Heterocycle-containing tranylcypromine derivatives endowed with high anti-LSD1 activity.

As regioisomers/bioisosteres of 1a, a 4-phenylbenzamide tranylcypromine (TCP) derivative previously disclosed by us, we report here the synthesis and biological evaluation of some (hetero)arylbenzoylamino TCP derivatives 1b-6, in which the 4-phenyl moiety of 1a was shifted at the benzamide C3 position or replaced by 2- or 3-furyl, 2- or 3-thienyl, or 4-pyridyl group, all at the benzamide C4 or C3 position. In anti-LSD1-CoREST assay, all the meta derivatives were more effective than the para analogues, with the meta thienyl analogs 4b and 5b being the most potent (IC50 values = 0.015 and 0.005 µM) and the most selective over MAO-B (selectivity indexes: 24.4 and 164). When tested in U937 AML and prostate cancer LNCaP cells, selected compounds 1a,b, 2b, 3b, 4b, and 5a,b displayed cell growth arrest mainly in LNCaP cells. Western blot analyses showed increased levels of H3K4me2 and/or H3K9me2 confirming the involvement of LSD1 inhibition in these assays.

Transdifferentiation of goat ear fibroblasts into lactating mammary epithelial cells induced by small molecule compounds.

Mammary epithelial cells are the only cells in the mammary glands that are capable of lactation and they are ideal for studying cellular and molecular biology mechanisms during growth, development and lactation of the mammary glands. The limiting factors in most of the currently available mammary epithelial cells are low cell viability, transgenerational efficiency and lactation function that renders them unsuitable for subsequent studies on mammary gland's cellular and lactation mechanisms and utilizing them as bioreactors. Hence, new methods are required to obtain mammary epithelial cells with high transgenerational efficiency and lactation function. In this study, transdifferentiation of goat ear fibroblasts (GEFs) into goat mammary epithelial cells (CiMECs) was induced in only eight days by five small molecule compounds, including 500 µg/mL VPA, 10 µM Tranylcypromine, 10 µM Forskolin, 1 µM TTNPB, 10 µM RepSox. Morphological observation, marker genes comparison, specific antigen expression and comparison of gene expression levels by transcriptome sequencing between the two types of cells that led to the primary deduction that CiMECs have similar biological properties to goat mammary epithelial cells (GMECs) and comparatively more lactation capacity. Therefore, we establish a novel reprogramming route to convert fibroblasts into CiMECs under fully chemically conditions. This study is expected to provide an in vitro platform for understanding cellular mechanisms such as mammary epithelial cells' fate determination and developmental differentiation, and also to find a new way to obtain a large number of functional mammary epithelial cells in vitro.

Role of CYP2A6 in Methimazole Bioactivation and Hepatotoxicity.

Methimazole (MMI) is a widely used antithyroid drug, but it can cause hepatotoxicity by unknown mechanisms. Previous studies showed that the hepatic metabolism of MMI produces N-methylthiourea, leading to liver damage. However, the specific enzyme responsible for the production of the toxic metabolite N-methylthiourea is still unclear. In this study, we screened cytochromes P450 (CYPs) in N-methylthiourea production from MMI. CYP2A6 was identified as the key enzyme in catalyzing MMI metabolism to produce N-methylthiourea. When mice were pretreated with a CYP2A6 inhibitor, formation of N-methylthiourea from MMI was remarkably reduced. Consistently, the CYP2A6 inhibitor prevented MMI-induced hepatotoxicity. These results demonstrated that CYP2A6 is essential in MMI bioactivation and hepatotoxicity.

Discovery of new tranylcypromine derivatives as highly potent LSD1 inhibitors.

Tranylcypromine (TCP)-based structural modifications lead to the discovery of new LSD1 inhibitors, of which compounds 26b and 29b effectively inhibit LSD1 with the IC50 values of 17 and 11 nM, respectively and also show good selectivity over MAO-B. Mechanistic studies showed that compound 29b concentration-dependently induced H3K4me1/2 accumulation in LSD1 overexpressed MGC-803 cells and also inhibited metastasis of MGC-803 cells. Collectively, both compounds could be promising lead compounds for further investigation.

New tranylcypromine derivatives containing sulfonamide motif as potent LSD1 inhibitors to target acute myeloid leukemia: Design, synthesis and biological evaluation.

