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1.
Alzheimers Res Ther ; 16(1): 70, 2024 04 04.
Article in English | MEDLINE | ID: mdl-38575959

ABSTRACT

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-ß protein (Aß) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aß pathology and tauopathy in vivo. METHODS: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model of a disease (Krabbe A) characterized by pronounced lysosomal dysfunction. Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau. RESULTS: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aß, manifesting as intense, exclusively intracellular aggregates; extracellular Aß deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatD-KO mice were found to develop prominent tauopathy by just ∼ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology present in aged JNPL3 mice. CatD-KO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (∼ 1250%) are present in CatD-KO mice but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aß42. CONCLUSIONS: Our findings support a major role for CatD in the proteostasis of both Aß and tau in vivo. To our knowledge, the CatD-KO mouse line is the only model to develop detectable Aß accumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aß42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.


Subject(s)
Alzheimer Disease , Tauopathies , Aged , Animals , Humans , Mice , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Cathepsin D , Disease Models, Animal , Mice, Knockout , Mice, Transgenic , tau Proteins/genetics , tau Proteins/metabolism , Tauopathies/genetics , Tauopathies/metabolism
2.
Res Sq ; 2023 Oct 23.
Article in English | MEDLINE | ID: mdl-37961253

ABSTRACT

Background: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-ß protein (Aß) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aß pathology and tauopathy in vivo. Methods: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model characterized by pronounced lysosomal dysfunction (Krabbe A). Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau. Results: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aß, manifesting as intense, exclusively intracellular aggregates; extracellular Aß deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatDKO mice were found to develop prominent tauopathy by just ~ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology in aged JNPL3 mice. CatDKO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (~ 1250%) are present in CatD-KO mice, but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aß42. Conclusions: Our findings support a major role for CatD in the proteostasis of both Aß and tau in vivo. To our knowledge, CatD-KO mice are the only model to develop detectable Aß acumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aß42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.

3.
Alzheimers Res Ther ; 12(1): 80, 2020 07 06.
Article in English | MEDLINE | ID: mdl-32631408

ABSTRACT

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid ß-protein (Aß) and the microtubule-associated protein, tau, and has been genetically linked to late-onset Alzheimer disease (AD). Here, we sought to examine the consequences of genetic deletion of CatD on Aß proteostasis in vivo and to more completely characterize the degradation of Aß42 and Aß40 by CatD. METHODS: We quantified Aß degradation rates and levels of endogenous Aß42 and Aß40 in the brains of CatD-null (CatD-KO), heterozygous null (CatD-HET), and wild-type (WT) control mice. CatD-KO mice die by ~ 4 weeks of age, so tissues from younger mice, as well as embryonic neuronal cultures, were investigated. Enzymological assays and surface plasmon resonance were employed to quantify the kinetic parameters (KM, kcat) of CatD-mediated degradation of monomeric human Aß42 vs. Aß40, and the degradation of aggregated Aß42 species was also characterized. Competitive inhibition assays were used to interrogate the relative inhibition of full-length human and mouse Aß42 and Aß40, as well as corresponding p3 fragments. RESULTS: Genetic deletion of CatD resulted in 3- to 4-fold increases in insoluble, endogenous cerebral Aß42 and Aß40, exceeding the increases produced by deletion of an insulin-degrading enzyme, neprilysin or both, together with readily detectable intralysosomal deposits of endogenous Aß42-all by 3 weeks of age. Quite significantly, CatD-KO mice exhibited ~ 30% increases in Aß42/40 ratios, comparable to those induced by presenilin mutations. Mechanistically, the perturbed Aß42/40 ratios were attributable to pronounced differences in the kinetics of degradation of Aß42 vis-à-vis Aß40. Specifically, Aß42 shows a low-nanomolar affinity for CatD, along with an exceptionally slow turnover rate that, together, renders Aß42 a highly potent competitive inhibitor of CatD. Notably, the marked differences in the processing of Aß42 vs. Aß40 also extend to p3 fragments ending at positions 42 vs. 40. CONCLUSIONS: Our findings identify CatD as the principal intracellular Aß-degrading protease identified to date, one that regulates Aß42/40 ratios via differential degradation of Aß42 vs. Aß40. The finding that Aß42 is a potent competitive inhibitor of CatD suggests a possible mechanistic link between elevations in Aß42 and downstream pathological sequelae in AD.


Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Alzheimer Disease/genetics , Animals , Cathepsin D/genetics , Mice , Peptide Fragments
4.
J Alzheimers Dis ; 49(1): 129-37, 2016.
Article in English | MEDLINE | ID: mdl-26444783

ABSTRACT

Age is the major risk factor for developing Alzheimer's disease (AD), and modifying age-related factors may help to delay the onset of the disease. The goal of this study was to investigate the relationship between age and the metabolic factors related to the risk of developing AD. The concentrations of insulin, amylin, and amyloid-ß peptide (Aß) in plasma were measured. We further measured the activity of serum Aß degradation by using fluorescein- and biotin-labeled Aß40. Apolipoprotein E4 allele (ApoE4) and cognitive impairment were characterized. Subjects were divided into three age groups: 60-70, 70-80, and ≥80 years old. We found that the older the subjects, the lower the concentration of insulin (p = 0.001) and the higher the concentration of Aß1-40 (p = 0.004) in plasma. However, age was not associated with the concentration of another pancreatic peptide, amylin, and only marginally with Aß1-42. These relationships remained in the absence of diabetes, cardiovascular disease, and stroke, and regardless of the presence of ApoE4 and cognitive impairment. Both age and ApoE4 were inversely associated with, while insulin was positively associated with, the activities of Aß degradation in serum. Our study suggested that low concentration of insulin and high concentration of Aß40 are aging factors related to the risk of AD.


Subject(s)
Aging , Alzheimer Disease/blood , Amyloid beta-Peptides/blood , Apolipoprotein E4/blood , Cognition Disorders/blood , Insulin/blood , Islet Amyloid Polypeptide/blood , Peptide Fragments/blood , Age Distribution , Aged , Aged, 80 and over , Biomarkers/blood , Cross-Sectional Studies , Female , Humans , Linear Models , Male , Middle Aged , Multivariate Analysis
5.
ACS Chem Biol ; 10(12): 2716-24, 2015 Dec 18.
Article in English | MEDLINE | ID: mdl-26398879

ABSTRACT

Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 µM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.


Subject(s)
Cytosol/chemistry , Drug Delivery Systems , Enzyme Inhibitors/chemistry , Extracellular Space/enzymology , Insulysin/antagonists & inhibitors , Sulfhydryl Compounds/pharmacology , Computer Simulation , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Inhibitory Concentration 50 , Insulin Antagonists/pharmacology , Insulysin/chemistry , Models, Biological , Molecular Structure , Structure-Activity Relationship , Sulfhydryl Compounds/chemistry
6.
Mol Neurodegener ; 9: 11, 2014 Mar 10.
Article in English | MEDLINE | ID: mdl-24607147

ABSTRACT

Recent genome-wide association studies (GWAS) of late-onset Alzheimer's disease (LOAD) have identified single nucleotide polymorphisms (SNPs) which show significant association at the well-known APOE locus and at nineteen additional loci. Among the functional, disease-associated variants at these loci, missense variants are particularly important because they can be readily investigated in model systems to search for novel therapeutic targets. It is now possible to perform a low-cost search for these "actionable" variants by genotyping the missense variants at known LOAD loci already cataloged on the Exome Variant Server (EVS). In this proof-of-principle study designed to explore the efficacy of this approach, we analyzed three rare EVS variants in APOE, p.L28P, p.R145C and p.V236E, in our case control series of 9114 subjects. p.R145C proved to be too rare to analyze effectively. The minor allele of p.L28P, which was in complete linkage disequilibrium (D' = 1) with the far more common APOE ϵ4 allele, showed no association with LOAD (P = 0.75) independent of the APOE ϵ4 allele. p.V236E was significantly associated with a marked reduction in risk of LOAD (P = 7.5 × 10⁻°5; OR = 0.10, 0.03 to 0.45). The minor allele of p.V236E, which was in complete linkage disequilibrium (D' = 1) with the common APOE ϵ3 allele, identifies a novel LOAD-associated haplotype (APOE ϵ3b) which is associated with decreased risk of LOAD independent of the more abundant APOE ϵ2, ϵ3 and ϵ4 haplotypes. Follow-up studies will be important to confirm the significance of this association and to better define its odds ratio. The ApoE p.V236E substitution is the first disease-associated change located in the lipid-binding, C-terminal domain of the protein. Thus our study (i) identifies a novel APOE missense variant which may profitably be studied to better understand how ApoE function may be modified to reduce risk of LOAD and (ii) indicates that analysis of protein-altering variants cataloged on the EVS can be a cost-effective way to identify actionable functional variants at recently discovered LOAD loci.


