the human population as well as the avian A/H5N1 virus that has become enzootic in poultry in Southeast Asia and that has recently spread to Europe. A complete assay involving extraction and amplification of the viral RNA was developed and tested. In a blind study of 72 influenza virus isolates, RNA from a wide range of influenza A and B viruses was amplified, hybridized, labeled with a fluorophore, and imaged. The entire analysis time was less than 12 h. The combined results for two assays provided the absolutely correct types and subtypes for an average of 72% of the isolates, the correct type and partially correct subtype information for 13% of the isolates, the correct type only for 10% of the isolates, false-negative signals for 4% of the isolates, and false-positive signals for 1% of the isolates. In the overwhelming majority of cases in which incomplete subtyping was observed, the failure was due to the nucleic acid amplification step rather than limitations in the microarray.

Comparison of the MChip to Viral Culture, Reverse Transcription-PCR, and the QuickVue Influenza A+B Test for Rapid Diagnosis of Influenza

ABSTRACT The performance of a diagnostic microarray (the MChip assay) for influenza was compared in a blind study to that of viral culture, reverse transcription (RT)-PCR, and the QuickVue Influenza A+B test. The patient sample data set was composed of 102 respiratory secretion specimens collected between 29 December 2005 and 2 February 2006 at Scott & White Hospital and Clinic in Temple, Texas. Samples were collected from a wide range of age groups by using direct collection, nasal/nasopharyngeal swabs, or nasopharyngeal aspiration. Viral culture and the QuickVue assay were performed at the Texas site at the time of collection. Aliquots for each sample, identified only by study numbers, were provided to the University of Colorado and Vanderbilt University teams for blinded analysis. When referenced to viral culture, the MChip exhibited a clinical sensitivity of 98% and a clinical specificity of 98%. When referenced to RT-PCR, the MChip assay exhibited a clinical sensitivity of 92% and a clinical specificity of 98%. While the MChip assay currently requires 7 to 8 h to complete the analysis, a significant advantage of the test for influenza virus-positive samples is simultaneous detection and full subtype identification for the two subtypes currently circulating in humans (A/H3N2 and A/H1N1) and avian (A/H5N1) viruses.

Robust Sequence Selection Method Used To Develop the FluChip Diagnostic Microarray for Influenza Virus

ABSTRACT DNA microarrays have proven to be powerful tools for gene expression analyses and are becoming increasingly attractive for diagnostic applications, e.g., for virus identification and subtyping. The selection of appropriate sequences for use on a microarray poses a challenge, particularly for highly mutable organisms such as influenza viruses, human immunodeficiency viruses, and hepatitis C viruses. The goal of this work was to develop an efficient method for mining large databases in order to identify regions of conservation in the influenza virus genome. From these regions of conservation, capture and label sequences capable of discriminating between different viral types and subtypes were selected. The salient features of the method were the use of phylogenetic trees for data reduction and the selection of a relatively small number of capture and label sequences capable of identifying a broad spectrum of influenza viruses. A detailed experimental evaluation of the selected sequences is described in a companion paper. The software is freely available under the General Public License at http://www.colorado.edu/chemistry/RGHP/software/ .

Toward the Characterization and Identification of γ‐Secretases Using Transition‐state Analogue Inhibitors

Abstract: The amyloid‐β protein (Aβ), strongly implicated in the etiology of Alzheimers disease (AD), is formed from the amyloid‐β precursor protein (APP) through sequential proteolysis by β‐ and γ‐secretases. Cleavage by γ‐secretase takes place within the middle of the single transmembrane region of APP and results primarily in 40‐ and 42‐amino acid Aβ C‐terminal variants, Aβ40 and Aβ42. The latter form of Aβ is highly fibrillogenic, is invariably elevated in autosomal‐dominant forms of AD, and is the major Aβ component found presymptomatically in cerebral deposits. Thus, blocking production of Aβ in general and Aβ42 in particular is considered an important therapeutic goal. We have developed transition‐state analogue inhibitors of γ‐secretase as molecular probes for characterizing the active site of this enzyme, as pharmacological tools for understanding its role in biology, and as affinity labels toward its definitive identification. Specifically, we found that: (1) difluoro ketone and difluoro alcohol peptidomimetics are effective inhibitors of γ‐secretase activity in APP‐transfected cells, strongly suggesting an aspartyl protease mechanism; (2) γ‐secretases that form Aβ40 and Aβ42 are pharmacologically distinct but are nevertheless closely similar; (3) large hydrophobic P1 substituents increase the inhibitory potency of these peptidomimetics, suggesting a large complementary S1 pocket for γ‐secretases; (4) Aβ42 production is increased several fold over control by these γ‐secretase inhibitors after replacement with inhibitor‐free media; (5) a bromoacetamide derivative of one of these analogues continues to inhibit total Aβ and Aβ42 production hours after replacement with compound‐free media and should help identify the target(s) of these protease transition‐state mimics.

Evaluation of MChip with Historic Subtype H1N1 Influenza A Viruses, Including the 1918 “Spanish Flu” Strain

ABSTRACT The robustness of a recently developed diagnostic microarray for influenza, the MChip, was evaluated with 16 historic subtype H1N1 influenza A viruses (A/H1N1), including A/Brevig Mission/1/1918. The matrix gene segments from all 16 viruses were successfully detected on the array. An artificial neural network trained with temporally related A/H1N1 viruses identified A/Brevig Mission/1/1918 as influenza virus A/H1N1 with 94% probability.

Transition-state analogue inhibitors of |[ggr]|-secretase bind directlyto presenilin-1

The β-amyloid precursor protein (β-APP), which is involved in the pathogenesis of Alzheimer’s disease, and the Notch receptor, which is responsible for critical signalling events during development, both undergo unusual proteolysis within their transmembrane domains by unknown γ-secretases. Here we show that an affinity reagent designed to interact with the active site of γ-secretase binds directly and specifically to heterodimeric forms of presenilins, polytopic proteins that are mutated in hereditary Alzheimer’s and are known mediators of γ-secretase cleavage of both β-APP and Notch. These results provide evidence that heterodimeric presenilins contain the active site of γ-secretase, and validate presenilins as principal targets for the design of drugs to treat and prevent Alzheimer’s disease.