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- Title
- Cyclic lipodepsipeptides as lead structures for the discovery of new antiobiotics.
- Creator
- Bionda, Nina., Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
- Abstract/Description
-
With antimicrobial resistance to current drugs steadily rising, the development of new antibiotics with novel mechanisms of action has become an imperative. The majority of life-threatening infections worldwide are caused by "ESKAPE" pathogens which are encountered in more than 40% of hospital-acquired infections, and are resistant to the majority of commonly used antibiotics. Naturally occurring cyclic depsipeptides, microbial secondary metabolites that contain one or more ester bonds in...
Show moreWith antimicrobial resistance to current drugs steadily rising, the development of new antibiotics with novel mechanisms of action has become an imperative. The majority of life-threatening infections worldwide are caused by "ESKAPE" pathogens which are encountered in more than 40% of hospital-acquired infections, and are resistant to the majority of commonly used antibiotics. Naturally occurring cyclic depsipeptides, microbial secondary metabolites that contain one or more ester bonds in addition to amide bonds, have emerged as an important source of pharmacologically active compounds or lead structures for the development of novel antibiotics. Some of those peptides are either already marketed (daptomycin) or in advanced stages of clinical development (ramoplanin). Structurally simple, yet potent, fusaricidin/LI-F and lysobactin families of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents capable of overco ming infections caused by multidrug-resistant bacteria. These natural products exhibit potent antimicrobial activity against a variety of clinically relevant fungi and Gram-positive bacteria. Therefore, access to these classes of natural products and their synthetic analogs, combined with elucidation of their mode of action represent important initial steps toward full exploitation of their antmicrobial potential. This dissertation describes a general approach toward the solid-phase synthesis of fusaricidin/LI-F and lysobactin analogs and an extensive structure-activity relationship (SAR) study. We have devised a simple and robust preparation strategy based on standard Fmoc solid-phase peptide synthesis protocols., The SAR study revealed key structural requirements for fusaricidin/LI-F and related cyclic lipopeptides antibacterial activity, including the presence of the guanidino moietly at the end of the lipidic tail, hydrophobic amino acid residues, and peptide conformation Moreover, substitution of the ester bond with an amide bond significantly improved stability under physiologically relevant conditions and reduced toxicity. In addition, we have shown that these antibacterial peptides exert their mode of action via a novel mechanism, which invloves bacterial membrane interactions, followed by peptide internalization. Altogether, the research described in this dissertation demonstrates that new antibiotics derived from fusaricidin/LI-F natural products, have the potential to meet the challenge of antibiotic resistance in Gram-positive bacteria.
Show less - Date Issued
- 2013
- PURL
- http://purl.flvc.org/FAU/3360768
- Subject Headings
- Microbial peptides, Drugs, Design, Peptides, Therapeutic use, Genetic engineering, Antibacterial agents, Peptide antibiotics, Research, Methodology, Peptide antibiotics, Analysis
- Format
- Document (PDF)
- Title
- Study of Cell Penetrating Peptide Uptake and Cancer Cell Discrimination with Raman Spectroscopy and Microscopy.
- Creator
- Cosme, Patrick Jason, Terentis, Andrew C., Florida Atlantic University, Charles E Schmidt College of Science, Department of Chemistry and Biochemistry
- Abstract/Description
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Cell penetrating peptides (CPPs) are short sequences of amino acids that excel in crossing the cellular membrane without inducing cytotoxicity Interest in these peptides stem from their ability to be attached, and grant their penetrating properties to, a variety of cargo In this work we have combined the application of Confocal Raman Microscopy (CRM) and Atomic Force Microscopy for the first time to examine the interactions of unlabeled Transportan (TP), one of the most well studied CPPs,...
Show moreCell penetrating peptides (CPPs) are short sequences of amino acids that excel in crossing the cellular membrane without inducing cytotoxicity Interest in these peptides stem from their ability to be attached, and grant their penetrating properties to, a variety of cargo In this work we have combined the application of Confocal Raman Microscopy (CRM) and Atomic Force Microscopy for the first time to examine the interactions of unlabeled Transportan (TP), one of the most well studied CPPs, with mammalian cells CRM’s capability to discriminate control and treated cell groups was verified by principal component analysis (PCA) and linear discriminant analysis (LDA) and was 93-100% accurate We’ve determined that at a concentration of 20 μM TP enters cells through a non-endocytotic mechanism, has a high affinity for the cytoplasm and membranes, and results in a significant increase in cellular stiffness Our work provides the first direct evidence of this cell-stiffening phenomenon SFTI-1, the smallest member of a bicyclic, cysteine rich class of CPPs, was examined by CRM to determine the potential role of cyclic structure on cellular uptake The peptide, along with monocyclic and linear analogs was heavy isotope labeled and incubated with mammalian cells at numerous concentrations and timespans Our work is the first SFTI-1 uptake study forgoing the use of fluorophore conjugates, which have been linked to artificial cellular uptake We demonstrate herein the absence of any CRM detectable uptake, providing the first evidence that SFTI-1 may not be a CPP Finally, CRM was applied to the discrimination of normal and basal cell carcinoma cells obtained from the same donor The use of patient matched cells avoids the normal biochemical variations that exist among individuals, ensuring that discrimination is based solely on the cell’s diseased state CRM spectra, analyzed by PCA and LDA, were capable of spectral discrimination with 100% accuracy Major differences in the cancerous cells were an increase in lipids and nucleic acids, and an overall decrease in protein We also demonstrate an enhancement in Raman signal through the use of an aluminum foil substrate, providing a practical approach for measuring cells with thin morphologies
Show less - Date Issued
- 2016
- PURL
- http://purl.flvc.org/fau/fd/FA00004756
- Subject Headings
- Peptides--Analysis, Peptides--Therapeutic use, Peptides--Physiological transport, Cellular signal transduction, Raman spectroscopy, Infrared spectroscopy
- Format
- Document (PDF)
- Title
- Study of cell penetrating peptides with Raman spectroscopy and microscopy.
- Creator
- Ye, Jing., Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
- Abstract/Description
-
Cell penetrating peptides (CPPs) have drawn the attention of researchers due to their ability to internalize large cargos into cells including cancer cells. The mechanism(s) with which the peptides enter the cell, however, is/are not clear and full of controversy. The peptide conformations and their microenvironment in live cells had been unknown until the development of a technique developed in our lab. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the...
Show moreCell penetrating peptides (CPPs) have drawn the attention of researchers due to their ability to internalize large cargos into cells including cancer cells. The mechanism(s) with which the peptides enter the cell, however, is/are not clear and full of controversy. The peptide conformations and their microenvironment in live cells had been unknown until the development of a technique developed in our lab. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 to 967 cm-1, thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals., On the basis of the position of the amide I vibrational band in the Raman spectra, the secondary structure of the peptide was found to be mainly random coil and b-strand in the cytoplasm, and possibly assembling as b-sheets in the nucleus. Next, label-free transportan was studied with the same methodology. The peptide, besides predominantly a-helix, adopted a significant portion of b-sheet conformation in the cytoplasm and nucleolus, which is different from the peptide in aqueous solution. The peptide microenvironment was also probed through H-bonding reported by the tyrosine Fermi doublet. Transportan displayed a tendency to accumulate in the cytoplasm over time which was unlike penetratin, which concentrated in the nucleus. The relative concentration of CPPs in various locations of live melanoma cells was directly estimated from the Raman spectra using average Phe concentration in the cell as an internal standard., The rapid entry and almost uniform cellular distribution of both peptides, as well as the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism of CPP internalization under the conditions of study was probably non-endocytotic. Last, transportan and penetratin were studied using polarized Raman spectroscopy for more detailed vibrational spectroscopic information of the two peptides in water and TFE solutions. The majority of the bands in the Raman spectra of the peptides were highly polarized, consistent with the high symmetry of aromatic ring side chain vibrational bands dispersed throughout the spectra. This work has provided new insights into the structure of CPPs in live cells and in solutions.
Show less - Date Issued
- 2011
- PURL
- http://purl.flvc.org/FAU/3342344
- Subject Headings
- Peptides, Analysis, Infrared spectroscopy, Raman spectroscopy, Cellular signal transduction
- Format
- Document (PDF)
- Title
- Assay development for lysyl hydroxylase.
- Creator
- Patel, Deepak A., Florida Atlantic University, Fields, Gregg B.
- Abstract/Description
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Hydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the...
Show moreHydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the corresponding hydroxylated (hydroxylysine-containing) peptide] using Fmoc solid-phase methodology. Peptides have been characterized by HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. A new method for efficient separation of lysine- from hydroxylysine-containing peptides by HPLC has been developed in both organic phase (1-anthroylnitrile as derivatizating reagent) and aqueous phase (dansyl chloride as derivatizating reagent). These reagents have been used to derivatize peptides prior to HPLC analysis. The products (di- and tetra-substituted lysine- and hydroxylysine-containing peptides) have been fully separated by HPLC and their structure confirmed by MALDI-TOF MS analysis. Efficient separation of derivatized peptides will allow for the convenient and rapid measurement of LH activity by HPLC methods.
Show less - Date Issued
- 2006
- PURL
- http://purl.flvc.org/fcla/dt/13384
- Subject Headings
- Biological transport, Proteins--Metabolism, Peptides--Analysis, Coenzymes, Bioorganic chemistry
- Format
- Document (PDF)
- Title
- Isolation and characterization of neuroactive peptides from the venom of cone snail species.
