95 results on '"Gardell SJ"'
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2. Vampire bat salivary plasminogen activator is quiescent in human plasma in the absence of fibrin unlike human tissue plasminogen activator
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Gardell, SJ, primary, Hare, TR, additional, Bergum, PW, additional, Cuca, GC, additional, O'Neill-Palladino, L, additional, and Zavodny, SM, additional
- Published
- 1990
- Full Text
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3. The pharmacokinetics of plasminogen activator inhibitor-1 in the rabbit
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Mayer, EJ, primary, Fujita, T, additional, Gardell, SJ, additional, Shebuski, RJ, additional, and Reilly, CF, additional
- Published
- 1990
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4. Genetic drivers of human plasma metabolites that determine mortality in heart failure patients with reduced ejection fraction.
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Revathi Venkateswaran V, She R, Gui H, Luzum JA, Bryson TD, Malouf ZE, Williams LK, Sabbah HN, Gardell SJ, and Lanfear DE
- Abstract
Background: Heart failure with reduced ejection fraction (HFrEF) remains a significant public health issue, with the disease advancing despite neurohormonal antagonism. Energetic dysfunction is a likely contributor to residual disease progression, and we have previously reported a strong association of plasma metabolite profiles with survival among patients with HFrEF. However, the genetic and biologic mechanisms that underlie the metabolite-survival association in HFrEF were uncertain., Methods and Results: We performed genetic mapping of the key metabolite parameters, followed by mediation analyses of metabolites and genotypes on survival, and genetic pathway analyses. Patients with HFrEF ( n = 1,003) in the Henry Ford Pharmacogenomic Registry (HFPGR; 500 self-reported Black/African race patients [AA], 503 self-reported White/European race patients [EA], and 249 deaths over a median of 2.7 years) with genome-wide genotyping and targeted metabolomic profiling of plasma were included. We tested genome-wide association (GWA) of single nucleotide polymorphisms (SNPs) with the prognostic metabolite profile (PMP) and its components; first stratified by race, and then combined via meta-analysis for the entire cohort. Seven independent loci were identified as GWA significant hits in AA patients (3 for PMP and 4 for individual metabolites), one of which was also significant in the entire cohort (rs944469). No genome wide significant hits were found in White/EA patients. Among these SNPs, only rs35792152, (a hit for 3.HBA) tended to be associated with mortality in standard survival analysis (HR = 1.436, p = 0.052). The mediation analyses indicated several significant associations between SNPs, metabolites, and mortality in AA patients. Functional annotation mapping (FUMA) implicated inflammation, DNA metabolic, and mRNA splicing processes., Conclusions: GWAS of key metabolites and survival along with FUMA pathway analysis revealed new candidate genes which unveiled molecular pathways that contribute to HF disease progression via metabolic and energetic abnormalities., Competing Interests: DL has received research funding or support from Akros, Astra Zeneca, Eli Lilly, Pfizer, Illumina, Janssen and SomaLogic, and has acted as consultant to ACI Clinical (Abbott Laboratories), AstraZeneca, Cytokinetics, Duke Clinical Research Institute (CONNECT-HF), Illumina, Janssen, Martin Pharmaceuticals, Ortho Clinical Diagnostics, Otsuka, and ARMGO. HS has received research funding from Novartis, and Impulse Dynamics Inc. and is consultant to Novartis, Impulse Dynamics, Vascular Dynamics, and ViCardia. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Revathi Venkateswaran, She, Gui, Luzum, Bryson, Malouf, Williams, Sabbah, Gardell and Lanfear.)
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- 2024
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5. The TAS1R2 G-protein-coupled receptor is an ambient glucose sensor in skeletal muscle that regulates NAD homeostasis and mitochondrial capacity.
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Serrano J, Boyd J, Brown IS, Mason C, Smith KR, Karolyi K, Maurya SK, Meshram NN, Serna V, Link GM, Gardell SJ, and Kyriazis GA
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- Animals, Humans, Male, Mice, Homeostasis, Mice, Inbred C57BL, Mice, Knockout, Mitochondria metabolism, Phosphorylation, Signal Transduction, Glucose metabolism, Muscle, Skeletal metabolism, NAD metabolism, Receptors, G-Protein-Coupled metabolism, Receptors, G-Protein-Coupled genetics
- Abstract
The bioavailability of nicotinamide adenine dinucleotide (NAD) is vital for skeletal muscle health, yet the mechanisms or signals regulating NAD homeostasis remain unclear. Here, we uncover a pathway connecting peripheral glucose sensing to the modulation of muscle NAD through TAS1R2, the sugar-sensing G protein-coupled receptor (GPCR) initially identified in taste perception. Muscle TAS1R2 receptor stimulation by glucose and other agonists induces ERK1/2-dependent phosphorylation and activation of poly(ADP-ribose) polymerase1 (PARP1), a major NAD consumer in skeletal muscle. Consequently, muscle-specific deletion of TAS1R2 (mKO) in male mice suppresses PARP1 activity, elevating NAD levels and enhancing mitochondrial capacity and running endurance. Plasma glucose levels negatively correlate with muscle NAD, and TAS1R2 receptor deficiency enhances NAD responses across the glycemic range, implicating TAS1R2 as a peripheral energy surveyor. These findings underscore the role of GPCR signaling in NAD regulation and propose TAS1R2 as a potential therapeutic target for maintaining muscle health., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)
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- 2024
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6. Comprehensive interrogation of human skeletal muscle reveals a dissociation between insulin resistance and mitochondrial capacity.
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Whytock KL, Pino MF, Sun Y, Yu G, De Carvalho FG, Yeo RX, Vega RB, Parmar G, Divoux A, Kapoor N, Yi F, Cornnell H, Patten DA, Harper ME, Gardell SJ, Smith SR, Walsh MJ, and Sparks LM
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- Humans, Mitochondria, Muscle, Skeletal metabolism, Obesity metabolism, Mitochondria, Muscle metabolism, Insulin Resistance physiology, Diabetes Mellitus, Type 2 metabolism
- Abstract
Insulin resistance and blunted mitochondrial capacity in skeletal muscle are often synonymous, however, this association remains controversial. The aim of this study was to perform an in-depth multifactorial comparison of skeletal muscle mitochondrial capacity between individuals who were lean and active (Active, n = 9), individuals with obesity (Obese, n = 9), and individuals with obesity, insulin resistance, and type 2 diabetes (T2D, n = 22). Mitochondrial capacity was assessed by ex vivo mitochondrial respiration with fatty-acid and glycolytic-supported protocols adjusted for mitochondrial content (mtDNA and citrate synthase activity). Supercomplex assembly was measured by Blue Native (BN)-PAGE and immunoblot. Tricarboxylic (TCA) cycle intermediates were assessed with targeted metabolomics. Exploratory transcriptomics and DNA methylation analyses were performed to uncover molecular differences affecting mitochondrial function among the three groups. We reveal no discernable differences in skeletal muscle mitochondrial content, mitochondrial capacity, supercomplex assembly, TCA cycle intermediates, and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (body mass index, age, and aerobic capacity). We highlight that lean, active individuals have greater mitochondrial content, mitochondrial capacity, supercomplex assembly, and TCA cycle intermediates. These phenotypical changes are reflected at the level of DNA methylation and gene transcription. The collective observation of comparable muscle mitochondrial capacity in individuals with obesity and T2D (vs. individuals without T2D) underscores a dissociation from skeletal muscle insulin resistance. Clinical trial number: NCT01911104. NEW & NOTEWORTHY Whether impaired mitochondrial capacity contributes to skeletal muscle insulin resistance is debated. Our multifactorial analysis shows no differences in skeletal muscle mitochondrial content, mitochondrial capacity, and mitochondrial molecular profiles between obese individuals with and without T2D that had comparable levels of confounding factors (BMI, age, aerobic capacity). We highlight that lean, active individuals have enhanced skeletal muscle mitochondrial capacity that is also reflected at the level of DNA methylation and gene transcription.
- Published
- 2023
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7. Exercise and ageing impact the kynurenine/tryptophan pathway and acylcarnitine metabolite pools in skeletal muscle of older adults.
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Hinkley JM, Yu GX, Standley RA, Distefano G, Tolstikov V, Narain NR, Greenwood BP, Karmacharya S, Kiebish MA, Carnero EA, Yi F, Vega RB, Goodpaster BH, Gardell SJ, and Coen PM
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- Young Adult, Humans, Aged, Kynurenic Acid, NAD metabolism, Muscle, Skeletal physiology, Exercise physiology, Kynurenine metabolism, Tryptophan metabolism
- Abstract
Exercise-induced perturbation of skeletal muscle metabolites is a probable mediator of long-term health benefits in older adults. Although specific metabolites have been identified to be impacted by age, physical activity and exercise, the depth of coverage of the muscle metabolome is still limited. Here, we investigated resting and exercise-induced metabolite distribution in muscle from well-phenotyped older adults who were active or sedentary, and a group of active young adults. Percutaneous biopsies of the vastus lateralis were obtained before, immediately after and 3 h following a bout of endurance cycling. Metabolite profile in muscle biopsies was determined by tandem mass spectrometry. Mitochondrial energetics in permeabilized fibre bundles was assessed by high resolution respirometry and fibre type proportion was assessed by immunohistology. We found that metabolites of the kynurenine/tryptophan pathway were impacted by age and activity. Specifically, kynurenine was elevated in muscle from older adults, whereas downstream metabolites of kynurenine (kynurenic acid and NAD
+ ) were elevated in muscle from active adults and associated with cardiorespiratory fitness and muscle oxidative capacity. Acylcarnitines, a potential marker of impaired metabolic health, were elevated in muscle from physically active participants. Surprisingly, despite baseline group difference, acute exercise-induced alterations in whole-body substrate utilization, as well as muscle acylcarnitines and ketone bodies, were remarkably similar between groups. Our data identified novel muscle metabolite signatures that associate with the healthy ageing phenotype provoked by physical activity and reveal that the metabolic responsiveness of muscle to acute endurance exercise is retained [NB]:AUTHOR: Please ensure that the appropriate material has been provide for Table S2, as well as for Figures S1 to S7, as also cited in the text with age regardless of activity levels. KEY POINTS: Kynurenine/tryptophan pathway metabolites were impacted by age and physical activity in human muscle, with kynurenine elevated in older muscle, whereas downstream products kynurenic acid and NAD+ were elevated in exercise-trained muscle regardless of age. Acylcarnitines, a marker of impaired metabolic health when heightened in circulation, were elevated in exercise-trained muscle of young and older adults, suggesting that muscle act as a metabolic sink to reduce the circulating acylcarnitines observed with unhealthy ageing. Despite the phenotypic differences, the exercise-induced response of various muscle metabolite pools, including acylcarnitine and ketone bodies, was similar amongst the groups, suggesting that older adults can achieve the metabolic benefits of exercise seen in young counterparts., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)- Published
- 2023
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8. Metabolic dysregulation impairs lymphocyte function during severe SARS-CoV-2 infection.
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Gurshaney S, Morales-Alvarez A, Ezhakunnel K, Manalo A, Huynh TH, Abe JI, Le NT, Weiskopf D, Sette A, Lupu DS, Gardell SJ, and Nguyen H
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- Humans, SARS-CoV-2, CD8-Positive T-Lymphocytes, COVID-19 Drug Treatment, COVID-19
- Abstract
Cellular metabolic dysregulation is a consequence of SARS-CoV-2 infection that is a key determinant of disease severity. However, how metabolic perturbations influence immunological function during COVID-19 remains unclear. Here, using a combination of high-dimensional flow cytometry, cutting-edge single-cell metabolomics, and re-analysis of single-cell transcriptomic data, we demonstrate a global hypoxia-linked metabolic switch from fatty acid oxidation and mitochondrial respiration towards anaerobic, glucose-dependent metabolism in CD8
+ Tc, NKT, and epithelial cells. Consequently, we found that a strong dysregulation in immunometabolism was tied to increased cellular exhaustion, attenuated effector function, and impaired memory differentiation. Pharmacological inhibition of mitophagy with mdivi-1 reduced excess glucose metabolism, resulting in enhanced generation of SARS-CoV-2- specific CD8+ Tc, increased cytokine secretion, and augmented memory cell proliferation. Taken together, our study provides critical insight regarding the cellular mechanisms underlying the effect of SARS-CoV-2 infection on host immune cell metabolism, and highlights immunometabolism as a promising therapeutic target for COVID-19 treatment., (© 2023. The Author(s).)- Published
- 2023
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9. Source of nicotinamide governs its metabolic fate in cultured cells, mice, and humans.
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Dutta T, Kapoor N, Mathew M, Chakraborty SS, Ward NP, Prieto-Farigua N, Falzone A, DeLany JP, Smith SR, Coen PM, DeNicola GM, and Gardell SJ
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- Humans, Mice, Animals, Nicotinamide Phosphoribosyltransferase metabolism, Cells, Cultured, Aging, Cytokines metabolism, NAD metabolism, Niacinamide pharmacology, Niacinamide metabolism
- Abstract
Metabolic routing of nicotinamide (NAM) to NAD
+ or 1-methylnicotinamide (MeNAM) has impacts on human health and aging. NAM is imported by cells or liberated from NAD+ . The fate of2 H4 -NAM in cultured cells, mice, and humans was determined by stable isotope tracing.2 H4 -NAM is an NAD+ precursor via the salvage pathway in cultured A549 cells and human PBMCs and in A549 cell xenografts and PBMCs from2 H4 -NAM-dosed mice and humans, respectively.2 H4 -NAM is a MeNAM precursor in A549 cell cultures and xenografts, but not isolated PBMCs. NAM released from NAD+ is a poor MeNAM precursor. Additional A549 cell tracer studies yielded further mechanistic insight. NAMPT activators promote NAD+ synthesis and consumption. Surprisingly, NAM liberated from NAD+ in NAMPT activator-treated A549 cells is also routed toward MeNAM production. Metabolic fate mapping of the dual NAM sources across the translational spectrum (cells, mice, humans) illuminates a key regulatory node governing NAD+ and MeNAM synthesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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10. The TAS1R2 sweet taste receptor regulates skeletal muscle mass and fitness.
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Serrano J, Boyd J, Mason C, Smith KR, Karolyi K, Kondo S, Brown IS, Maurya SK, Meshram NN, Serna V, Gilger J, Branch DA, Gardell SJ, Baskin KK, Ayala JE, Pratley RE, Goodpaster BH, Coen PM, and Kyriazis GA
- Abstract
Muscle fitness and mass deteriorate under the conditions of obesity and aging for reasons yet to be fully elucidated. Herein, we describe a novel pathway linking peripheral nutrient sensing and skeletal muscle function through the sweet taste receptor TAS1R2 and the involvement of ERK2-PARP1-NAD signaling axis. Muscle-specific deletion of TAS1R2 (mKO) in mice produced elevated NAD levels due to suppressed PARP1 activity, improved mitochondrial function, increased muscle mass and strength, and prolonged running endurance. Deletion of TAS1R2 in obese or aged mice also ameliorated the decline in muscle mass and fitness arising from these conditions. Remarkably, partial loss-of-function of TAS1R2 (rs35874116) in older, obese humans recapitulated the healthier muscle phenotype displayed by mKO mice in response to exercise training. Our findings show that inhibition of the TAS1R2 signaling in skeletal muscle is a promising therapeutic approach to preserve muscle mass and function., Competing Interests: Competing interests The authors declare no competing interests
- Published
- 2023
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11. NAD + and human muscle health.
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Gardell SJ and Coen PM
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- Humans, NAD, Muscle, Skeletal physiology
- Published
- 2022
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12. Discovery of 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea as a potent NAMPT (nicotinamide phosphoribosyltransferase) activator with attenuated CYP inhibition.
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Akiu M, Tsuji T, Sogawa Y, Terayama K, Yokoyama M, Tanaka J, Asano D, Sakurai K, Sergienko E, Sessions EH, Gardell SJ, Pinkerton AB, and Nakamura T
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- Animals, Dose-Response Relationship, Drug, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors chemistry, Humans, Mice, Mice, Inbred C57BL, Molecular Structure, Structure-Activity Relationship, Urea analogs & derivatives, Urea chemistry, Cytochrome P-450 Enzyme System metabolism, Cytokines metabolism, Drug Discovery, Enzyme Inhibitors pharmacology, Nicotinamide Phosphoribosyltransferase metabolism, Urea pharmacology
- Abstract
Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the NAD
+ salvage pathway. Since NAD+ plays a pivotal role in many biological processes including metabolism and aging, activation of NAMPT is an attractive therapeutic target for treatment of diverse array of diseases. Herein, we report the continued optimization of novel urea-containing derivatives which were identified as potent NAMPT activators. Early optimization of HTS hits afforded compound 12, with a triazolopyridine core, as a lead compound. CYP direct inhibition (DI) was identified as an issue of concern, and was resolved through modulation of lipophilicity to culminate in 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea (21), which showed potent NAMPT activity accompanied with attenuated CYP DI towards multiple CYP isoforms., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2021
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13. Metabolic adaptation characterizes short-term resistance to weight loss induced by a low-calorie diet in overweight/obese individuals.
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Whytock KL, Corbin KD, Parsons SA, Pachori A, Bock CP, Jones KP, Smith JS, Yi F, Xie H, Petucci CJ, Gardell SJ, and Smith SR
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- Adult, Biomarkers, Body Composition, Female, Humans, Male, Middle Aged, Oxidation-Reduction, Retrospective Studies, Time Factors, Adaptation, Physiological physiology, Diet, Reducing, Energy Intake, Energy Metabolism physiology, Overweight, Weight Loss
- Abstract
Background: Low-calorie diet (LCD)-induced weight loss demonstrates response heterogeneity. Physiologically, a decrease in energy expenditure lower than what is predicted based on body composition (metabolic adaptation) and/or an impaired capacity to increase fat oxidation may hinder weight loss. Understanding the metabolic components that characterize weight loss success is important for optimizing weight loss strategies., Objectives: We tested the hypothesis that overweight/obese individuals who had lower than expected weight loss in response to a 28-d LCD would be characterized by 1) impaired fat oxidation and 2) whole-body metabolic adaptation. We also characterized the molecular mechanisms associated with weight loss success/failure., Methods: This was a retrospective comparison of participants who met their predicted weight loss targets [overweight/obese diet sensitive (ODS), n = 23, females = 21, males = 2] and those that did not [overweight/obese diet resistant (ODR), n = 14, females = 12, males = 2] after a 28-d LCD (900-1000 kcal/d). We used whole-body (energy expenditure and fat oxidation) and tissue-specific measurements (metabolic proteins in skeletal muscle, gene expression in adipose tissue, and metabolites in serum) to detect metabolic properties and biomarkers associated with weight loss success., Results: The ODR group had greater mean ± SD metabolic adaptation (-175 ± 149 kcal/d; +119%) than the ODS group (-80 ± 108 kcal/d) after the LCD (P = 0.030). Mean ± SD fat oxidation increased similarly for both groups from baseline (0.0701 ± 0.0206 g/min) to day 28 (0.0869 ± 0.0269 g/min; P < 0.001). A principal component analysis factor comprised of serum 3-hydroxybutyric acid, citrate, leucine/isoleucine, acetyl-carnitine, and 3-hydroxylbutyrlcarnitine was associated with weight loss success at day 28 (std. β = 0.674, R2 = 0.479, P < 0.001)., Conclusions: Individuals who achieved predicted weight loss targets after a 28-d LCD were characterized by reduced metabolic adaptation. Accumulation of metabolites associated with acetyl-CoA excess and enhanced ketogenesis was identified in the ODS group.This trial was registered at clinicaltrials.gov as NCT01616082., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)
- Published
- 2021
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14. Optimization of a urea-containing series of nicotinamide phosphoribosyltransferase (NAMPT) activators.
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Pinkerton AB, Sessions EH, Hershberger P, Maloney PR, Peddibhotla S, Hopf M, Sergienko E, Ma CT, Smith LH, Jackson MR, Tanaka J, Tsuji T, Akiu M, Cohen SE, Nakamura T, and Gardell SJ
- Subjects
- Dose-Response Relationship, Drug, Humans, Molecular Structure, Structure-Activity Relationship, Urea analogs & derivatives, Urea chemistry, Cytokines metabolism, Nicotinamide Phosphoribosyltransferase metabolism, Urea pharmacology
- Abstract
NAD
+ is a crucial cellular factor that plays multifaceted roles in wide ranging biological processes. Low levels of NAD+ have been linked to numerous diseases including metabolic disorders, cardiovascular disease, neurodegeneration, and muscle wasting disorders. A novel strategy to boost NAD+ is to activate nicotinamide phosphoribosyltransferase (NAMPT), the putative rate-limiting step in the NAD+ salvage pathway. We previously showed that NAMPT activators increase NAD+ levels in vitro and in vivo. Herein we describe the optimization of our NAMPT activator prototype (SBI-0797812) leading to the identification of 1-(4-((4-chlorophenyl)sulfonyl)phenyl)-3-(oxazol-5-ylmethyl)urea (34) that showed far more potent NAMPT activation and improved oral bioavailability., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2021
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15. Boosting NAD + with a small molecule that activates NAMPT.