Subject(s)
Alzheimer Disease/genetics , Apolipoproteins E/genetics , Genetic Predisposition to Disease/genetics , Polymorphism, Single Nucleotide , Age of Onset , Aged , Case-Control Studies , Female , Genome-Wide Association Study , Genotype , Humans , Male , Middle Aged
7.
PLoS One ; 8(6): e64164, 2013.
Article in English | MEDLINE | ID: mdl-23750206

ABSTRACT

Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. LRRTM3 is a nested gene within α-T catenin (CTNNA3) and resides at the linkage peak for late-onset Alzheimer's disease (LOAD) risk and plasma amyloid ß (Aß) levels. In-vitro knock-down of LRRTM3 was previously shown to decrease secreted Aß, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of LRRTM3 had lower BACE1 and higher CTNNA3 mRNA levels, but no change in APP. Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking LRRTM3 in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5'UTR-Intron 1 of LRRTM3 that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02-0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.


Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Aspartic Acid Endopeptidases/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Polymorphism, Single Nucleotide , Alzheimer Disease/pathology , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Aspartic Acid Endopeptidases/genetics , Cell Line, Tumor , Gene Expression Regulation , Gene Knockdown Techniques , Genetic Loci/genetics , Genetic Predisposition to Disease/genetics , Humans , Intracellular Space/metabolism , Membrane Proteins/deficiency , Mice , Nerve Tissue Proteins/deficiency , Protein Binding
8.
J Med Chem ; 56(6): 2246-55, 2013 Mar 28.
Article in English | MEDLINE | ID: mdl-23437776

ABSTRACT

Insulin-degrading enzyme (IDE) is an atypical zinc-metallopeptidase that degrades insulin and the amyloid ß-protein and is strongly implicated in the pathogenesis of diabetes and Alzheimer's disease. We recently developed the first effective inhibitors of IDE, peptide hydroxamates that, while highly potent and selective, are relatively large (MW > 740) and difficult to synthesize. We present here a facile synthetic route that yields enantiomerically pure derivatives comparable in potency to the parent compounds. Through the generation of truncated variants, we identified a compound with significantly reduced size (MW = 455.5) that nonetheless retains good potency (ki = 78 ± 11 nM) and selectivity for IDE. Notably, the potency of these inhibitors was found to vary as much as 60-fold in a substrate-specific manner, an unexpected finding for active site-directed inhibitors. Collectively, our findings demonstrate that potent, small-molecule IDE inhibitors can be developed that, in certain instances, can be highly substrate selective.


Subject(s)
Drug Design , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Insulysin/antagonists & inhibitors , Insulysin/metabolism , Peptides/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/metabolism , Insulysin/chemistry , Molecular Docking Simulation , Protein Conformation , Stereoisomerism , Substrate Specificity
9.
Chem Res Toxicol ; 25(12): 2725-36, 2012 Dec 17.
Article in English | MEDLINE | ID: mdl-23035985

ABSTRACT

The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA) includes induction of chemopreventive mechanisms and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the "NO-specific" 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM donor, bioactivated by cellular esterase activity to release salicylates, NO(3)(-), and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery.


