2014
Leurs, Ulrike; Lohse, Brian; Ming, Shonoi; Cole, Philip A; Clausen, Rasmus P; Kristensen, Jesper L; Rand, Kasper D
In: Anal Chem, vol. 86, no. 23, pp. 11734-41, 2014, ISSN: 1520-6882.
Abstract | Links | BibTeX | Tags: Binding Sites, Deuterium Exchange Measurement, Histone Demethylases, Humans, Jumonji Domain-Containing Histone Demethylases, Ligands, Mass Spectrometry, Models, Molecular, Molecular Structure, Peptides
@article{1299975,
title = {Dissecting the binding mode of low affinity phage display peptide ligands to protein targets by hydrogen/deuterium exchange coupled to mass spectrometry},
author = {Ulrike Leurs and Brian Lohse and Shonoi Ming and Philip A Cole and Rasmus P Clausen and Jesper L Kristensen and Kasper D Rand},
doi = {10.1021/ac503137u},
issn = {1520-6882},
year = {2014},
date = {2014-12-01},
journal = {Anal Chem},
volume = {86},
number = {23},
pages = {11734-41},
abstract = {Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, nonbinding control peptides, and statistical analysis of replicate data. We show that HDX-MS can identify regions in the two epigenetic regulator proteins KDM4C and KDM1A that are perturbed through weak interactions with PD-identified peptides. Two peptides cause reduced HDX on opposite sides of the active site of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches.},
keywords = {Binding Sites, Deuterium Exchange Measurement, Histone Demethylases, Humans, Jumonji Domain-Containing Histone Demethylases, Ligands, Mass Spectrometry, Models, Molecular, Molecular Structure, Peptides},
pubstate = {published},
tppubtype = {article}
}
Phage display (PD) is frequently used to discover peptides capable of binding to biological protein targets. The structural characterization of peptide-protein complexes is often challenging due to their low binding affinities and high structural flexibility. Here, we investigate the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, nonbinding control peptides, and statistical analysis of replicate data. We show that HDX-MS can identify regions in the two epigenetic regulator proteins KDM4C and KDM1A that are perturbed through weak interactions with PD-identified peptides. Two peptides cause reduced HDX on opposite sides of the active site of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions and pave the way for understanding and optimizing the binding of peptide scaffolds identified through PD and similar ligand discovery approaches.
Leurs, Ulrike; Lohse, Brian; Rand, Kasper D; Ming, Shonoi; Riise, Erik S; Cole, Philip A; Kristensen, Jesper L; Clausen, Rasmus P
Substrate- and cofactor-independent inhibition of histone demethylase KDM4C Journal Article
In: ACS Chem Biol, vol. 9, no. 9, pp. 2131-8, 2014, ISSN: 1554-8937.
Abstract | Links | BibTeX | Tags: Amino Acid Sequence, Catalytic Domain, Cell Line, Coenzymes, Deuterium Exchange Measurement, Enzyme Inhibitors, High-Throughput Screening Assays, Histone Demethylases, Humans, Inhibitory Concentration 50, Jumonji Domain-Containing Histone Demethylases, Molecular Sequence Data, Peptide Library
@article{1299981,
title = {Substrate- and cofactor-independent inhibition of histone demethylase KDM4C},
author = {Ulrike Leurs and Brian Lohse and Kasper D Rand and Shonoi Ming and Erik S Riise and Philip A Cole and Jesper L Kristensen and Rasmus P Clausen},
doi = {10.1021/cb500374f},
issn = {1554-8937},
year = {2014},
date = {2014-09-01},
journal = {ACS Chem Biol},
volume = {9},
number = {9},
pages = {2131-8},
abstract = {Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging, as the active sites of KDM1A-B and KDM4A-D histone demethylases are highly conserved. Most inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide sequence or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation, and chemical modifications. Hydrogen/deuterium exchange mass spectrometry revealed that the peptide-based inhibitors target KDM4C through substrate-independent interactions located on the surface remote from the active site within less conserved regions of KDM4C. The sites discovered in this study provide a new approach of targeting KDM4C through substrate- and cofactor-independent interactions and may be further explored to develop potent selective inhibitors and biological probes for the KDM4 family.},
keywords = {Amino Acid Sequence, Catalytic Domain, Cell Line, Coenzymes, Deuterium Exchange Measurement, Enzyme Inhibitors, High-Throughput Screening Assays, Histone Demethylases, Humans, Inhibitory Concentration 50, Jumonji Domain-Containing Histone Demethylases, Molecular Sequence Data, Peptide Library},
pubstate = {published},
tppubtype = {article}
}
Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging, as the active sites of KDM1A-B and KDM4A-D histone demethylases are highly conserved. Most inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide sequence or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation, and chemical modifications. Hydrogen/deuterium exchange mass spectrometry revealed that the peptide-based inhibitors target KDM4C through substrate-independent interactions located on the surface remote from the active site within less conserved regions of KDM4C. The sites discovered in this study provide a new approach of targeting KDM4C through substrate- and cofactor-independent interactions and may be further explored to develop potent selective inhibitors and biological probes for the KDM4 family.
Prusevich, Polina; Kalin, Jay H; Ming, Shonoi A; Basso, Manuela; Givens, Jeffrey; Li, Xin; Hu, Jianfei; Taylor, Martin S; Cieniewicz, Anne M; Hsiao, Po-Yuan; Huang, Rong; Roberson, Heather; Adejola, Nkosi; Avery, Lindsay B; Casero, Robert A; Taverna, Sean D; Qian, Jiang; Tackett, Alan J; Ratan, Rajiv R; McDonald, Oliver G; Feinberg, Andrew P; Cole, Philip A
A selective phenelzine analogue inhibitor of histone demethylase LSD1 Journal Article
In: ACS Chem Biol, vol. 9, no. 6, pp. 1284-93, 2014, ISSN: 1554-8937.
Abstract | Links | BibTeX | Tags: Animals, Blotting, Cell Survival, Cells, Cultured, DNA Methylation, Embryo, Enzyme Inhibitors, Fetus, Histone Demethylases, Histones, Humans, Mammalian, Monoamine Oxidase, Neurons, Phenelzine, Rats, Sprague-Dawley, Western
@article{1299983,
title = {A selective phenelzine analogue inhibitor of histone demethylase LSD1},
author = {Polina Prusevich and Jay H Kalin and Shonoi A Ming and Manuela Basso and Jeffrey Givens and Xin Li and Jianfei Hu and Martin S Taylor and Anne M Cieniewicz and Po-Yuan Hsiao and Rong Huang and Heather Roberson and Nkosi Adejola and Lindsay B Avery and Robert A Casero and Sean D Taverna and Jiang Qian and Alan J Tackett and Rajiv R Ratan and Oliver G McDonald and Andrew P Feinberg and Philip A Cole},
doi = {10.1021/cb500018s},
issn = {1554-8937},
year = {2014},
date = {2014-06-01},
journal = {ACS Chem Biol},
volume = {9},
number = {6},
pages = {1284-93},
abstract = {Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease.},
keywords = {Animals, Blotting, Cell Survival, Cells, Cultured, DNA Methylation, Embryo, Enzyme Inhibitors, Fetus, Histone Demethylases, Histones, Humans, Mammalian, Monoamine Oxidase, Neurons, Phenelzine, Rats, Sprague-Dawley, Western},
pubstate = {published},
tppubtype = {article}
}
Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease.