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Peter B. Dervan - One of the best experts on this subject based on the ideXlab platform.
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single position substitution of hairpin pyrrole imidazole Polyamides imparts distinct dna binding profiles across the human genome
PLOS ONE, 2020Co-Authors: Paul B Finn, Devesh Bhimsaria, Asfa Ali, Asuka Eguchi, Aseem Z Ansari, Peter B. DervanAbstract:Regulating desired loci in the genome with sequence-specific DNA-binding molecules is a major goal for the development of precision medicine. Pyrrole–imidazole (Py–Im) Polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide–DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This method, termed COSMIC-seq, confirms the ability of hairpin-Polyamides, with similar architectures but differing at a single ring position, to retain in vitro specificities and display distinct genome-wide binding profiles. These results underpin the development of Py-Im Polyamides as DNA-targeting molecules that mediate their regulatory or remedial functions at desired genomic loci.
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Characterization and Solubilization of Pyrrole−Imidazole Polyamide Aggregates
2016Co-Authors: A E. Hargrove, Jevgenij A. Raskatov, Jordan L. Meier, David C. Montgomery, Peter B. DervanAbstract:ABSTRACT: To optimize the biological activity of pyrrole−imidazole polyamide DNA-binding molecules, we characterized the aggregation propensity of these compounds through dynamic light scattering and fractional solubility analysis. Nearly all studied Polyamides were found to form measurable particles 50−500 nm in size under biologically relevant conditions, while HPLC-based analyses revealed solubility trends in both core sequences and peripheral substituents that did not correlate with overall ionic charge. The solubility of both hairpin and cyclic Polyamides was increased upon addition of carbohydrate solubilizing agents, in particular, 2-hydroxypropyl-β-cyclodextrin (HpβCD). In mice, the use of HpβCD allowed for improved injection conditions and subsequent investigations of the availability of Polyamides in mouse plasma to human cells. The results of these studies will influence the further design of Py-Im Polyamides and facilitate their study in animal models. N-Methylpyrrole (Py) and N-methylimidazole (Im) polya-mides are heterocycle-based oligomers that bind the minor groove of DNA in a sequence-specific manner.1−3 Inves-tigations of Py-Im polyamide biological properties hav
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guiding the design of synthetic dna binding molecules with massively parallel sequencing
Journal of the American Chemical Society, 2012Co-Authors: Jordan L. Meier, Ian F Korf, David J Segal, Peter B. DervanAbstract:Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 1012-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for Polyamides containing β/Im pairs. These insights allow the redesign of hairpin Polyamides with different turn units capable of distinguishing 5′-WCGCGW-3′ from 5′-WGCGCW-3′. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.
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in vivo imaging of pyrrole imidazole Polyamides with positron emission tomography
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Daniel A Harki, Nagichettiar Satyamurthy, David B Stout, Michael E Phelps, Peter B. DervanAbstract:The biodistribution profiles in mice of two pyrrole-imidazole Polyamides were determined by PET. Pyrrole-imidazole Polyamides are a class of small molecules that can be programmed to bind a broad repertoire of DNA sequences, disrupt transcription factor-DNA interfaces, and modulate gene expression pathways in cell culture experiments. The 18F-radiolabeled Polyamides were prepared by oxime ligation between 4-[18F]-fluorobenzaldehyde and a hydroxylamine moiety at the polyamide C terminus. Small animal PET imaging of radiolabeled Polyamides administered to mice revealed distinct differences in the biodistribution of a 5-ring β-linked polyamide versus an 8-ring hairpin, which exhibited better overall bioavailability. In vivo imaging of pyrrole-imidazole Polyamides by PET is a minimum first step toward the translation of polyamide-based gene regulation from cell culture to small animal studies.
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α-Diaminobutyric Acid-Linked Hairpin Polyamides
Bioorganic & Medicinal Chemistry, 2007Co-Authors: Michelle E. Farkas, Sherry M. Tsai, Peter B. DervanAbstract:A hairpin polyamide-chlorambucil conjugate linked by α-diaminobutyric acid (α-DABA) has been shown to have interesting biological properties in cellular and small animal models. Remarkably, this new class of hairpin Polyamides has not been previously characterized with regard to energetics and sequence specificity. Herein we present a series of pyrrole-imidazole hairpin Polyamides linked by α-DABA and compare them to Polyamides containing the standard γ-DABA turn unit. The α-DABA hairpins have overall decreased binding affinities. However, α-DABA polyamide-chlorambucil conjugates are sequence-specific DNA alkylators with increased specificities. Affinity cleavage studies of α-DABA polyamide-EDTA conjugates confirmed their preference for binding DNA in a forward hairpin conformation. In contrast, an unsubstituted glycine-linked polyamide prefers to bind in an extended binding mode. Thus, substitution on the turn unit locks the α-DABA polyamide into the forward hairpin binding motif.
