Corrigendum to “Structure–activity study for (bis)ureidopropyl- and (bis)thioureidopropyldiamine LSD1 inhibitors with 3-5-3 and 3-6-3 carbon backbone architectures” [Bioorg. Med. Chem. 23 (2015) 1601–1612](S0968089615000735)(10.1016/j.bmc.2015.01.049)

Shannon Lyn Nowotarski, Boobalan Pachaiyappan, Steven L. Holshouser, Craig J. Kutz, Youxuan Li, Yi Huang, Shiv K. Sharma, Robert A. Casero, Patrick M. Woster

Research output: Contribution to journalComment/debate

1 Citation (Scopus)

Abstract

The Authors regret that in Fig. 4 on page 1605, a Lineweaver-Burk graph was inserted that indicated compound 6d competitively inhibited lysine-specific demethylase 1 (LSD1) with a Ki of 2.4 μM. However, the data obtained in the absence of inhibitor was inadvertently omitted from the figure. The correct graph is shown in the figure, generated as described below. Since the original publication of this manuscript, we have encountered problems with the assay procedure that was used to gather the data for the kinetic analysis of compound 6d. The LSD1 assay procedures in the manuscript were conducted using a commercially available LSD1 assay kit (BPS Bioscience, San Diego, CA, kit #50106). The assay employs a 21-mer peptide substrate analogous to the tail found on histone 3, containing dimethylated lysine at position 4. Recombinant LSD1 and the peptide substrate are co-incubated, and the activity of the enzyme is determined by converting the LSD1 by-product H 2 O 2 to a radical cation using horseradish peroxidase (HRP). The radical cation is then quantitated with Amplex Red. The first issue, which is manageable, is that there is no reagent in the kit with which to quench the enzymatic reaction, and thus the timing of each step in the procedure is critical to obtaining reliable results. It is now known that the LSD1 reaction can be quenched using the 21-mer peptide in which dimethyllysine 4 is replaced by a methionine. 1 This peptide is a potent inhibitor of LSD1, however, this methodology was not available at the time our manuscript was published. A second issue we and other groups have encountered is the potential for LSD1 inhibitors to inhibit HRP. This effect would lead to an artificially high value for % inhibition of LSD1, since it would prevent HRP from metabolizing H 2 O 2 . In light of these limitations to the commercial assay kit, we decided to repeat the procedure that was used in our kinetic studies. This experiment was repeated 4 times, with nearly identical results. The data shown in the figure above are typical of these experiments. At a low concentration of 6d (0.625 μM) the Lineweaver-Burk plot suggested competitive inhibition, and a Ki of 0.7 μM was calculated. At higher concentrations of 6d (1.25 and 2.5 μM) the corresponding lines were much steeper, and did not correlate with the data from the lower concentration. This effect was not detected for 6d until we assayed the compound at concentrations higher than those reported in our manuscript. We postulate that this anomaly is due to inhibition of HRP by 6d, and thus the kinetic data obtained with this assay is unreliable. It is concluded that the commercial LSD1 assay kit we used is suitable for initial screening of new compounds, but not for determining kinetic constants for active inhibitors unless the 21-mer inhibitor is used as a quenching agent, and inhibition of HRP is ruled out.

Original languageEnglish (US)
Pages (from-to)3728-3729
Number of pages2
JournalBioorganic and Medicinal Chemistry
Volume26
Issue number12
DOIs
StatePublished - Jul 23 2018

Fingerprint

Lysine
Carbon
Assays
Horseradish Peroxidase
Manuscripts
Peptides
Kinetics
Cations
corrigendum
Substrates
Methionine
Histones
Byproducts
Publications
Quenching
Screening
Emotions
Experiments
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry

Cite this

@article{751ee318698a4b03b556995423890951,
title = "Corrigendum to “Structure–activity study for (bis)ureidopropyl- and (bis)thioureidopropyldiamine LSD1 inhibitors with 3-5-3 and 3-6-3 carbon backbone architectures” [Bioorg. Med. Chem. 23 (2015) 1601–1612](S0968089615000735)(10.1016/j.bmc.2015.01.049)",
abstract = "The Authors regret that in Fig. 4 on page 1605, a Lineweaver-Burk graph was inserted that indicated compound 6d competitively inhibited lysine-specific demethylase 1 (LSD1) with a Ki of 2.4 μM. However, the data obtained in the absence of inhibitor was inadvertently omitted from the figure. The correct graph is shown in the figure, generated as described below. Since the original publication of this manuscript, we have encountered problems with the assay procedure that was used to gather the data for the kinetic analysis of compound 6d. The LSD1 assay procedures in the manuscript were conducted using a commercially available LSD1 assay kit (BPS Bioscience, San Diego, CA, kit #50106). The assay employs a 21-mer peptide substrate analogous to the tail found on histone 3, containing dimethylated lysine at position 4. Recombinant LSD1 and the peptide substrate are co-incubated, and the activity of the enzyme is determined by converting the LSD1 by-product H 2 O 2 to a radical cation using horseradish peroxidase (HRP). The radical cation is then quantitated with Amplex Red. The first issue, which is manageable, is that there is no reagent in the kit with which to quench the enzymatic reaction, and thus the timing of each step in the procedure is critical to obtaining reliable results. It is now known that the LSD1 reaction can be quenched using the 21-mer peptide in which dimethyllysine 4 is replaced by a methionine. 1 This peptide is a potent inhibitor of LSD1, however, this methodology was not available at the time our manuscript was published. A second issue we and other groups have encountered is the potential for LSD1 inhibitors to inhibit HRP. This effect would lead to an artificially high value for {\%} inhibition of LSD1, since it would prevent HRP from metabolizing H 2 O 2 . In light of these limitations to the commercial assay kit, we decided to repeat the procedure that was used in our kinetic studies. This experiment was repeated 4 times, with nearly identical results. The data shown in the figure above are typical of these experiments. At a low concentration of 6d (0.625 μM) the Lineweaver-Burk plot suggested competitive inhibition, and a Ki of 0.7 μM was calculated. At higher concentrations of 6d (1.25 and 2.5 μM) the corresponding lines were much steeper, and did not correlate with the data from the lower concentration. This effect was not detected for 6d until we assayed the compound at concentrations higher than those reported in our manuscript. We postulate that this anomaly is due to inhibition of HRP by 6d, and thus the kinetic data obtained with this assay is unreliable. It is concluded that the commercial LSD1 assay kit we used is suitable for initial screening of new compounds, but not for determining kinetic constants for active inhibitors unless the 21-mer inhibitor is used as a quenching agent, and inhibition of HRP is ruled out.",
author = "Nowotarski, {Shannon Lyn} and Boobalan Pachaiyappan and Holshouser, {Steven L.} and Kutz, {Craig J.} and Youxuan Li and Yi Huang and Sharma, {Shiv K.} and Casero, {Robert A.} and Woster, {Patrick M.}",
year = "2018",
month = "7",
day = "23",
doi = "10.1016/j.bmc.2018.05.032",
language = "English (US)",
volume = "26",
pages = "3728--3729",
journal = "Bioorganic and Medicinal Chemistry",
issn = "0968-0896",
publisher = "Elsevier Limited",
number = "12",

}

Corrigendum to “Structure–activity study for (bis)ureidopropyl- and (bis)thioureidopropyldiamine LSD1 inhibitors with 3-5-3 and 3-6-3 carbon backbone architectures” [Bioorg. Med. Chem. 23 (2015) 1601–1612](S0968089615000735)(10.1016/j.bmc.2015.01.049). / Nowotarski, Shannon Lyn; Pachaiyappan, Boobalan; Holshouser, Steven L.; Kutz, Craig J.; Li, Youxuan; Huang, Yi; Sharma, Shiv K.; Casero, Robert A.; Woster, Patrick M.

In: Bioorganic and Medicinal Chemistry, Vol. 26, No. 12, 23.07.2018, p. 3728-3729.

Research output: Contribution to journalComment/debate

TY - JOUR

T1 - Corrigendum to “Structure–activity study for (bis)ureidopropyl- and (bis)thioureidopropyldiamine LSD1 inhibitors with 3-5-3 and 3-6-3 carbon backbone architectures” [Bioorg. Med. Chem. 23 (2015) 1601–1612](S0968089615000735)(10.1016/j.bmc.2015.01.049)

AU - Nowotarski, Shannon Lyn

AU - Pachaiyappan, Boobalan

AU - Holshouser, Steven L.

AU - Kutz, Craig J.

AU - Li, Youxuan

AU - Huang, Yi

AU - Sharma, Shiv K.

AU - Casero, Robert A.

AU - Woster, Patrick M.

