Publications by authors named "Micha Fridman"

48 Publications

Azide-Functionalized Derivatives of the Virulence-Associated Sugar Pseudaminic Acid: Chiral Pool Synthesis and Labeling of Bacteria.

Authors:
Daniel B Werz Amol M Vibhute Hideki Tamai Dana Logviniuk Peter G Jones Micha Fridman

Chemistry 2021 Mar 26. Epub 2021 Mar 26.

Tel Aviv University Raymond and Beverly Sackler Faculty of Exact Sciences, Organic Chemistry, ISRAEL.

Pseudaminic acid (Pse) is a significant prokaryotic monosaccharide found in important Gram-negative and Gram-positive bacteria. This unique sugar serves as a component of cell-surface-associated glycans or glycoproteins and is associated with their virulence. We report the synthesis of azidoacetamido-functionalized Pse derivatives as part of a search for Pse-derived metabolic labeling reagents. The synthesis was initiated with D-glucose (Glc), which served as a cost-effective chiral pool starting material. Key synthetic steps involve the conversion of C1 of Glc into the terminal methyl group of Pse, and inverting deoxyaminations at C3 and C5 of Glc followed by backbone elongation with a three-carbon unit using the Barbier reaction. Metabolic labeling experiments revealed that, of the four Pse derivatives, ester-protected C5 azidoacetamido-Pse successfully labeled cells of Pse-expressing Gram-positive and Gram-negative strains. No labeling was observed in cells of non-Pse-expressing strains. The ester-protected and C5 azidoacetamido-functionalized Pse is thus a useful reagent for the identification of bacteria expressing this unique virulence-associated nonulosonic acid.
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http://dx.doi.org/10.1002/chem.202100443DOI Listing
March 2021

Design Guidelines for Cationic Pillar[n]arenes that Prevent Biofilm Formation by Gram-Positive Pathogens.

Authors:
Dana Kaizerman-Kane Maya Hadar Roymon Joseph Dana Logviniuk Yossi Zafrani Micha Fridman Yoram Cohen

ACS Infect Dis 2021 03 3;7(3):579-585. Epub 2021 Mar 3.

School of Chemistry, Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.

Bacterial biofilms are a major threat to human health, causing persistent infections that lead to millions of fatalities worldwide every year. Biofilms also cause billions of dollars of damage annually by interfering with industrial processes. Recently, cationic pillararenes were found to be potent inhibitors of biofilm formation in Gram-positive bacteria. To identify the structural features of pillararenes that result in antibiofilm activity, we evaluated the activity of 16 cationic pillar[5]arene derivatives including that of the first cationic water-soluble pillar[5]arene-based rotaxane. Twelve of the derivatives were potent inhibitors of biofilm formation by Gram-positive pathogens. Structure activity analyses of our pillararene derivatives indicated that positively charged head groups are critical for the observed antibiofilm activity. Although certain changes in the lipophilicity of the substituents on the positively charged head groups are tolerated, dramatic elevation in the hydrophobicity of the substituents or an increase in steric bulk on these positive charges abolishes the antibiofilm activity. An increase in the overall positive charge from 10 to 20 did not affect the activity significantly, but pillararenes with 5 positive charges and 5 long alkyl chains had reduced activity. Surprisingly, the cavity of the pillar[n]arene is not essential for the observed activity, although the macrocyclic structure of the pillar[n]arene core, which facilitates the clustering of the positive charges, appears important. Interestingly, the compounds found to be efficient inhibitors of biofilm formation were nonhemolytic at concentrations that are ∼100-fold of their MBIC (the minimal concentration of a compound at which at least 50% inhibition of biofilm formation was observed compared to untreated cells). The structure-activity relationship guidelines established here pave the way for a rational design of potent cationic pillar[n]arene-based antibiofilm agents.
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http://dx.doi.org/10.1021/acsinfecdis.0c00662DOI Listing
March 2021

Fresh Molecular Concepts to Extend the Lifetimes of Old Antimicrobial Drugs.

Authors:
Qais Z Jaber Micha Fridman

Chem Rec 2021 Feb 19. Epub 2021 Feb 19.

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel.

Antimicrobial drug development generally initiates with target identification and mode of action studies. Often, emergence of resistance and/or undesired side effects that are discovered only after prolonged clinical use, result in discontinuation of clinical use. Since the cost and time required for improvement of existing drugs are considerably lower than those required for the development of novel drugs, academic and pharmaceutical company researchers pursue this direction. In this account we describe selected examples of how chemical probes generated from antimicrobial drugs and chemical and enzymatic modifications of these drugs have been used to modify modes of action, block mechanisms of resistance, or reduce side effects, improving performance. These examples demonstrate how new and comprehensive mechanistic insights can be translated into fresh concepts for development of next-generation antimicrobial agents.
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http://dx.doi.org/10.1002/tcr.202100014DOI Listing
February 2021

Combining Colistin and Fluconazole Synergistically Increases Fungal Membrane Permeability and Antifungal Cidality.

Authors:
Maayan Bibi Sarah Murphy Raphael I Benhamou Alex Rosenberg Adi Ulman Tihana Bicanic Micha Fridman Judith Berman

ACS Infect Dis 2021 02 20;7(2):377-389. Epub 2021 Jan 20.

Shmunis School of Biomedical and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 6997801, Israel.