Subject(s)
Aspirin/analogs & derivatives , Aspirin/pharmacology , Indolequinones/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Prodrugs/pharmacology , Animals , Cell Line, Tumor , DNA Damage , Glutathione/metabolism , Glutathione S-Transferase pi/metabolism , Humans , Kelch-Like ECH-Associated Protein 1 , Mice , NAD(P)H Dehydrogenase (Quinone)/metabolism , Nitric Oxide/metabolism
10.
Mol Neurodegener ; 7: 46, 2012 Sep 17.
Article in English | MEDLINE | ID: mdl-22986058

ABSTRACT

BACKGROUND: Proteases that degrade the amyloid ß-protein (Aß) have emerged as key players in the etiology and potential treatment of Alzheimer's disease (AD), but it is unlikely that all such proteases have been identified. To discover new Aß-degrading proteases (AßDPs), we conducted an unbiased, genome-scale, functional cDNA screen designed to identify proteases capable of lowering net Aß levels produced by cells, which were subsequently characterized for Aß-degrading activity using an array of downstream assays. RESULTS: The top hit emerging from the screen was ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2), a rather unexpected finding given the well-established role of its close homolog, BACE1, in the production of Aß. BACE2 is known to be capable of lowering Aß levels via non-amyloidogenic processing of APP. However, in vitro, BACE2 was also found to be a particularly avid AßDP, with a catalytic efficiency exceeding all known AßDPs except insulin-degrading enzyme (IDE). BACE1 was also found to degrade Aß, albeit ~150-fold less efficiently than BACE2. Aß is cleaved by BACE2 at three peptide bonds-Phe19-Phe20, Phe20-Ala21, and Leu34-Met35--with the latter cleavage site being the initial and principal one. BACE2 overexpression in cultured cells was found to lower net Aß levels to a greater extent than multiple, well-established AßDPs, including neprilysin (NEP) and endothelin-converting enzyme-1 (ECE1), while showing comparable effectiveness to IDE. CONCLUSIONS: This study identifies a new functional role for BACE2 as a potent AßDP. Based on its high catalytic efficiency, its ability to degrade Aß intracellularly, and other characteristics, BACE2 represents a particulary strong therapeutic candidate for the treatment or prevention of AD.


Subject(s)
Alzheimer Disease/enzymology , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Amyloid/metabolism , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Alzheimer Disease/genetics , Cells, Cultured , Endothelin-Converting Enzymes , Humans , Insulysin/metabolism , Metalloendopeptidases/metabolism , Neprilysin/metabolism
11.
J Alzheimers Dis ; 29(2): 329-40, 2012.
Article in English | MEDLINE | ID: mdl-22232014

ABSTRACT

Sporadic Alzheimer's disease (AD) patients have low amyloid-ß peptide (Aß) clearance in the central nervous system. The peripheral Aß clearance may also be important but its role in AD remains unclear. We aimed to study the Aß degrading proteases including insulin degrading enzyme (IDE), angiotensin converting enzyme (ACE) and others in blood. Using the fluorogenic substrate V (a substrate of IDE and other metalloproteases), we showed that human serum degraded the substrate V, and the activity was inhibited by adding increasing dose of Aß. The existence of IDE activity was demonstrated by the inhibition of insulin, amylin, or EDTA, and further confirmed by immunocapture of IDE using monoclonal antibodies. The involvement of ACE was indicated by the ability of the ACE inhibitor, lisinopril, to inhibit the substrate V degradation. To test the variations of substrate V degradation in humans, we used serum samples from a homebound elderly population with cognitive diagnoses. Compared with the elderly who had normal cognition, those with probable AD and amnestic mild cognitive impairment (amnestic MCI) had lower peptidase activities. Probable AD or amnestic MCI as an outcome remained negatively associated with serum substrate V degradation activity after adjusting for the confounders. The elderly with probable AD had lower serum substrate V degradation activity compared with those who had vascular dementia. The blood proteases mediating Aß degradation may be important for the AD pathogenesis. More studies are needed to specify each Aß degrading protease in blood as a useful biomarker and a possible treatment target for AD.