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Gardell SJ, Hopf M, Khan A, Dispagna M, Hampton Sessions E, Falter R, Kapoor N, Brooks J, Culver J, Petucci C, Ma CT, Cohen SE, Tanaka J, Burgos ES, Hirschi JS, Smith SR, Sergienko E, and Pinkerton AB
- Subjects
- A549 Cells, Animals, Biocatalysis drug effects, Enzyme Activators administration & dosage, Enzyme Activators chemistry, Humans, Intracellular Space drug effects, Intracellular Space metabolism, Liver drug effects, Liver metabolism, Mice, Molecular Structure, Phosphorylation drug effects, Small Molecule Libraries administration & dosage, Small Molecule Libraries chemistry, Enzyme Activators pharmacology, NAD metabolism, Nicotinamide Mononucleotide metabolism, Nicotinamide Phosphoribosyltransferase metabolism, Small Molecule Libraries pharmacology
- Abstract
Pharmacological strategies that boost intracellular NAD
+ are highly coveted for their therapeutic potential. One approach is activation of nicotinamide phosphoribosyltransferase (NAMPT) to increase production of nicotinamide mononucleotide (NMN), the predominant NAD+ precursor in mammalian cells. A high-throughput screen for NAMPT activators and hit-to-lead campaign yielded SBI-797812, a compound that is structurally similar to active-site directed NAMPT inhibitors and blocks binding of these inhibitors to NAMPT. SBI-797812 shifts the NAMPT reaction equilibrium towards NMN formation, increases NAMPT affinity for ATP, stabilizes phosphorylated NAMPT at His247, promotes consumption of the pyrophosphate by-product, and blunts feedback inhibition by NAD+ . These effects of SBI-797812 turn NAMPT into a "super catalyst" that more efficiently generates NMN. Treatment of cultured cells with SBI-797812 increases intracellular NMN and NAD+ . Dosing of mice with SBI-797812 elevates liver NAD+ . Small molecule NAMPT activators such as SBI-797812 are a pioneering approach to raise intracellular NAD+ and realize its associated salutary effects.- Published
- 2019
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16. Measurement of Pyridine Nucleotides in Biological Samples Using LC-MS/MS.
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Petucci C, Culver JA, Kapoor N, Sessions EH, Divlianska D, and Gardell SJ
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- Animals, Cells, Cultured, Chromatography, High Pressure Liquid methods, Metabolomics standards, NAD chemistry, NAD metabolism, NADP chemistry, NADP metabolism, Oxidation-Reduction, Oxygen Isotopes chemistry, Reference Standards, Metabolomics methods, NAD analysis, NADP analysis, Tandem Mass Spectrometry methods
- Abstract
Pyridine nucleotides which include NAD
+ , NADH, NADP, and NADPH play vital roles in many different biological processes. These metabolites can be accurately quantified in a wide variety of biological samples using LC-MS/MS. The quality and precision of these measurements was enhanced using heavy isotope-labeled internal standards and carefully crafted protocols for sample processing.- Published
- 2019
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17. Metabolomics Analyses of Muscle Atrophy Induced by Hind Limb Unloading.
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Gardell SJ, Zhang X, Kapoor N, Petucci C, and Coen PM
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- Animals, Chromatography, High Pressure Liquid methods, Disease Models, Animal, Hindlimb Suspension adverse effects, Humans, Mice, Muscle, Skeletal pathology, Muscular Atrophy etiology, Tandem Mass Spectrometry methods, Metabolomics methods, Muscle, Skeletal metabolism, Muscular Atrophy pathology
- Abstract
Acute periods of contractile inactivity cause skeletal muscle atrophy along with profound alterations in tissue metabolism. Hind limb unloading via tail suspension is a commonly used rodent model of muscle atrophy. Here, we describe a sample preparation and LC-MS/MS approach for quantifying specific panels of acylcarnitines, amino acids, and organic acids in small (~8 mg) samples of atrophied mouse soleus following a period of hind limb unloading.
- Published
- 2019
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18. Impaired Mitochondrial Energetics Characterize Poor Early Recovery of Muscle Mass Following Hind Limb Unloading in Old Mice.
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Zhang X, Trevino MB, Wang M, Gardell SJ, Ayala JE, Han X, Kelly DP, Goodpaster BH, Vega RB, and Coen PM
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- Aging genetics, Animals, Energy Metabolism, Fatty Acids metabolism, Hindlimb Suspension physiology, Insulin Resistance, Male, Metabolome, Mice, Mice, Inbred C57BL, Muscular Atrophy genetics, Muscular Atrophy metabolism, Muscular Atrophy pathology, Sarcopenia genetics, Sarcopenia metabolism, Sarcopenia pathology, Transcriptome, Aging metabolism, Aging pathology, Hindlimb Suspension adverse effects, Mitochondria, Muscle metabolism, Muscle, Skeletal metabolism, Muscle, Skeletal pathology
- Abstract
The progression of age-related sarcopenia can be accelerated by impaired recovery of muscle mass following periods of disuse due to illness or immobilization. However, the mechanisms underlying poor recovery of aged muscle following disuse remain to be delineated. Recent evidence suggests that mitochondrial energetics play an important role in regulation of muscle mass. Here, we report that 22- to 24-month-old mice with low muscle mass and low glucose clearance rate also display poor early recovery of muscle mass following 10 days of hind limb unloading. We used unbiased and targeted approaches to identify changes in energy metabolism gene expression, metabolite pools and mitochondrial phenotype, and show for the first time that persistent mitochondrial dysfunction, dysregulated fatty acid β-oxidation, and elevated H2O2 emission occur concomitantly with poor early recovery of muscle mass following a period of disuse in old mice. Importantly, this is linked to more severe whole-body insulin resistance, as determined by insulin tolerance test. The findings suggest that muscle fuel metabolism and mitochondrial energetics could be a focus for mining therapeutic targets to improve recovery of muscle mass following periods of disuse in older animals.
- Published
- 2018
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19. Elevated Nicotinamide Phosphoribosyl Transferase in Skeletal Muscle Augments Exercise Performance and Mitochondrial Respiratory Capacity Following Exercise Training.
- Author
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Brouwers B, Stephens NA, Costford SR, Hopf ME, Ayala JE, Yi F, Xie H, Li JL, Gardell SJ, Sparks LM, and Smith SR
- Abstract
Mice overexpressing NAMPT in skeletal muscle (NamptTg mice) develop higher exercise endurance and maximal aerobic capacity (VO
2 max) following voluntary exercise training compared to wild-type (WT) mice. Here, we aimed to investigate the mechanisms underlying by determining skeletal muscle mitochondrial respiratory capacity in NamptTg and WT mice. Body weight and body composition, tissue weight (gastrocnemius, quadriceps, soleus, heart, liver, and epididymal white adipose tissue), skeletal muscle and liver glycogen content, VO2 max, skeletal muscle mitochondrial respiratory capacity (measured by high-resolution respirometry), skeletal muscle gene expression (measured by microarray and qPCR), and skeletal muscle protein content (measured by Western blot) were determined following 6 weeks of voluntary exercise training (access to running wheel) in 13-week-old male NamptTg (exercised NamptTg) mice and WT (exercised WT) mice. Daily running distance and running time during the voluntary exercise training protocol were recorded. Daily running distance ( p = 0.51) and running time ( p = 0.85) were not significantly different between exercised NamptTg mice and exercised WT mice. VO2 max was higher in exercised NamptTg mice compared to exercised WT mice ( p = 0.02). Body weight ( p = 0.92), fat mass ( p = 0.49), lean mass ( p = 0.91), tissue weight (all p > 0.05), and skeletal muscle ( p = 0.72) and liver ( p = 0.94) glycogen content were not significantly different between exercised NamptTg mice and exercised WT mice. Complex I oxidative phosphorylation (OXPHOS) respiratory capacity supported by fatty acid substrates ( p < 0.01), maximal (complex I+II) OXPHOS respiratory capacity supported by glycolytic ( p = 0.02) and fatty acid ( p < 0.01) substrates, and maximal uncoupled respiratory capacity supported by fatty acid substrates ( p < 0.01) was higher in exercised NamptTg mice compared to exercised WT mice. Transcriptomic analyses revealed differential expression for genes involved in oxidative metabolism in exercised NamptTg mice compared to exercised WT mice, specifically, enrichment for the gene set related to the SIRT3-mediated signaling pathway. SIRT3 protein content correlated with NAMPT protein content ( r = 0.61, p = 0.04). In conclusion, NamptTg mice develop higher exercise capacity following voluntary exercise training compared to WT mice, which is paralleled by higher mitochondrial respiratory capacity in skeletal muscle. The changes in SIRT3 targets suggest that these effects are due to remodeling of mitochondrial function.- Published
- 2018
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20. Skeletal muscle overexpression of nicotinamide phosphoribosyl transferase in mice coupled with voluntary exercise augments exercise endurance.
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Costford SR, Brouwers B, Hopf ME, Sparks LM, Dispagna M, Gomes AP, Cornnell HH, Petucci C, Phelan P, Xie H, Yi F, Walter GA, Osborne TF, Sinclair DA, Mynatt RL, Ayala JE, Gardell SJ, and Smith SR
- Subjects
- Animals, Cytokines genetics, Diet, High-Fat, Mice, Mice, Inbred C57BL, Mitochondria metabolism, Muscle, Skeletal physiology, NAD metabolism, Nicotinamide Phosphoribosyltransferase genetics, Cytokines metabolism, Muscle, Skeletal metabolism, Nicotinamide Phosphoribosyltransferase metabolism, Oxygen Consumption, Physical Conditioning, Animal
- Abstract
Objective: Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway that produces nicotinamide adenine dinucleotide (NAD
+ ), an essential co-substrate regulating a myriad of signaling pathways. We produced a mouse that overexpressed NAMPT in skeletal muscle (NamptTg) and hypothesized that NamptTg mice would have increased oxidative capacity, endurance performance, and mitochondrial gene expression, and would be rescued from metabolic abnormalities that developed with high fat diet (HFD) feeding., Methods: Insulin sensitivity (hyperinsulinemic-euglycemic clamp) was assessed in NamptTg and WT mice fed very high fat diet (VHFD, 60% by kcal) or chow diet (CD). The aerobic capacity (VO2 max) and endurance performance of NamptTg and WT mice before and after 7 weeks of voluntary exercise training (running wheel in home cage) or sedentary conditions (no running wheel) were measured. Skeletal muscle mitochondrial gene expression was also measured in exercised and sedentary mice and in mice fed HFD (45% by kcal) or low fat diet (LFD, 10% by kcal)., Results: NAMPT enzyme activity in skeletal muscle was 7-fold higher in NamptTg mice versus WT mice. There was a concomitant 1.6-fold elevation of skeletal muscle NAD+ . NamptTg mice fed VHFD were partially protected against body weight gain, but not against insulin resistance. Notably, voluntary exercise training elicited a 3-fold higher exercise endurance in NamptTg versus WT mice. Mitochondrial gene expression was higher in NamptTg mice compared to WT mice, especially when fed HFD. Mitochondrial gene expression was higher in exercised NamptTg mice than in sedentary WT mice., Conclusions: Our studies have unveiled a fascinating interaction between elevated NAMPT activity in skeletal muscle and voluntary exercise that was manifest as a striking improvement in exercise endurance., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)- Published
- 2018
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21. Targeted Metabolomic Profiling of Plasma and Survival in Heart Failure Patients.
