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Development and Optimization of AlphaScreen Assay for Discovery of Galectin-1/-3 Inhibitors
Title
Development and Optimization of AlphaScreen Assay for Discovery of Galectin-1/-3 Inhibitors.
Creator
Rivero, Yaima, Fitzgerald, Forrest, Cudic, Mare
Abstract/Description
Galectin-1 is a beta-galactoside-binding protein implicated in regulating apoptosis, cell proliferation and cell differentiation. The objective of our research was to develop and optimize an assay format for the discovery of new inhibitors of galectin-1 using AlphaScreen technology in a competitive binding configuration. Our efforts were hampered by the weak binding affinities of galectin-1 for its binding ligands (μM range). Consequently, the AlphaScreen assay could not have been developed...
Show moreGalectin-1 is a beta-galactoside-binding protein implicated in regulating apoptosis, cell proliferation and cell differentiation. The objective of our research was to develop and optimize an assay format for the discovery of new inhibitors of galectin-1 using AlphaScreen technology in a competitive binding configuration. Our efforts were hampered by the weak binding affinities of galectin-1 for its binding ligands (μM range). Consequently, the AlphaScreen assay could not have been developed at the level that would satisfy the guidelines from the National Chemical Genomics Center. Nevertheless, we have optimized the AlphaScreen assay for galectin-3, another member of galectin family, and screened an FDA approved oncology drug library (n = 101) from the NIH. We identified three possible inhibitors of galectin-3. These compounds showed a positive activity in dose response experiments: bleomycin (IC50 = 25.7 nM), cisplatin (IC50 = 127 nM), and mitoxantrone HCl (IC50 = 662 nM).
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Date Issued
2018
PURL
http://purl.flvc.org/fau/fd/FAU_SR00000050
Subject Headings
College students --Research --United States.
Format
Document (PDF)
Roles of adjuvant and route of vaccination in antibody response and protection engendered by a synthetic matrix protein 2-based influenza A virus vaccine in the mouse
Title
Roles of adjuvant and route of vaccination in antibody response and protection engendered by a synthetic matrix protein 2-based influenza A virus vaccine in the mouse.
Creator
Mozdzanowska, Krystyna, Zharikova, Darya, Cudic, Mare, Otvos, Laszlo, Gerhard, Walter
Date Issued
2007-10-27
PURL
http://purl.flvc.org/fcla/dt/3327982
Format
Document (PDF)
Characterization of Disulfide Constrained Natural Peptides
Title
Characterization of Disulfide Constrained Natural Peptides.
Creator
Hoggard, Mickelene F., Cudic, Mare, Florida Atlantic University, Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
The use of peptide drugs has gained popularity recently. Peptides are attractive drug targets due to their high specificity and potency towards their biological targets. A drawback for peptide drugs is a lack of stability for oral delivery. Two classes of disulfide-rich peptides, conotoxins and cyclotides, have been shown to have higher stability than linear peptides thanks to their disulfide connectivity. Conotoxins are present in the venom of cone snails, a carnivorous marine mollusk that...
Show moreThe use of peptide drugs has gained popularity recently. Peptides are attractive drug targets due to their high specificity and potency towards their biological targets. A drawback for peptide drugs is a lack of stability for oral delivery. Two classes of disulfide-rich peptides, conotoxins and cyclotides, have been shown to have higher stability than linear peptides thanks to their disulfide connectivity. Conotoxins are present in the venom of cone snails, a carnivorous marine mollusk that preys upon fish, worms, or other mollusks. Conotoxins are promising drugs leads with great prospects in the treatment of diseases and disorders such as chronic pain, multiple sclerosis and Parkinson’s and Alzheimer’s diseases. Cyclotides, which are cyclic cysteine knot containing peptides, isolated from the Violaceae (violet), Rubiaceae (coffee), and Cucurbitaceae (cucurbit) families and they have a wide range of biological activities, such as anti-HIV, uterotonic, and antimicrobial. P-superfamily framework IX conotoxins (C-C- C-CXC- C) contain the same cysteine framework, homologous sequences, and similar 3D structures to cyclotides. The knot containing conotoxins have been identified in several Conus species, but this work focuses on those from Conus brunneus, Conus purpurascens, and Conus gloriamaris. The cysteine knot motif of cyclotides and P-superfamily conotoxins is characterized by a cyclic backbone and six-conserved cysteine residues that form the three-disulfide bridges of the “knot”. This motif provides cyclotides and conotoxins with superior stability against thermal, chemical, and enzymatic degradation; marking them as potential frameworks for peptide drug delivery. Presented are details on the isolation of conotoxins and cyclotides, from Viola tricolor, and the characterization of their activity in the well-characterized Drosophila melanogaster giant fiber system (GFS) neuronal circuit, which contains GAP, acetylcholine, and glutamate synapses. The transcriptomes of two Conus brunneus specimens were assembled and mined for P-superfamily framework IX conotoxins. Eleven mature P-superfamily framework IX conotoxins were identified in the crude venom.