Subject(s)
Alzheimer Disease/blood , Amyloid beta-Peptides/blood , Cognitive Dysfunction/blood , Insulysin/blood , Peptide Fragments/blood , Peptidyl-Dipeptidase A/blood , Serum/enzymology , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Apolipoprotein E4/genetics , Brain/pathology , Cognitive Dysfunction/genetics , Female , Fluorescent Dyes/metabolism , Humans , Islet Amyloid Polypeptide/blood , Magnetic Resonance Imaging , Male , Neuropsychological Tests , Psychiatric Status Rating Scales , Statistics, Nonparametric , Time Factors
12.
ACS Med Chem Lett ; 2(9): 656-661, 2011 Sep 08.
Article in English | MEDLINE | ID: mdl-21927645

ABSTRACT

Hybrid nitrate drugs have been reported to provide NO bioactivity to ameliorate side effects or to provide ancillary therapeutic activity. Hybrid nitrate selective serotonin reuptake inhibitors (NO-SSRIs) were prepared to improve the therapeutic profile of this drug class. A synthetic strategy for use of a thiocarbamate linker was developed, which in the case of NO-fluoxetine facilitated hydrolysis to fluoxetine at pH 7.4 within 7 hours. In cell culture, NO-SSRIs were weak inhibitors of the serotonin transporter, however, in the forced swimming task (FST) in rats, NO-fluoxetine demonstrated classical antidepressant activity. Comparison of NO-fluoxetine, with fluoxetine, and an NO-chimera nitrate developed for Alzheimer's disease (GT-1061), was made in the step through passive avoidance (STPA) test of learning and memory in rats treated with scopolamine as an amnesic agent. Fluoxetine was inactive, whereas NO-fluoxetine and GT-1061 both restored long-term memory. GT-1061 also produced antidepressant behavior in FST. These data support the potential for NO-SSRIs to overcome the lag in onset of therapeutic action and provide co-therapy of neuropathologies concomitant with depression.

13.
PLoS One ; 6(6): e20818, 2011.
Article in English | MEDLINE | ID: mdl-21695259

ABSTRACT

BACKGROUND: Insulin-degrading enzyme (IDE) is widely recognized as the principal protease responsible for the clearance and inactivation of insulin, but its role in glycemic control in vivo is poorly understood. We present here the first longitudinal characterization, to our knowledge, of glucose regulation in mice with pancellular deletion of the IDE gene (IDE-KO mice). METHODOLOGY: IDE-KO mice and wild-type (WT) littermates were characterized at 2, 4, and 6 months of age in terms of body weight, basal glucose and insulin levels, and insulin and glucose tolerance. Consistent with a functional role for IDE in insulin clearance, fasting serum insulin levels in IDE-KO mice were found to be ∼3-fold higher than those in wild-type (WT) controls at all ages examined. In agreement with previous observations, 6-mo-old IDE-KO mice exhibited a severe diabetic phenotype characterized by increased body weight and pronounced glucose and insulin intolerance. In marked contrast, 2-mo-old IDE-KO mice exhibited multiple signs of improved glycemic control, including lower fasting glucose levels, lower body mass, and modestly enhanced insulin and glucose tolerance relative to WT controls. Biochemically, the emergence of the diabetic phenotype in IDE-KO mice correlated with age-dependent reductions in insulin receptor (IR) levels in muscle, adipose, and liver tissue. Primary adipocytes harvested from 6-mo-old IDE-KO mice also showed functional impairments in insulin-stimulated glucose uptake. CONCLUSIONS: Our results indicate that the diabetic phenotype in IDE-KO mice is not a primary consequence of IDE deficiency, but is instead an emergent compensatory response to chronic hyperinsulinemia resulting from complete deletion of IDE in all tissues throughout life. Significantly, our findings provide new evidence to support the idea that partial and/or transient inhibition of IDE may constitute a valid approach to the treatment of diabetes.


