36 results on '"M. Flattery"'
Search Results
2. Ibrexafungerp: An orally active β-1,3-glucan synthesis inhibitor
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Dongfang Meng, Dann L. Parker, Robin Kirwan, M A Powles, Jennifer Nielsen Kahn, James M. Apgar, Fred Racine, Paul A. Liberator, Amy M. Flattery, Andrew Galgoci, Kingsley H. Nelson, James M. Balkovec, Andrew S. Misura, Weiming Fan, Donald M. Sperbeck, Michael Robert Peel, Robert A. Giacobbe, Mark L. Greenlee, Ahmed Mamai, Jasminka Dragovic, Kenneth J. Wildonger, Hao Liu, Robert R. Wilkening, Mary Motyl, Shu Lee, Ming-Jo Hsu, Charles Gill, and George K. Abruzzo
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β 1 3 glucan ,Antifungal Agents ,beta-Glucans ,medicine.drug_class ,Clinical Biochemistry ,Triazole ,Substituent ,Pharmaceutical Science ,Phases of clinical research ,Administration, Oral ,Carboxamide ,Pharmacology ,01 natural sciences ,Biochemistry ,chemistry.chemical_compound ,Mice ,Structure-Activity Relationship ,Drug Discovery ,Candida albicans ,medicine ,Animals ,Aspergillosis ,Glycosides ,Molecular Biology ,Natural product ,010405 organic chemistry ,Organic Chemistry ,Candidiasis ,Enfumafungin ,Triterpenes ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Disease Models, Animal ,Orally active ,Aspergillus ,chemistry ,Molecular Medicine ,Half-Life - Abstract
We previously reported medicinal chemistry efforts that identified MK-5204, an orally efficacious β-1,3-glucan synthesis inhibitor derived from the natural product enfumafungin. Further extensive optimization of the C2 triazole substituent identified 4-pyridyl as the preferred replacement for the carboxamide of MK-5204, leading to improvements in antifungal activity in the presence of serum, and increased oral exposure. Reoptimizing the aminoether at C3 in the presence of this newly discovered C2 substituent, confirmed that the (R) t-butyl, methyl aminoether of MK-5204 provided the best balance of these two key parameters, culminating in the discovery of ibrexafungerp, which is currently in phase III clinical trials. Ibrexafungerp displayed significantly improved oral efficacy in murine infection models, making it a superior candidate for clinical development as an oral treatment for Candida and Aspergillus infections.
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- 2020
3. Regenerative peripheral nerve interfaces for real-time, proportional control of a Neuroprosthetic hand
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Andrej Nedic, Melanie G. Urbanchek, Patrick J. Buchanan, Christopher M. Frost, Stephen W.P. Kemp, Paul S. Cederna, Jana D. Moon, Shane M. Flattery, Theodore A. Kung, Daniel C. Ursu, Cheryl A. Hassett, and R. Brent Gillespie
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Male ,030506 rehabilitation ,Neuroprosthetics ,Movement ,Rat model ,Proportional control ,Health Informatics ,Artificial Limbs ,Electromyography ,Signal ,lcsh:RC321-571 ,03 medical and health sciences ,0302 clinical medicine ,Amputees ,Peripheral nerve ,Peripheral nerve interface ,Medicine ,Animals ,Peripheral Nerves ,Muscle, Skeletal ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Prosthetics ,medicine.diagnostic_test ,business.industry ,Research ,Rehabilitation ,Signal Processing, Computer-Assisted ,Peripheral nerve Interface ,Hindlimb ,Nerve Regeneration ,Rats ,Regenerative medicine ,0305 other medical science ,business ,030217 neurology & neurosurgery ,Common peroneal nerve ,Biomedical engineering - Abstract
Introduction Regenerative peripheral nerve interfaces (RPNIs) are biological constructs which amplify neural signals and have shown long-term stability in rat models. Real-time control of a neuroprosthesis in rat models has not yet been demonstrated. The purpose of this study was to: a) design and validate a system for translating electromyography (EMG) signals from an RPNI in a rat model into real-time control of a neuroprosthetic hand, and; b) use the system to demonstrate RPNI proportional neuroprosthesis control. Methods Animals were randomly assigned to three experimental groups: (1) Control; (2) Denervated, and; (3) RPNI. In the RPNI group, the extensor digitorum longus (EDL) muscle was dissected free, denervated, transferred to the lateral thigh and neurotized with the residual end of the transected common peroneal nerve. Rats received tactile stimuli to the hind-limb via monofilaments, and electrodes were used to record EMG. Signals were filtered, rectified and integrated using a moving sample window. Processed EMG signals (iEMG) from RPNIs were validated against Control and Denervated group outputs. Results Voluntary reflexive rat movements produced signaling that activated the prosthesis in both the Control and RPNI groups, but produced no activation in the Denervated group. Signal-to-Noise ratio between hind-limb movement and resting iEMG was 3.55 for Controls and 3.81 for RPNIs. Both Control and RPNI groups exhibited a logarithmic iEMG increase with increased monofilament pressure, allowing graded prosthetic hand speed control (R2 = 0.758 and R2 = 0.802, respectively). Conclusion EMG signals were successfully acquired from RPNIs and translated into real-time neuroprosthetic control. Signal contamination from muscles adjacent to the RPNI was minimal. RPNI constructs provided reliable proportional prosthetic hand control. Electronic supplementary material The online version of this article (10.1186/s12984-018-0452-1) contains supplementary material, which is available to authorized users.
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- 2018
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4. Can We Make Small Molecules Lean? Optimization of a Highly Lipophilic TarO Inhibitor
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Terry Roemer, James R. Tata, Charles G. Garlisi, Mihirbaran Mandal, Christina B. Madsen-Duggan, Ginny D. Ho, Todd Mayhood, Jin Wu, Marc A. Labroli, John P. Caldwell, Alexei V. Buevich, Emma R. Parmee, Hao Wang, Jing Su, Paul Tawa, Sandra Koseoglu, Xiujuan Wen, Sang Ho Lee, Kallol Basu, Payal R. Sheth, Shu-Wei Yang, David G. McLaren, Haifeng Tang, Zheng Tan, Reynalda deJesus, Jenny Liu, Erika Walsh, Sookhee Ha, Christopher M. Tan, Li Xiao, Diane Rindgen, Amy M. Flattery, Debra Mcguinness, Christine Yang, Charles Gill, Paul L. Walsh, and Lianzhu Liang
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Methicillin-Resistant Staphylococcus aureus ,0301 basic medicine ,030106 microbiology ,Microbial Sensitivity Tests ,Small Molecule Libraries ,Mice ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,In vivo ,Drug Discovery ,Animals ,Structure–activity relationship ,Solubility ,Mice, Inbred BALB C ,Teichoic acid ,Ligand efficiency ,biology ,Lipids ,Small molecule ,Enzyme assay ,chemistry ,Biochemistry ,Lipophilicity ,biology.protein ,Molecular Medicine ,Female - Abstract
We describe our optimization efforts to improve the physicochemical properties, solubility, and off-target profile of 1, an inhibitor of TarO, an early stage enzyme in the biosynthetic pathway for wall teichoic acid (WTA) synthesis. Compound 1 displayed a TarO IC50 of 125 nM in an enzyme assay and possessed very high lipophilicity (clogP = 7.1) with no measurable solubility in PBS buffer. Structure–activity relationship (SAR) studies resulted in a series of compounds with improved lipophilic ligand efficiency (LLE) consistent with the reduction of clogP. From these efforts, analog 9 was selected for our initial in vivo study, which in combination with subefficacious dose of imipenem (IPM) robustly lowered the bacterial burden in a neutropenic Staphylococci murine infection model. Concurrent with our in vivo optimization effort using 9, we further improved LLE as exemplified by a much more druglike analog 26.
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- 2017
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5. Use of Translational Pharmacokinetic/Pharmacodynamic Infection Models To Understand the Impact of Neutropenia on the Efficacy of Tedizolid Phosphate
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Shawn Flanagan, Hwa-Ping Feng, Jin Wu, Amy M. Flattery, Christopher M. Tan, Jenny Liu, Jianying Xiao, Lianzhu Liang, and Charles Gill
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Methicillin-Resistant Staphylococcus aureus ,Neutropenia ,medicine.drug_class ,Antibiotics ,Microbial Sensitivity Tests ,Pharmacology ,medicine.disease_cause ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Pharmacokinetics ,medicine ,Animals ,Potency ,Pharmacology (medical) ,Oxazoles ,0303 health sciences ,030306 microbiology ,business.industry ,Linezolid ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,medicine.disease ,Organophosphates ,Anti-Bacterial Agents ,Disease Models, Animal ,Infectious Diseases ,chemistry ,Staphylococcus aureus ,Pharmacodynamics ,Staphylococcal Skin Infections ,Tedizolid ,business - Abstract
Tedizolid phosphate, the prodrug of the active antibiotic tedizolid, is an oxazolidinone for the treatment of acute bacterial skin and skin structure infections. Studies in a mouse thigh infection model demonstrated that tedizolid has improved potency and pharmacokinetics/pharmacodynamics (PK/PD) compared with those of linezolid. Subsequent studies showed that the efficacy of tedizolid was enhanced in immunocompetent (IC) mice compared with neutropenic (immunosuppressed [IS]) mice, with stasis at clinically relevant doses being achieved only in the presence of granulocytes. The tedizolid label therefore contains a warning about its use in neutropenic patients. This study reevaluated the PK/PD of tedizolid and linezolid in the mouse thigh infection model in IC and IS mice using a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 33591) and a methicillin-susceptible S. aureus (MSSA) strain (ATCC 29213). The antistaphylococcal effect of doses ranging from 1 to 150 mg/kg of body weight tedizolid (once daily) or linezolid (twice daily) was determined at 24, 48, and 72 h after initiating treatment. In IC mice, stasis was achieved in the absence of antibiotics, and both tedizolid and linezolid reduced the burden further beyond a static effect. In IS mice, tedizolid achieved stasis against MRSA ATCC 33591 and MSSA ATCC 29213 at 72 h at a human clinical dose of 200 mg, severalfold lower than that in earlier studies. Linezolid achieved a static effect against MRSA ATCC 33591 in IS mice at a dose lower than that used clinically. This study demonstrates that, with time, both tedizolid and linezolid at clinically relevant exposures achieve stasis in neutropenic mice with an MRSA or MSSA thigh infection.