- Author
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Lanfear DE, Gibbs JJ, Li J, She R, Petucci C, Culver JA, Tang WHW, Pinto YM, Williams LK, Sabbah HN, and Gardell SJ
- Subjects
- Aged, Biomarkers blood, Cause of Death trends, Female, Heart Failure mortality, Heart Failure physiopathology, Humans, Male, Prognosis, Stroke Volume, Survival Rate trends, United States epidemiology, Heart Failure blood, Metabolome physiology, Metabolomics methods, Risk Assessment methods
- Abstract
Objectives: This study sought to derive and validate plasma metabolite associations with survival in heart failure (HF) patients., Background: Profiling of plasma metabolites to predict the course of HF appears promising, but validation and incremental value of these profiles are less established., Methods: Patients (n = 1,032) who met Framingham HF criteria with a history of reduced ejection fraction were randomly divided into derivation and validation cohorts (n = 516 each). Amino acids, organic acids, and acylcarnitines were quantified using mass spectrometry in fasting plasma samples. We derived a prognostic metabolite profile (PMP) in the derivation cohort using Lasso-penalized Cox regression. Validity was assessed by 10-fold cross validation in the derivation cohort and by standard testing in the validation cohort. The PMP was analyzed as both a continuous variable (PMPscore) and dichotomized at the median (PMPcat), in univariate and multivariate models adjusted for clinical risk score and N-terminal pro-B-type natriuretic peptide., Results: Overall, 48% of patients were African American, 35% were women, and the average age was 69 years. After a median follow-up of 34 months, there were 256 deaths (127 and 129 in derivation and validation cohorts, respectively). Optimized modeling defined the 13 metabolite PMPs, which was cross validated as both the PMPscore (hazard ratio [HR]: 3.27; p < 2 × 10
-16 ) and PMPcat (HR: 3.04; p = 2.93 × 10-8 ). The validation cohort showed similar results (PMPscore HR: 3.9; p < 2 × 10-16 and PMPcat HR: 3.99; p = 3.47 × 10-9 ). In adjusted models, PMP remained associated with mortality in the cross-validated derivation cohort (PMPscore HR: 1.63; p = 0.0029; PMPcat HR: 1.47; p = 0.081) and the validation cohort (PMPscore HR: 1.54; p = 0.037; PMPcat HR: 1.69; p = 0.043)., Conclusions: Plasma metabolite profiles varied across HF subgroups and were associated with survival incremental to conventional predictors. Additional investigation is warranted to define mechanisms and clinical applications., (Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)- Published
- 2017
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22. Use of Ion Chromatography/Mass Spectrometry for Targeted Metabolite Profiling of Polar Organic Acids.
- Author
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Petucci C, Zelenin A, Culver JA, Gabriel M, Kirkbride K, Christison TT, and Gardell SJ
- Abstract
Organic acids (OAs) serve as metabolites that play pivotal roles in a host of different metabolic and regulatory pathways. The polar nature of many OAs poses a challenge to their measurement using widely practiced analytical methods. In this study, a targeted metabolomics method was developed using ion chromatography/triple quadrupole mass spectrometry (IC/MS) to quantitate 28 polar OAs with limits of quantitation ranging from 0.25 to 50 μM. The interday assay precisions ranged from 1% to 19%, with accuracies ranging from 82% to 115%. The IC/MS assay was used to quantitate OAs in quadriceps muscle from sedentary mice compared to fatigued mice subjected to either a low intensity, long duration (LILD) or high intensity, short duration (HISD) forced treadmill regimen. Among the OAs examined, significant differences were detected for hippuric acid, malic acid, fumaric acid, and 2-ketoglutaric acid between the sedentary and fatigued mice. In conclusion, the IC/MS method enabled the separation and quantitative survey of a broad range of polar OAs that are difficult to analyze by chromatographic techniques.
- Published
- 2016
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23. Presence of arachidonoyl-carnitine is associated with adverse cardiometabolic responses in hypertensive patients treated with atenolol.
- Author
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Weng L, Gong Y, Culver J, Gardell SJ, Petucci C, Morse AM, Frye RF, Turner ST, Chapman A, Boerwinkle E, Gums J, Beitelshees AL, Borum PR, Johnson JA, Garrett TJ, McIntyre LM, and Cooper-DeHoff RM
- Abstract
Introduction: Atenolol, a commonly prescribed β blocker for hypertension, is also associated with adverse cardiometabolic effects such as hyperglycemia and dyslipidemia. Knowledge of the mechanistic underpinnings of these adverse effects of atenolol is incomplete., Objective: We sought to identify biomarkers associated with risk for these untoward effects of atenolol. We measured baseline blood serum levels of acylcarnitines (ACs) that are involved in a host of different metabolic pathways, to establish associations with adverse cardiometabolic responses after atenolol treatment., Methods: Serum samples from Caucasian hypertensive patients (n = 224) who were treated with atenolol in the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) study were interrogated using a quantitative LC/MS assay for a large number of unique ACs in serum. For the 23 ACs that were detected in serum from ≥80 % of all patients, we conducted linear regression for changes in cardiometabolic factors with baseline AC levels, baseline cardiometabolic factors, age, sex, and BMI as covariates. For the 5 ACs that were detected in serum from 20 to 79 % of the patients, we similarly modeled changes in cardiometabolic factors, but with specifying the AC as present/absent in the regression., Results: Among the 28 ACs, the presence (vs. absence) of arachidonoyl-carnitine (C20:4) was significantly associated with increased glucose (p = 0.0002), and was nominally associated with decreased plasma HDL-C (p = 0.017) and with less blood pressure (BP) lowering (p = 0.006 for systolic BP, p = 0.002 for diastolic BP), after adjustment., Conclusion: Serum level of C20:4 is a promising biomarker to predict adverse cardiometabolic responses including glucose and poor antihypertensive response to atenolol., Competing Interests: Conflict of interest: The authors declare that they have no conflict of interest.
- Published
- 2016
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24. The Failing Heart Relies on Ketone Bodies as a Fuel.
- Author
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Aubert G, Martin OJ, Horton JL, Lai L, Vega RB, Leone TC, Koves T, Gardell SJ, Krüger M, Hoppel CL, Lewandowski ED, Crawford PA, Muoio DM, and Kelly DP
- Subjects
- Animals, Female, Gene Expression Profiling methods, Heart Failure diet therapy, Mice, Mice, Inbred C57BL, Diet, Ketogenic methods, Fatty Acids metabolism, Heart Failure metabolism, Heart Failure pathology, Ketone Bodies metabolism
- Abstract
Background: Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure., Methods and Results: Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of β-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified., Conclusions: These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure., (© 2016 American Heart Association, Inc.)
- Published
- 2016
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25. Rotenone induces reductive stress and triacylglycerol deposition in C2C12 cells.
- Author
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He Q, Wang M, Petucci C, Gardell SJ, and Han X
- Subjects
- 3T3-L1 Cells, ATP Citrate (pro-S)-Lyase metabolism, Animals, Cell Line, Electron Transport Complex I metabolism, Enzyme Activation drug effects, HEK293 Cells, Humans, Mice, Mitochondria, Muscle drug effects, Mitochondria, Muscle metabolism, Muscle Cells metabolism, Obesity metabolism, Obesity pathology, Oxidation-Reduction, Oxidative Stress drug effects, Oxygen Consumption drug effects, Transfection, Muscle Cells drug effects, Obesity complications, Rotenone pharmacology, Triglycerides metabolism
- Abstract
Environmental rotenone is associated with Parkinson's disease due to its inhibitory property to the complex I of mitochondrial respiration chain. Although environmental pollution has been postulated as a causal factor for the increasing prevalence of obesity, the role of rotenone in the pathogenesis of obesity has not been studied. We employed muscle-derived cell C2C12 as a model and shotgun lipidomics as a tool for lipid analysis and found that treatment with rotenone led to the profound deposition of intracellular triacylglycerol (TAG) in a time- and dose-dependent fashion. The TAG deposition resulted from complex I inhibition. Further studies revealed that rotenone induced mitochondrial stress as shown by decreased mitochondrial oxygen consumption rate, increased NADH/NAD+ ratio (i.e., reductive stress) and mitochondrial metabolites. We demonstrated that rotenone activated fatty acid de novo synthesis and TAG synthesis and ultimately resulted in intracellular TAG deposition. These studies suggested that increased mitochondrial stresses might be an underlying mechanism responsible for TAG accumulation manifest in obesity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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26. Cardiovascular drug discovery in the academic setting: building infrastructure, harnessing strengths, and seeking synergies.
- Author
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Gardell SJ, Roth GP, and Kelly DP
- Subjects
- Animals, Cooperative Behavior, Diffusion of Innovation, Drug Industry, Humans, Interinstitutional Relations, Molecular Targeted Therapy, Organizational Objectives, Program Development, Academies and Institutes organization & administration, Cardiovascular Agents therapeutic use, Cardiovascular Diseases drug therapy, Drug Discovery organization & administration, Organizations, Nonprofit organization & administration, Translational Research, Biomedical organization & administration
- Abstract
The flow of innovative, effective, and safe new drugs from pharmaceutical laboratories for the treatment and prevention of cardiovascular disease has slowed to a trickle. While the need for breakthrough cardiovascular disease drugs is still paramount, the incentive to develop these agents has been blunted by burgeoning clinical development costs coupled with a heightened risk of failure due to the unprecedented nature of the emerging drug targets and increasingly challenging regulatory environment. A fuller understanding of the drug targets and employing novel biomarker strategies in clinical trials should serve to mitigate the risk. In any event, these current challenges have evoked changing trends in the pharmaceutical industry, which have created an opportunity for non-profit biomedical research institutions to play a pivotal partnering role in early stage drug discovery. The obvious strengths of academic research institutions is the breadth of their scientific programs and the ability and motivation to "go deep" to identify and characterize new target pathways that unlock the specific mysteries of cardiovascular diseases--leading to a bounty of novel therapeutic targets and prescient biomarkers. However, success in the drug discovery arena within the academic environment is contingent upon assembling the requisite infrastructure, annexing the talent to interrogate and validate the drug targets, and building translational bridges with pharmaceutical organizations and patient-oriented researchers.
- Published
- 2010
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27. Effect of the small molecule plasminogen activator inhibitor-1 (PAI-1) inhibitor, PAI-749, in clinical models of fibrinolysis.
- Author
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Lucking AJ, Visvanathan A, Philippou H, Fraser S, Grant PJ, Connolly TM, Gardell SJ, Feuerstein GZ, Fox KA, Booth NA, and Newby DE
- Subjects
- Adult, Cross-Over Studies, Double-Blind Method, Humans, Models, Biological, Fibrinolysis drug effects, Indoles pharmacology, Plasminogen Activator Inhibitor 1, Tetrazoles pharmacology
- Abstract
Background: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor-1 (PAI-1). PAI-749 is a small molecule inhibitor of PAI-1 with proven antithrombotic efficacy in several preclinical models., Objective: To assess the effect of PAI-749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma-based and whole blood-based models of fibrinolysis., Methods: In a double-blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue-type plasminogen activator (t-PA) in the presence of PAI-749 or control. t-PA-mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins., Results: There was a dose-dependent reduction in ex vivo thrombus formation by t-PA (P < 0.0001). PAI-749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t-PA. Inhibition of PAI-1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05)., Conclusions: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI-749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood-based systems.
- Published
- 2010
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28. LXR ligand lowers LDL cholesterol in primates, is lipid neutral in hamster, and reduces atherosclerosis in mouse.