The increasing emergence of drug-resistant fungal pathogens, together with the limited number of available antifungal drugs, presents serious clinical challenges to treating systemic, life-threatening infections. Repurposing existing drugs to augment the antifungal activity of well-tolerated antifungals is a promising antifungal strategy with the potential to be implemented rapidly. Here, we explored the mechanism by which colistin, a positively charged lipopeptide antibiotic, enhances the antifungal activity of fluconazole, the most widely used orally available antifungal. In a range of susceptible and drug-resistant isolates and species, colistin was primarily effective at reducing fluconazole tolerance, a property of subpopulations of cells that grow slowly in the presence of a drug and may promote the emergence of persistent infections and resistance. Clinically relevant concentrations of colistin synergized with fluconazole, reducing fluconazole minimum inhibitory concentration 4-fold. Combining fluconazole and colistin also increased survival in a infection, especially for a highly fluconazole-tolerant isolate. Mechanistically, colistin increased permeability to fluorescent antifungal azole probes and to intracellular dyes, accompanied by an increase in cell death that was dependent upon pharmacological or genetic inhibition of the ergosterol biosynthesis pathway. The positive charge of colistin is critical to its antifungal, and antibacterial, activity: colistin directly binds to several eukaryotic membrane lipids (, l-α-phosphatidylinositol, l-α-phosphatidyl-l-serine, and l-α-phosphatidylethanolamine) that are enriched in the membranes of ergosterol-depleted cells. These results support the idea that colistin binds to fungal membrane lipids and permeabilizes fungal cells in a manner that depends upon the degree of ergosterol depletion.
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http://dx.doi.org/10.1021/acsinfecdis.0c00721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887753PMC
February 2021

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

Authors:
Alexander Zimmermann Qais Z Jaber Johannes Koch Steffen Riebe Cecilia Vallet Kateryna Loza Matthias Hayduk Kfir B Steinbuch Shirley K Knauer Micha Fridman Jens Voskuhl

Chembiochem 2021 Jan 7. Epub 2021 Jan 7.

Faculty of chemistry (Organic Chemistry) and, Centre for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, Universitätsstrasse 7, 45117, Essen, Germany.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.
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http://dx.doi.org/10.1002/cbic.202000725DOI Listing
January 2021

Serum Prevents Interactions between Antimicrobial Amphiphilic Aminoglycosides and Plasma Membranes.

Authors:
Dana Logviniuk Micha Fridman

ACS Infect Dis 2020 12 11;6(12):3212-3223. Epub 2020 Nov 11.

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 6997801, Israel.

Antimicrobial cationic amphiphiles have broad-spectrum activity, and microbes do not readily develop resistance to these agents, highlighting their clinical and industrial potential. Cationic amphiphiles perturb the integrity of membranes leading to cell death, and the lack of discrimination between microbial and mammalian plasma membranes is thought to be one of the main barriers of using these agents for the treatment of systemic infections. Here, we describe the synthesis and study of 20 antimicrobial cationic amphiphiles that are derivatives of the aminoglycoside nebramine with different numbers of alkyl chain ethers that differ in length and degree of unsaturation. We determined antifungal activities and evaluated hemoglobin release from red blood cells as a measure of membrane selectivity and analyzed how serum influences these activities. Microscopic images revealed morphological transformations of red blood cells from the normal double-disc shape to empty ghost cells upon treatment with the cationic amphiphiles. Antifungal activity, hemolysis, and morphological changes in red blood cells decreased as the percentage of serum in the culture medium was increased. In images of red blood cells treated with fluorescently labeled amphiphilic nebramine probes, the accumulation of the cationic amphiphiles in the membranes decreased as serum concentration increased. This suggests that, in addition to its known effect of preventing the deformability of red blood cells, serum prevents interactions between cationic amphiphiles and the plasma membrane. The results of this study indicate that biological activities of cationic amphiphiles are abrogated in serum. Thus, these agents are suitable for external and industrial uses but probably not for effective treatment of systemic infections.
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http://dx.doi.org/10.1021/acsinfecdis.0c00588DOI Listing
December 2020

Elevated Vacuolar Uptake of Fluorescently Labeled Antifungal Drug Caspofungin Predicts Echinocandin Resistance in Pathogenic Yeast.

Authors:
Qais Z Jaber Maayan Bibi Ewa Ksiezopolska Toni Gabaldon Judith Berman Micha Fridman

ACS Cent Sci 2020 Oct 9;6(10):1698-1712. Epub 2020 Sep 9.

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 6997801, Israel.

Echinocandins are the newest class of antifungal drugs in clinical use. These agents inhibit β-glucan synthase, which catalyzes the synthesis of β-glucan, an essential component of the fungal cell wall, and have a high clinical efficacy and low toxicity. Echinocandin resistance is largely due to mutations in the gene encoding β-glucan synthase, but the mode of action is not fully understood. We developed fluorescent probes based on caspofungin, the first clinically approved echinocandin, and studied their cellular biology in species, the most common cause of human fungal infections worldwide. Fluorescently labeled caspofungin probes, like the unlabeled drug, were most effective against metabolically active cells. The probes rapidly accumulated in vacuoles, as shown by colocalization with vacuolar proteins and vacuole-specific stains. The uptake of fluorescent caspofungin is facilitated by endocytosis: The labeled drug formed vesicles similar to fluorescently labeled endocytic vesicles, the vacuolar accumulation of fluorescent caspofungin was energy-dependent, and inhibitors of endocytosis reduced its uptake. In a panel comprised of isogenic strains carrying different β-glucan synthase mutations as well as clinical isolates, resistance correlated with increased fluorescent drug uptake into vacuoles. Fluorescent drug uptake also associated with elevated levels of chitin, a sugar polymer that increases cell-wall rigidity. Monitoring the intracellular uptake of fluorescent caspofungin provides a rapid and simple assay that can enable the prediction of echinocandin resistance, which is useful for research applications as well as for selecting the appropriate drugs for treatments of invasive fungal infections.
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http://dx.doi.org/10.1021/acscentsci.0c00813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596861PMC
October 2020

The relationship between the structure and toxicity of aminoglycoside antibiotics.

