ABSTRACT
Epigenetic modulators such as lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) are drug targets for cancer, neuropsychiatric disease, or inflammation, but inhibitors of these enzymes exhibit considerable side effects. For a potential local treatment with reduced systemic toxicity, we present here soft drug candidates as new LSD1 and HDAC inhibitors. A soft drug is a compound that is degraded in vivo to less active metabolites after having achieved its therapeutic function. This has been successfully applied for corticosteroids in the clinic, but soft drugs targeting epigenetic enzymes are scarce, with the HDAC inhibitor remetinostat being the only example. We have developed new methyl ester-containing inhibitors targeting LSD1 or HDACs and compared the biological activities of these to their respective carboxylic acid cleavage products. In vitro activity assays, cellular experiments, and a stability assay identified potent HDAC and LSD1 soft drug candidates that are superior to their corresponding carboxylic acids in cellular models.
ABSTRACT
The original version of this Article contained an error in the Acknowledgements section.
ABSTRACT
LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.
Subject(s)
Cell Differentiation/drug effects , DNA-Binding Proteins/metabolism , Histone Demethylases/antagonists & inhibitors , Leukemia, Myeloid, Acute/pathology , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Tranylcypromine/pharmacology , Animals , Antidepressive Agents/pharmacology , DNA-Binding Proteins/genetics , Gene Expression Regulation, Leukemic , Histone Demethylases/genetics , Histone Demethylases/metabolism , Histone Demethylases/physiology , Humans , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Mice, Knockout , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Tumor Cells, CulturedABSTRACT
FAD-dependent lysine-specific demethylase 1 (LSD1) is overexpressed or deregulated in many cancers such as AML and prostate cancer and hence is a promising anticancer target with first inhibitors in clinical trials. Clinical candidates are N-substituted derivatives of the dual LSD1-/monoamine oxidase-inhibitor tranylcypromine (2-PCPA) with a basic amine function in the N-substituent. These derivatives are selective over monoamine oxidases. So far, only very limited information on structure-activity studies about this important class of LSD1 inhibitors is published in peer reviewed journals. Here, we show that N-substituted 2-PCPA derivatives without a basic function or even a polar group are still potent inhibitors of LSD1 in vitro and effectively inhibit colony formation of leukemic cells in culture. Yet, these lipophilic inhibitors also block the structurally related monoamine oxidases (MAO-A and MAO-B), which may be of interest for the treatment of neurodegenerative disorders, but this property is undesired for applications in cancer treatment. The introduction of a polar, non-basic function led to optimized structures that retain potent LSD1 inhibitors but exhibit selectivity over MAOs and are highly potent in the suppression of colony formation of cultured leukemic cells. Cellular target engagement is shown via a Cellular Thermal Shift Assay (CETSA) for LSD1.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Tranylcypromine/analogs & derivatives , Tranylcypromine/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Differentiation/drug effects , Cell Line, Tumor , Histone Demethylases/metabolism , Humans , Leukemia/drug therapy , Leukemia/metabolism , Leukemia/pathology , Mice , Models, Molecular , Monoamine Oxidase Inhibitors/chemistry , Monoamine Oxidase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
Spontaneous electron transport to molecular oxygen led to regeneration of oxidised nicotinamide cofactor in cell lysates that contain an alcohol dehydrogenase, a quinone reductase and a quinone mediator. This concept allows the efficient oxidation of alcohols in the presence of alcohol dehydrogenase-containing E. coli lysates and catalytic amounts of the quinone lawsone.
Subject(s)
Alcohol Dehydrogenase/metabolism , Alcohols/metabolism , Escherichia coli/enzymology , Quinones/metabolism , Biocatalysis , Electron Transport , Oxidation-ReductionABSTRACT
Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.
Subject(s)
Histone Demethylases/antagonists & inhibitors , Histones/chemistry , Peptides/chemistry , Animals , Antibodies/chemistry , Biotinylation , Drug Discovery , Epigenomics , Fluorescence , Histone Demethylases/chemistry , Humans , Immunoassay , Insecta , Kinetics , Lysine/chemistry , Mass Spectrometry , Protein Processing, Post-Translational , Reproducibility of Results , Spectrometry, FluorescenceABSTRACT
Lysine demethylases play an important role in epigenetic regulation and thus in the development of diseases like cancer or neurodegenerative disorders. As the lysine specific demethylase 1 (LSD1/KDM1) has been strongly connected to androgen and estrogen dependent gene expression, it serves as a promising target for the therapy of hormone dependent cancer. Here, we report on the discovery of new small molecule inhibitors of LSD1 containing a propargylamine warhead, starting out from lysine containing substrate analogues. On the basis of these substrate mimicking inhibitors, we were able to increase potency by a combination of similarity-based virtual screening and subsequent synthetic optimization resulting in more druglike LSD1 inhibitors that led to histone hypermethylation in breast cancer cells.