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- 2019
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6. Novel orally active inhibitors of β-1,3-glucan synthesis derived from enfumafungin
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James M. Balkovec, Robert A. Giacobbe, Jennifer Nielsen Kahn, Ming Jo Hsu, Weiming Fan, Ahmed Mamai, Michael Robert Peel, James M. Apgar, Amy M. Flattery, Paul A. Liberator, Fred Racine, Bahanu Habulihaz, Mary Motyl, Kingsley H. Nelson, Robert R. Wilkening, Robin Kirwan, M A Powles, George K. Abruzzo, Andrew Galgoci, Charles Gill, Andrew S. Misura, Shu Lee, Jasminka Dragovic, Hao Liu, and Mark L. Greenlee
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Antifungal Agents ,beta-Glucans ,Clinical Biochemistry ,Administration, Oral ,Pharmaceutical Science ,Microbial Sensitivity Tests ,Biochemistry ,Mice ,Structure-Activity Relationship ,chemistry.chemical_compound ,Pharmacokinetics ,Candida albicans ,Drug Discovery ,Animals ,Moiety ,Potency ,Structure–activity relationship ,Glycosides ,Molecular Biology ,chemistry.chemical_classification ,Natural product ,biology ,Terpenes ,Aspergillus fumigatus ,Organic Chemistry ,Candidiasis ,Glycoside ,biology.organism_classification ,Triterpenes ,chemistry ,Glucosyltransferases ,Molecular Medicine ,Echinocandins ,Half-Life - Abstract
The clinical success of the echinocandins, which can only be administered parentally, has validated β-1,3-glucan synthase (GS) as an antifungal target. Semi-synthetic modification of enfumafungin, a triterpene glycoside natural product, was performed with the aim of producing a new class of orally active GS inhibitors. Replacement of the C2 acetoxy moiety with various heterocycles did not improve GS or antifungal potency. However, replacement of the C3 glycoside with an aminoether moiety dramatically improved oral pharmacokinetic (PK) properties while maintaining GS and antifungal potency. Installing an aminotetrazole at C2 in conjunction with an N-alkylated aminoether at C3 produced derivatives with significantly improved GS and antifungal potency that exhibited robust oral efficacy in a murine model of disseminated candidiasis.
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- 2015
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7. Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting
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Paul Mann, Amy M. Flattery, Hao Wang, Li Xiao, Xinwei Sher, Megan D. McCurry, Nicholas Murgolo, John A. Howe, Juliana C. Malinverni, Terry Roemer, Artjohn Villafania, Andrew Galgoci, Matthias Mack, Charles Gill, Todd Mayhood, and Christopher M. Barbieri
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0301 basic medicine ,Riboswitch ,Methicillin-Resistant Staphylococcus aureus ,Models, Molecular ,Staphylococcus aureus ,Flavin Mononucleotide ,Protein Conformation ,Riboflavin ,030106 microbiology ,Clinical Biochemistry ,Flavin mononucleotide ,Biology ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Drug Discovery ,Gene expression ,medicine ,Escherichia coli ,Animals ,Homeostasis ,Molecular Targeted Therapy ,Molecular Biology ,Pharmacology ,Base Sequence ,RNA ,Small molecule ,Anti-Bacterial Agents ,Metabolic pathway ,030104 developmental biology ,Pyrimidines ,chemistry ,Molecular Medicine - Abstract
Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide a mechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism.
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- 2016
8. Disease Progression and Resolution in Rodent Models of Clostridium difficile Infection and Impact of Antitoxin Antibodies and Vancomycin
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Amy M. Flattery, Alex G. Therien, Peter Warn, Todd A. Black, Zuo Zhang, David F. Corbett, Lorraine D. Hernandez, Abdul Sattar, and Pia Thommes
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0301 basic medicine ,medicine.drug_class ,media_common.quotation_subject ,Antibiotics ,Bacterial Toxins ,Disease ,Gut flora ,Bioinformatics ,03 medical and health sciences ,Enterotoxins ,Mice ,Cricetulus ,Bacterial Proteins ,Vancomycin ,Medicine ,Animals ,Humans ,Pharmacology (medical) ,Mechanisms of Action: Physiological Effects ,media_common ,Pharmacology ,biology ,business.industry ,Clostridioides difficile ,Convalescence ,Antibodies, Monoclonal ,Clostridium difficile ,biology.organism_classification ,Antibodies, Neutralizing ,Survival Analysis ,Anti-Bacterial Agents ,Gastrointestinal Microbiome ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,Infectious Diseases ,Bezlotoxumab ,Immunology ,Clostridium Infections ,Disease Progression ,Antitoxins ,Antitoxin ,business ,Broadly Neutralizing Antibodies ,medicine.drug - Abstract
Clostridium difficile causes infections of the colon in susceptible patients. Specifically, gut dysbiosis induced by treatment with broad-spectrum antibiotics facilitates germination of ingested C. difficile spores, expansion of vegetative cells, and production of symptom-causing toxins TcdA and TcdB. The current standard of care for C. difficile infections (CDI) consists of administration of antibiotics such as vancomycin that target the bacterium but also perpetuate gut dysbiosis, often leading to disease recurrence. The monoclonal antitoxin antibodies actoxumab (anti-TcdA) and bezlotoxumab (anti-TcdB) are currently in development for the prevention of recurrent CDI. In this study, the effects of vancomycin or actoxumab/bezlotoxumab treatment on progression and resolution of CDI were assessed in mice and hamsters. Rodent models of CDI are characterized by an early severe phase of symptomatic disease, associated with high rates of morbidity and mortality; high intestinal C. difficile burden; and a disrupted intestinal microbiota. This is followed in surviving animals by gradual recovery of the gut microbiota, associated with clearance of C. difficile and resolution of disease symptoms over time. Treatment with vancomycin prevents disease initially by inhibiting outgrowth of C. difficile but also delays microbiota recovery, leading to disease relapse following discontinuation of therapy. In contrast, actoxumab/bezlotoxumab treatment does not impact the C. difficile burden but rather prevents the appearance of toxin-dependent symptoms during the early severe phase of disease, effectively preventing disease until the microbiota (the body's natural defense against C. difficile ) has fully recovered. These data provide insight into the mechanism of recurrence following vancomycin administration and into the mechanism of recurrence prevention observed clinically with actoxumab/bezlotoxumab.
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- 2016
9. TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci
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Zheng Tan, Mihir Mandal, Michelle Batchlett, Reshma Kuvelkar, Paul Mann, Todd Mayhood, Sookhee Ha, Paul Zuck, Yan Hou, Xiujuan Wen, Christine Yang, Christopher M. Tan, Carl J. Balibar, Hao Wang, Terry Roemer, Charles Gill, Jenny Liu, Amy M. Flattery, Payal R. Sheth, Jing Xiao, Kristine Devito, Jianying Xiao, Jing Su, Marc A. Labroli, Charles G. Garlisi, Shu-Wei Yang, Nicholas Murgolo, Sang Ho Lee, Lianzhu Liang, Paul Tawa, Sandra Koseoglu, and Xinwei Sher
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Methicillin-Resistant Staphylococcus aureus ,Models, Molecular ,0301 basic medicine ,medicine.drug_class ,Phenotypic screening ,Antibiotics ,Treatment outcome ,Growth inhibitory ,Microbial Sensitivity Tests ,beta-Lactams ,Bioinformatics ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Cell Wall ,medicine ,Wall teichoic acid biosynthesis ,Animals ,Dicloxacillin ,Mice, Inbred BALB C ,business.industry ,Treatment options ,General Medicine ,Staphylococcal Infections ,biochemical phenomena, metabolism, and nutrition ,Biosynthetic Pathways ,Teichoic Acids ,Phenotype ,Treatment Outcome ,030104 developmental biology ,chemistry ,Staphylococcus aureus ,Lactam ,Female ,business - Abstract
The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci.
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- 2016
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10. Comparison of Lipoprotein Separation and Lipid Analysis Methodologies for Human and Cynomolgus Monkey Plasma Samples
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David G. McLaren, Richard Raubertas, Neil S. Geoghagen, Seongah Han, Thomas P. Roddy, Gail Forrest, Brian K. Hubbard, Jose Castro-Perez, Ray Rosa, Amy M. Flattery, Sang Ho Lee, Jing Li, Douglas G. Johns, and Vivienne Mendoza
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Adult ,Male ,Lipoproteins ,Pharmaceutical Science ,High density ,Chemistry Techniques, Analytical ,Mass Spectrometry ,Enzymatic Assays ,chemistry.chemical_compound ,Genetics ,Animals ,Humans ,Triglycerides ,Genetics (clinical) ,Autoanalysis ,Plasma samples ,Chemistry ,Cholesterol ,Cholesterol, HDL ,Reproducibility of Results ,Fast protein liquid chromatography ,Cholesterol, LDL ,Repeatability ,Middle Aged ,Lipids ,Macaca fascicularis ,Biochemistry ,Molecular Medicine ,Female ,lipids (amino acids, peptides, and proteins) ,Ultracentrifuge ,Cardiology and Cardiovascular Medicine ,Ultracentrifugation ,Biomarkers ,Chromatography, Liquid ,Lipoprotein - Abstract
To assess cardiovascular risk in both clinical and basic research settings, it is imperative to be able to accurately measure plasma lipid levels. Here, methods commonly used to measure lipoproteins and lipids: ultracentrifugation (UC), fast protein liquid chromatography (FPLC), Roche auto-analyzer, and enzymatic assays were tested and compared. Plasma samples from 20 healthy humans and 22 cynomolgus monkeys were analyzed for their total cholesterol (TC), cholesterol in low density lipoproteins (LDL) and high density lipoproteins (HDL), and triglycerides (TG). Major lipid classes from UC and FPLC separated lipoprotein fractions from human plasma were further characterized by liquid chromatography-mass spectrometry analysis. All the tested methods showed acceptable performance with Roche analyzer among the best in approximate dilution linearity and recovery for most lipids as well as in repeatability between measurements of the same samples. TC, LDL, HDL, and TG values measured in human vs. monkey were-183.9 ± 35.5 (mean ± SD) vs. 105.6 ± 24.6 mg/dl, 106.0 ± 30.1 vs. 42.8 ± 13.0 mg/dl, 50.0 ± 11.4 vs. 53.4 ± 14.8 mg/dl, and 107.6 ± 50.7 vs. 58.0 ± 52.3 mg/dl. While no single method was uniformly the best, we recommend the Roche analyzer for routine measurements. UC or FPLC separation is needed for further functional characterization for specific lipid fraction. We have shown athero-protective profile in cynomolgus monkey compared with humans.