Authors:
Moriah Jospe-Kaufman Liza Siomin Micha Fridman

Bioorg Med Chem Lett 2020 07 25;30(13):127218. Epub 2020 Apr 25.

School of Chemistry, Raymond and Beverley Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 6997801, Israel. Electronic address:

Aminoglycoside antibiotics, used to treat persistent gram-negative infections, tuberculosis, and life-threatening infections in neonates and patients with cystic fibrosis, can infer acute kidney injury and irreversible hearing loss. The full repertoire of cellular targets and processes leading to the toxicity of aminoglycosides is not fully resolved, making it challenging to devise rational directions to circumvent their adverse effects. As a result, there has been very limited effort to rationally address the issue of aminoglycoside-induced toxicity. Here we provide an overview of the reported effects of aminoglycosides on cells of the inner ear and on kidney tubular epithelial cells. We describe selected examples for structure-toxicity relationships established by evaluation of both natural and semisynthetic aminoglycosides. The various assays and models used to evaluate these antibiotics and recent progress in development of safer aminoglycoside antibiotics are discussed.
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http://dx.doi.org/10.1016/j.bmcl.2020.127218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194799PMC
July 2020

Bromopyrrole Alkaloids of the Sponge Collected Near the Israeli Mediterranean Coastline.

Authors:
Dimitry Kovalerchik Ravindra Pal Singh Pnina Schlesinger Aseel Mahajni Sigal Shefer Micha Fridman Micha Ilan Shmuel Carmeli

J Nat Prod 2020 02 19;83(2):374-384. Epub 2020 Feb 19.

Raymond and Beverly Sackler School of Chemistry and Faculty of Exact Sciences , Tel Aviv University , Ramat-Aviv 69978 , Israel.

Chemical investigation of the Mediterranean Sea sponge, , collected off the Tel Aviv coast, yielded eight new bromopyrrole metabolites, agesamine C (), dioroidamide A (), slagenin D (), (-)-monobromoagelaspongin (), (-)-11-deoxymonobromoagelaspongin (), (-)-11--methylmonobromoagelaspongin (), -dispacamide (), and pyrrolosine (), along with 18 known bromopyrrole alkaloids and a known bromotyrosine derivative. The structures of the new metabolites were elucidated by analysis of the spectroscopic and spectrometric data, including 1D and 2D NMR, ECD, and high-resolution mass spectrometry. The sponge extract exhibited antimicrobial activity against pathogenic and environmental bacteria, and quorum sensing inhibitory activity (QSI) against . QSI guided separation of the extract established oroidin, benzosceptrin C, and 4,5-dibromopyrrole-2-carboxamide as the active components. The latter compounds were tested for inhibition of growth and biofilm formation in PAO1. The most active and available compound, oroidin, was assayed for inhibition of growth and biofilm formation in bacteria that were isolated from the sponge and its environment.
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http://dx.doi.org/10.1021/acs.jnatprod.9b00863DOI Listing
February 2020

Chemical Modifications Reduce Auditory Cell Damage Induced by Aminoglycoside Antibiotics.

Authors:
Sivan Louzoun Zada Bar Ben Baruch Luba Simhaev Hamutal Engel Micha Fridman

J Am Chem Soc 2020 02 29;142(6):3077-3087. Epub 2020 Jan 29.

School of Chemistry, Raymond and Beverley Sackler Faculty of Exact Sciences , Tel Aviv University , Tel Aviv , Israel , 6997801.

Although aminoglycoside antibiotics are effective against Gram-negative infections, these drugs often cause irreversible hearing damage. Binding to the decoding site of the eukaryotic ribosomes appears to result in ototoxicity, but there is evidence that other effects are involved. Here, we show how chemical modifications of apramycin and geneticin, considered among the least and most toxic aminoglycosides, respectively, reduce auditory cell damage. Using molecular dynamics simulations, we studied how modified aminoglycosides influence the essential freedom of movement of the decoding site of the ribosome, the region targeted by aminoglycosides. By determining the ratio of a protein translated in mitochondria to that of a protein translated in the cytoplasm, we showed that aminoglycosides can paradoxically elevate rather than reduce protein levels. We showed that certain aminoglycosides induce rapid plasma membrane permeabilization and that this nonribosomal effect can also be reduced through chemical modifications. The results presented suggest a new paradigm for the development of safer aminoglycoside antibiotics.
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http://dx.doi.org/10.1021/jacs.9b12420DOI Listing
February 2020

Antifungal activity, mode of action variability, and subcellular distribution of coumarin-based antifungal azoles.

Authors:
Rebecca Elias Raphael I Benhamou Qais Z Jaber Orly Dorot Sivan Louzoun Zada Keren Oved Edward Pichinuk Micha Fridman

Eur J Med Chem 2019 Oct 2;179:779-790. Epub 2019 Jul 2.

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel. Electronic address:

Azole antifungals inhibit the biosynthesis of ergosterol, the fungal equivalent of cholesterol in mammalian cells. Here we report an investigation of the activity of coumarin-substituted azole antifungals. Screening against a panel of Candida pathogens, including a mutant lacking CYP51, the target of antifungal azoles, revealed that this enzyme is inhibited by triazole-based antifungals, whereas imidazole-based derivatives have more than one mode of action. The imidazole-bearing antifungals more effectively reduced trailing growth associated with persistence and/or recurrence of fungal infections than triazole-based derivatives. The imidazole derivatives were more toxic to mammalian cells and more potently inhibited the activity of CYP3A4, which is one of the main causes of azole toxicity. Using live cell imaging, we showed that regardless of the type of azole ring fluorescent 7-diethylaminocoumarin-based azoles localized to the endoplasmic reticulum, the organelle that harbors CYP51. This study suggests that the coumarin is a promising scaffold for development of novel azole-based antifungals that effectively localize to the fungal cell endoplasmic reticulum.
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http://dx.doi.org/10.1016/j.ejmech.2019.07.003DOI Listing
October 2019

Guiding Drugs to Target-Harboring Organelles: Stretching Drug-Delivery to a Higher Level of Resolution.

Authors:
Sivan Louzoun-Zada Qais Z Jaber Micha Fridman

Angew Chem Int Ed Engl 2019 10 12;58(44):15584-15594. Epub 2019 Aug 12.

School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel.

The ratio between the dose of drug required for optimal efficacy and the dose that causes toxicity is referred to as the therapeutic window. This ratio can be increased by directing the drug to the diseased tissue or pathogenic cell. For drugs targeting fungi and malignant cells, the therapeutic window can be further improved by increasing the resolution of drug delivery to the specific organelle that harbors the drug's target. Organelle targeting is challenging and is, therefore, an under-exploited strategy. Here we provide an overview of recent advances in control of the subcellular distribution of small molecules with the focus on chemical modifications. Highlighted are recent examples of active and passive organelle-specific targeting by incorporation of organelle-directing molecular determinants or by chemical modifications of the pharmacophore. The outstanding potential that lies in the development of organelle-specific drugs is becoming increasingly apparent.
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http://dx.doi.org/10.1002/anie.201906284DOI Listing
October 2019

Fluorescent Tracking of the Endoplasmic Reticulum in Live Pathogenic Fungal Cells.

Authors:
Raphael I Benhamou Qais Z Jaber Ido M Herzog Yael Roichman Micha Fridman

ACS Chem Biol 2018 12 26;13(12):3325-3332. Epub 2018 Nov 26.

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences , Tel Aviv University , Tel Aviv 6997801 , Israel.

In fungal cells, the endoplasmic reticulum (ER) harbors several of the enzymes involved in the biosynthesis of ergosterol, an essential membrane component, making this organelle the site of action of antifungal azole drugs, used as a first-line treatment for fungal infections. This highlights the need for specific fluorescent labeling of this organelle in cells of pathogenic fungi. Here we report on the development and evaluation of a collection of fluorescent ER trackers in a panel of Candida, considered the most frequently encountered pathogen in fungal infections. These trackers enabled imaging of the ER in live fungal cells. Organelle specificity was associated with the expression of the target enzyme of antifungal azoles that resides in the ER; specific ER labeling was not observed in mutant cells lacking this enzyme. Labeling of live Candida cells with a combination of a mitotracker and one of the novel fungal ER trackers revealed sites of contact between the ER and mitochondria. These fungal ER trackers therefore offer unique molecular tools for the study of the ER and its interactions with other organelles in live cells of pathogenic fungi.
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http://dx.doi.org/10.1021/acschembio.8b00782DOI Listing
December 2018

Cationic Amphiphiles Induce Macromolecule Denaturation and Organelle Decomposition in Pathogenic Yeast.

Authors:
Qais Z Jaber Raphael I Benhamou Ido M Herzog Bar Ben Baruch Micha Fridman

Angew Chem Int Ed Engl 2018 12 11;57(50):16391-16395. Epub 2018 Nov 11.

School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel.

Cationic amphiphiles are a large and diverse class of antimicrobial agents. Although their mode of action is not fully resolved, it is generally accepted that these antimicrobials perturb the structural integrity of the plasma membrane leading to the microbial cell disruption. Here we report on the development of inherently fluorescent antifungal cationic amphiphiles and on the study of their effects on cells of Candida, one of the most common fungal pathogens in humans. Fluorescent images of Candida yeast cells that express a fluorescent reporter protein revealed that the cationic amphiphiles rapidly accumulated in the cytosol and led to structural changes in proteins and DNA. Using fluorescent organelle-specific dyes, we showed that these antifungal agents also caused organelle disassembly in Candida cells. The results of this study indicate that, in designing antifungal cationic amphiphiles for clinical use, the intracellular activities of these molecules must be addressed to avoid undesired side effects to mammalian cells.
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http://dx.doi.org/10.1002/anie.201809410DOI Listing
December 2018

Derivatives of Ribosome-Inhibiting Antibiotic Chloramphenicol Inhibit the Biosynthesis of Bacterial Cell Wall.

Authors:
Sivan Louzoun Zada Keith D Green Sanjib K Shrestha Ido M Herzog Sylvie Garneau-Tsodikova Micha Fridman

ACS Infect Dis 2018 07 1;4(7):1121-1129. Epub 2018 May 1.

Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry , Tel Aviv University , Tel Aviv , 6997801 , Israel.

Here, we describe the preparation and evaluation of α,β-unsaturated carbonyl derivatives of the bacterial translation inhibiting antibiotic chloramphenicol (CAM). Compared to the parent antibiotic, two compounds containing α,β-unsaturated ketones (1 and 4) displayed a broader spectrum of activity against a panel of Gram-positive pathogens with a minimum inhibitory concentration range of 2-32 μg/mL. Interestingly, unlike the parent CAM, these compounds do not inhibit bacterial translation. Microscopic evidence and metabolic labeling of a cell wall peptidoglycan suggested that compounds 1 and 4 caused extensive damage to the envelope of Staphylococcus aureus cells by inhibition of the early stage of cell wall peptidoglycan biosynthesis. Unlike the effect of membrane-disrupting antimicrobial cationic amphiphiles, these compounds did not rapidly permeabilize the bacterial membrane. Like the parent antibiotic CAM, compounds 1 and 4 had a bacteriostatic effect on S. aureus. Both compounds 1 and 4 were cytotoxic to immortalized nucleated mammalian cells; however, neither caused measurable membrane damage to mammalian red blood cells. These data suggest that the reported CAM-derived antimicrobial agents offer a new molecular scaffold for development of novel bacterial cell wall biosynthesis inhibiting antibiotics.
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http://dx.doi.org/10.1021/acsinfecdis.8b00078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536003PMC
July 2018

Bacterial-derived exopolysaccharides enhance antifungal drug tolerance in a cross-kingdom oral biofilm.

Authors:
Dongyeop Kim Yuan Liu Raphael I Benhamou Hiram Sanchez Áurea Simón-Soro Yong Li Geelsu Hwang Micha Fridman David R Andes Hyun Koo

ISME J 2018 06 18;12(6):1427-1442. Epub 2018 Apr 18.

Biofilm Research Laboratory, Department of Orthodontics and Divisions of Pediatric Dentistry & Community Oral Health, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Fungal-bacterial interactions generate unique biofilms that cause many infections in humans. Candida albicans interact with Streptococcus mutans in dental biofilms associated with severe childhood tooth-decay, a prevalent pediatric oral disease. Current modalities are ineffective and primarily based on antimicrobial monotherapies despite the polymicrobial nature of the infection. Here, we show that the combination of clinically used topical antifungal fluconazole with povidone iodine (PI) can completely suppress C. albicans carriage and mixed-biofilm formation without increasing bacterial killing activity in vivo. We unexpectedly found that the inclusion of PI enhanced fluconazole efficacy by potently disrupting the assembly of a protective bacterial exopolysaccharide (EPS) matrix through inhibition of α-glucan synthesis by S. mutans exoenzyme (GtfB) bound on the fungal surface. Further analyses revealed that the EPS produced in situ directly bind and sequester fluconazole, reducing uptake and intracellular transportation of the drug. Conversely, inhibition of GtfB activity by PI, enzymatic degradation of the α-glucan matrix or co-culturing with gtfB-defective S. mutans re-established antifungal susceptibility. Hence, topical antifungal has limitations in mixed oral biofilms due to enhanced C. albicans tolerance to fluconazole afforded by the shielding effect of bacterial-derived EPS. The data provide new insights for treatment of C. albicans in cross-kingdom biofilms, indicating that EPS inhibitors may be required for enhanced killing efficacy and optimal anti-biofilm activity.
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http://dx.doi.org/10.1038/s41396-018-0113-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955968PMC
June 2018

Localizing Antifungal Drugs to the Correct Organelle Can Markedly Enhance their Efficacy.

Authors:
Raphael I Benhamou Maayan Bibi Judith Berman Micha Fridman

Angew Chem Int Ed Engl 2018 05 2;57(21):6230-6235. Epub 2018 May 2.

School of Chemistry, Raymond&Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel.

A critical aspect of drug design is optimal target inhibition by specifically delivering the drug molecule not only to the target tissue or cell but also to its therapeutically active site within the cell. This study demonstrates, as a proof of principle, that drug efficacy can be increased considerably by a structural modification that targets it to the relevant organelle. Specifically, by varying the fluorescent dye segment an antifungal azole was directed from the fungal cell mitochondria to the endoplasmic reticulum (ER), the organelle that harbors the drug target. The ER-localized azole displayed up to two orders of magnitude improved antifungal activity and also dramatically reduced the growth of drug-tolerant fungal subpopulations in a panel of Candida species, which are the most prevalent causes of serious human fungal infections. The principle underlying the "target organelle localization" approach provides a new paradigm to improve drug potency and replenish the limited pipeline of antifungal drugs.
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http://dx.doi.org/10.1002/anie.201802509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035955PMC
May 2018

Increased Degree of Unsaturation in the Lipid of Antifungal Cationic Amphiphiles Facilitates Selective Fungal Cell Disruption.

Authors:
Kfir B Steinbuch Raphael I Benhamou Lotan Levin Reuven Stein Micha Fridman

ACS Infect Dis 2018 05 15;4(5):825-836. Epub 2018 Feb 15.

School of Chemistry, Raymond and Beverley Sackler Faculty of Exact Sciences , Tel Aviv University , Tel Aviv , Israel 6997801.

Antimicrobial cationic amphiphiles derived from aminoglycosides act through cell membrane permeabilization but have limited selectivity for microbial cell membranes. Herein, we report that an increased degree of unsaturation in the fatty acid segment of antifungal cationic amphiphiles derived from the aminoglycoside tobramycin significantly reduced toxicity to mammalian cells. A collection of tobramycin-derived cationic amphiphiles substituted with C lipid chains varying in degree of unsaturation and double bond configuration were synthesized. All had potent activity against a panel of important fungal pathogens including strains with resistance to a variety of antifungal drugs. The tobramycin-derived cationic amphiphile substituted with linolenic acid with three cis double bonds (compound 6) was up to an order of magnitude less toxic to mammalian cells than cationic amphiphiles composed of lipids with a lower degree of unsaturation and than the fungal membrane disrupting drug amphotericin B. Compound 6 was 12-fold more selective (red blood cell hemolysis relative to antifungal activity) than compound 1, the derivative with a fully saturated lipid chain. Notably, compound 6 disrupted the membranes of fungal cells without affecting the viability of cocultured mammalian cells. This study demonstrates that the degree of unsaturation and the configuration of the double bond in lipids of cationic amphiphiles are important parameters that, if optimized, result in compounds with broad spectrum and potent antifungal activity as well as reduced toxicity toward mammalian cells.
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http://dx.doi.org/10.1021/acsinfecdis.7b00272DOI Listing
May 2018

Structural insights of lincosamides targeting the ribosome of Staphylococcus aureus.