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Date Issued
2018
PURL
http://purl.flvc.org/fau/fd/FA00005955
Subject Headings
Peptide drugs, Cyclotides, Conotoxins
Format
Document (PDF)
TUMOR-ASSOCIATED MUC1-TN GLYCOPEPTIDE INTERACTIONS WITH MACROPHAGE GALACTOSE LECTIN
Title
TUMOR-ASSOCIATED MUC1-TN GLYCOPEPTIDE INTERACTIONS WITH MACROPHAGE GALACTOSE LECTIN.
Creator
Beckwith, Donella Marie, Cudic, Mare, Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
Abstract/Description
The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. Mucin 1 (MUC1), the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern in many cancers. The presence of truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play key roles in tumor initiations, progression, and metastasis. Accumulating evidence...
Show moreThe transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. Mucin 1 (MUC1), the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern in many cancers. The presence of truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play key roles in tumor initiations, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with escape of immune defenses. Human macrophage galactose-type lectin (hMGL, HML, CD301 or CLEC10A), a C-type lectin expressed by antigen presenting cells (APC), is a receptor of mucin-type TACAs, -GalNAc (Thomsen nouvelle antigen; Tn; CD175) and its 2,6-sialylated derivative (sTn; CD175s). To date, the relative contributions of these glycans, as well as underlying peptide backbone, and different degrees of valency, on binding thermodynamics and kinetics with hMGL remains elusive. In order to discern the subtle utility of these distinct features, chemical syntheses of the MUC1, HGVTSAPDTRPAPGSTAPPA tandem repeat sequence, and its site-specific serine (Ser) and threonine (Thr) glycosylated analogs were carried out. Circular dichroism (CD) spectroscopy experiments detected increasing structural order of the Thr glycopeptides compared to its nonglycosylated analogs. Isothermal titration calorimetry (ITC) data analysis of lectin binding to the Thr glycopeptides invariably showed enthalpy-driven processes. Affinity enhancement of the Thr glycopeptides for hMGL occurred relative to free GalNAc, revealing an increasing trend in affinity by one order of magnitude, for mono- (KD = 6-8 μM) to triglycosylated (KD = 600 nM) MUC1 peptides. To delineate the relevance of the solvent structure in the protein carbohydrate recognition process, experiments in D2O were performed, exposing enthalpy-entropy compensation differences. KinITC analysis highlighted prolonged complex lifetimes. Furthermore, atomic force microscopy (AFM) based dynamic single-molecule force spectroscopy (SMFS) provided molecular level insight into the energy landscapes governing recognition of the MUC1(Tn)-hMGL complexes. In summary, our results suggest that contact with hMGL critically depends on the type of TACA, nature of the vicinity surrounding the glycan, and its density. This highlights the importance and current efforts in design of prophylactic and therapeutic cancer vaccines with special emphasis on the synthetic glycopeptide vaccines.
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Date Issued
2021
PURL
http://purl.flvc.org/fau/fd/FA00013750
Subject Headings
Antigens, Tumor-Associated, Carbohydrate, Mucin-1, Cancer vaccines, Glycopeptides
Format
Document (PDF)
Development of a convenient peptide-based assay for lysyl hydroxylase
Title
Development of a convenient peptide-based assay for lysyl hydroxylase.
Creator
Cudic, Mare, Patel, Deepak A., Lauer-Fields, Janelle L., Brew, Keith, Fields, Gregg B.
Date Issued
2007-07-03
PURL
http://purl.flvc.org/fau/flvc_fau_islandoraimporter_10.1002_bip.20799_1638210547
Format
Document (PDF)
Engineered Sarafotoxins as Tissue Inhibitor of Metalloproteinases-like Matrix Metalloproteinase Inhibitors
Title
Engineered Sarafotoxins as Tissue Inhibitor of Metalloproteinases-like Matrix Metalloproteinase Inhibitors.
Creator
Lauer-Fields, Janelle L., Cudic, Mare, Wei, Shuo, Mari, Frank, Fields, Gregg B., Brew, Keith
Date Issued
2007-09
PURL
http://purl.flvc.org/fau/flvc_fau_islandoraimporter_10.1074_jbc.M611612200_1638212835
Format
Document (PDF)
MULTIVALENT PROTEIN GLYCOSYLATION: A DRIVING FORCE OF CANCER PROGRESSION AND ALZHEIMER’S DISEASE PATHOGENESIS
Title
MULTIVALENT PROTEIN GLYCOSYLATION: A DRIVING FORCE OF CANCER PROGRESSION AND ALZHEIMER’S DISEASE PATHOGENESIS.
Creator
Singh, YashoNandini, Cudic, Maré, Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
Abstract/Description
Glycosylation is a frequent and heterogeneous post-translational protein modification occurring in all domains of life. Aberrant cell-surface glycosylation is shown to mediate several processes involved in tumor cell proliferation, adhesion, and metastasis. Recent findings linked altered protein glycosylation to Alzheimer’s disease (AD) pathogenesis. One key obstacle in studying functional consequences of glycosylation has been the lack of structurally defined glycopeptide or protein model...
Show moreGlycosylation is a frequent and heterogeneous post-translational protein modification occurring in all domains of life. Aberrant cell-surface glycosylation is shown to mediate several processes involved in tumor cell proliferation, adhesion, and metastasis. Recent findings linked altered protein glycosylation to Alzheimer’s disease (AD) pathogenesis. One key obstacle in studying functional consequences of glycosylation has been the lack of structurally defined glycopeptide or protein model compounds for biochemical studies at the molecular level. For tumor progression, studies are crucial towards understanding the glycan-lectin recognition process tied to deciphering the information contained in glycan structures and for AD, foundational studies are necessary for understanding the role of O-glycosylation in protein processing and its fate toward the amyloid pathway. In chapter 1, a highly O-glycosylated transmembrane and cancer-associated mucin protein, MUC1, is used as a model for designing synthetic tools for exploring its role in metastasis via association with lectins and specificity of anti-MUC1 antibodies. This dissertation for the first time presents a MUC1-based positional scanning synthetic glycopeptide combinatorial library that varies in the number and location of tumor-associated Tn antigen. The importance of defined structural complexity for evaluating glycan density and glycosylation patterns for binding to Tn-specific plant lectins and anti-MUC1 (mouse) monoclonal antibodies was revealed using an enzyme-linked lectin assay (ELLA). Chapter 2 addressed the growing significance of peptide lectinomimics for recognizing tumor-specific glycans. Fluorescently labelled alanine scan analogues of odorranalectin (OL), a cyclic peptide that exhibits lectin like properties, were screened for binding BSA-conjugated monosaccharides using ELLA. Results revealed that Lys5, Phe7, Tyr9, Gly12, Leu14, and Thr17 were crucial for binding BSA-L-fucose, BSA-Dgalactose and BSA-N-acetyl-D-galactosamine. The thermodynamics of binding of the selected alanine analogues was evaluated by isothermal titration calorimetry. The thermodynamic profile of interactions with asialofetuin exhibits shift to an entropy-driven mechanism compared to fucoidan, which displayed an enthalpy-entropy compensation, typically associated with the carbohydrate-lectin recognition process. Chapter 3 focused on amyloid-precursor protein (APP) O-glycosylation and its role in AD pathogenesis. We synthesized native and Swedish-mutated (glyco)peptides with O-GalNAc moiety on Thr663 and/or Ser667 or Tyr681 to explore the role of glycosylation on conformation, secretase activity, and aggregation kinetics of Aβ40. Our results show that conformation is strongly dependent on external conditions such as buffer ions and solvent polarity as well as internal modifications of (glyco)peptides such as length, O-glycosylation, and Swedish mutation (Lys670Asn/Met671Leu). Furthermore, the level of β-secretase activity significantly increased for the glycopeptides containing the Swedish mutation compared to their nonglycosylated and native counterparts. Lastly, glycopeptides impacted the kinetics of Aβ40 aggregation by significantly increasing the lag phase and delaying aggregation onset, however, this effect was less pronounced for its Swedish-mutated counterparts.
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Date Issued
2022
PURL
http://purl.flvc.org/fau/fd/FA00013949
Subject Headings
Glycosylation, Alzheimer Disease, Lectins
Format
Document (PDF)
Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential
Title
Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential.
Creator
Lohmueller, Jason J., Sato, Shuji, Popova, Lana, Chu, Isabel M., Tucker, Meghan A., Barberena, Roberto, Innocenti, Gregory M., Cudic, Mare, Ham, James D., Cheung, Wan Cheung, Polakiewicz, Roberto D., Finn, Olivera J.
Abstract/Description
MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a...
Show moreMUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a wide range of binding affinities. These antibodies bind hypoglycosylated MUC1 on human cancer cell lines and tumor tissues but show no reactivity against fully-glycosylated MUC1 on normal cells and tissues. We found that several antibodies activate complement-mediated cytotoxicity and that T cells carrying chimeric antigen receptors with the antibody variable regions kill MUC1+ target cells, express activation markers, and produce interferon gamma. Fully-human and tumor-specific, these antibodies are candidates for further testing and development as immunotherapeutic drugs.
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Date Issued
2016-10-22
PURL
http://purl.flvc.org/fau/fd/FAUIR000012
Format
Citation
The Roles of Substrate Thermal Stability and P2 and P1′ Subsite Identity on Matrix Metalloproteinase Triple-helical Peptidase Activity and Collagen Specificity
Title
The Roles of Substrate Thermal Stability and P2 and P1′ Subsite Identity on Matrix Metalloproteinase Triple-helical Peptidase Activity and Collagen Specificity.
Creator
Minond, Dmitriy, Lauer-Fields, Janelle L., Cudic, Mare, Overall, Christopher M., Pei, Duanqing, Brew, Keith, Visse, Robert, Nagase, Hideaki, Fields, Gregg B.
Date Issued
2006-12
PURL
http://purl.flvc.org/fau/flvc_fau_islandoraimporter_10.1074_jbc.M606004200_1638203001
Format
Document (PDF)