- Creator
- Cano, Herminsul, Florida Atlantic University, Mari, Frank
- Abstract/Description
-
The mam objective of the work described in this thesis is isolation and characterization of novel neuroactive peptides from the venom of cone snail species. The first section is an introduction about cone snails. The first chapter is dedicated to the analysis of the milked venom obtained from three different specimens of C. ermineus which is the only fish-hunter cone snail from the east Atlantic region. MALDI-TOF mass spectrometry analysis of two specimens showed an identical profile with all...
Show moreThe mam objective of the work described in this thesis is isolation and characterization of novel neuroactive peptides from the venom of cone snail species. The first section is an introduction about cone snails. The first chapter is dedicated to the analysis of the milked venom obtained from three different specimens of C. ermineus which is the only fish-hunter cone snail from the east Atlantic region. MALDI-TOF mass spectrometry analysis of two specimens showed an identical profile with all components of the venom being novel conopeptides. The third specimen showed a mass spectrometry profile with molecular weights corresponding to already reported conotoxins plus one additional new conopeptide. Ten new conotoxins were isolated from C. ermineus; seven of them have sequences corresponding to A-superfamily of conotoxins, specifically a-conotoxins family. Six of these seven conotoxins are the first a4/4 conotoxins isolated from the milked venom from any fish-hunter cone snail specimens; the other one is a a4/7 conotoxin similar sequence to the already reported a-EI from C. ermineus. Two more conotoxins that belong to the 0-superfamily have the same amino acid sequence with the only difference being a hydroxyproline residue instead of a proline at position 21 of the sequence. In the second chapter, four specimens of C. purpurascens, the only fish-hunter of the Eastern Pacific region were analyzed. One of the specimens was sacrified and the crude venom was dissected-out of the venom duct. For the three remaining live specimens the venom was obtained by the "milking" procedure. Mass spectrometry profiles were compared between dissected and milked venom and between milked venom from different specimens. Analysis showed both similarities and differences in the profiles of the dissected and the three milked venoms. A comparison of the three milked venoms found some differences. This analysis showed that one specimen expressed two isomorphs of a putative a4/4-conotoxin; the only difference was the presence of proline instead of hydroxyproline at position seven in the amino acid sequence. These a4/4-conotoxins are the second report of this sub-class of conotoxin from the milked venom of cone snails and they have sequence homology to the a4/4 conotoxins isolated from C. ermineus. The analysis of the MALDI-TOF MS/MS spectra of the Leu-contryphan-P conopeptide from C. purpurascens revealed that conotoxins with a single disulfide bond in the sequence behave as a linear peptide in the mass spectrometry experiment exhibiting a good fragmentation pattern. Using this information by comparing the MS/MS spectra we were able to identify L-contryphan-P conopeptide lacking the first Gly residue in the sequence. In the third chapter, three conotoxins with sequence homology to the omega-superfamily were isolated from the crude dissected venom of the worm-hunter cone snail C. vexillum. The precursor of one of these conotoxins was already characterized by another research group. Analysis and comparison of this precursor with already known precursor allowed us to hypothesize that these conotoxins were ro-conotoxins. Two of the three conotoxins have the same amino acid sequence with hydroxyproline instead a proline in the structure. These conotoxins were the first ones isolated from the venom duct of these cone snail species. Several conotoxins had been reported from C. vexillum but they were isolated using eDNA cloning techniques. Chapter four shows the analysis of the worm-hunter cone snail C. pseudoarantius crude venom. Eight novel conotoxins were isolated from the pooled duct dissected venom from different specimens. The first was a a4/3-conotoxin with a carboxyglutamate residue present at position one in the sequence. Five more conotoxins with conotoxin frameworks and sequences similar to M-superfamily of conotoxins were also found; additionally, two more novel conotoxins with sequence homology to o-conotoxins from the S-superfamily were isolated. All the above conotoxins were analyzed by comparison of their structures against sequences of known conotoxins. All 23 conotoxins found in this research are novel conopeptides isolated from cone snail specimens. Future work on the activity these conotoxins will be important in the search for possible drugs in treatment of many diseases.
Show less - Date Issued
- 2005
- PURL
- http://purl.flvc.org/fcla/dt/12185
- Subject Headings
- Peptides--Structure, Gastropoda--Venom, Conus, Mass spectrometry--Analysis
- Format
- Document (PDF)
- Title
- Using ATR-IR spectroscopy to study the conformation of cell-penetrating peptides.
- Creator
- Fontoura, Luiza, Rezler, Evonne
- Date Issued
- 2012-04-06
- PURL
- http://purl.flvc.org/fcla/dt/3351389
- Subject Headings
- Cell-Penetrating Peptides, Antennapedia Homeodomain Protein, Homeodomain Proteins --chemistry, Spectroscopy, Fourier Transform Infrared --methods, Spectrum Analysis, Amides --chemistry, Carrier Progeins
- Format
- Document (PDF)