PY - 2018/7/23

Y1 - 2018/7/23

N2 - The Authors regret that in Fig. 4 on page 1605, a Lineweaver-Burk graph was inserted that indicated compound 6d competitively inhibited lysine-specific demethylase 1 (LSD1) with a Ki of 2.4 μM. However, the data obtained in the absence of inhibitor was inadvertently omitted from the figure. The correct graph is shown in the figure, generated as described below. Since the original publication of this manuscript, we have encountered problems with the assay procedure that was used to gather the data for the kinetic analysis of compound 6d. The LSD1 assay procedures in the manuscript were conducted using a commercially available LSD1 assay kit (BPS Bioscience, San Diego, CA, kit #50106). The assay employs a 21-mer peptide substrate analogous to the tail found on histone 3, containing dimethylated lysine at position 4. Recombinant LSD1 and the peptide substrate are co-incubated, and the activity of the enzyme is determined by converting the LSD1 by-product H 2 O 2 to a radical cation using horseradish peroxidase (HRP). The radical cation is then quantitated with Amplex Red. The first issue, which is manageable, is that there is no reagent in the kit with which to quench the enzymatic reaction, and thus the timing of each step in the procedure is critical to obtaining reliable results. It is now known that the LSD1 reaction can be quenched using the 21-mer peptide in which dimethyllysine 4 is replaced by a methionine. 1 This peptide is a potent inhibitor of LSD1, however, this methodology was not available at the time our manuscript was published. A second issue we and other groups have encountered is the potential for LSD1 inhibitors to inhibit HRP. This effect would lead to an artificially high value for % inhibition of LSD1, since it would prevent HRP from metabolizing H 2 O 2 . In light of these limitations to the commercial assay kit, we decided to repeat the procedure that was used in our kinetic studies. This experiment was repeated 4 times, with nearly identical results. The data shown in the figure above are typical of these experiments. At a low concentration of 6d (0.625 μM) the Lineweaver-Burk plot suggested competitive inhibition, and a Ki of 0.7 μM was calculated. At higher concentrations of 6d (1.25 and 2.5 μM) the corresponding lines were much steeper, and did not correlate with the data from the lower concentration. This effect was not detected for 6d until we assayed the compound at concentrations higher than those reported in our manuscript. We postulate that this anomaly is due to inhibition of HRP by 6d, and thus the kinetic data obtained with this assay is unreliable. It is concluded that the commercial LSD1 assay kit we used is suitable for initial screening of new compounds, but not for determining kinetic constants for active inhibitors unless the 21-mer inhibitor is used as a quenching agent, and inhibition of HRP is ruled out.

AB - The Authors regret that in Fig. 4 on page 1605, a Lineweaver-Burk graph was inserted that indicated compound 6d competitively inhibited lysine-specific demethylase 1 (LSD1) with a Ki of 2.4 μM. However, the data obtained in the absence of inhibitor was inadvertently omitted from the figure. The correct graph is shown in the figure, generated as described below. Since the original publication of this manuscript, we have encountered problems with the assay procedure that was used to gather the data for the kinetic analysis of compound 6d. The LSD1 assay procedures in the manuscript were conducted using a commercially available LSD1 assay kit (BPS Bioscience, San Diego, CA, kit #50106). The assay employs a 21-mer peptide substrate analogous to the tail found on histone 3, containing dimethylated lysine at position 4. Recombinant LSD1 and the peptide substrate are co-incubated, and the activity of the enzyme is determined by converting the LSD1 by-product H 2 O 2 to a radical cation using horseradish peroxidase (HRP). The radical cation is then quantitated with Amplex Red. The first issue, which is manageable, is that there is no reagent in the kit with which to quench the enzymatic reaction, and thus the timing of each step in the procedure is critical to obtaining reliable results. It is now known that the LSD1 reaction can be quenched using the 21-mer peptide in which dimethyllysine 4 is replaced by a methionine. 1 This peptide is a potent inhibitor of LSD1, however, this methodology was not available at the time our manuscript was published. A second issue we and other groups have encountered is the potential for LSD1 inhibitors to inhibit HRP. This effect would lead to an artificially high value for % inhibition of LSD1, since it would prevent HRP from metabolizing H 2 O 2 . In light of these limitations to the commercial assay kit, we decided to repeat the procedure that was used in our kinetic studies. This experiment was repeated 4 times, with nearly identical results. The data shown in the figure above are typical of these experiments. At a low concentration of 6d (0.625 μM) the Lineweaver-Burk plot suggested competitive inhibition, and a Ki of 0.7 μM was calculated. At higher concentrations of 6d (1.25 and 2.5 μM) the corresponding lines were much steeper, and did not correlate with the data from the lower concentration. This effect was not detected for 6d until we assayed the compound at concentrations higher than those reported in our manuscript. We postulate that this anomaly is due to inhibition of HRP by 6d, and thus the kinetic data obtained with this assay is unreliable. It is concluded that the commercial LSD1 assay kit we used is suitable for initial screening of new compounds, but not for determining kinetic constants for active inhibitors unless the 21-mer inhibitor is used as a quenching agent, and inhibition of HRP is ruled out.

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