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- 2011
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11. Chronic Antagonism of the Mineralocorticoid Receptor Ameliorates Hypertension and End Organ Damage in a Rodent Model of Salt-Sensitive Hypertension
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Zadok Ruben, Yonghua Zhu, Huawei Zhao, Daphne Szeto, Michael J. Forrest, Wanda Sharif-Rodriguez, Olga Urosevic-Price, Martin Crook, Xiaoyan Zhou, Yi Pan, Gail Forrest, Amy M. Flattery, Sophie Roy, and Li Wang
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Male ,medicine.medical_specialty ,hypertension ,Hypertension, Renal ,Physiology ,End organ damage ,Blood Pressure ,Spironolactone ,Pharmacology ,Kidney ,end organ protection ,Electrolytes ,chemistry.chemical_compound ,Mineralocorticoid receptor ,Heart Rate ,Internal medicine ,Internal Medicine ,medicine ,Animals ,mineralocorticoid receptor antagonist ,Sodium Chloride, Dietary ,eplerenone ,Aldosterone ,Dahl salt-sensitive rats ,Mineralocorticoid Receptor Antagonists ,Rats, Inbred Dahl ,Proteinuria ,business.industry ,Articles ,Organ Size ,General Medicine ,medicine.disease ,Rats ,Eplerenone ,Disease Models, Animal ,Receptors, Mineralocorticoid ,Endocrinology ,Blood pressure ,medicine.anatomical_structure ,chemistry ,Creatinine ,Chronic Disease ,Disease Progression ,medicine.symptom ,business ,medicine.drug - Abstract
We investigated the effects of chronic mineralocorticoid receptor blockade with eplerenone on the development and progression of hypertension and end organ damage in Dahl salt-sensitive rats. Eplerenone significantly attenuated the progressive rise in systolic blood pressure (SBP) (204 ± 3 vs. 179±3 mmHg, p < 0.05), reduced proteinuria (605.5 ± 29.6 vs. 479.7 ± 26.1 mg/24h, p < 0.05), improved injury scores of glomeruli, tubules, renal interstitium, and vasculature in Dahl salt-sensitive rats fed a high-salt diet. These results demonstrate that mineralocorticoid receptor antagonism provides target organ protection and attenuates the development of elevated blood pressure (BP) in a model of salt-sensitive hypertension.
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- 2011
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12. Efficacy of Caspofungin in a Juvenile Mouse Model of Central Nervous System Candidiasis
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George K. Abruzzo, Rena Zhang, Mary Ann Powles, Emily Hickey, Charles Gill, Andrew S. Misura, Joan D. Ellis, John Ronan, Andrew Galgoci, Punam Sandhu, and Amy M. Flattery
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Antifungal Agents ,Echinocandin ,Biology ,Kidney ,Echinocandins ,Lipopeptides ,Mice ,chemistry.chemical_compound ,Central Nervous System Fungal Infections ,Caspofungin ,Amphotericin B ,medicine ,Animals ,Pharmacology (medical) ,Candida albicans ,Mycosis ,Pharmacology ,Neonatal Candidiasis ,Candidiasis ,Brain ,bacterial infections and mycoses ,medicine.disease ,Disseminated Candidiasis ,biology.organism_classification ,Infectious Diseases ,chemistry ,Immunology ,Encephalitis ,medicine.drug - Abstract
Neonatal candidiasis is an increasingly common occurrence causing significant morbidity and mortality and a higher risk of dissemination to the central nervous system (CNS) than that seen with older patients. The current understanding of optimal antifungal therapy in this setting is limited. We have developed a model of disseminated candidiasis with CNS involvement in juvenile mice to assess the efficacy of the echinocandin caspofungin relative to amphotericin B (AmB). Juvenile mice were inoculated intravenously with 5.64 × 10 4 CFU of Candida albicans MY1055. Treatment with caspofungin at 1, 2, 4, and 8 mg/kg of body weight/day, AmB at 1 mg/kg/day, or a vehicle control (VC) was initiated 30 h after infection and continued for 7 days. Pharmacokinetic parameters for caspofungin were also determined. Culture and histology showed evidence of disseminated candidiasis with multifocal encephalitis at the start of antifungal therapy. Survival was 100% in all treated groups, while mortality was 100% in the VC by day 11 after infection. By day 5, all mice in the caspofungin treatment (four doses) groups showed reductions in kidney and brain burden relative to the VC, while AmB treatment reduced kidney burden but gave no reduction of brain fungal burden. Systemic levels of caspofungin were similar in infected and uninfected mice, while brain levels were higher in infected animals. In this juvenile mouse model, caspofungin demonstrated dose-dependent activity, equivalent to or better than that of AmB at 1 mg/kg, against disseminated candidiasis with CNS involvement.
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- 2011
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13. Validation of Human ApoB and ApoAI Immunoturbidity Assays for Non-human Primate Dyslipidemia and Atherosclerosis Research
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Ying Chen, Kenny K. Wong, Zhu Chen, Amy M. Flattery, Neil S. Geoghagen, Alice C. Stefanni, Alison M. Strack, Brian K. Hubbard, Li Wang, Olga Urosevic-Price, Yi Pan, Thomas P. Roddy, Theresa McLaughlin, Michael E. Lassman, and Weizhen Wu
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Simvastatin ,Apolipoprotein B ,Pharmaceutical Science ,Enzyme-Linked Immunosorbent Assay ,Pharmacology ,Mass Spectrometry ,Drug treatment ,Spike recovery ,Nephelometry and Turbidimetry ,Predictive Value of Tests ,Genetics ,medicine ,Animals ,Humans ,Genetics (clinical) ,Apolipoproteins B ,Dyslipidemias ,Immunoassay ,Non human primate ,Apolipoprotein A-I ,biology ,Reproducibility of Results ,Lipid metabolism ,Haplorhini ,Elisa assay ,Atherosclerosis ,medicine.disease ,humanities ,Human genetics ,Disease Models, Animal ,Calibration ,Immunology ,biology.protein ,Molecular Medicine ,Electrophoresis, Polyacrylamide Gel ,lipids (amino acids, peptides, and proteins) ,Hydroxymethylglutaryl-CoA Reductase Inhibitors ,Cardiology and Cardiovascular Medicine ,Biomarkers ,Dyslipidemia - Abstract
Emerging evidence suggests apolipoprotein B (apoB) and apolipoprotein AI (apoAI) are strong risk predictors for atherosclerosis. Non-human primates (NHP), including rhesus monkeys, cynomolgus monkeys, and African green monkeys, are important preclinical species for studying dyslipidemia and atherosclerosis as they more closely resemble humans in lipid metabolism and disease physiology compared to lower species such as rodents. However, no commercial assays are currently available for measuring apoB and apoAI in NHP. We therefore evaluated analytical methods for routinely measuring apoB and apoAI in our NHP dyslipidemia and atherosclerosis research. Since NHP apoB and apoAI sequences are likely highly similar to human, we focused on the clinically validated and widely utilized human apoB and apoAI immunoturbidity assays. We carried out technical validation of these assays with NHP samples and leveraged orthogonal technical platforms including mass spectrometry, independent ELISA assay, and absolute quantitation via SDS-PAGE for further characterization. Analysis of purified lipoproteins demonstrated that the immunoturbidity assays detect NHP apoAI and apoB, with good dilution linearity and spike recovery from NHP plasma. Orthogonal studies showed apoAI correlated with protein concentration and apoB levels correlated with LC/MS and an independent ELISA. NHP samples from a drug treatment study were analyzed with the immunoturbidity assays and levels of apoB and apoAI fit our understanding of biology and expectations from literature. These studies serve as important technical and biological validation of the immunoturbidity assays for NHP samples, and demonstrate that these assays provide a high-throughput, fully automated analytical platform for NHP samples. Our studies pave the way for future translational research in NHP for developing therapies for treating dyslipidemia and atherosclerosis.
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- 2011
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14. Efficacy of Caspofungin against Aspergillus flavus , Aspergillus terreus , and Aspergillus nidulans
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Cameron M. Douglas, Charles Gill, Andrew S. Misura, Ming-Jo Hsu, George K. Abruzzo, T. C. Wang, Emily Hickey, Paul A. Liberator, Amy M. Flattery, J. Nielsen Kahn, Barbara A. Pelak, and Joel Bowman
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Antifungal Agents ,Time Factors ,Echinocandin ,Aspergillus flavus ,Microbial Sensitivity Tests ,Peptides, Cyclic ,Aspergillus nidulans ,Microbiology ,Echinocandins ,Lipopeptides ,Mice ,chemistry.chemical_compound ,Caspofungin ,Amphotericin B ,polycyclic compounds ,medicine ,Animals ,Experimental Therapeutics ,Pharmacology (medical) ,Aspergillus terreus ,skin and connective tissue diseases ,Pharmacology ,Aspergillus ,Dose-Response Relationship, Drug ,biology ,Fungi imperfecti ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,biology.organism_classification ,Survival Analysis ,Disease Models, Animal ,Treatment Outcome ,Infectious Diseases ,chemistry ,Mice, Inbred DBA ,Female ,medicine.drug - Abstract
The echinocandin caspofungin is a potent inhibitor of the activity of 1,3-β- d -glucan synthase from Aspergillus flavus , Aspergillus terreus , and Aspergillus nidulans . In murine models of disseminated infection, caspofungin prolonged survival and reduced the kidney fungal burden. Caspofungin was at least as effective as amphotericin B against these filamentous fungi in vivo.
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- 2006
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15. Toxin-Mediated Paracellular Transport of Antitoxin Antibodies Facilitates Protection against Clostridium difficile Infection
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Zuo Zhang, Xinhua Chen, Ciaran P. Kelly, S. Kramer, Amy M. Flattery, Alex G. Therien, Shi-Juan Chen, Lorraine D. Hernandez, Fred Racine, H. Cape, Philip Lipari, and Jon D. Polishook
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Male ,Bacterial Toxins ,Immunology ,Fc receptor ,Receptors, Fc ,Microbiology ,Immunoglobulin G ,Enterotoxins ,Organ Culture Techniques ,Bacterial Proteins ,Animals ,Transcellular ,Mice, Knockout ,Mesocricetus ,biology ,Clostridioides difficile ,Histocompatibility Antigens Class I ,Immunization, Passive ,Bacterial Infections ,Antibodies, Bacterial ,Antibodies, Neutralizing ,Author Corrections ,Gut Epithelium ,Mice, Inbred C57BL ,Disease Models, Animal ,Infectious Diseases ,Bezlotoxumab ,Paracellular transport ,Clostridium Infections ,biology.protein ,Female ,Parasitology ,Antitoxins ,Antibody ,Antitoxin - Abstract
The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile . Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab′) 2 fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens.
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- 2014
16. Kibdelomycin Is a Potent and Selective Agent against Toxigenic Clostridium difficile
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Lynn Miesel, Amy M. Flattery, Jon D. Polishook, James R. Osmolski, Jing Lan, Lianzhu Liang, Fangbiao Li, Philip Lipari, Dale N. Gerding, Sheo B. Singh, David W. Hecht, Jenny Liu, and David B. Olsen
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Drug ,Male ,media_common.quotation_subject ,Hamster ,Microbial Sensitivity Tests ,Biology ,Microbiology ,Mice ,Cricetinae ,Kibdelomycin ,medicine ,Animals ,Pharmacology (medical) ,Experimental Therapeutics ,Colitis ,media_common ,Pharmacology ,chemistry.chemical_classification ,Clostridioides difficile ,Clostridium difficile ,medicine.disease ,Anti-Bacterial Agents ,Diarrhea ,Infectious Diseases ,Enzyme ,chemistry ,Clostridium Infections ,medicine.symptom ,Anaerobic exercise - Abstract
Clostridium difficile is the causative agent of C. difficile -associated diarrhea (CDAD), with increased risk in elderly populations. Kibdelomycin, a novel natural-product inhibitor of type II topoisomerase enzymes, was evaluated for activity against C. difficile and gastrointestinal anaerobic organisms. Toxigenic C. difficile isolates ( n = 168) from U.S. hospitals and anaerobic Gram-positive and Gram-negative organisms ( n = 598) from Chicago-area hospitals were tested. Kibdelomycin showed potent activity against toxigenic C. difficile (MIC 90 = 0.25 μg/ml) and most Gram-positive aerobic organisms but had little activity against Bacteroides species (MIC 50 > 32 μg/ml; n = 270). Potent anti- C. difficile activity was also observed in the hamster model of C. difficile colitis. Dosing at 1.6 mg/kg (twice-daily oral dose) resulted in protection from a lethal infection and a 2-log reduction in C. difficile cecal counts. A 6.25-mg/kg twice-daily oral dose completely eliminated detectable C. difficile counts in cecal contents. A single 6.25-mg/kg oral dose showed that cecal contents were exposed to the drug at >2 μM (eightfold higher than the MIC), with no significant plasma exposure. These findings support further exploration of kibdelomycin for development of an anti- C. difficile agent.
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- 2014
17. The Discovery of Enfumafungin, a Novel Antifungal Compound Produced by an Endophytic Hormonema Species Biological Activity and Taxonomy of the Producing Organisms
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Myra B. Kurtz, Francisca Vicente, Maria Teresa Diez, Antonio González del Val, Robert E. Schwartz, Fernando Pelaez, Amy M. Flattery, Gerald F. Bills, George K. Abruzzo, Isabel Martán, Gonzalo Platas, Angela Basilio, Robert A. Giacobbe, Maria S. Meinz, Angeles Cabello, Janet C. Onishi, and Li Kong
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Antifungal Agents ,Molecular Sequence Data ,Fungus ,Applied Microbiology and Biotechnology ,Microbiology ,Plant use of endophytic fungi in defense ,Aspergillus fumigatus ,Mice ,chemistry.chemical_compound ,Animals ,Glycosides ,Mycological Typing Techniques ,Ribosomal DNA ,Ecology, Evolution, Behavior and Systematics ,Candida ,Aspergillus ,Dose-Response Relationship, Drug ,biology ,Terpenes ,Candidiasis ,Fungi ,Fungi imperfecti ,biology.organism_classification ,Caspofungin Acetate ,DNA Fingerprinting ,Triterpenes ,chemistry ,Juniperus ,Caspofungin - Abstract
In a screening of natural products with antifungal activity derived from endophytic fungi, we detected a potent activity in a culture belonging to the form-genus Hormonema, isolated from leaves of Juniperus communis. The compound is a new triterpene glycoside, showing an antifungal activity highly potent in vitro against Candida and Aspergillus and with moderate efficacy in an in vivo mouse model of disseminated candidiasis. The agent is especially interesting since its antifungal spectrum and its effect on morphology of Aspergillus fumigatus is comparable to that of the glucan synthase inhibitor pneumocandin B,,, the natural precursor of the clinical candidate MK-0991 (caspofungin acetate). An additional search for other Hormonema isolates producing improved titers or derivatives resulted in the isolation of two more strains recovered from the same plant host showing identical activity. The producing isolates were compared with other non-producing Hormonema strains by DNA fingerprinting and sequencing of the rDNA internal transcribed spacers. Comparison of rDNA sequences with other fungal species suggests that the producing fungus could be an undetermined Kabatina species. Kabatina is a coelomycetous genus whose members are known to produce Hormonema-like states in culture.
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- 2000
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18. Efficacy of the Echinocandin Caspofungin against Disseminated Aspergillosis and Candidiasis in Cyclophosphamide-Induced Immunosuppressed Mice
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Jeffrey G. Smith, C. Leighton, Charles Gill, Amy M. Flattery, V. B. Pikounis, Leopold Kong, George K. Abruzzo, Hugh Rosen, and Bartizal Kenneth F
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Antifungal Agents ,Echinocandin ,Microbial Sensitivity Tests ,Biology ,Aspergillosis ,Peptides, Cyclic ,Microbiology ,Echinocandins ,Immunocompromised Host ,Lipopeptides ,Mice ,chemistry.chemical_compound ,Caspofungin ,Amphotericin B ,polycyclic compounds ,medicine ,Animals ,Experimental Therapeutics ,Pharmacology (medical) ,skin and connective tissue diseases ,Cyclophosphamide ,Mycosis ,Candida ,Immunosuppression Therapy ,Pharmacology ,Mice, Inbred ICR ,Candidiasis ,bacterial infections and mycoses ,Caspofungin Acetate ,medicine.disease ,Anti-Bacterial Agents ,Disease Models, Animal ,Aspergillus ,Treatment Outcome ,Infectious Diseases ,chemistry ,Pneumocandin Bo ,Female ,Peptides ,Fluconazole ,medicine.drug - Abstract
The in vivo efficacy of the echinocandin antifungal caspofungin acetate (caspofungin; MK-0991) was evaluated in models of disseminated aspergillosis and candidiasis in mice with cyclophosphamide (CY)-induced immunosuppression. Caspofungin is a 1,3-β- d -glucan synthesis inhibitor efficacious against a number of clinically relevant fungi including Aspergillus and Candida species. Models of CY-induced transient or chronic leukopenia were used with once daily administration of therapy initiated 24 h after microbial challenge. Caspofungin was effective in treating disseminated aspergillosis in mice that were transiently leukopenic (significant prolongation of survival at doses of ≥0.125 mg/kg of body weight and a 50% protective dose [PD 50 ] of 0.245 mg/kg/day at 28 days after challenge) or chronically leukopenic (50 to 100% survival at doses of ≥0.5 mg/kg and PD 50 s ranging from 0.173 to 0.400 mg/kg/day). Caspofungin was effective in the treatment and sterilization of Candida infections in mice with transient leukopenia with a 99% effective dose based on reduction in log 10 CFU of Candida albicans /gram of kidneys of 0.119 mg/kg and 80 to 100% of the caspofungin-treated mice having sterile kidneys at caspofungin doses from 0.25 to 2.0 mg/kg. In Candida -infected mice with chronic leukopenia, caspofungin was effective at all dose levels tested (0.25 to 1.0 mg/kg), with the log 10 CFU of C. albicans /gram of kidneys of caspofungin-treated mice being significantly lower (>99% reduction) than that of sham-treated mice from day 4 to day 28 after challenge. Also, 70 to 100% of the caspofungin-treated, chronic leukopenic mice had sterile kidneys at caspofungin doses of 0.5 to 1.0 mg/kg from day 8 to 28 after challenge. Sterilization of Candida infections by caspofungin in the absence of host leukocytes provides compelling in vivo evidence for fungicidal activity against C. albicans . Further human clinical trials with caspofungin against serious fungal infections are in progress.