- Author
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Quinet EM, Basso MD, Halpern AR, Yates DW, Steffan RJ, Clerin V, Resmini C, Keith JC, Berrodin TJ, Feingold I, Zhong W, Hartman HB, Evans MJ, Gardell SJ, DiBlasio-Smith E, Mounts WM, LaVallie ER, Wrobel J, Nambi P, and Vlasuk GP
- Subjects
- Animals, Atherosclerosis metabolism, Caco-2 Cells, Cricetinae, Disease Models, Animal, Humans, Indazoles blood, Indazoles chemistry, Ligands, Liver enzymology, Liver metabolism, Liver X Receptors, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Orphan Nuclear Receptors metabolism, Atherosclerosis drug therapy, Cholesterol, LDL drug effects, Cholesterol, LDL metabolism, Indazoles pharmacology, Lipid Metabolism drug effects, Macaca fascicularis metabolism, Orphan Nuclear Receptors agonists
- Abstract
Liver X receptors (LXRs) are ligand-activated transcription factors that coordinate regulation of gene expression involved in several cellular functions but most notably cholesterol homeostasis encompassing cholesterol transport, catabolism, and absorption. WAY-252623 (LXR-623) is a highly selective and orally bioavailable synthetic modulator of LXR, which demonstrated efficacy for reducing lesion progression in the murine LDLR(-/-) atherosclerosis model with no associated increase in hepatic lipogenesis either in this model or Syrian hamsters. In nonhuman primates with normal lipid levels, WAY-252623 significantly reduced total (50-55%) and LDL-cholesterol (LDLc) (70-77%) in a time- and dose-dependent manner as well as increased expression of the target genes ABCA1/G1 in peripheral blood cells. Statistically significant decreases in LDLc were noted as early as day 7, reached a maximum by day 28, and exceeded reductions observed for simvastatin alone (20 mg/kg). Transient increases in circulating triglycerides and liver enzymes reverted to baseline levels over the course of the study. Complementary microarray analysis of duodenum and liver gene expression revealed differential activation of LXR target genes and suggested no direct activation of hepatic lipogenesis. WAY-252623 displays a unique and favorable pharmacological profile suggesting synthetic LXR ligands with these characteristics may be suitable for evaluation in patients with atherosclerotic dyslipidemia.
- Published
- 2009
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29. Selective Kv1.5 blockers: development of (R)-1-(methylsulfonylamino)-3-[2-(4-methoxyphenyl)ethyl]-4-(4-methoxyphenyl)-2-imidazolidinone (KVI-020/WYE-160020) as a potential treatment for atrial arrhythmia.
- Author
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Blass BE, Fensome A, Trybulski E, Magolda R, Gardell SJ, Liu K, Samuel M, Feingold I, Huselton C, Jackson CM, Djandjighian L, Ho D, Hennan J, and Janusz JM
- Subjects
- Animals, Anti-Arrhythmia Agents pharmacokinetics, Anti-Arrhythmia Agents pharmacology, Cell Line, Cricetinae, Cricetulus, Dogs, Humans, Imidazolidines pharmacokinetics, Imidazolidines pharmacology, In Vitro Techniques, Microsomes, Liver metabolism, Patch-Clamp Techniques, Solubility, Stereoisomerism, Structure-Activity Relationship, Sulfonamides pharmacokinetics, Sulfonamides pharmacology, Anti-Arrhythmia Agents chemical synthesis, Atrial Fibrillation drug therapy, Imidazolidines chemical synthesis, Kv1.5 Potassium Channel antagonists & inhibitors, Sulfonamides chemical synthesis
- Abstract
Atrial fibrillation is the most prevalent form of cardiac arrhythmia. Current treatments extend the atrial effective refractory period by nonselective blockade of cardiac ion channels. An alternative approach selectively targeting the Kv1.5 ion channel offers the opportunity for therapeutic benefit with decreased risk of adverse cardiovascular events. KVI-020 (4g) successfully demonstrated antiarrhythmic efficacy in a canine arrhythmia model, and these findings support its utility as an antiarrhythmic agent.
- Published
- 2009
- Full Text
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30. GAP-134 ([2S,4R]-1-[2-aminoacetyl]4-benzamidopyrrolidine-2-carboxylic acid) prevents spontaneous ventricular arrhythmias and reduces infarct size during myocardial ischemia/reperfusion injury in open-chest dogs.
- Author
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Hennan JK, Swillo RE, Morgan GA, Rossman EI, Kantrowitz J, Butera J, Petersen JS, Gardell SJ, and Vlasuk GP
- Subjects
- Animals, Anti-Arrhythmia Agents administration & dosage, Benzamides administration & dosage, Coronary Circulation drug effects, Disease Models, Animal, Dogs, Dose-Response Relationship, Drug, Infusions, Intravenous, Myocardial Infarction etiology, Myocardial Infarction pathology, Myocardial Infarction physiopathology, Myocardial Reperfusion Injury complications, Myocardial Reperfusion Injury pathology, Myocardial Reperfusion Injury physiopathology, Proline administration & dosage, Proline pharmacology, Tachycardia, Ventricular etiology, Tachycardia, Ventricular pathology, Tachycardia, Ventricular physiopathology, Time Factors, Ventricular Premature Complexes etiology, Ventricular Premature Complexes pathology, Ventricular Premature Complexes physiopathology, Anti-Arrhythmia Agents pharmacology, Benzamides pharmacology, Myocardial Infarction prevention & control, Myocardial Reperfusion Injury drug therapy, Myocardium pathology, Proline analogs & derivatives, Tachycardia, Ventricular prevention & control, Ventricular Premature Complexes prevention & control
- Abstract
The antiarrhythmic dipeptide, GAP-134, ([2S,4R]-1[2-aminoacetyl]-4-benzamido-pyrrolidine-2-carboxylic acid) was evaluated in canine ischemia/reperfusion model. In dogs subjected to 60-minute ischemia and 4-hour reperfusion, GAP-134 was administered 10 minutes before reperfusion as a bolus + intravenous (IV) infusion. The doses administered were 0.25 microg/kg bolus + 0.19 microg/kg per hour infusion; 2.5 microg/kg + 1.9 microg/kg per hour; 25 mg/kg + 19 mg/kg per hour; 75 mg/kg + 57 mg/kg per hour. Ventricular ectopy was quantified during reperfusion, including premature ventricular contractions (PVC) and ventricular tachycardia (VT). Total incidence of VT was reduced significantly with the 2 highest doses of GAP-134 (1.7 + 0.8; 2.2 + 1.4 events; P < .05) compared to controls (23.0 + 6.1). Total PVCs were reduced significantly from 11.1 + 1.6% in control animals to 2.0% + 0.7% and 1.8% + 0.8% after the 2 highest doses of GAP-134. Infarct size, expressed as percentage of left ventricle, was reduced significantly from 19.0% + 3.5% in controls to 7.9% + 1.5% and 7.1% + 0.8% (P < .05) at the 2 highest doses of GAP-134. GAP-134 is an effective antiarrhythmic agent with potential to reduce ischemia/reperfusion injury.
- Published
- 2009
- Full Text
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31. Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR-/- and apoE-/- mice.
- Author
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Hartman HB, Gardell SJ, Petucci CJ, Wang S, Krueger JA, and Evans MJ
- Subjects
- Animals, Apolipoproteins E genetics, Atherosclerosis genetics, Atherosclerosis metabolism, Atherosclerosis pathology, Azepines pharmacology, Bile Acids and Salts metabolism, Cholesterol 7-alpha-Hydroxylase antagonists & inhibitors, Cholesterol 7-alpha-Hydroxylase genetics, Dyslipidemias genetics, Dyslipidemias metabolism, Dyslipidemias prevention & control, Female, Gene Expression drug effects, Indoles pharmacology, Lipids blood, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Receptors, Cytoplasmic and Nuclear antagonists & inhibitors, Receptors, Cytoplasmic and Nuclear deficiency, Receptors, Cytoplasmic and Nuclear genetics, Receptors, Cytoplasmic and Nuclear metabolism, Receptors, LDL genetics, Steroid 12-alpha-Hydroxylase antagonists & inhibitors, Steroid 12-alpha-Hydroxylase genetics, Apolipoproteins E deficiency, Atherosclerosis prevention & control, Receptors, Cytoplasmic and Nuclear agonists, Receptors, LDL deficiency
- Abstract
The role of farnesoid X receptor (FXR) in the development of atherosclerosis has been unclear. Here, LDL receptor (LDLR(-/-)) or apolipoprotein E (apoE(-/-)) female or male mice were fed a Western diet and treated with a potent synthetic FXR agonist, WAY-362450. Activation of FXR blocked diet-induced hypertriglyceridemia and elevations of non-HDL cholesterol and produced a near complete inhibition of aortic lesion formation. WAY-362450 also induced small heterodimer partner (SHP) expression and repressed cholesterol 7alpha-hydroxylase (CYP7A1) and sterol 12 alpha-hydroxylase (CYP8B1) expression. To determine if SHP was essential for these protective activities, LDLR(-/-)SHP(-/-) and apoE(-/-)SHP(-/-) mice were similarly treated with WAY-362450. Surprisingly, a notable sex difference was observed in these mice. In male LDLR(-/-)SHP(-/-) or apoE(-/-)SHP(-/-) mice, WAY-362450 still repressed CYP7A1 and CYP8B1 expression by 10-fold and still strongly reduced non-HDL cholesterol levels and aortic lesion area. In contrast, in the female LDLR(-/-)SHP(-/-) or apoE(-/-)SHP(-/-) mice, WAY-362450 only slightly repressed CYP7A1 and CYP8B1 expression and did not reduce non-HDL cholesterol or aortic lesion size. WAY-362450 inhibition of hypertriglyceridemia remained intact in LDLR(-/-) or apoE(-/-) mice lacking SHP of both sexes. These results suggest that activation of FXR protects against atherosclerosis in the mouse, and this protective effect correlates with repression of bile acid synthetic genes, with mechanistic differences between male and female mice.
- Published
- 2009
- Full Text
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32. The gap junction modifier, GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], improves conduction and reduces atrial fibrillation/flutter in the canine sterile pericarditis model.