Authors:
Donna Matzov Zohar Eyal Raphael I Benhamou Moran Shalev-Benami Yehuda Halfon Miri Krupkin Ella Zimmerman Haim Rozenberg Anat Bashan Micha Fridman Ada Yonath

Nucleic Acids Res 2017 Sep;45(17):10284-10292

Department of Structural Biology, The Weizmann Institute of Science, Rehovot 7610001, Israel.

Antimicrobial resistance within a wide range of pathogenic bacteria is an increasingly serious threat to global public health. Among these pathogenic bacteria are the highly resistant, versatile and possibly aggressive bacteria, Staphylococcus aureus. Lincosamide antibiotics were proved to be effective against this pathogen. This small, albeit important group of antibiotics is mostly active against Gram-positive bacteria, but also used against selected Gram-negative anaerobes and protozoa. S. aureus resistance to lincosamides can be acquired by modifications and/or mutations in the rRNA and rProteins. Here, we present the crystal structures of the large ribosomal subunit of S. aureus in complex with the lincosamides lincomycin and RB02, a novel semisynthetic derivative and discuss the biochemical aspects of the in vitro potency of various lincosamides. These results allow better understanding of the drugs selectivity as well as the importance of the various chemical moieties of the drug for binding and inhibition.
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http://dx.doi.org/10.1093/nar/gkx658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622323PMC
September 2017

Tuning the Effects of Bacterial Membrane Permeability through Photo-Isomerization of Antimicrobial Cationic Amphiphiles.

Authors:
Joana Salta Raphael I Benhamou Ido M Herzog Micha Fridman

Chemistry 2017 Sep 18;23(52):12724-12728. Epub 2017 Aug 18.

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801, Israel.

Several important antimicrobial drugs act by permeabilizing cell membranes. In this study, we showed that the intensity of membrane permeability caused by antimicrobial cationic amphiphiles can be modified not only by their concentration but also through light-induced isomerization of their lipid segment. Two types of photo-isomerizable cationic amphiphiles were developed and the effects of photo-isomerization on bacterial growth and membrane permeability were evaluated. One photo-isomer inhibited cell growth and division, whereas the other photo-isomer led to a rapid and lethal bacterial membrane-disrupting effect. The switch from "on" to "off" can be obtained by either the cis- or trans-isomer depending on the bacterial strain and the type of cationic amphiphile. These cationic amphiphiles offer a novel tool for research and industrial applications that require light-controlled bacterial membrane permeabilization.
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http://dx.doi.org/10.1002/chem.201703010DOI Listing
September 2017

Real-Time Imaging of the Azole Class of Antifungal Drugs in Live Candida Cells.

Authors:
Raphael I Benhamou Maayan Bibi Kfir B Steinbuch Hamutal Engel Maayan Levin Yael Roichman Judith Berman Micha Fridman

ACS Chem Biol 2017 07 12;12(7):1769-1777. Epub 2017 May 12.

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University , Tel Aviv, 6997801, Israel.

Azoles are the most commonly used class of antifungal drugs, yet where they localize within fungal cells and how they are imported remain poorly understood. Azole antifungals target lanosterol 14α-demethylase, a cytochrome P450, encoded by ERG11 in Candida albicans, the most prevalent fungal pathogen. We report the synthesis of fluorescent probes that permit visualization of antifungal azoles within live cells. Probe 1 is a dansyl dye-conjugated azole, and probe 2 is a Cy5-conjugated azole. Docking computations indicated that each of the probes can occupy the active site of the target cytochrome P450. Like the azole drug fluconazole, probe 1 is not effective against a mutant that lacks the target cytochrome P450. In contrast, the azole drug ketoconazole and probe 2 retained some antifungal activity against mutants lacking the target cytochrome P450, implying that both act against more than one target. Both fluorescent azole probes colocalized with the mitochondria, as determined by fluorescence microscopy with MitoTracker dye. Thus, these fluorescent probes are useful molecular tools that can lead to detailed information about the activity and localization of the important azole class of antifungal drugs.
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http://dx.doi.org/10.1021/acschembio.7b00339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7030953PMC
July 2017

Effects of 5-O-Ribosylation of Aminoglycosides on Antimicrobial Activity and Selective Perturbation of Bacterial Translation.

Authors:
Ido M Herzog Sivan Louzoun Zada Micha Fridman

J Med Chem 2016 09 19;59(17):8008-18. Epub 2016 Aug 19.

School of Chemistry, Raymond and Beverley Sackler Faculty of Exact Sciences, Tel Aviv University , Tel Aviv 6997801, Israel.

We studied six pairs of aminoglycosides and their corresponding ribosylated derivatives synthesized by attaching a β-O-linked ribofuranose to the 5-OH of the deoxystreptamine ring of the parent pseudo-oligosaccharide antibiotic. Ribosylation of the 4,6-disubstituted 2-deoxystreptamine aminoglycoside kanamycin B led to improved selectivity for inhibition of prokaryotic relative to cytosolic eukaryotic in vitro translation. For the pseudodisaccharide aminoglycoside scaffolds neamine and nebramine, ribosylated derivatives were both more potent antimicrobials and more selective to inhibition of prokaryotic translation. On the basis of the results of this study, we suggest that modification of the 5-OH position of the streptamine ring of other natural or semisynthetic pseudodisaccharide aminoglycoside scaffolds containing an equatorial amine at the 2' sugar position with a β-O-linked ribofuranose is a promising avenue for the development of novel aminoglycoside antibiotics with improved efficacy and reduced toxicity.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00793DOI Listing
September 2016

Phosphonium pillar[5]arenes as a new class of efficient biofilm inhibitors: importance of charge cooperativity and the pillar platform.