Volker Sieber - One of the best experts on this subject based on the ideXlab platform.
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new bio Polyamides from terpenes α pinene and 3 carene as valuable resources for lactam production
Macromolecular Rapid Communications, 2019Co-Authors: Paul N. Stockmann, Dominik L. Pastoetter, Marion Woelbing, Claudia Falcke, Malte Winnacker, Harald Strittmatter, Volker SieberAbstract:The synthesis and polymerization of two β-lactams and two e-lactams derived from the terpenes α-pinene and (+)-3-carene are reported. The new biopolymers can be considered as polyamide 2 (PA2) and polyamide 6 (PA6)-types with aliphatic stereoregular side chains, which lead to remarkable new properties. The macromolecules are investigated by gel permeation chromatography (GPC), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and infrared (IR). The (+)-3-carene-derived PA6-type is of particular interest, since it reaches a molecular weight of over 30 kDa, which is the highest value for lactam-based Polyamides derived from terpenes reported to date. Additionally, a glass transition temperature (Tg ) of 120 °C is observed, surpassing the glass transition temperature of PA6 by 60 °C. The absence of a melting point (Tm ) indicates high amorphicity, another novelty for terpene-based Polyamides, which might give transparent bio-Polyamides access to new fields of application.
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New Bio-Polyamides From Terpenes: α-Pinene and (+)-3-Carene as Valuable Resources for Lactam Production
Macromolecular rapid communications, 2019Co-Authors: Paul N. Stockmann, Dominik L. Pastoetter, Marion Woelbing, Claudia Falcke, Malte Winnacker, Harald Strittmatter, Volker SieberAbstract:The synthesis and polymerization of two β-lactams and two e-lactams derived from the terpenes α-pinene and (+)-3-carene are reported. The new biopolymers can be considered as polyamide 2 (PA2) and polyamide 6 (PA6)-types with aliphatic stereoregular side chains, which lead to remarkable new properties. The macromolecules are investigated by gel permeation chromatography (GPC), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and infrared (IR). The (+)-3-carene-derived PA6-type is of particular interest, since it reaches a molecular weight of over 30 kDa, which is the highest value for lactam-based Polyamides derived from terpenes reported to date. Additionally, a glass transition temperature (Tg ) of 120 °C is observed, surpassing the glass transition temperature of PA6 by 60 °C. The absence of a melting point (Tm ) indicates high amorphicity, another novelty for terpene-based Polyamides, which might give transparent bio-Polyamides access to new fields of application.
James K. Bashkin - One of the best experts on this subject based on the ideXlab platform.
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A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid.
Journal of Virology, 2018Co-Authors: Ryan H. Gumpper, Carlos H. Castaneda, M. José Scuderi, James K. Bashkin, Ming LuoAbstract:Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that Polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of Polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, Polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.IMPORTANCE Negative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing Polyamides as antiviral drugs against NSVs.
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promoter scanning of the human cox 2 gene with 8 ring Polyamides unexpected weakening of polyamide dna binding and selectivity by replacing an internal n me pyrrole with β alanine
Biochimie, 2013Co-Authors: James K. Bashkin, Karl Aston, Joseph Ramos, Kevin J Koeller, Rupesh Nanjunda, Cynthia M Dupureur, David W WilsonAbstract:Abstract Rules for polyamide–DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal β-alanines as polyamide building blocks in place of N-methylpyrrole; β-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long Polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327–339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with β-alanine in 8-ring Polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with β-alanine to generate a β/Im pairing in two 8-ring Polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that β-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide–DNA recognition.
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hpv episome levels are potently decreased by pyrrole imidazole Polyamides
Antiviral Research, 2011Co-Authors: Terri G. Edwards, James K. Bashkin, Kevin J Koeller, Urszula Slomczynska, Kam Fok, Michael J. Helmus, Chris FisherAbstract:Abstract Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N -methylpyrrole–imidazole Polyamides of the hairpin type, polyamide 1 (PA 1 ) and polyamide 25 (PA 25 ). Both Polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4–18 cells), and HPV31 (in HPV31 maintaining cells). From a library of Polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication ( ori ), four Polyamides were identified that possessed apparent IC 50 s ⩽ 150 nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA 1 and PA 25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA 1 , PA 25 , and related Polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our Polyamides, is accompanied by significant cellular toxicity.