Lysine-specific demethylase 1 (LSD1) is frequently elevated in acute myeloid leukemia (AML) and often leads to tumorigenesis. In recent years, numerous LSD1 inhibitors based on tranylcypromine (TCP) scaffolding have reached clinical trials. Most TCP derivatives were modified at the amino site of cyclopropane motif. Herein, we for the first time introduced a sulfonamide group in TCP benzene ring of series a compounds and performed a systematical study on structure and activity relationships by varying sulfonamide groups. The introduction of sulfonamide significantly increased the targeting capacity of TCP against LSD1. Moreover, we discovered that the Boc attached LSD1 inhibitors (labelled as series b compounds) substantially improved their anti-proliferation capacity towards AML cells. The intracellular thermal shift and LC-MS/MS results implied that Boc enhanced the drug lipophilicity and might be removed under the cancerous acidic environment to release the real pharmacophore, evidenced by the fact that a structurally similar but acidic inert pivaloyl to replace Boc dramatically dropped the cellular anti-proliferation effect. Finally, a benzyl group installed at the amino site to appropriately increase lipophilicity led to trans-4-(2-(benzylamino)-cyclopropyl)-N,N-diethylbenzenesulfonamide a10 that showed better anti-proliferation activity in AML cells and enzymatic inhibition against LSD1. Taken together, our work offers a novel TCP-based structure and provides a prodrug strategy for the discovery of potent LSD1 inhibitors by having appropriate lipophilicity.

Prediction of Severity of Drug-Drug Interactions Caused by Enzyme Inhibition and Activation.

Drug-drug interactions (DDIs) severity assessment is a crucial problem because polypharmacy is increasingly common in modern medical practice. Many DDIs are caused by alterations of the plasma concentrations of one drug due to another drug inhibiting and/or inducing the metabolism or transporter-mediated disposition of the victim drug. Accurate assessment of clinically relevant DDIs for novel drug candidates represents one of the significant tasks of contemporary drug research and development and is important for practicing physicians. This work is a development of our previous investigations and aimed to create a model for the severity of DDIs prediction. PASS program and PoSMNA descriptors were implemented for prediction of all five classes of DDIs severity according to OpeRational ClassificAtion (ORCA) system: contraindicated (class 1), provisionally contraindicated (class 2), conditional (class 3), minimal risk (class 4), no interaction (class 5). Prediction can be carried out both for known drugs and for new, not yet synthesized substances using only their structural formulas. Created model provides an assessment of DDIs severity by prediction of different ORCA classes from the first most dangerous class to the fifth class when DDIs do not take place in the human organism. The average accuracy of DDIs class prediction is about 0.75.

Surfactant-assisted electromembrane extraction combined with cyclodextrin-modified capillary electrophoresis for the separation and quantification of Tranylcypromine enantiomers in biological samples.

Surfactant-assisted electromembrane extraction coupled with cyclodextrin-modified capillary electrophoresis was developed for the separation and determination of Tranylcypromine enantiomers in biological samples. This combination would provide a new strategy for selective and sensitive determination of target analytes. The addition of surfactant in the donor solution improved the analyte transport into the lumen of hollow fiber that resulted in an enhancement in the analytes migration into acceptor solution. Optimization of the variables, affecting proposed method, was carried out and best results were achieved with a 175 V potential as driving force of the electromembrane extraction, 2-nitrophenyloctylether as the supported liquid membrane, donor solution containing 0.2 mM Triton X-100 with pH 3 and 0.1 M HCl for acceptor solution. Then, the extract was analyzed using cyclodextrin-modified capillary electrophoresis method for separation of Tranylcypromine enantiomers. The best results were obtained with a phosphate running buffer (100 mM, pH 2.0) containing 7% w/v hydroxypropyl-α-cyclodextrin. Under the optimum conditions, a low limit of detection (3.03 ng/mL), good linearity (R2  > 0.9953), and relative standard deviations below 4.0% (n = 5) were obtained. Finally, this procedure was applied to determine the concentration of Tranylcypromine enantiomers in urine samples with satisfactory results.

Design, Synthesis, and In Vitro Evaluation of Novel Histone H3 Peptide-Based LSD1 Inactivators Incorporating α,α-Disubstituted Amino Acids with γ-Turn-Inducing Structures.