Subject(s)
Aging/blood , Blood Glucose/metabolism , Gene Deletion , Insulin Resistance , Insulysin/genetics , Animals , Blood Glucose/drug effects , Body Weight/drug effects , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/complications , Hyperinsulinism/blood , Hyperinsulinism/complications , Insulin/blood , Insulin/pharmacology , Insulysin/metabolism , Mice , Mice, Knockout , Phenotype
14.
J Med Chem ; 54(7): 2293-306, 2011 Apr 14.
Article in English | MEDLINE | ID: mdl-21405086

ABSTRACT

Poor blood-brain barrier penetration of nonsteroidal anti-inflammatory drugs (NSAIDs) has been blamed for the failure of the selective amyloid lowering agent (SALA) R-flurbiprofen in phase 3 clinical trials for Alzheimer's disease (AD). NO-donor NSAIDs (NO-NSAIDs) provide an alternative, gastric-sparing approach to NSAID SALAs, which may improve bioavailability. NSAID analogues were studied for anti-inflammatory activity and for SALA activity in N2a neuronal cells transfected with human amyloid precursor protein (APP). Flurbiprofen (1) analogues were obtained with enhanced anti-inflammatory and antiamyloidogenic properties compared to 1, however, esterification led to elevated Aß(1-42) levels. Hybrid nitrate prodrugs possessed superior anti-inflammatory activity and reduced toxicity relative to the parent NSAIDs, including clinical candidate CHF5074. Although hybrid nitrates elevated Aß(1-42) at higher concentration, SALA activity was observed at low concentrations (≤1 µM): both Aß(1-42) and the ratio of Aß(1-42)/Aß(1-40) were lowered. This biphasic SALA activity was attributed to the intact nitrate drug. For several compounds, the selective modulation of amyloidogenesis was tested using an immunoprecipitation MALDI-TOF approach. These data support the development of NO-NSAIDs as an alternative approach toward a clinically useful SALA.


Subject(s)
Amyloid beta-Peptides/metabolism , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Nitrates/chemistry , Amyloid beta-Peptides/chemistry , Amyloidosis/drug therapy , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/toxicity , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Cell Line, Tumor , Humans , Mice , Neurons/drug effects , Neurons/metabolism , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/toxicity
15.
PLoS One ; 5(5): e10504, 2010 May 07.
Article in English | MEDLINE | ID: mdl-20498699

ABSTRACT

BACKGROUND: Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. METHODOLOGY/PRINCIPAL FINDINGS: We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are approximately 10(6) times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-à-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's "closed," inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. CONCLUSIONS/SIGNIFICANCE: The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes.


Subject(s)
Drug Design , Enzyme Inhibitors/pharmacology , Insulin/metabolism , Insulysin/antagonists & inhibitors , Animals , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Enzyme Inhibitors/analysis , Enzyme Inhibitors/chemistry , Extracellular Space/drug effects , Extracellular Space/metabolism , HeLa Cells , Humans , Insulysin/chemistry , Models, Molecular , Peptide Library , Protein Binding/drug effects , Signal Transduction/drug effects
16.
J Neurochem ; 111(3): 766-76, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19702655

ABSTRACT

The non-steroidal anti-inflammatory drug flurbiprofen is a selective amyloid lowering agent which has been studied clinically in Alzheimer's disease. HCT-1026 is an ester prodrug of flurbiprofen incorporating a nitrate carrier moiety that in vivo provides NO bioactivity and an improved safety profile. In vitro, HCT-1026 retained the cyclooxygenase inhibitory and non-steroidal anti-inflammatory drug activity of flurbiprofen, but at concentrations at which levels of amyloid-beta 1-42 amino acid were lowered by flurbiprofen, amyloid-beta 1-42 amino acid levels were elevated 200% by HCT-1026. Conversely, at lower concentrations, HCT-1026 behaved as a selective amyloid lowering agent with greater potency than flurbiprofen. The difference in concentration-responses between flurbiprofen and HCT-1026 in vitro suggests different cellular targets; and in no case did a combination of nitrate drug with flurbiprofen provide similar actions. In vivo, HCT-1026 was observed to reverse cognitive deficits induced by scopolamine in two behavioral assays; activity that was also shown by a classical nitrate drug, but not by flurbiprofen. The ability to restore aversive memory and spatial working and reference memory after cholinergic blockade has been demonstrated by other agents that stimulate NO/cGMP signaling. These observations add positively to the preclinical profile of HCT-1026 and NO chimeras in Alzheimer's disease.