- Author
-
Rossman EI, Liu K, Morgan GA, Swillo RE, Krueger JA, Gardell SJ, Butera J, Gruver M, Kantrowitz J, Feldman HS, Petersen JS, Haugan K, and Hennan JK
- Subjects
- Animals, Anti-Arrhythmia Agents pharmacology, Anti-Arrhythmia Agents therapeutic use, Atrial Fibrillation physiopathology, Atrial Flutter physiopathology, Benzamides pharmacology, Connexin 43 metabolism, Dipeptides adverse effects, Dipeptides pharmacology, Disease Models, Animal, Dogs, Electric Conductivity, Female, Gap Junctions physiology, Heart Atria drug effects, Heart Atria metabolism, Heart Atria physiopathology, Heart Conduction System physiology, Male, Molecular Structure, Oligopeptides pharmacology, Oligopeptides therapeutic use, Pericarditis physiopathology, Postoperative Complications drug therapy, Postoperative Complications physiopathology, Proline pharmacology, Proline therapeutic use, Rats, Rats, Sprague-Dawley, Refractory Period, Electrophysiological drug effects, Atrial Fibrillation drug therapy, Atrial Flutter drug therapy, Benzamides therapeutic use, Dipeptides therapeutic use, Gap Junctions drug effects, Heart Conduction System drug effects, Pericarditis drug therapy, Proline analogs & derivatives
- Abstract
Gap junction uncoupling can alter conduction pathways and promote cardiac re-entry mechanisms that potentiate many supraventricular arrhythmias, such as atrial fibrillation (AF) and atrial flutter (AFL). Our objective was to determine whether GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], a small dipeptide gap junction modifier, can improve conduction and ultimately prevent AF/AFL. In rat atrial strips subjected to metabolic stress, GAP-134 prevented significantly conduction velocity slowing at 10 nM compared with vehicle (p < 0.01). In the canine sterile pericarditis model, conduction time (CT; n = 5), atrial effective refractory period (AERP; n = 3), and AF/AFL duration/inducibility (n = 16) were measured 2 to 3 days postoperatively in conscious dogs. CT was significantly faster after GAP-134 infusion (average plasma concentration, 250 nM) at cycle lengths of 300 ms (66.2 +/- 1.0 versus 62.0 +/- 1.0 ms; p < 0.001) and 200 ms (64.4 +/- 0.9 versus 61.0 +/- 1.3 ms; p < 0.001). No significant changes in AERP were noted after GAP-134 infusion. The mean number of AF/AFL inductions per animal was significantly decreased after GAP-134 infusion (2.7 +/- 0.6 versus 1.6 +/- 0.8; p < 0.01), with total AF/AFL burden being decreased from 12,280 to 6063 s. Western blot experiments showed no change in connexin 43 expression. At concentrations exceeding those described in the AF/AFL experiments, GAP-134 had no effect on heart rate, blood pressure, or any electrocardiogram parameters. In conclusion, GAP-134 shows consistent efficacy on measures of conduction and AF/AFL inducibility in the canine sterile pericarditis model. These findings, along with its oral bioavailability, underscore its potential antiarrhythmic efficacy.
- Published
- 2009
- Full Text
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33. A synthetic farnesoid X receptor (FXR) agonist promotes cholesterol lowering in models of dyslipidemia.
- Author
-
Evans MJ, Mahaney PE, Borges-Marcucci L, Lai K, Wang S, Krueger JA, Gardell SJ, Huard C, Martinez R, Vlasuk GP, and Harnish DC
- Subjects
- Animals, Apolipoprotein A-I blood, Apolipoprotein A-I genetics, Azepines chemistry, Cells, Cultured, Cholesterol pharmacology, DNA-Binding Proteins metabolism, Disease Models, Animal, Dyslipidemias complications, Female, Fructose pharmacology, Humans, Hyperglycemia complications, Hyperglycemia genetics, Hyperinsulinism complications, Hyperinsulinism genetics, Indoles chemistry, Kidney cytology, Male, Mice, Mice, Inbred C57BL, Mice, Mutant Strains, Mice, Transgenic, Rats, Rats, Sprague-Dawley, Receptors, Cytoplasmic and Nuclear metabolism, Receptors, LDL genetics, Receptors, Leptin genetics, Transcription Factors metabolism, Triglycerides blood, Azepines pharmacology, Cholesterol blood, DNA-Binding Proteins agonists, Dyslipidemias drug therapy, Dyslipidemias metabolism, Indoles pharmacology, Receptors, Cytoplasmic and Nuclear agonists, Transcription Factors agonists
- Abstract
The nuclear hormone receptor farnesoid X receptor (FXR) plays a critical role in the regulation of bile acid, triglyceride (TG), and cholesterol homeostasis. WAY-362450 (FXR-450/XL335) is a potent synthetic FXR agonist as characterized in luciferase reporter assays and in mediating FXR target gene regulation in primary human and immortalized mouse hepatocytes. In vivo, WAY-362450 dose dependently decreased serum TG levels after 7 days of oral dosing in western diet-fed low-density lipoprotein receptor-/- mice and in the diabetic mouse strains KK-Ay and db/db comparable to that achieved with the peroxisome proliferator activated receptor-alpha agonist, fenofibrate. WAY-362450 treatment also reduced serum cholesterol levels via reductions in LDLc, VLDLc, and HDLc lipoprotein fractions that were not accompanied by hepatic cholesterol accumulation. This cholesterol lowering was dependent on FXR as demonstrated in a hypothyroid-induced hypercholesterolemia setting in FXR-/- mice. In fructose-fed models, WAY-362450 also decreased TG and VLDLc levels in rats and hamsters but significantly increased HDLc levels in rats while reducing HDLc levels in hamsters. The differential effect of WAY-362450 on HDLc is likely due to a murine-specific induction of endothelial lipase and scavenger receptor-BI that does not occur in rats. These studies demonstrate a consistent ability of WAY-362450 to lower both serum TG and cholesterol levels and suggest that synthetic FXR agonists may have clinical utility in the treatment of mixed dyslipidemia.
- Published
- 2009
- Full Text
- View/download PDF
34. Indazole-based liver X receptor (LXR) modulators with maintained atherosclerotic lesion reduction activity but diminished stimulation of hepatic triglyceride synthesis.
- Author
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Wrobel J, Steffan R, Bowen SM, Magolda R, Matelan E, Unwalla R, Basso M, Clerin V, Gardell SJ, Nambi P, Quinet E, Reminick JI, Vlasuk GP, Wang S, Feingold I, Huselton C, Bonn T, Farnegardh M, Hansson T, Nilsson AG, Wilhelmsson A, Zamaratski E, and Evans MJ
- Subjects
- Animals, Arteriosclerosis metabolism, Cell Differentiation drug effects, Cell Line, Cricetinae, Crystallography, X-Ray, DNA-Binding Proteins metabolism, Humans, Hydrogen Bonding, Indazoles chemical synthesis, Indazoles chemistry, Ligands, Liver drug effects, Liver X Receptors, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Animal, Models, Molecular, Molecular Structure, Orphan Nuclear Receptors, Receptors, Cytoplasmic and Nuclear metabolism, Recombinant Proteins drug effects, Recombinant Proteins metabolism, Structure-Activity Relationship, Triglycerides blood, Arteriosclerosis drug therapy, DNA-Binding Proteins agonists, Indazoles pharmacology, Liver metabolism, Receptors, Cytoplasmic and Nuclear agonists, Triglycerides biosynthesis
- Abstract
A series of substituted 2-benzyl-3-aryl-7-trifluoromethylindazoles were prepared as LXR modulators. These compounds were partial agonists in transactivation assays when compared to 1 (T0901317) and were slightly weaker with respect to potency and efficacy on LXRalpha than on LXRbeta. Lead compounds in this series 12 (WAY-252623) and 13 (WAY-214950) showed less lipid accumulation in HepG2 cells than potent full agonists 1 and 3 (WAY-254011) but were comparable in efficacy to 1 and 3 with respect to cholesterol efflux in THP-1 foam cells, albeit weaker in potency. Compound 13 reduced aortic lesion area in LDLR knockout mice equivalently to 3 or positive control 2 (GW3965). In a 7-day hamster model, compound 13 showed a lesser propensity for plasma TG elevation than 3, when the compounds were compared at doses in which they elevated ABCA1 and ABCG1 gene expression in duodenum and liver at equal levels. In contrast to results previously published for 2, the lack of TG effect of 13 correlated with its inability to increase liver fatty acid synthase (FAS) gene expression, which was up-regulated 4-fold by 3. These results suggest indazoles such as 13 may have an improved profile for potential use as a therapeutic agent.
- Published
- 2008
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35. Effect of tiplaxtinin (PAI-039), an orally bioavailable PAI-1 antagonist, in a rat model of thrombosis.
- Author
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Hennan JK, Morgan GA, Swillo RE, Antrilli TM, Mugford C, Vlasuk GP, Gardell SJ, and Crandall DL
- Subjects
- Administration, Oral, Animals, Biological Availability, Disease Models, Animal, Indoleacetic Acids administration & dosage, Indoleacetic Acids pharmacokinetics, Male, Platelet Aggregation drug effects, Prothrombin Time, Rats, Rats, Sprague-Dawley, Indoleacetic Acids pharmacology, Plasminogen Activator Inhibitor 1, Thrombosis prevention & control
- Abstract
Objective: To assess the antithrombotic and profibrinolytic effects of tiplaxtinin (PAI-039), an orally bioavailable antagonist of PAI-1, in rat models of thrombosis., Methods and Results: Carotid artery and vena cava vascular injury was produced by application of FeCl3 and blood flow was monitored using ultrasonic technology. To assess efficacy in a thrombosis prevention paradigm, PAI-039 was administered orally 90 min before injury (1-30 mg kg(-1)). To assess efficacy in a thrombosis treatment paradigm, vascular injury and stable thrombus formation were followed 4 h later by recovery and PAI-039 administration. PAI-039 prevented carotid artery occlusion in 20, 68 and 60% of animals pretreated with 0.3, 1.0 and 3.0 mg kg(-1), respectively. Time to occlusive thrombosis was increased from 18.2 +/- 4.6 min in controls to 32.5 +/- 8.7 (P = ns), 46.1 +/- 7.0 (P < 0.05), and 41.6 +/- 11.3 min (P < 0.05) in the respective PAI-039 treatment groups. In the vena cava protocol, PAI-039 pretreatment significantly reduced thrombus weight at PAI-039 doses of 3, 10 and 30 mg kg(-1). When PAI-039 was dosed in a treatment paradigm 4 h after stable arterial and venous thrombosis, a significant reduction in thrombus weight was observed 24 h later at PAI-039 doses of 3, 10 and 30 mg kg(-1). PAI-039 (10, 30 and 100 mg kg(-1)) had no effect on platelet aggregation in response to ADP or collagen and was not associated with increased bleeding or prolonged prothrombin time. In animals bearing no vascular injury, PAI-039 had no effect on circulating, low-levels of PAI-1 activity. In contrast, circulating PAI-1 activity increased 5-fold following the induction of vascular injury, which was completely neutralized by PAI-039., Conclusions: PAI-039 exerts antithrombotic efficacy in rat models of arterial and venous vascular injury without effecting platelet aggregation.
- Published
- 2008
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36. Enhanced clearance of Abeta in brain by sustaining the plasmin proteolysis cascade.
- Author
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Jacobsen JS, Comery TA, Martone RL, Elokdah H, Crandall DL, Oganesian A, Aschmies S, Kirksey Y, Gonzales C, Xu J, Zhou H, Atchison K, Wagner E, Zaleska MM, Das I, Arias RL, Bard J, Riddell D, Gardell SJ, Abou-Gharbia M, Robichaud A, Magolda R, Vlasuk GP, Bjornsson T, Reinhart PH, and Pangalos MN
- Subjects
- Animals, Humans, Immunohistochemistry, Mice, Mice, Transgenic, Plasminogen Inactivators metabolism, Tissue Plasminogen Activator antagonists & inhibitors, Tissue Plasminogen Activator metabolism, Amyloid beta-Peptides metabolism, Brain metabolism, Fibrinolysin metabolism, Fibrinolytic Agents metabolism
- Abstract
The amyloid hypothesis states that a variety of neurotoxic beta-amyloid (Abeta) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Abeta production and clearance. Enzymes responsible for the degradation of Abeta are not well understood, and, thus far, it has not been possible to enhance Abeta catabolism by pharmacological manipulation. We provide evidence that Abeta catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Abeta levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Abeta oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Abeta. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Abeta levels, restore long-term potentiation deficits in hippocampal slices from transgenic Abeta-producing mice, and reverse cognitive deficits in these mice.
