Journal article
Structure-activity study for (bis) ureidopropyl- and (bis) thioureidopropyldiamine LSD1 inhibitors with 3-5-3 and 3-6-3 carbon backbone architectures (vol 23, pg 1601, 2015)
Bioorganic & medicinal chemistry, Vol.26(12), pp.3728-3729
07/23/2018
DOI: 10.1016/j.bmc.2018.05.032
PMID: 29859749
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 H2O2 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 H2O2. 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.
Details
- Title: Subtitle
- Structure-activity study for (bis) ureidopropyl- and (bis) thioureidopropyldiamine LSD1 inhibitors with 3-5-3 and 3-6-3 carbon backbone architectures (vol 23, pg 1601, 2015)
- Creators
- Shannon L. Nowotarski - Sidney Kimmel Comprehensive Cancer CenterBoobalan Pachaiyappan - Medical University of South CarolinaSteven L. Holshouser - Medical University of South CarolinaCraig J. Kutz - Medical University of South CarolinaYouxuan Li - Medical University of South CarolinaYi Huang - Sidney Kimmel Comprehensive Cancer CenterShiv K. Sharma - Eugene Applebaum College of Pharmacy and Health SciencesRobert A. Casero - Sidney Kimmel Comprehensive Cancer CenterPatrick M. Woster - Medical University of South Carolina
- Resource Type
- Journal article
- Publication Details
- Bioorganic & medicinal chemistry, Vol.26(12), pp.3728-3729
- Publisher
- Elsevier
- DOI
- 10.1016/j.bmc.2018.05.032
- PMID
- 29859749
- ISSN
- 0968-0896
- eISSN
- 1464-3391
- Number of pages
- 2
- Language
- English
- Date published
- 07/23/2018
- Academic Unit
- Internal Medicine
- Record Identifier
- 9984383316402771
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