Subject(s)
Amyloid beta-Peptides/metabolism , Cognition Disorders/chemically induced , Flurbiprofen/analogs & derivatives , Neuroprotective Agents/pharmacology , Peptide Fragments/metabolism , Scopolamine , Amyloid beta-Protein Precursor/genetics , Animals , Avoidance Learning/drug effects , Cell Line, Tumor , Cognition Disorders/drug therapy , Disease Models, Animal , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay/methods , Flurbiprofen/pharmacology , Humans , Immunoprecipitation/methods , Lipopolysaccharides/pharmacology , Male , Maze Learning/drug effects , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuroblastoma , Nitric Oxide Synthase Type II/metabolism , Prostaglandins/metabolism , Rats , Rats, Long-Evans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Tetrazolium Salts , Thiazoles , Transfection/methods
17.
J Neurochem ; 111(3): 683-95, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19702658

ABSTRACT

Gamma-secretase modulators (GSMs) include selected non-steroidal anti-inflammatory drugs such as flurbiprofen that selectively lowers the neurotoxic amyloid-beta peptide Abeta(1-42). GSMs are attractive targets for Alzheimer's disease, in contrast to 'inverse GSMs,' such as fenofibrate, which selectively increase the level of Abeta(1-42). A methodology for screening of Abeta modulating drugs was developed utilizing an Abeta-producing neuroblastoma cell line stably transfected with mutant human amyloid precursor protein, immunoprecipitation of Abeta peptides, and mass spectroscopic quantitation of Abeta(1-37)/Abeta(1-38)/Abeta(1-40)/Abeta(1-42) using an Abeta internal standard. The unexpected conclusion of this work was that in this system, drug effects are independent of gamma-secretase. The methodology recapitulated reported results for modulation of Abeta by GSMs. However, control experiments in which exogenous Abeta(1-40)/Abeta(1-42) was added (i) to drug-treated wild-type cells or (ii) to conditioned media from these wild-type cells, gave comparable patterns of Abeta modulation. These results, suggesting that drugs modulate the ability of cell-derived factors to degrade Abeta, was interrogated by adding protease inhibitors and performing molecular weight cut-off fractionation. The results confirmed that modulation of Abeta(1-40)/Abeta(1-42) was mediated by selective proteolysis. Treatment of N2a cells with flurbiprofen or fenofibric acid selectively enhanced Abeta(1-42) clearance by extracellular proteolysis; treatment with HCT-1026 or fenofibrate (esters of flurbiprofen and fenobric acid) inhibited clearance of Abeta(1-40) and Abeta(1-42).


Subject(s)
Amyloid beta-Peptides/drug effects , Amyloid beta-Peptides/metabolism , Enzyme Inhibitors/pharmacology , Fenofibrate/pharmacology , Flurbiprofen/pharmacology , Peptide Fragments/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay/methods , Flurbiprofen/analogs & derivatives , Humans , Immunoprecipitation , Mice , Neuroblastoma/pathology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Transfection/methods
18.
Bioorg Med Chem Lett ; 19(1): 264-74, 2009 Jan 01.
Article in English | MEDLINE | ID: mdl-19013792

ABSTRACT

A series of transition state analogues of beta-secretases 1 and 2 (BACE1, 2) inhibitors containing fused-ring or biaryl moieties were designed computationally to probe the S2 pocket, synthesized, and tested for BACE1 and BACE2 inhibitory activity. It has been shown that unlike the biaryl analogs, the fused-ring moiety is successfully accommodated in the BACE1 binding site resulting in the ligands with excellent inhibitory activity. Ligand 5b reduced 65% of Abeta40 production in N2a cells stably transfected with Swedish human APP.


Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Models, Molecular , Protease Inhibitors/chemical synthesis , Amyloid beta-Peptides/biosynthesis , Amyloid beta-Protein Precursor/genetics , Binding Sites , Cell Line , Computer Simulation , Drug Design , Humans , Ligands , Protease Inhibitors/pharmacology , Transfection
19.
Mol Pharmacol ; 74(5): 1381-91, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18676677

ABSTRACT

Nonsteroidal anti-inflammatory drugs (NSAIDs) have shown promise in colorectal cancer (CRC), but they are compromised by gastrotoxicity. NO-NSAIDs are hybrid nitrates conjugated to an NSAID designed to exploit the gastroprotective properties of NO bioactivity. The NO chimera ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)benzoate (GT-094), a novel nitrate containing an NSAID and disulfide pharmacophores, is effective in vivo in rat models of CRC and is a lead compound for design of agents of use in CRC. Preferred chemopreventive agents possess 1) antiproliferative and 2) anti-inflammatory actions and 3) the ability to induce cytoprotective phase 2 enzymes. To determine the contribution of each pharmacophore to the biological activity of GT-094, these three biological activities were studied in vitro in compounds that deconstructed the structural elements of the lead GT-094. The anti-inflammatory and antiproliferative actions of GT-094 in vivo were recapitulated in vitro, and GT-094 was seen to induce phase 2 enzymes via the antioxidant responsive element. In the variety of colon, macrophage-like, and liver cell lines studied, the evidence from structure-activity relationships was that the disulfide structural element of GT-094 is the dominant contributor in vitro to the anti-inflammatory activity, antiproliferation, and enzyme induction. The results provide a direction for lead compound refinement. The evidence for a contribution from the NO mimetic activity of nitrates in vitro was equivocal, and combinations of nitrates with acetylsalicylic acid were inactive.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anticarcinogenic Agents/pharmacology , Cell Proliferation/drug effects , Cytoprotection/drug effects , Nitric Oxide/pharmacology , Animals , Blotting, Western , Cell Line , Enzyme Induction , Humans , Mice , Nitric Oxide/chemistry , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction
20.
Nitric Oxide ; 19(2): 115-24, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18485921

ABSTRACT

Properties of the NO-ASA family of NO-donating NSAIDs (NO-NSAIDs), notably NCX 4016 (mNO-ASA) and NCX 4040 (pNO-ASA), reported in more than one hundred publications, have included positive preclinical data in cancer chemoprevention and therapy. Evidence is presented that the antiproliferative, the chemopreventive (antioxidant/electrophile response element (ARE) activation), and the anti-inflammatory activity of NO-ASA in cell cultures is replicated by X-ASA derivatives that are incapable of acting as NO donors. pBr-ASA and mBr-ASA are conisogenic with NO-ASA, but are not NO donors. The biological activity of pNO-ASA is replicated by pBr-ASA; and both pNO-ASA and pBr-ASA are bioactivated to the same quinone methide electrophile. The biological activity of mNO-ASA is replicated by mBr-ASA; mNO-ASA and mBr-ASA are bioactivated to different benzyl electrophiles. The observed activity is likely initiated by trapping of thiol biomolecules by the quinone and benzyl electrophiles, leading to depletion of GSH and modification of Cys-containing sensor proteins. Whereas all NO-NSAIDs containing the same structural "linker" as NCX 4040 and NCX 4016 are anticipated to possess activity resulting from bioactivation to electrophilic metabolites, this expectation does not extend to other linker structures. Nitrates require metabolic bioactivation to liberate NO bioactivity, which is often poorly replicated in vitro, and NO bioactivity provided by NO-NSAIDs in vivo provides proven therapeutic benefits in mitigation of NSAID gastrotoxicity. The in vivo properties of X-ASA drugs await discovery.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Neoplasms/prevention & control , Nitric Oxide Donors/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antineoplastic Agents/pharmacology , Aspirin/analogs & derivatives , Aspirin/pharmacology , Aspirin/therapeutic use , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Chemoprevention/methods , Humans , Macrophages , Mice , Neoplasms/drug therapy , Nitrates/metabolism , Nitric Oxide/metabolism , Nitric Oxide Donors/therapeutic use , Nitro Compounds/pharmacology , Nitro Compounds/therapeutic use
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