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- 2000
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19. In Vivo Efficacy of a Novel Oxazolidinone Compound in Two Mouse Models of Infection
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Amy M. Flattery, Andrew S. Misura, Ken Bartizal, Charles Gill, George K. Abruzzo, and Emily Hickey
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medicine.drug_class ,Ratón ,Antibiotics ,Colony Count, Microbial ,Administration, Oral ,Biology ,Staphylococcal infections ,medicine.disease_cause ,Microbiology ,Mice ,chemistry.chemical_compound ,In vivo ,Acetamides ,medicine ,Animals ,Localized infection ,heterocyclic compounds ,Experimental Therapeutics ,Pharmacology (medical) ,Oxazolidinones ,Antibacterial agent ,Pharmacology ,Mice, Inbred C3H ,Dose-Response Relationship, Drug ,Linezolid ,Staphylococcal Infections ,biochemical phenomena, metabolism, and nutrition ,bacterial infections and mycoses ,medicine.disease ,Anti-Bacterial Agents ,Infectious Diseases ,chemistry ,Staphylococcus aureus ,Injections, Intravenous ,bacteria ,Methicillin Resistance - Abstract
A novel oxazolidinone, AM 7359, was evaluated in two mouse models of Staphylococcus aureus infection. AM 7359 and linezolid were equally efficacious in a methicillin-susceptible S. aureus organ burden model and a methicillin-resistant S. aureus localized infection model. However, AM 7359 was eightfold more efficacious than linezolid against a linezolid- and methicillin-resistant S. aureus strain in this localized (thigh) infection model.
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- 2007
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20. Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors
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Myra B. Kurtz, Amy M. Flattery, Cameron M. Douglas, J A D'Ippolito, George K. Abruzzo, Janet C. Onishi, Bartizal Kenneth F, Maria S. Meinz, W Li, G J Shei, Aaron P. Mitchell, and Jean A. Marrinan
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Antifungal Agents ,Saccharomyces cerevisiae Proteins ,Genotype ,Molecular Sequence Data ,Mutant ,Locus (genetics) ,Biology ,Microbiology ,Fungal Proteins ,Echinocandins ,Mice ,Plasmid ,Candida albicans ,Animals ,Pharmacology (medical) ,Amino Acid Sequence ,Gene conversion ,Enzyme Inhibitors ,Allele ,Pharmacology ,Base Sequence ,Membrane Proteins ,Drug Resistance, Microbial ,biology.organism_classification ,Molecular biology ,Corpus albicans ,Phenotype ,Infectious Diseases ,Glucosyltransferases ,Schizosaccharomyces pombe Proteins ,Research Article - Abstract
Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.
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- 1997
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21. In vitro preclinical evaluation studies with the echinocandin antifungal MK-0991 (L-743,872)
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Jeffrey G. Smith, James M. Balkovec, Patricia M. Scott, Aileen Bouffard, Amy M. Flattery, Li Kong, James F. Dropinski, Claire E. Leighton, Ken Bartizal, Charles Gill, and George K. Abruzzo
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Antifungal Agents ,Echinocandin ,Drug Evaluation, Preclinical ,Microbial Sensitivity Tests ,Hemolysis ,Peptides, Cyclic ,Microbiology ,Aspergillus fumigatus ,Candida tropicalis ,Echinocandins ,Lipopeptides ,Mice ,Caspofungin ,Amphotericin B ,medicine ,Animals ,Humans ,Pharmacology (medical) ,Candida albicans ,Candida ,Pharmacology ,Cryptococcus neoformans ,biology ,Caspofungin Acetate ,biology.organism_classification ,Corpus albicans ,Anti-Bacterial Agents ,Aspergillus ,Infectious Diseases ,Peptides ,Research Article ,medicine.drug - Abstract
The echinocandin MK-0991, formerly L-743,872, is a water-soluble lipopeptide that has been demonstrated in preclinical studies to have potent activity against Candida spp., Aspergillus fumigatus, and Pneumocystis carinii. An extensive in vitro biological evaluation of MK-0991 was performed to better define the potential activities of this novel compound. Susceptibility testing with MK-0991 against approximately 200 clinical isolates of Candida, Cryptococcus neoformans, and Aspergillus isolates was conducted to determine MICs and minimum fungicidal concentrations MF(s). The MFC at which 90% of isolates are inhibited for 40 C. albicans clinical isolates was 0.5 microg/ml. Susceptibility testing with panels of antifungal agent-resistant species of Candida and C. neoformans isolates indicated that the MK-0991 MFCs for these isolates are comparable to those obtained for susceptible isolates. Growth kinetic studies of MK-0991 against Candida albicans and Candida tropicalis isolates showed that the compound exhibited fungicidal activity (i.e., a 99% reduction in viability) within 3 to 7 h at concentrations ranging from 0.06 to 1 microg/ml (0.25 to 4 times the MIC). Drug combination studies with MK-0991 plus amphotericin B found that this combination was not antagonistic against C. albicans, C. neoformans, or A. fumigatus in vitro. Studies with 0 to 50% pooled human or mouse serum established that fungal susceptibility to MK-0991 was not significantly influenced by the presence of human or mouse serum. Results from resistance induction studies suggested that the susceptibility of C. albicans was not altered by repeated exposure (40 passages) to MK-0991. Erythrocyte hemolysis studies with MK-0991 with washed and unwashed human or mouse erythrocytes indicated minimal hemolytic potential with this compound. These favorable results of preclinical studies support further studies with MK-0991 with humans.
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- 1997
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22. Evaluation of Experimental Therapeutics in a New Mouse Model ofHelicobacter felisUtilizing 16S rRNA Polymerase Chain Reaction for Detection
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Bartizal Kenneth F, Helmut Kropp, Amy M. Flattery, Lynn L. Silver, Jeffrey G. Smith, Leopold Kong, George K. Abruzzo, Charles Gill, and Patricia M. Scott
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Spirillaceae ,Polymerase Chain Reaction ,Helicobacter Infections ,law.invention ,Mice ,law ,Helicobacter ,RNA, Ribosomal, 16S ,Gene expression ,Animals ,Humans ,Polymerase chain reaction ,Helicobacter pylori ,biology ,Felis ,Gastroenterology ,Ribosomal RNA ,Anti-Ulcer Agents ,biology.organism_classification ,16S ribosomal RNA ,Virology ,Anti-Bacterial Agents ,Disease Models, Animal ,Immunology ,Helicobacter felis ,Drug Therapy, Combination ,Omeprazole - Abstract
Background: A new mouse model of Helicobacter felis infection, which mimics the human infection observed with H. pylori, has recently been developed utilizing polymerase chain reaction (PCR) based on the 16S rRNA gene sequence for detection of infection. Methods: We tested several therapeutic regimens in this model, including some currently utilized in the clinic and some shown ineffective in the clinic. Results: The therapeutic results obtained by PCR with this model are consistent with results observed in the published human H. pylori clinical trials and also with results obtained in another H. felis mouse model utilizing culture and histology. Conclusions: These results support further use of this new model in screening for new therapeutic regimens for the management of Helicobacter disease.
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- 1997
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23. Characterization of echinocandin-resistant mutants of Candida albicans: genetic, biochemical, and virulence studies
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James A. Milligan, Cameron M. Douglas, W Li, Bartizal Kenneth F, Amy M. Flattery, Myra B. Kurtz, George K. Abruzzo, K Nollstadt, and Jean A. Marrinan
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Antifungal Agents ,Echinocandin ,Immunology ,Mutant ,Mutagenesis (molecular biology technique) ,Virulence ,Microbial Sensitivity Tests ,Spheroplasts ,Peptides, Cyclic ,Microbiology ,Fungal Proteins ,Lethal Dose 50 ,Echinocandins ,Mice ,Candida albicans ,medicine ,Animals ,Genes, Dominant ,Pyrans ,Fungal protein ,Dose-Response Relationship, Drug ,biology ,Drug Resistance, Microbial ,biology.organism_classification ,Corpus albicans ,Anti-Bacterial Agents ,Infectious Diseases ,Glucosyltransferases ,Mice, Inbred DBA ,Mutation ,Female ,Parasitology ,Peptides ,Research Article ,medicine.drug - Abstract
The pneumocandins are potent antifungal agents of the echinocandin class which are under development for use as broad-spectrum antimycotic therapy. One important consideration for any new therapeutic class for treating serious fungal infections is the potential for drug resistance development. In this study we have isolated and characterized four independent spontaneous Candida albicans mutants resistant to the potent semisynthetic pneumocandin L-733,560. These mutants have many of the properties of FKS1/ETG1 echinocandin-resistant mutants of Saccharomyces cerevisiae, including (i) cross-resistance to other 1,3-beta-D-glucan synthase inhibitors, such as papulacandin and echinocandins, but no change in sensitivity to other antifungal agents; (ii) in vitro glucan synthase activity that is more resistant to pneumocandins than the wild-type parent enzyme; and (iii) semidominant drug resistance in spheroplast fusion strains. The mutants were compared with C. albicans echinocandin-resistant mutants isolated by mutagenesis by L. Beckford and D. Kerridge (mutant M-2) (abstr. PS3.11, in Proceedings of the XI Congress of the International Society for Human and Animal Mycology, Montreal, Canada, 1992) and by A. Cassone, R. E. Mason, and D. Kerridge (mutant CA-2) (Sabouraudia 19:97-110, 1981). All of the strains had resistant enzyme activity in vitro. M-2 grew poorly and had low levels of enzyme activity. In contrast, CA-2 and the spontaneous mutants grew as well as the parents and had normal levels of glucan synthase activity. These results suggest that these resistant mutants may have alterations in glucan synthase. CA-2 was unable to form germ tubes, an ability retained by the spontaneous mutants. The virulence of the spontaneous mutants was unimpaired in a mouse model of disseminated candidiasis, while M-2 and CA-2 were 2 orders of magnitude less virulent than their parent strains. Significantly, mice challenged with the spontaneous mutant CAI4R1 responded therapeutically to lower levels of L-733,560 than would he predicted by the increase in in vitro susceptibility.