Lysine-specific demethylase 1 (LSD1) mainly removes methyl groups of mono- or di-methylated lysine residues at the fourth position of histone H3 to epigenetically regulate the expression of genes associated with several diseases, such as cancer. Therefore, LSD1 inactivators are expected to be used as therapeutic agents. In this study, to identify novel peptide-based LSD1 inactivators, we focused on the X-ray structure of LSD1 complexed with a H3 peptide-based suicide substrate. It has been proposed that a methylated histone substrate forms three consecutive γ-turn structures in the active pocket of LSD1. Based on this, we designed and synthesized novel histone H3 peptide-based LSD1 inactivators 2a⁻c by incorporating various α,α-disubstituted amino acids with γ-turn-inducing structures. Among synthetic peptides 2a⁻c, peptide 2b incorporating two 1-aminocyclohexanecarboxylic acids at both sides of a lysine residue bearing a trans-2-phenylcyclopropylamine (PCPA) moiety, which is a pharmacophore for LSD1 inactivation, was the most potent and selective LSD1 inactivator. These findings are useful for the further development of histone H3 peptide-based LSD1 inactivators.

Towards the development of activity-based probes for detection of lysine-specific demethylase-1 activity.

The implications of lysine-specific demethylase-1 (LSD1) in tumorigenesis have urged scientists to develop diagnostic tools in order to explore the function of this enzyme. In this work, we present our efforts on the development of tranylcypromine (TCP)-based functionalized probes for activity-based protein profiling (ABPP) of LSD1 activity. Biotinylated forms of selected compounds enabled dose-dependent enzyme labeling of recombinant LSD1. However, treatment with LSD1 inhibitors did not clearly reduce the LSD1 labeling efficiency thus indicating that labeling using these probes is not activity dependent. This calls for alternative strategies to develop probes for ABPP of the enzyme LSD1.

Gram-Scale Synthesis of Chiral Cyclopropane-Containing Drugs and Drug Precursors with Engineered Myoglobin Catalysts Featuring Complementary Stereoselectivity.

Engineered hemoproteins have recently emerged as promising systems for promoting asymmetric cyclopropanations, but variants featuring predictable, complementary stereoselectivity in these reactions have remained elusive. In this study, a rationally driven strategy was implemented and applied to engineer myoglobin variants capable of providing access to 1-carboxy-2-aryl-cyclopropanes with high trans-(1R,2R) selectivity and catalytic activity. The stereoselectivity of these cyclopropanation biocatalysts complements that of trans-(1S,2S)-selective variants developed here and previously. In combination with whole-cell biotransformations, these stereocomplementary biocatalysts enabled the multigram synthesis of the chiral cyclopropane core of four drugs (Tranylcypromine, Tasimelteon, Ticagrelor, and a TRPV1 inhibitor) in high yield and with excellent diastereo- and enantioselectivity (98-99.9% de; 96-99.9% ee). These biocatalytic strategies outperform currently available methods to produce these drugs.

Targeting Cancer with PCPA-Drug Conjugates: LSD1 Inhibition-Triggered Release of 4-Hydroxytamoxifen.

Targeting cancer with small molecule prodrugs should help overcome problems associated with conventional cancer-targeting methods. Herein, we focused on lysine-specific demethylase 1 (LSD1) to trigger the controlled release of anticancer drugs in cancer cells, where LSD1 is highly expressed. Conjugates of the LSD1 inhibitor trans-2-phenylcyclopropylamine (PCPA) were used as novel prodrugs to selectively release anticancer drugs by LSD1 inhibition. As PCPA-drug conjugate (PDC) prototypes, we designed PCPA-tamoxifen conjugates 1 a and 1 b, which released 4-hydroxytamoxifen in the presence of LSD1 in vitro. Furthermore, 1 a and 1 b inhibited the growth of breast cancer cells by the simultaneous inhibition of LSD1 and the estrogen receptor without exhibiting cytotoxicity toward normal cells. These results demonstrate that PDCs provide a useful prodrug method that may facilitate the selective release of drugs in cancer cells.

An overview of phenylcyclopropylamine derivatives: biochemical and biological significance and recent developments.

trans-2-Phencylcyclopropylamine (2-PCPA), a potent, clinically used antidepressant, affects monoamine neurotransmitter levels by inhibiting the main metabolizing enzymes, monoamine oxidases (MAOs). However, the antidepressant action of this compound was not fully explained by its effects on MAOs due to its wide variety of biological effects. 2-PCPA also affects depression-associated pathophysiological pathways, and linked with increased levels of trace amines in brain, upregulation of GABAB receptors (where GABA is gamma amino butyric acid), modulation of phospholipid metabolism, and interference with various cytochrome P450 (CYP) enzymes. Consequently, despite its adverse effects and limited clinical applicability, 2-PCPA has attracted interest as a structural scaffold for the development of mechanism-based inhibitors of various enzymes, including lysine-specific demethylase 1 (LSD1), which is a possible target for cancer chemotherapy. In the recent years, many reports have appeared in the literature based on 2-PCPA scaffold and their potential medicinal implications. This review mainly focuses on the medicinal chemistry aspects including drug design, structure-activity relationships (SAR), biological and biochemical properties, and mechanism of actions of 2-PCPA and its derivatives. Furthermore, we also highlight recent advance in this area and discuss their future applications for beneficial therapeutic effects.