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HPV episome levels are potently decreased by pyrrole–imidazole Polyamides
Antiviral research, 2011Co-Authors: Terri G. Edwards, James K. Bashkin, Kevin J Koeller, Urszula Slomczynska, Kam Fok, Michael J. Helmus, Chris FisherAbstract:Abstract Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N -methylpyrrole–imidazole Polyamides of the hairpin type, polyamide 1 (PA 1 ) and polyamide 25 (PA 25 ). Both Polyamides have potent anti-HPV activity against three different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4–18 cells), and HPV31 (in HPV31 maintaining cells). From a library of Polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication ( ori ), four Polyamides were identified that possessed apparent IC 50 s ⩽ 150 nM with no evidence of cytotoxicity. We report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA 1 and PA 25 are currently in preclinical development as antiviral compounds for treatment of HPV-related disease, including cervical dysplasia. PA 1 , PA 25 , and related Polyamides offer promise as antiviral agents and as tools to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to our Polyamides, is accompanied by significant cellular toxicity.
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Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells
Bioorganic & medicinal chemistry letters, 2003Co-Authors: Kathleen S. Crowley, Dennis P. Phillion, Scott S. Woodard, Barbara A. Schweitzer, Megh Singh, Hossein Shabany, Barry L. Burnette, Paul J. Hippenmeyer, Monique Heitmeier, James K. BashkinAbstract:The intracellular distribution of fluorescent-labeled Polyamides was examined in live cells. We showed that BODIPY-labeled Polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.
Hiroshi Sugiyama - One of the best experts on this subject based on the ideXlab platform.
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progress and prospects of pyrrole imidazole polyamide fluorophore conjugates as sequence selective dna probes
ChemBioChem, 2012Co-Authors: Thangavel Vaijayanthi, Toshikazu Bando, Ganesh N Pandian, Hiroshi SugiyamaAbstract:Recently, the versatility of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide conjugates, which have been developed from the DNA-binding antibiotics distamycin A and netropsin, has been shown. These synthetic small molecules can permeate cells to bind with duplex DNA in a sequence-specific manner, and hence can influence gene expression in vivo. Accordingly, several reports demonstrating the sequence specificity and biological activity of Py-Im Polyamides have accumulated. However, the benefits of Py-Im Polyamides, in particular those conjugated with fluorophores, has been overlooked. Moreover, clear directions for the employment of these attractive artificial small molecules have not yet been shown. Here, we present a detailed overview of the current and prospective applications of Py-Im polyamide-fluorophore conjugates, including sequence-specific recognition with fluorescence emission properties, and their potential roles in biological imaging.
Paul N. Stockmann - One of the best experts on this subject based on the ideXlab platform.
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new bio Polyamides from terpenes α pinene and 3 carene as valuable resources for lactam production
Macromolecular Rapid Communications, 2019Co-Authors: Paul N. Stockmann, Dominik L. Pastoetter, Marion Woelbing, Claudia Falcke, Malte Winnacker, Harald Strittmatter, Volker SieberAbstract:The synthesis and polymerization of two β-lactams and two e-lactams derived from the terpenes α-pinene and (+)-3-carene are reported. The new biopolymers can be considered as polyamide 2 (PA2) and polyamide 6 (PA6)-types with aliphatic stereoregular side chains, which lead to remarkable new properties. The macromolecules are investigated by gel permeation chromatography (GPC), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and infrared (IR). The (+)-3-carene-derived PA6-type is of particular interest, since it reaches a molecular weight of over 30 kDa, which is the highest value for lactam-based Polyamides derived from terpenes reported to date. Additionally, a glass transition temperature (Tg ) of 120 °C is observed, surpassing the glass transition temperature of PA6 by 60 °C. The absence of a melting point (Tm ) indicates high amorphicity, another novelty for terpene-based Polyamides, which might give transparent bio-Polyamides access to new fields of application.
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New Bio-Polyamides From Terpenes: α-Pinene and (+)-3-Carene as Valuable Resources for Lactam Production
Macromolecular rapid communications, 2019Co-Authors: Paul N. Stockmann, Dominik L. Pastoetter, Marion Woelbing, Claudia Falcke, Malte Winnacker, Harald Strittmatter, Volker SieberAbstract:The synthesis and polymerization of two β-lactams and two e-lactams derived from the terpenes α-pinene and (+)-3-carene are reported. The new biopolymers can be considered as polyamide 2 (PA2) and polyamide 6 (PA6)-types with aliphatic stereoregular side chains, which lead to remarkable new properties. The macromolecules are investigated by gel permeation chromatography (GPC), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and infrared (IR). The (+)-3-carene-derived PA6-type is of particular interest, since it reaches a molecular weight of over 30 kDa, which is the highest value for lactam-based Polyamides derived from terpenes reported to date. Additionally, a glass transition temperature (Tg ) of 120 °C is observed, surpassing the glass transition temperature of PA6 by 60 °C. The absence of a melting point (Tm ) indicates high amorphicity, another novelty for terpene-based Polyamides, which might give transparent bio-Polyamides access to new fields of application.