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- 1996
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24. A sensitive and specific PCR method to detect Helicobacter felis in a conventional mouse model
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C. Cioffe, C M Thompson, Lynn L. Silver, L. Lynch, Patricia M. Scott, K. A. Bartizal, Helmut Kropp, Amy M. Flattery, David Bramhill, Charles Gill, C. Bonfiglio, Leopold Kong, George K. Abruzzo, and Jeffrey G. Smith
- Subjects
DNA, Bacterial ,Male ,Microbiology (medical) ,medicine.drug_class ,Molecular Sequence Data ,Clinical Biochemistry ,Immunology ,Antibiotics ,DNA, Ribosomal ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Helicobacter Infections ,Microbiology ,law.invention ,Bacterial genetics ,Mice ,law ,Helicobacter ,RNA, Ribosomal, 16S ,medicine ,Gastric mucosa ,Animals ,Germ-Free Life ,Immunology and Allergy ,Polymerase chain reaction ,DNA Primers ,biology ,Felis ,Ribosomal RNA ,biology.organism_classification ,Anti-Bacterial Agents ,Disease Models, Animal ,RNA, Bacterial ,medicine.anatomical_structure ,Gastric Mucosa ,Helicobacter felis ,Drug Therapy, Combination ,Female ,Research Article - Abstract
Although many detection methods have been used to determine Helicobacter colonization in small animal models, the sensitivity and specificity of these detection methods are limited. To improve the Helicobacter felis conventional mouse model for accurate evaluation of therapeutic regimens, we developed a PCR for detection of, and a competitive PCR for quantitation of, H. felis in viral antibody-free (VAF) mice. The PCR was based on the H. felis 16S rRNA gene. An internal control DNA was used for competitive quantitation of the PCR. VAF conventional Swiss-Webster mice were infected with an H. felis culture by oral gavage. At various times after H. felis challenge and therapy, stomach mucosa was collected and evaluated by PCR. PCR detected approximately 50 to 100 H. felis cells per mouse stomach and showed no cross-reaction with other bacteria commonly found in mouse stomachs. Colonization of H. felis in the mouse stomach was confirmed by culture isolation from germfree mice and histological examination of VAF mice. Response to therapy in this H. felis model correlated well with results seen in human clinical trials with H. pylori. A model utilizing PCR detection which may be useful for discovering new antibiotics and/or vaccines against Helicobacter ulcer disease has been developed.
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- 1996
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25. Use of anti-CD4+ hybridoma cells to induce Pneumocystis carinii in mice
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D C McFadden, Bartizal Kenneth F, M A Powles, Amy M. Flattery, Dennis M. Schmatz, and Jeffrey G. Smith
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CD4-Positive T-Lymphocytes ,Male ,medicine.drug_class ,Immunology ,Population ,Spleen ,Monoclonal antibody ,Microbiology ,Dexamethasone ,Lymphocyte Depletion ,Immunocompromised Host ,Mice ,Subcutaneous injection ,In vivo ,medicine ,Animals ,education ,Mice, Inbred C3H ,education.field_of_study ,Hybridomas ,biology ,Pneumocystis ,Pneumonia, Pneumocystis ,CD4 Lymphocyte Count ,Disease Models, Animal ,Infectious Diseases ,medicine.anatomical_structure ,Pneumocystis carinii ,biology.protein ,Parasitology ,Antibody ,CD8 ,Research Article - Abstract
A reduction of peripheral CD4+ cell levels has been correlated with the onset of Pneumocystis carinii pneumonia in AIDS patients. Most in vivo drug discovery and development for P. carinii have been conducted in corticosteroid-treated rats. There is need for the development of new small animal models with more selective methods of immunosuppression. This study outlines a new mouse model in which specific depletion of the CD4+ T-lymphocyte population was achieved by subcutaneous injection of G.K1.5 hybridoma cells into C3HeB/FeJ mice. A significant reduction in splenic CD4+ cells was maintained over a 10-week period following a single injection of cells. Circulating anti-CD4+ antibody was detected throughout the 10-week period in hybridoma-injected mice, while circulating antibody was undetectable 4 weeks after repeated injection of purified monoclonal antibody. There was no significant increase in the CD8+ cell populations of the hybridoma-injected mice. P. carinii cysts increased in the lungs of CD4+ T-cell-depleted mice, with the number of cysts detected comparable to levels in dexamethasone-treated mice. High levels of cysts were detected when CD4+ cell populations in the spleen remained below 5% and decreased when CD4+ populations increased above the 5% level. In mice whose CD4+ population was not reduced below 5%, there was no significant increase in P. carinii cysts detected. This study presents a new mouse model with specific immunosuppression requiring a minimum of animal manipulation for use in discovery and development of potential new therapeutics for P. carinii pneumonia.
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- 1994
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26. A rapid method for cross-species quantitation of apolipoproteins A1, B48 and B100 in plasma by ultra-performance liquid chromatography/tandem mass spectrometry
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Michael E, Lassman, Theresa M, McLaughlin, Elizabeth P, Somers, Alice C, Stefanni, Zhu, Chen, Beth Ann, Murphy, Kathleen K, Bierilo, Amy M, Flattery, Kenneth K, Wong, Jose M, Castro-Perez, Brian K, Hubbard, and Thomas P, Roddy
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Apolipoprotein A-I ,Macaca mulatta ,Peptide Fragments ,Rats ,Mice ,Dogs ,Species Specificity ,Cardiovascular Diseases ,Tandem Mass Spectrometry ,Cricetinae ,Gene Knockdown Techniques ,Apolipoprotein B-100 ,Linear Models ,Animals ,Humans ,Computer Simulation ,Trypsin ,Amino Acid Sequence ,RNA, Small Interfering ,Apolipoprotein B-48 ,Chromatography, High Pressure Liquid - Abstract
Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.
- Published
- 2011
27. Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates
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Hongxia Fan, David S. Perlin, Amy M. Flattery, Ming-Jo Hsu, Rosemarie Kelly, Gary Chrebet, Elizabeth A. Register, Hedy Teppler, Cameron M. Douglas, Steven Park, George K. Abruzzo, J. Robles, Charles Gill, Stephen A. Parent, W Li, Myra B. Kurtz, Valmik K. Vyas, and J. Nielsen Kahn
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Antifungal Agents ,Echinocandin ,Mutant ,Mutagenesis (molecular biology technique) ,Microbial Sensitivity Tests ,Peptides, Cyclic ,Microbiology ,chemistry.chemical_compound ,Echinocandins ,Lipopeptides ,Mice ,Caspofungin ,Drug Resistance, Fungal ,Mechanisms of Resistance ,Candida krusei ,Candida albicans ,medicine ,polycyclic compounds ,Animals ,Humans ,Pharmacology (medical) ,skin and connective tissue diseases ,Candida ,Pharmacology ,biology ,Candidiasis ,biology.organism_classification ,bacterial infections and mycoses ,Corpus albicans ,Infectious Diseases ,chemistry ,Amino Acid Substitution ,Glucosyltransferases ,Laboratories ,medicine.drug - Abstract
An association between reduced susceptibility to echinocandins and changes in the 1,3-β- d -glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Ca fks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei .
- Published
- 2005
28. Inactivation of Kex2p diminishes the virulence of Candida albicans
- Author
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Alan Kuo, Julie Johnson Blake, Charles Gill, Myra B. Kurtz, Amy M. Flattery, Nina Agabian, George K. Abruzzo, and George Newport
- Subjects
Saccharomyces cerevisiae Proteins ,Mutant ,Genes, Fungal ,Molecular Sequence Data ,Virulence ,Biology ,Biochemistry ,Microbiology ,Cell Line ,Fungal Proteins ,Mice ,Candida albicans ,Animals ,Secretion ,Amino Acid Sequence ,Subtilisins ,Molecular Biology ,Sequence Homology, Amino Acid ,Hydrolysis ,Macrophages ,Cell Biology ,biology.organism_classification ,Phenotype ,Corpus albicans ,Bacterial adhesin ,Mutation ,Kexin ,Proprotein Convertases - Abstract
Deletion of the kexin gene (KEX2) in Candida albicans has a pleiotropic effect on phenotype and virulence due partly to a defect in the expression of two major virulence factors: the secretion of active aspartyl proteinases and the formation of hyphae. kex2/kex2 mutants are highly attenuated in a mouse systemic infection model and persist within cultured macrophages for at least 24 h without causing damage. Pathology is modest, with little disruption of kidney matrix. The infecting mutant cells are largely confined to glomeruli, and are aberrant in morphology. The complex phenotype of the deletion mutants reflects a role for kexin in a wide range of cellular processes. Taking advantage of the specificity of Kex2p cleavage, an algorithm we developed to scan the 9168 open reading frames in Assembly 6 of the C. albicans genome identified 147 potential substrates of Kex2p. These include all previously identified substrates, including eight secreted aspartyl proteinases, the exoglucanase Xog1p, the immunodominant antigen Mp65, and the adhesin Hwp1p. Other putative Kex2p substrates identified include several adhesins, cell wall proteins, and hydrolases previously not implicated in pathogenesis. Kexins also process fungal mating pheromones; a modification of the algorithm identified a putative mating pheromone with structural similarities to Saccharomyces cerevisiae alpha-factor.
- Published
- 2002
29. Antimicrobial activity of ergokonin A from Trichoderma longibrachiatum
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Gonzalo Platas, Mark Rosenbach, Maria Teresa Diez, Myra B. Kurtz, Kenneth E. Wilson, Angeles Cabello, Fernando Pelaez, Leopold Kong, George K. Abruzzo, John R. Thompson, Janet C. Onishi, Amy M. Flattery, Angela Basilio, María Francisca Vicente, Robert A. Giacobbe, Sarah Dreikorn, A. Tsipouras, and Maria S. Meinz
- Subjects
Antifungal Agents ,Hypha ,Trichoderma longibrachiatum ,Fungus ,Microbial Sensitivity Tests ,Applied Microbiology and Biotechnology ,Aspergillus fumigatus ,Microbiology ,Mice ,Hypocrea ,Candida albicans ,DNA, Ribosomal Spacer ,Animals ,Mode of action ,Trichoderma ,biology ,Bacteria ,Candidiasis ,Fungi ,General Medicine ,Fungi imperfecti ,Sequence Analysis, DNA ,biology.organism_classification ,Yeast ,RNA, Ribosomal, 5.8S ,Sterols ,Water Microbiology ,Biotechnology - Abstract
Aims: Natural fungal products were screened for antifungal compounds. The mode of action of one of the hits found and the taxonomy of the producing organism were analysed. Methods and Results: An extract from a Trichoderma species showed a more potent activity in an agar-based assay against the null mutant fks1::HIS strain than against the wild-type strain, suggesting that it could contain a glucan synthesis inhibitor. The active component was identified as the known compound ergokonin A. The compound exhibited activity against Candida and Aspergillus species, but was inactive against Cryptococcus species. It induced alterations in the hyphal morphology of Aspergillus fumigatus. The identification of the producing isolate was confirmed by sequencing of the rDNA internal transcribed spacers and comparison with the sequences of other Trichoderma species. The analysis showed that the producing fungus had a high homology with other strains classified as Trichoderma longibrachiatum and its teleomorph Hypocrea schweinitzii. Conclusions: The antifungal activity spectrum of ergokonin A and the morphology alterations induced on A. fumigatus are consistent with glucan synthesis as the target for ergokonin A. The production of ergokonin A is not uncommon, but is probably restricted to Trichoderma species. Significance and Impact of the Study: The discovery that ergokonin A could be an inhibitor of glucan synthesis, having a structure very different to other inhibitors, increases the likelihood that orally active agents with this fungal-specific mode of action may be developed.
- Published
- 2001
30. Rustmicin, a potent antifungal agent, inhibits sphingolipid synthesis at inositol phosphoceramide synthase
- Author
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Rosemary A. Thornton, Suzanne M. Mandala, Herbert G. Bull, Sarah Dreikorn, George K. Abruzzo, Myra B. Kurtz, James A. Milligan, Mark Rosenbach, Charles Gill, Amy M. Flattery, Margarita Garcia-Calvo, Guy H. Harris, and Bartizal Kenneth F
- Subjects
Ceramide ,Antifungal Agents ,Mice, Inbred Strains ,Saccharomyces cerevisiae ,Biochemistry ,Glycosphingolipids ,Fungal Proteins ,chemistry.chemical_compound ,Lactones ,Mice ,Animals ,Inositol ,Enzyme Inhibitors ,Molecular Biology ,IC50 ,Cryptococcus neoformans ,chemistry.chemical_classification ,Sphingolipids ,biology ,ATP synthase ,Molecular Structure ,Fungi ,Membrane Proteins ,Cell Biology ,Cryptococcosis ,biology.organism_classification ,Sphingolipid ,Enzyme ,chemistry ,Hexosyltransferases ,biology.protein ,Efflux ,Cell Division - Abstract
Rustmicin is a 14-membered macrolide previously identified as an inhibitor of plant pathogenic fungi by a mechanism that was not defined. We discovered that rustmicin inhibits inositol phosphoceramide synthase, resulting in the accumulation of ceramide and the loss of all of the complex sphingolipids. Rustmicin has potent fungicidal activity against clinically important human pathogens that is correlated with its sphingolipid inhibition. It is especially potent against Cryptococcus neoformans, where it inhibits growth and sphingolipid synthesis at concentrations
- Published
- 1998
31. Evaluation of the echinocandin antifungal MK-0991 (L-743,872): efficacies in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis
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James M. Balkovec, Aileen Bouffard, Bartizal Kenneth F, Helmut Kropp, Jeffrey G. Smith, Amy M. Flattery, James F. Dropinski, Leopold Kong, Hugh Rosen, George K. Abruzzo, V. B. Pikounis, and Charles Gill
- Subjects
Antifungal Agents ,Drug Evaluation, Preclinical ,Administration, Oral ,Aspergillosis ,Candida parapsilosis ,Peptides, Cyclic ,Microbiology ,Candida tropicalis ,chemistry.chemical_compound ,Echinocandins ,Lipopeptides ,Mice ,Caspofungin ,Candida krusei ,medicine ,Animals ,Pharmacology (medical) ,Candida albicans ,Pharmacology ,biology ,Candida lusitaniae ,Candidiasis ,Cryptococcosis ,medicine.disease ,biology.organism_classification ,Disseminated Candidiasis ,Anti-Bacterial Agents ,Disease Models, Animal ,Infectious Diseases ,chemistry ,Mice, Inbred DBA ,Female ,Kidney Diseases ,Peptides ,Injections, Intraperitoneal ,Research Article - Abstract
The in vivo activity of the Merck antifungal echinocandin drug candidate MK-0991 (L-743,872) was evaluated in mouse models of disseminated candidiasis, aspergillosis, and cryptococcosis. The echinocandins are potent inhibitors of 1,3-beta-D-glucan synthase. Two models of disseminated candidiasis were used. In a Candida albicans mouse survival model with both DBA/2N and CD-1 mice, estimates of the 50% effective doses (ED50s) of MK-0991 were 0.04 and 0.10 mg/kg of body weight/dose at 21 days after challenge, respectively. In a C. albicans target organ assay (TOA) with DBA/2N mice, MK-0991 at levels of > or =0.09 mg/kg/dose significantly reduced the numbers of C. albicans CFU/g of kidneys compared to the numbers in the kidneys of control mice from 1 to 28 days after challenge. Even when given as a single intraperitoneal dose either 30 min or 24 h after challenge, MK-0991 was effective and significantly reduced the numbers of C. albicans CFU/g of kidney compared to those in the controls. MK-0991 was >300-fold less active when it was administered orally than when it was administered parenterally. MK-0991 was efficacious in mouse TOAs against other C. albicans strains and Candida species including Candida tropicalis, Candida (Torulopsis) glabrata, Candida lusitaniae, Candida parapsilosis, and Candida krusei. MK-0991 was ineffective against disseminated Cryptococcus neoformans infections. In the model of disseminated aspergillosis in mice, MK-0991 at doses of > or =0.02 mg/kg/dose significantly prolonged the survival of DBA/2N mice, with estimates of the ED50 and ED90 of MK-0991 being 0.03 and 0.12 mg/kg/dose, respectively, at 28 days after challenge. MK-0991 is a potent, parenterally administered therapeutic agent against disseminated candidiasis and aspergillosis that warrants further investigation in human clinical trials.
- Published
- 1997
32. New model of oropharyngeal and gastrointestinal colonization by Candida albicans in CD4+ T-cell-deficient mice for evaluation of antifungal agents
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George K. Abruzzo, Jeffrey G. Smith, Bartizal Kenneth F, Charles Gill, and Amy M. Flattery
- Subjects
CD4-Positive T-Lymphocytes ,Nystatin ,Antifungal Agents ,Population ,Oropharynx ,Microbial Sensitivity Tests ,Peptides, Cyclic ,Microbiology ,chemistry.chemical_compound ,Mice ,Candida albicans ,medicine ,Animals ,Pharmacology (medical) ,education ,Fluconazole ,Pharmacology ,education.field_of_study ,Gastrointestinal tract ,biology ,Aminoglycoside ,Antibodies, Monoclonal ,biology.organism_classification ,Corpus albicans ,Anti-Bacterial Agents ,Disease Models, Animal ,Infectious Diseases ,Ketoconazole ,chemistry ,Mice, Inbred DBA ,Pneumocandin Bo ,Female ,Peptides ,Digestive System ,medicine.drug ,Research Article - Abstract
A new model for the evaluation of antifungal compounds against oropharyngeal and gastrointestinal mucosal colonization by Candida albicans was developed. To simulate the immune deficiency observed in AIDS patients, mice were depleted of CD4+ T lymphocytes by the injection of either GK1.5 hybridoma cells or purified anti-CD4+ T lymphocytes by the injection of either GK1.5 hybridoma cells or purified anti-CD4+ monoclonal antibody derived from GK1.5 hybridoma cells in tissue culture. Fluorescence-activated cell sorter analysis of splenic lymphocytes confirmed the elimination of the CD4+ T-cell population. Gentamicin, a broad-spectrum, nonabsorbable aminoglycoside antibiotic, was given via the drinking water to reduce the normal gastrointestinal microflora, allowing less competition for colonization of the gastrointestinal tract by the C. albicans isolates. Mice were challenged by gavage and swabbing their oral mucosae with a pure culture of C. albicans. Gentamicin was withdrawn 3 days postchallenge, and antifungal compounds were administered via the drinking water ad libitum at concentrations ranging from 25 to 400 micrograms/ml. L-693989, a water-soluble phosphorylated cyclic lipopeptide prodrug of pneumocandin Bo, and L-733560, a semisynthetic derivative of pneumocandin Bo, are inhibitors of 1,3-beta-D-glucan synthesis that exhibit potent in vivo anti-Candida spp. and anti-Pneumocystis carinii activities. The efficacies of L-693989, L-733560, fluconazole, ketoconazole, and nystatin were evaluated in this new oropharyngeal and gastrointestinal model of mucosal colonization. L-693989, L-733560, fluconazole, and ketoconazole showed superior efficacies in reducing the numbers of C. albicans CFU per gram of feces and the numbers of oral CFU relative to those in sham-treated controls in this model, while nystatin was moderately effective in reducing oral and fecal colonization by C. albicans in this model.