CD86 expression as a surrogate cellular biomarker for pharmacological inhibition of the histone demethylase lysine-specific demethylase 1.

There is a lack of rapid cell-based assays that read out enzymatic inhibition of the histone demethylase LSD1 (lysine-specific demethylase 1). Through transcriptome analysis of human acute myeloid leukemia THP1 cells treated with a tranylcypromine-derivative inhibitor of LSD1 active in the low nanomolar range, we identified the cell surface marker CD86 as a sensitive surrogate biomarker of LSD1 inhibition. Within 24h of enzyme inhibition, there was substantial and dose-dependent up-regulation of CD86 expression, as detected by quantitative polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay. Thus, the use of CD86 expression may facilitate screening of compounds with putative LSD1 inhibitory activities in cellular assays.

The opposite effect of isotype-selective monoamine oxidase inhibitors on adipogenesis in human bone marrow mesenchymal stem cells.

Adiponectin production during adipocyte differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) can be used to evaluate the pharmacological activity of anti-diabetic drugs to improve insulin sensitivity. Monoamine oxidase (MAO) inhibitors such as phenelzine and pargyline inhibit adipogenesis in murine pre-adipocytes. In this study, however, we found that selective MAO-A inhibitors, moclobemide and Ro41-1049, and a selective MAO-B inhibitor, selegiline, promoted adiponectin production during adipocyte differentiation in hBM-MSCs, which suggested the anti-diabetic potential of these drugs. In contrast, non-selective MAO inhibitors, phenelzine and tranylcypromine, inhibited adipocyte differentiation of hBM-MSCs. Concomitant treatments of MAO-A and MAO-B selective inhibitors did not change the stimulatory effect on adiponectin production in hBM-MSCs. Taken together, the opposite effects of isotype-selective MAO inhibitors on adiponectin production during adipogenesis in hBM-MSCs may not be directly associated with the inhibitory effects of MAO, suggested that the structure of MAO inhibitors may contain a novel anti-diabetic pharmacophore.

In silico and intuitive predictions of CYP46A1 inhibition by marketed drugs with subsequent enzyme crystallization in complex with fluvoxamine.

Cytochrome P450 46A1 (cholesterol 24-hydroxylase) is an important brain enzyme that may be inhibited by structurally distinct pharmaceutical agents both in vitro and in vivo. To identify additional inhibitors of CYP46A1 among U.S. Food and Drug Administration-approved therapeutic agents, we used in silico and intuitive predictions and evaluated some of the predicted binders in the enzyme and spectral binding assays. We tested a total of 298 marketed drugs for the inhibition of CYP46A1-mediated cholesterol hydroxylation in vitro and found that 13 of them reduce CYP46A1 activity by >50%. Of these 13 inhibitors, 7 elicited a spectral response in CYP46A1 with apparent spectral K(d) values in a low micromolar range. One of the identified tight binders, the widely used antidepressant fluvoxamine, was cocrystallized with CYP46A1. The structure of this complex was determined at a 2.5 Å resolution and revealed the details of drug binding to the CYP46A1 active site. The NH(2)-containing arm of the Y-shaped fluvoxamine coordinates the CYP46A1 heme iron, whereas the methoxy-containing arm points away from the heme group and has multiple hydrophobic interactions with aliphatic amino acid residues. The CF(3)-phenyl ring faces the entrance to the substrate access channel and has contacts with the aromatic side chains. The crystal structure suggests that only certain drug conformers can enter the P450 substrate access channel and reach the active site. Once inside the active site, the conformer probably further adjusts its configuration and elicits the movement of the protein side chains.

Novel colorimetric assay of LSD1 activity using gold nanoparticles.

We have developed a simple and sensitive strategy for colorimetric LSD1 enzyme activity assay using avidin modified gold nanoparticles. The strategy is based on the vivid color change of a gold nanoparticle solution from red to violet upon addition of a test solution of peptide-antibody treated with LSD1. Thus, the presence of LSD1 in a sample can be determined by simple visual inspection with the naked eye. In addition, a wide range of LSD1 concentrations (13 pM to 0.13 µM) were quantitatively determined by spectrophotometry, which shows the possibility of quantitative analysis of the over expression levels of LSD1 in cancer tissue samples.