- Published
- 2008
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37. Small-molecule ghrelin receptor antagonists improve glucose tolerance, suppress appetite, and promote weight loss.
- Author
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Esler WP, Rudolph J, Claus TH, Tang W, Barucci N, Brown SE, Bullock W, Daly M, Decarr L, Li Y, Milardo L, Molstad D, Zhu J, Gardell SJ, Livingston JN, and Sweet LJ
- Subjects
- Animals, Appetite Depressants pharmacology, Cells, Cultured, Drug Evaluation, Preclinical, Ghrelin antagonists & inhibitors, Hypoglycemic Agents pharmacology, Hypoglycemic Agents therapeutic use, Male, Mice, Mice, Inbred C57BL, Models, Biological, Piperidines pharmacology, Piperidines therapeutic use, Quinazolinones pharmacology, Quinazolinones therapeutic use, Rats, Rats, Sprague-Dawley, Rats, Wistar, Appetite drug effects, Appetite Depressants therapeutic use, Glucose Intolerance drug therapy, Receptors, Ghrelin antagonists & inhibitors, Weight Loss drug effects
- Abstract
Ghrelin, through action on its receptor, GH secretagogue receptor type 1a (GHS-R1a), exerts a variety of metabolic functions including stimulation of appetite and weight gain and suppression of insulin secretion. In the present study, we examined the effects of novel small-molecule GHS-R1a antagonists on insulin secretion, glucose tolerance, and weight loss. Ghrelin dose-dependently suppressed insulin secretion from dispersed rat islets. This effect was fully blocked by a GHS-R1a antagonist. Consistent with this observation, a single oral dose of a GHS-R1a antagonist improved glucose homeostasis in an ip glucose tolerance test in rat. Improvement in glucose tolerance was attributed to increased insulin secretion. Daily oral administration of a GHS-R1a antagonist to diet-induced obese mice led to reduced food intake and weight loss (up to 15%) due to selective loss of fat mass. Pair-feeding experiments indicated that weight loss was largely a consequence of reduced food intake. The impact of a GHS-R1a antagonist on gastric emptying was also examined. Although the GHS-R1a antagonist modestly delayed gastric emptying at the highest dose tested (10 mg/kg), delayed gastric emptying does not appear to be a requirement for weight loss because lower doses produced weight loss without an effect on gastric emptying. Consistent with the hypothesis that ghrelin regulates feeding centrally, the anorexigenic effects of potent GHS-R1a antagonists in mice appeared to correspond with their brain exposure. These observations demonstrate that GHS-R1a antagonists have the potential to improve the diabetic condition by promoting glucose-dependent insulin secretion and promoting weight loss.
- Published
- 2007
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38. Quinazolinone derivatives as orally available ghrelin receptor antagonists for the treatment of diabetes and obesity.
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Rudolph J, Esler WP, O'connor S, Coish PD, Wickens PL, Brands M, Bierer DE, Bloomquist BT, Bondar G, Chen L, Chuang CY, Claus TH, Fathi Z, Fu W, Khire UR, Kristie JA, Liu XG, Lowe DB, McClure AC, Michels M, Ortiz AA, Ramsden PD, Schoenleber RW, Shelekhin TE, Vakalopoulos A, Tang W, Wang L, Yi L, Gardell SJ, Livingston JN, Sweet LJ, and Bullock WH
- Subjects
- Administration, Oral, Animals, Binding, Competitive, Blood Glucose analysis, Cell Line, Eating drug effects, Glucose Tolerance Test, Humans, Male, Mice, Mice, Inbred C57BL, Quinazolinones chemistry, Quinazolinones pharmacology, Radioligand Assay, Rats, Rats, Wistar, Stereoisomerism, Structure-Activity Relationship, Weight Loss drug effects, Diabetes Mellitus drug therapy, Obesity drug therapy, Quinazolinones chemical synthesis, Receptors, Ghrelin antagonists & inhibitors
- Abstract
The peptide hormone ghrelin is the endogenous ligand for the type 1a growth hormone secretagogue receptor (GHS-R1a) and the only currently known circulating appetite stimulant. GHS-R1a antagonism has therefore been proposed as a potential approach for obesity treatment. More recently, ghrelin has been recognized to also play a role in controlling glucose-induced insulin secretion, which suggests another possible benefit for a GHS-R1a antagonist, namely, the role as an insulin secretagogue with potential value for diabetes treatment. In our laboratories, piperidine-substituted quinazolinone derivatives were identified as a new class of small-molecule GHS-R1a antagonists. Starting from an agonist with poor oral bioavailability, optimization led to potent, selective, and orally bioavailable antagonists. In vivo efficacy evaluation of selected compounds revealed suppression of food intake and body weight reduction as well as glucose-lowering effects mediated by glucose-dependent insulin secretion.
- Published
- 2007
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39. Neutralization of plasminogen activator inhibitor I (PAI-1) by the synthetic antagonist PAI-749 via a dual mechanism of action.
- Author
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Gardell SJ, Krueger JA, Antrilli TA, Elokdah H, Mayer S, Orcutt SJ, Crandall DL, and Vlasuk GP
- Subjects
- Biopolymers metabolism, Electrophoresis, Polyacrylamide Gel, Fluorescence, Humans, Hydrolysis, Micelles, Vitronectin metabolism, Vitronectin pharmacology, Indoles pharmacology, Plasminogen Activator Inhibitor 1 metabolism, Tetrazoles pharmacology
- Abstract
PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC(50) values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K(d) (254 nM) was similar to the IC(50) (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.
- Published
- 2007
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40. A presenilin-independent aspartyl protease prefers the gamma-42 site cleavage.
- Author
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Lai MT, Crouthamel MC, DiMuzio J, Pietrak BL, Donoviel DB, Bernstein A, Gardell SJ, Li YM, and Hazuda D
- Subjects
- Amyloid Precursor Protein Secretases, Animals, Blastocyst metabolism, Carbamates pharmacology, Cell Membrane metabolism, Cells, Cultured, DNA genetics, Dipeptides pharmacology, Endopeptidases metabolism, Hydrogen-Ion Concentration, Luminescent Measurements, Membrane Proteins genetics, Membrane Proteins physiology, Mice, Neurons enzymology, Neurons metabolism, Presenilin-1, Presenilin-2, Transfection, Amyloid beta-Peptides metabolism, Aspartic Acid Endopeptidases metabolism
- Abstract
beta-Amyloid peptides (Abeta40 and Abeta42) are the major constituents of amyloid plaques, which are one of the hallmarks of Alzheimer's disease (AD). The Abeta is derived from sequential cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. gamma-Secretase consists of at least four proteins where presenilins (PS1 and PS2 or PS) are the catalytic subunit involved in the gamma-site cleavage of APP. Secretion of both Abeta40 and Abeta42 is significantly reduced in PS1 knock-out cells and completely abolished in cells deficient for both PS1 and PS2. Consequently, both the PS proteins play essential roles in the production of the secretory of Abeta from cells. Recent studies in primary neurons, however, suggest that PSs are not required for intracellular Abeta42 accumulation; thus the intracellular Abeta42 appears to be generated in a PS-independent manner. Here we present the first biochemical evidence indicating that Abeta, especially Abeta42, can be generated in the absence of PS based on an in vitrogamma-secretase assay employing membranes prepared from PS-deficient Blastocyst-derived (BD) cells. This PS-independent gamma-secretase (PSIG) activity is sensitive to the changes in pH and displays an optimal activity at pH 6.0. Pepstatin A is a potent inhibitor for this proteolytic activity with IC50 of 1.2 nm and 0.4 nm for Abeta40 and Abeta42 generation, respectively. These results indicate that these PS-independent gamma-site cleavages are mediated by an aspartyl protease. More importantly, the PSIG activity displays a distinct preference in mediating the 42-site cleavage over the 40-site cleavage, thereby generating Abeta42 as the predominant product.
- Published
- 2006
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41. P2 pyridine N-oxide thrombin inhibitors: a novel peptidomimetic scaffold.
- Author
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Nantermet PG, Burgey CS, Robinson KA, Pellicore JM, Newton CL, Deng JZ, Selnick HG, Lewis SD, Lucas BJ, Krueger JA, Miller-Stein C, White RB, Wong B, McMasters DR, Wallace AA, Lynch JJ Jr, Yan Y, Chen Z, Kuo L, Gardell SJ, Shafer JA, Vacca JP, and Lyle TA
- Subjects
- Hydrogen Bonding, Models, Molecular, Molecular Mimicry, Antithrombins chemistry, Pyrimidines chemistry
- Abstract
In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).
- Published
- 2005
- Full Text
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42. Electrochemiluminescence assay for basic carboxypeptidases: inhibition of basic carboxypeptidases and activation of thrombin-activatable fibrinolysis inhibitor.
- Author
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Mao SS, Colussi D, Bailey CM, Bosserman M, Burlein C, Gardell SJ, and Carroll SS
- Subjects
- Animals, Carboxypeptidase B2 blood, Carboxypeptidase B2 isolation & purification, Dogs, Electrochemistry, Enzyme Activation, Enzyme Precursors blood, Enzyme Precursors isolation & purification, Enzyme Precursors metabolism, Fibrinolysis, Humans, Luminescent Measurements, Lung, Mice, Rabbits, Rats, Thrombin, Carboxypeptidase B2 metabolism, Carboxypeptidases analysis, Carboxypeptidases antagonists & inhibitors
- Abstract
Carboxypeptidases catalyze the removal of the C-terminal amino acid residues in peptides and proteins and exert important biological functions. Assays for carboxypeptidase activity that rely on change of absorbance generally suffer from low sensitivity and are difficult to adapt to high-throughput screening. We have developed a sensitive, robust assay for basic carboxypeptidase activity that makes use of electrochemiluminescent (ECL) detection of reaction product. In this assay, a peptide substrate contains the epitope for antibody (G2-10) binding which is masked by a C-terminal arginine. Carboxypeptidase activity exposes the epitope, allowing the binding of ruthenylated G2-10 which is then detected using ECL. High sensitivity allowed detection limits of 1-2 pM enzyme for carboxypeptidase B and activated thrombin-activatable fibrinolysis inhibitor (TAFIa). The inhibition of several basic carboxypeptidases by commercially available inhibitors was studied. This antibody-based method can be extended to other sensitive detection techniques such as amplified luminescent proximity homogeneous assay. The high sensitivity of the assay allowed the determination of the activatable levels of TAFI in human and other animal plasma in the presence of epsilon -aminocaproic acid, an active-site inhibitor that stabilizes TAFIa. A method to isolate in situ activated TAFIa from human serum in the presence of epsilon -aminocaproic acid was also developed.
- Published
- 2003
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43. Presenilin-1 and presenilin-2 exhibit distinct yet overlapping gamma-secretase activities.
- Author
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Lai MT, Chen E, Crouthamel MC, DiMuzio-Mower J, Xu M, Huang Q, Price E, Register RB, Shi XP, Donoviel DB, Bernstein A, Hazuda D, Gardell SJ, and Li YM
- Subjects
- Affinity Labels, Alzheimer Disease, Amyloid Precursor Protein Secretases, Animals, Aspartic Acid Endopeptidases, Blastocyst enzymology, Cell Membrane enzymology, Cloning, Molecular, Enzyme Inhibitors pharmacology, Membrane Proteins antagonists & inhibitors, Mice, Mice, Knockout, Presenilin-1, Presenilin-2, Recombinant Proteins metabolism, Substrate Specificity, Endopeptidases metabolism, Membrane Proteins metabolism
- Abstract
Presenilin-1 (PS1) and presenilin 2 (PS2) are proposed to be transmembrane aspartyl proteases that cleave amyloid precursor protein and Notch. PS1- and PS2-mediated activities were individually characterized using blastocyst-derived (BD) cells and membranes from PS1+/--PS2-/- and PS1-/-PS2+/+ mice, respectively. The relative amounts of PS1 and PS2 in the various BD cells were determined from the intensities of the anti-PS1 and anti-PS2 immunoblot signals by comparison with standard curves using radiolabeled PS1 and PS2 standards produced by in vitro transcription and translation. Cellular membranes from wild type, PS1-/-PS2+/+, and PS1+/--PS2-/- but not PS1-/-PS2-/- BD cells generated the Abeta40 and Abeta42 products from the C100FLAG substrate. PS1-associated gamma-secretase displays considerably higher specific activity than PS2-associated gamma-secretase. Moreover, the PS1+/-PS2-/- BD cells and corresponding membranes exhibited much higher gamma-secretase activity as compared with other BD cells and membranes. The PS1-mediated gamma-secretase activity correlated better with the amount of PS1 that is modifiable by a photoactivated active site-directed gamma-secretase inhibitor rather than total PS1; hence, only a small portion (<14%) of the PS1 in wild-type membranes appears to be engaged in an active gamma-secretase complex. This finding suggests that PS1 may serve other biological functions in addition to that associated with its gamma-secretase activity. Furthermore, the PS1 gamma-secretase complex and the PS2 gamma-secretase complex activities can be discriminated on the basis of their susceptibility to inhibition by a potent gamma-secretase inhibitor. The distinct yet overlapping enzymatic properties of the PS1 gamma-secretase complex and the PS2 gamma-secretase complex imply that these two putative aspartyl class proteases may contribute to different biological processes.
- Published
- 2003
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44. Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity.
- Author
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Ferrer M, Zuck P, Kolodin G, Mao SS, Peltier RR, Bailey C, Gardell SJ, Strulovici B, and Inglese J
- Subjects
- Aminocaproates chemistry, Aminocaproates pharmacology, Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Biotin chemistry, Carboxypeptidase B analysis, Carboxypeptidase B antagonists & inhibitors, Carboxypeptidase B chemistry, Enzyme Inhibitors pharmacology, Europium chemistry, Fluorescence Resonance Energy Transfer, Miniaturization, Oligopeptides chemistry, Oligopeptides metabolism, Phycocyanin chemistry, Sensitivity and Specificity, Streptavidin chemistry, Swine, Carboxypeptidase B metabolism, Fluorescent Antibody Technique methods
- Abstract
An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR-OH) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-OH) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu(3+)-cryptate ([Eu(3+)]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z(')>0.5) assay for high-throughput screening.
- Published
- 2003
- Full Text
- View/download PDF
45. Pharmacokinetic optimization of 3-amino-6-chloropyrazinone acetamide thrombin inhibitors. Implementation of P3 pyridine N-oxides to deliver an orally bioavailable series containing P1 N-benzylamides.
- Author
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Burgey CS, Robinson KA, Lyle TA, Nantermet PG, Selnick HG, Isaacs RC, Lewis SD, Lucas BJ, Krueger JA, Singh R, Miller-Stein C, White RB, Wong B, Lyle EA, Stranieri MT, Cook JJ, McMasters DR, Pellicore JM, Pal S, Wallace AA, Clayton FC, Bohn D, Welsh DC, Lynch JJ Jr, Yan Y, Chen Z, Kuo L, Gardell SJ, Shafer JA, and Vacca JP
- Subjects
- Animals, Biological Availability, Chemical Phenomena, Chemistry, Physical, Crystallography, X-Ray, Dogs, Half-Life, Humans, In Vitro Techniques, Injections, Intravenous, Macaca mulatta, Microsomes, Liver metabolism, Models, Molecular, Oxides chemistry, Rats, Solubility, Structure-Activity Relationship, Thrombosis chemically induced, Thrombosis drug therapy, Acetamides chemical synthesis, Acetamides pharmacology, Pyrazines chemical synthesis, Pyrazines pharmacology, Pyridines chemistry, Thrombin antagonists & inhibitors
- Abstract
In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors. An expedited investigation of the P1 SAR with respect to oral bioavailability, plasma half-life, and human liver microsome stability revealed 5 as the best candidate for advanced evaluation.
- Published
- 2003
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46. Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines.
- Author
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Burgey CS, Robinson KA, Lyle TA, Sanderson PE, Lewis SD, Lucas BJ, Krueger JA, Singh R, Miller-Stein C, White RB, Wong B, Lyle EA, Williams PD, Coburn CA, Dorsey BD, Barrow JC, Stranieri MT, Holahan MA, Sitko GR, Cook JJ, McMasters DR, McDonough CM, Sanders WM, Wallace AA, Clayton FC, Bohn D, Leonard YM, Detwiler TJ Jr, Lynch JJ Jr, Yan Y, Chen Z, Kuo L, Gardell SJ, Shafer JA, and Vacca JP
- Subjects
- Acetamides chemical synthesis, Acetamides pharmacology, Administration, Oral, Animals, Anticoagulants chemical synthesis, Anticoagulants pharmacology, Biological Availability, Crystallography, X-Ray, Dogs, Macaca mulatta, Models, Molecular, Protease Inhibitors chemistry, Protease Inhibitors pharmacology, Pyrazines chemical synthesis, Pyrazines pharmacology, Pyridines chemical synthesis, Pyridines pharmacology, Rats, Structure-Activity Relationship, Acetamides pharmacokinetics, Anticoagulants pharmacokinetics, Protease Inhibitors chemical synthesis, Pyrazines pharmacokinetics, Pyridines pharmacokinetics, Thrombin antagonists & inhibitors
- Abstract
Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.
- Published
- 2003
- Full Text
- View/download PDF
47. gamma-Secretase: characterization and implication for Alzheimer disease therapy.
- Author
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Xu M, Lai MT, Huang Q, DiMuzio-Mower J, Castro JL, Harrison T, Nadin A, Neduvelil JG, Shearman MS, Shafer JA, Gardell SJ, and Li YM
- Subjects
- Amyloid Precursor Protein Secretases, Aspartic Acid Endopeptidases, Carbamates pharmacology, Dipeptides pharmacology, Endopeptidases analysis, Enzyme Activation, Enzyme Inhibitors pharmacology, Enzyme Inhibitors therapeutic use, Escherichia coli enzymology, Escherichia coli genetics, Humans, In Vitro Techniques, Presenilin-1, Presenilin-2, Reproducibility of Results, Sensitivity and Specificity, Alzheimer Disease drug therapy, Alzheimer Disease metabolism, Endopeptidases metabolism, Immunoenzyme Techniques methods, Membrane Proteins metabolism
- Abstract
gamma-Secretase is a membrane-bound protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C-termini of the Abeta peptides which are believed to play a central role in the neuropathology of Alzheimer's disease. An in vitro gamma-secretase assay using a recombinant substrate C100Flag has been developed to facilitate the characterization and identification of this enigmatic protease. Biochemical studies establish that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex. Moreover, the fact that the photoreactive active gamma-secretase inhibitor directed to the active site labels PS1 suggests that PS1 contains the active site of the protease. Presenilin/gamma-secretase as a potential target for AD therapy and its role in regulated intramembrane proteolysis are discussed.
- Published
- 2002
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- View/download PDF
48. Abetapp secretases are co-expressed with Abetapp in the pancreatic islets.
- Author
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Figueroa DJ, Shi XP, Gardell SJ, and Austin CP
- Abstract
Amyloid beta protein precursor is cleaved by beta- and gamma-secretases to produce Abeta peptides which deposit in amyloid plaques in Alzheimer's disease (AD) brain. A recently identified beta-site cleaving enzyme (BACE) appears to fulfill the requirements for beta-secretase, and presenilin-1 (PS1) appears to constitute the catalytic component of gamma-secretase. Each protein has a close homologue (BACE2 and PS2, respectively), whose roles in AbetaPP cleavage remain uncertain. All four of these genes have been reported to be expressed in the human pancreas, but the cell types expressing these genes remains unknown. We demonstrate here the cell-specific expression of AbetaPP, BACE, BACE2, PS1, and PS2 in the human pancreas. The insulin-producing betacells were found to express AbetaPP, BACE and PS2 at high levels, and PS1 at a lower level. The other islet cell types expressed none of these five genes. By contrast, the exocrine ductal cells of the pancreas expressed AbetaPP and BACE2 selectively. These results suggest that secretase inhibitors under development for the treatment of AD, particularly those that target BACE, may have potential for adverse effects on pancreatic beta cell function, and therefore glycemic control.
- Published
- 2001
- Full Text
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49. The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.
- Author
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Shi XP, Chen E, Yin KC, Na S, Garsky VM, Lai MT, Li YM, Platchek M, Register RB, Sardana MK, Tang MJ, Thiebeau J, Wood T, Shafer JA, and Gardell SJ
- Subjects
- Amino Acid Sequence, Amyloid Precursor Protein Secretases, Animals, Aspartic Acid Endopeptidases antagonists & inhibitors, Aspartic Acid Endopeptidases isolation & purification, Baculoviridae metabolism, Binding Sites, Catalysis, Cell Line, Concanavalin A pharmacology, Culture Media, Conditioned metabolism, DNA, Complementary metabolism, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Endopeptidases metabolism, Furin, Guanidine metabolism, Humans, Immunoblotting, Insecta, Kinetics, Molecular Sequence Data, Precipitin Tests, Protein Denaturation, Protein Folding, Protein Precursors isolation & purification, Protein Structure, Tertiary, Recombinant Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Subtilisins pharmacology, Time Factors, Aspartic Acid Endopeptidases chemistry, Protein Precursors chemistry
- Abstract
beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.
- Published
- 2001
- Full Text
- View/download PDF
50. Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1.
- Author
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Li YM, Xu M, Lai MT, Huang Q, Castro JL, DiMuzio-Mower J, Harrison T, Lellis C, Nadin A, Neduvelil JG, Register RB, Sardana MK, Shearman MS, Smith AL, Shi XP, Yin KC, Shafer JA, and Gardell SJ
- Subjects
- Alzheimer Disease enzymology, Alzheimer Disease metabolism, Amyloid Precursor Protein Secretases, Amyloid beta-Protein Precursor metabolism, Aspartic Acid Endopeptidases, Binding Sites, Biotin, Enzyme Inhibitors, HeLa Cells, Humans, Membrane Proteins antagonists & inhibitors, Photoaffinity Labels, Photochemistry, Presenilin-1, Presenilin-2, Endopeptidases metabolism, Membrane Proteins metabolism
- Abstract
Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.
- Published
- 2000
- Full Text
- View/download PDF
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