Authors:
Roymon Joseph Dana Kaizerman Ido M Herzog Maya Hadar Mark Feldman Micha Fridman Yoram Cohen

Chem Commun (Camb) 2016 Aug;52(70):10656-9

School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

Biofilm formation, which frequently occurs in microbial infections and often reduces the efficacy of antibiotics, also perturbs many industrial and domestic processes. We found that a new class of water soluble pillar[5]arenes bearing phosphonium moieties (1, 2) and their respective ammonium analogues (3, 4) inhibit biofilm formation with IC50 values in the range of 0.67-1.66 μM. These compounds have no antimicrobial activity, do not damage red blood cell membranes, and do not affect mammalian cell viability in culture. Comparison of the antibiofilm activities of the phosphonium-decorated pillar[5]arene derivatives 1 and 2 with their respective ammonium counterparts 3 and 4 and their monomers 5 and 6, demonstrate that while positive charges, charge cooperativity and the pillararene platform are essential for the observed antibiofilm activity the nature of the charges is not.
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http://dx.doi.org/10.1039/c6cc05170gDOI Listing
August 2016

Antifungal Imidazole-Decorated Cationic Amphiphiles with Markedly Low Hemolytic Activity.

Authors:
Raphael I Benhamou Kfir B Steinbuch Micha Fridman

Chemistry 2016 Aug 30;22(32):11148-51. Epub 2016 Jun 30.

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, 6997801, Tel Aviv, Israel.

Herein we report that an imidazole-decorated cationic amphiphile derived from the pseudo-disaccharide nebramine has potent antifungal activity against strains of Candida glabrata pathogens. In combination with the natural bis-benzylisoquinoline alkaloid tetrandrine the reported antifungal cationic amphiphile demonstrated synergistic antifungal activity against Candida albicans pathogens. This unique membrane disruptor caused no detectible mammalian red blood cell hemolysis at concentrations up to more than two orders of magnitude greater than its minimal inhibitory concentrations against the tested C. glabrata strains. We provide evidence that potency against C. glabrata may be associated with differences between the drug efflux pumps of C. albicans and C. glabrata. Imidazole decorated-cationic amphiphiles show promise for the development of less toxic membrane-disrupting antifungal drugs and drug combinations.
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http://dx.doi.org/10.1002/chem.201602198DOI Listing
August 2016

Cationic Pillararenes Potently Inhibit Biofilm Formation without Affecting Bacterial Growth and Viability.

Authors:
Roymon Joseph Alissa Naugolny Mark Feldman Ido M Herzog Micha Fridman Yoram Cohen

J Am Chem Soc 2016 Jan 14;138(3):754-7. Epub 2016 Jan 14.

School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University , Tel Aviv 69978, Israel.

It is estimated that up to 80% of bacterial infections are accompanied by biofilm formation. Since bacteria in biofilms are less susceptible to antibiotics than are bacteria in the planktonic state, biofilm-associated infections pose a major health threat, and there is a pressing need for antibiofilm agents. Here we report that water-soluble cationic pillararenes differing in the quaternary ammonium groups efficiently inhibited the formation of biofilms by clinically important Gram-positive pathogens. Biofilm inhibition did not result from antimicrobial activity; thus, the compounds should not inhibit growth of natural bacterial flora. Moreover, none of the cationic pillararenes caused detectable membrane damage to red blood cells or toxicity to human cells in culture. The results indicate that cationic pillararenes have potential for use in medical applications in which biofilm formation is a problem.
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http://dx.doi.org/10.1021/jacs.5b11834DOI Listing
January 2016

Di-N-Methylation of Anti-Gram-Positive Aminoglycoside-Derived Membrane Disruptors Improves Antimicrobial Potency and Broadens Spectrum to Gram-Negative Bacteria.

Authors:
Raphael I Benhamou Pazit Shaul Ido M Herzog Micha Fridman

Angew Chem Int Ed Engl 2015 Nov 29;54(46):13617-21. Epub 2015 Sep 29.

School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, 6997801 (Israel).

The effect of di-N-methylation of bacterial membrane disruptors derived from aminoglycosides (AGs) on antimicrobial activity is reported. Di-N-methylation of cationic amphiphiles derived from several diversely structured AGs resulted in a significant increase in hydrophobicity compared to the parent compounds that improved their interactions with membrane lipids. The modification led to an enhancement in antibacterial activity and a broader antimicrobial spectrum. While the parent compounds were either modestly active or inactive against Gram-negative pathogens, the corresponding di-N-methylated compounds were potent against the tested Gram-negative as well as Gram-positive bacterial strains. The reported modification offers a robust strategy for the development of broad-spectrum membrane-disrupting antibiotics for topical use.
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http://dx.doi.org/10.1002/anie.201506814DOI Listing
November 2015

Exploring the Effects of Glycosylation and Etherification of the Side Chains of the Anticancer Drug Mitoxantrone.

Authors:
Pazit Shaul Kfir B Steinbuch Eran Blacher Reuven Stein Micha Fridman

ChemMedChem 2015 Sep 31;10(9):1528-38. Epub 2015 Jul 31.

School of Chemistry, Beverly and Raymond Sackler Faculty of Exact, Sciences, Tel Aviv University, Tel Aviv 6997801 (Israel).