An Update of Lysine Specific Demethylase 1 Inhibitor: A Patent Review (2016-2020).

BACKGROUND: As a FAD (Flavin Adenine Dinucleotide) - dependent histone demethylase discovered in 2004, LSD1 (lysine-specific demethylase 1) was reported to be overexpressed in diverse tumors, regulating target genes transcription associated with cancer development. Hence, LSD1 targeted inhibitors may represent a new insight in anticancer drug discovery. For these reasons, researchers in both the pharmaceutical industry and academia have been actively pursuing LSD1 inhibitors in the quest for new anti-cancer drugs. OBJECTIVES: This review summaries patents about LSD1 inhibitors in recent 5 years in the hope of providing a reference for LSD1 researchers to develop new modulators of LSD1 with higher potency and fewer adverse effects. METHODS: This review collects LSD1 inhibitors disclosed in patents since 2016. The primary ways of patent searching are Espacenet®, Google Patents, and CNKI. RESULTS: This review covers dozens of patents related to LSD1 inhibitors in recent five years. The compound structures are mainly divided into TCP (Tranylcypromine) derivatives, imidazole derivatives, pyrimidine derivatives, and other natural products and peptides. Meanwhile, the compounds that have entered the clinical phase are also described. CONCLUSION: Most of the compounds in these patents have been subjected to activity analysis with LSD1 and multi-cell lines, showing good antitumor activity in vitro and in vivo. These patents exhibited the structural diversity of LSD1 inhibitors and the potential of natural products as novel LSD1 inhibitors.

Structural basis of drug binding to CYP46A1, an enzyme that controls cholesterol turnover in the brain.

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.

Enantioselective synthesis of tranylcypromine analogues as lysine demethylase (LSD1) inhibitors.

Asymmetric cyclopropanation of styrenes by tert-butyl diazoacetate followed by ester hydrolysis and Curtius rearrangement gave a series of tranylcypromine analogues as single enantiomers. The o,- m- and p-bromo analogues were all more active than tranylcypromine in a LSD1 enzyme assay. The m- and p-bromo analogues were micromolar growth inhibitors of the LNCaP prostate cancer cell line as were the corresponding biphenyl analogues prepared from the bromide by Suzuki crosscoupling.

QSAR Modeling, Molecular Docking and Molecular Dynamics Simulations Studies of Lysine-Specific Demethylase 1 (LSD1) Inhibitors as Anticancer Agents.

BACKGROUND: Histone Lysine Demetylases1 (LSD1) is a promising medication to treat cancer, which plays a crucial role in epigenetic modulation of gene expression. Inhibition of LSD1with small molecules has emerged as a vital mechanism to treat cancer. OBJECTIVE: In the present research, molecular modeling investigations, such as CoMFA, CoMFA-RF, CoMSIA and HQSAR, molecular docking and Molecular Dynamics (MD) simulations were carried out on some tranylcypromine derivatives as LSD1 inhibitors. METHODS: The QSAR models were carried out on a series of Tranylcypromine derivatives as data set via the SYBYL-X2.1.1 program. Molecular docking and MD simulations were carried out by the MOE software and the SYBYL program, respectively. The internal and external predictability performances related to the generated models for these LSD1 inhibitors were justified by evaluating cross-validated correlation coefficient (q2), noncross- validated correlation coefficient (r2ncv) and predicted correlation coefficient (r2pred) of the training and test set molecules, respectively. RESULTS: The CoMFA (q2, 0.670; r2ncv, 0.930; r2pred, 0.968), CoMFA-RF (q2, 0.694; r2ncr, 0.926; r2pred, 0.927), CoMSIA (q2, 0.834; r2ncv, 0.956; r2pred, 0.958) and HQSAR models (q2, 0.854; r2ncv, 0.900; r2pred, 0.728) for training as well as the test set of LSD1 inhibition resulted in significant findings. CONCLUSION: These QSAR models were found to be perfect and strong with better predictability. Contour maps of all models were generated and it was proven by molecular docking studies and molecular dynamics simulation that the hydrophobic, electrostatic and hydrogen bonding fields are crucial in these models for improving the binding affinity and determining the structure-activity relationship. These theoretical results are possibly beneficial to design new strong LSD1 inhibitors with enhanced activity to treat cancer.