- Published
- 1996
33. PCR detection of colonization by Helicobacter pylori in conventional, euthymic mice based on the 16S ribosomal gene sequence
- Author
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David Bramhill, Amy M. Flattery, Charles Gill, Leopold Kong, George K. Abruzzo, C. Cioffe, Bartizal Kenneth F, Patricia M. Scott, Jeffrey G. Smith, and C M Thompson
- Subjects
Microbiology (medical) ,DNA, Bacterial ,medicine.medical_specialty ,Time Factors ,Clinical Biochemistry ,Immunology ,Molecular Sequence Data ,Mice, Transgenic ,Mice, SCID ,DNA, Ribosomal ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Microbiology ,Helicobacter Infections ,chemistry.chemical_compound ,Mice ,RNA, Ribosomal, 16S ,medicine ,Immunology and Allergy ,Animals ,Germ-Free Life ,Humans ,Colonization ,Helicobacter ,DNA Primers ,biology ,Base Sequence ,Helicobacter pylori ,Stomach ,Ribosomal RNA ,biology.organism_classification ,Disease Models, Animal ,RNA, Bacterial ,medicine.anatomical_structure ,chemistry ,Gastric Mucosa ,Mice, Inbred DBA ,Histopathology ,Bacteria ,DNA ,Research Article - Abstract
Many animal models of Helicobacter infection have been described, including infection in rhesus monkeys, ferrets, gnotobiotic piglets, and mice. These animal models utilize a combination of detection methods, including culture, urease testing, and histopathology, all of which may be unreliable, insensitive, or labor-intensive. Development of new animal models of Helicobacter pylori requires new methods of detection with increased sensitivity and specificity. We have developed sensitive and specific PCR primers based on the 16S ribosomal gene sequence of H. pylori. The primers detected single-copy 16S DNA representing 0.2 cell of pure H. pylori (2 cells in the presence of mouse stomach mucosal DNA) and did not cross-react with closely related bacteria. We were able to detect colonization by H. pylori in conventional, euthymic, outbred mice up to 4 weeks postinoculation with a high percentage of isolates tested. One isolate of H. pylori was detected by PCR in 100% of the mice at 6 months and 60% of the mice 1 year after inoculation. Approximately 10(3) to 10(4) H. pylori cells per stomach were detected by utilizing this PCR methodology semiquantitatively. These primers and PCR methodology have facilitated detection of H. pylori colonization in conventional, euthymic mice, colonization which may not have been detectable by other methods.
- Published
- 1996
34. In vivo evaluation of three acid-stable azalide compounds, L-701,677, L-708,299 and L-708,365 compared to erythromycin, azithromycin and clarithromycin
- Author
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Robert R. Wilkening, Helmut Kropp, Amy M. Flattery, Jesse J. Jackson, George K. Abruzzo, Jeffrey G. Smith, Ken Bartizal, Charles Gill, Kothandaraman Shankaran, and Li Kong
- Subjects
medicine.drug_class ,Antibiotics ,Drug Evaluation, Preclinical ,Erythromycin ,Biology ,Azalide ,Azithromycin ,medicine.disease_cause ,Microbiology ,Mice ,Drug Stability ,Clarithromycin ,Streptococcal Infections ,Drug Discovery ,medicine ,Animals ,Antibacterial agent ,Pharmacology ,Molecular Structure ,Bacterial Infections ,biochemical phenomena, metabolism, and nutrition ,Staphylococcal Infections ,Anti-Bacterial Agents ,Klebsiella Infections ,Staphylococcus aureus ,Mice, Inbred DBA ,Streptococcus pyogenes ,Female ,Macrolides ,medicine.drug ,Half-Life - Abstract
L-701, 677, L-708, 299 and L-708, 365 are novel azalide derivatives of erythromycin that exhibit improved acid stability over erythromycin, azithromycin and clarithromycin. The half-life in aqueous solution at pH = 2.1 of these compounds ranged from 0.3 hour for erythromycin to 16.2 hours for L-708, 299. The rank order of half-life in acid solution from most to least stable was L-708, 299>L-701, 677>L-708, 365 > azithromycin = clarithromycin>erythromycin. In a disseminated Streptococcus pyogenes mouse infection model, azithromycin and L-708, 365 were slightly more efficacious than clarithromycin, L-701, 677 and L-708, 299; all 5 compounds being more active than erythromycin. In a Klebsiella pneumoniae pulmonary challenge mouse model, azithromycin, L-701, 677, L-708, 299 and L-708, 365 were all equal in efficacy and at least four-fold more active than clarithromycin and erythromycin. Clarithromycin, L-708, 365 and interestingly erythromycin, showed greater bacterial clearance than azitnromycin, L-701, 677 and L-708, 299 in a localized infection model that measured clearance of Staphylococcus aureus from mouse thigh tissues. Our results indicate that L-701, 677, L-708, 299 and L-708, 365 exhibit improved acid stability and were at least equally efficacious as presently marketed macrolide/azalide antibiotics.
- Published
- 1995
35. Evaluation of water-soluble pneumocandin analogs L-733560, L-705589, and L-731373 with mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis
- Author
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Helmut Kropp, Amy M. Flattery, V. B. Pikounis, D Krupa, Jeffrey G. Smith, Bartizal Kenneth F, Leopold Kong, George K. Abruzzo, and Charles Gill
- Subjects
Antifungal Agents ,Aspergillosis ,Peptides, Cyclic ,Aspergillus fumigatus ,Microbiology ,Mice ,Amphotericin B ,medicine ,Animals ,Pharmacology (medical) ,Candida albicans ,Mycosis ,Pharmacology ,biology ,Candidiasis ,Cryptococcosis ,medicine.disease ,biology.organism_classification ,Disseminated Candidiasis ,Anti-Bacterial Agents ,Infectious Diseases ,Mice, Inbred DBA ,Ketoconazole ,Female ,Peptides ,medicine.drug ,Research Article - Abstract
The activities of the water-soluble pneumocandin derivatives L-733560, L-705589, and L-731373 were evaluated in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis and were compared with those of commercially available antifungal agents. Pneumocandins are inhibitors of 1,3-beta-D-glucan synthesis. In the aspergillosis model, L-733560 and L-705589 significantly prolonged the survival of DBA/2N mice challenged intravenously with Aspergillus fumigatus conidia. L-733560 and L-705589 exhibited efficacies comparable to that of amphotericin B (AMB) with 90% effective doses of 0.48, 0.12, and 0.36 mg/kg of body weight, respectively. Two mouse models of disseminated candidiasis were used to evaluate these compounds. In both models, mice were challenged intravenously with Candida albicans. In a C. albicans survival model with DBA/2N and CD-1 mice, the efficacy of L-733560 was comparable to that of AMB, while L-731373 and L-705589 were somewhat less active. In a previously described C. albicans target organ kidney assay, the pneumocandin analogs and AMB at doses of > or = 0.09 mg/kg were effective in sterilizing kidneys, while fluconazole and ketoconazole were considerably less active and did not sterilize kidneys when they were used at concentrations of < or = 100 mg/kg. Although orally administered L-733560 showed activity in both candidiasis models, its efficacy was reduced compared with that of parenterally administered drug. In a disseminated cryptococcosis mouse model that measures the number of CFU of Cryptococcus neoformans per gram of brain and spleen, L-733560 at 10 mg/kg was ineffective in reducing the counts in organs, while AMB at 0.31 mg/kg sterilized the organs. These results indicate that the pneumocandins may be beneficial as potent parenterally administered therapeutic agents for disseminated aspergillosis and candidiasis.
- Published
- 1995
36. PAP Inhibitor with In Vivo Efficacy Identified by Candida albicans Genetic Profiling of Natural Products
- Author
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Robert A. Giacobbe, Yang Li, Tara Ransom, Gonzalo Platas, Bo Jiang, Douglas Wisniewski, Fariba Rahkhoodaee, Amy M. Flattery, Roberto Rodriguez-Suarez, Phil Youngman, Kenneth E. Wilson, Scott P. Salowe, Jennifer Nielsen Kahn, Gerald F. Bills, Terry Roemer, Hao Wang, Nick Martel, Ming Jo Hsu, Steve Trosok, Judyann Wiltsie, Maria Teresa Diez, Daniel Gauvin, Paul A. Liberator, Guy H. Harris, Craig A. Parish, Susan Sillaots, John J. Allocco, Sarah Kauffman, John Davison, Karynn Veillette, Li Zhang, Wenqi Hu, Fernando Pelaez, Jeff Becker, Deming Xu, George K. Abruzzo, and Charles Gill
- Subjects
Heterozygote ,Antifungal Agents ,Molecular Sequence Data ,Clinical Biochemistry ,Drug Evaluation, Preclinical ,Microbial Sensitivity Tests ,Saccharomyces cerevisiae ,Drug resistance ,Complex Mixtures ,Polyadenylation ,Biochemistry ,Microbiology ,Mice ,chemistry.chemical_compound ,Drug Resistance, Fungal ,In vivo ,Candida albicans ,Drug Discovery ,Animals ,Amino Acid Sequence ,RNA, Messenger ,Molecular Biology ,Alleles ,Polymerase ,Pharmacology ,Biological Products ,Natural product ,Deoxyadenosines ,biology ,Drug discovery ,Aspergillus fumigatus ,Candidiasis ,Polynucleotide Adenylyltransferase ,General Medicine ,biology.organism_classification ,Disseminated Candidiasis ,Treatment Outcome ,CHEMBIO ,DNA profiling ,chemistry ,Fermentation ,Mutation ,biology.protein ,Molecular Medicine ,RNA - Abstract
SummaryNatural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.
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