Herein we report the synthesis and biological evaluation of symmetric and asymmetric analogues of the DNA intercalating drug mitoxantrone (MTX) in which the side chains of the parent drug were modified through glycosylation or methyl etherification. Several analogues with glycosylated side chains exhibited higher DNA affinity than the parent MTX. The most potent in vitro cytotoxicity was observed for MTX analogue 8 (1,4-dimethoxy-5,8-bis[2-(2-methoxyethylamino)ethylamino]anthracene-9,10-dione) with methoxy ether containing side chains. Treatment of melanoma-bearing mice with MTX or analogue 8 decreased the intraperitoneal tumor burden relative to untreated mice; the effect of 8 was less pronounced than that of MTX. In vitro metabolism assays of MTX with rabbit liver S9 fraction gave rise to several metabolites; almost no metabolites were detected for MTX analogue 8. The results presented indicate that derivatization of the MTX side chain primary hydroxy groups may result in a significant improvement in DNA affinity and lower susceptibility to the formation of potentially toxic metabolites.
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http://dx.doi.org/10.1002/cmdc.201500274DOI Listing
September 2015

One-Pot Chemoenzymatic Cascade for Labeling of the Epigenetic Marker 5-Hydroxymethylcytosine.

Authors:
Gil Nifker Michal Levy-Sakin Yifat Berkov-Zrihen Tamar Shahal Tslil Gabrieli Micha Fridman Yuval Ebenstein

Chembiochem 2015 Sep 27;16(13):1857-1860. Epub 2015 Jul 27.

Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel Aviv 6997801 (Israel).

The epigenetic DNA modification 5-hydroxymethylcytosine (5-hmC) is important for the regulation of gene expression during development and in tumorigenesis. 5-hmC can be selectively glycosylated by T4 β-glucosyltransferase (β-GT); introduction of an azide on the attached sugar provides a chemical handle for isolation or fluorescent tagging of 5-hmC residues by click chemistry. This approach has not been broadly adopted because of the challenging synthesis and limited commercial availability of the glycosylation substrate, 6-deoxy-6-azido-α-D-glucopyranoside. We report the enzyme-assisted synthesis of this precursor by the uridylyltransferase from Pasteurella multocida (PmGlmU). We were able to directly label 5-hmC in genomic DNA by an enzymatic cascade involving successive action of PmGlmU and β-GT. This is a facile and cost-effective one-pot chemoenzymatic methodology for 5-hmC analysis.
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http://dx.doi.org/10.1002/cbic.201500329DOI Listing
September 2015

Tobramycin and nebramine as pseudo-oligosaccharide scaffolds for the development of antimicrobial cationic amphiphiles.

Authors:
Yifat Berkov-Zrihen Ido M Herzog Raphael I Benhamou Mark Feldman Kfir B Steinbuch Pazit Shaul Shachar Lerer Avigdor Eldar Micha Fridman

Chemistry 2015 Mar 4;21(11):4340-9. Epub 2015 Feb 4.

School of Chemistry, Raymond & Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, 6997801 (Israel).

Antimicrobial cationic amphiphiles derived from aminoglycoside pseudo-oligosaccharide antibiotics interfere with the structure and function of bacterial membranes and offer a promising direction for the development of novel antibiotics. Herein, we report the design and synthesis of cationic amphiphiles derived from the pseudo-trisaccharide aminoglycoside tobramycin and its pseudo-disaccharide segment nebramine. Antimicrobial activity, membrane selectivity, mode of action, and structure-activity relationships were studied. Several cationic amphiphiles showed marked antimicrobial activity, and one amphiphilic nebramine derivative proved effective against all of the tested strains of bacteria; furthermore, against several of the tested strains, this compound was well over an order of magnitude more potent than the parent antibiotic tobramycin, the membrane-targeting antimicrobial peptide mixture gramicidin D, and the cationic lipopeptide polymyxin B, which are in clinical use.
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http://dx.doi.org/10.1002/chem.201406404DOI Listing
March 2015

Antimycobacterial activity of DNA intercalator inhibitors of Mycobacterium tuberculosis primase DnaG.

Authors:
Chathurada Gajadeera Melisa J Willby Keith D Green Pazit Shaul Micha Fridman Sylvie Garneau-Tsodikova James E Posey Oleg V Tsodikov

J Antibiot (Tokyo) 2015 Mar 24;68(3):153-7. Epub 2014 Sep 24.

Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY, USA.

Owing to the rise in drug resistance in tuberculosis combined with the global spread of its causative pathogen, Mycobacterium tuberculosis (Mtb), innovative anti mycobacterial agents are urgently needed. Recently, we developed a novel primase-pyrophosphatase assay and used it to discover inhibitors of an essential Mtb enzyme, primase DnaG (Mtb DnaG), a promising and unexplored potential target for novel antituberculosis chemotherapeutics. Doxorubicin, an anthracycline antibiotic used as an anticancer drug, was found to be a potent inhibitor of Mtb DnaG. In this study, we investigated both inhibition of Mtb DnaG and the inhibitory activity against in vitro growth of Mtb and M. smegmatis (Msm) by other anthracyclines, daunorubicin and idarubicin, as well as by less cytotoxic DNA intercalators: aloe-emodin, rhein and a mitoxantrone derivative. Generally, low-μM inhibition of Mtb DnaG by the anthracyclines was correlated with their low-μM minimum inhibitory concentrations. Aloe-emodin displayed threefold weaker potency than doxorubicin against Mtb DnaG and similar inhibition of Msm (but not Mtb) in the mid-μM range, whereas rhein (a close analog of aloe-emodin) and a di-glucosylated mitoxantrone derivative did not show significant inhibition of Mtb DnaG or antimycobacterial activity. Taken together, these observations strongly suggest that several clinically used anthracyclines and aloe-emodin target mycobacterial primase, setting the stage for a more extensive exploration of this enzyme as an antibacterial target.
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http://dx.doi.org/10.1038/ja.2014.131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687077PMC
March 2015