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.

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.

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.

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.

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.

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.

Tranylcypromine Based Lysine-Specific Demethylase 1 Inhibitor: Summary and Perspective.

Histone lysine-specific demethylase 1 (LSD1/KDM1A) has become an important and promising anticancer target since it was first identified in 2004 and specially demethylates lysine residues of histone H3K4me1/2 and H3K9me1/2. LSD1 is ubiquitously overexpressed in diverse cancers, and abrogation of LSD1 results in inhibition of proliferation, invasion, and migration in cancer cells. Over the past decade, a number of biologically active small-molecule LSD1 inhibitors have been developed. To date, six trans-2-phenylcyclopropylamine (TCP)-based LSD1 inhibitors (including TCP, ORY-1001, GSK-2879552, INCB059872, IMG-7289, and ORY-2001) that covalently bind to the flavin adenine dinucleotide (FAD) within the LSD1 catalytic cavity have already entered into clinical trials. Here, we provide an overview about the structures, activities, and structure-activity relationship (SAR) of TCP-based LSD1 inhibitors that mainly covers the literature from 2008 to date. The opportunities, challenges, and future research directions in this emerging and promising field are also discussed.

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.

Development and Structural Evaluation of N-Alkylated trans-2-Phenylcyclopropylamine-Based LSD1 Inhibitors.

Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyzes the demethylation of histone H3 and regulates gene expression. Because it is implicated in the regulation of diseases such as acute myeloid leukemia, potent LSD1-specific inhibitors have been pursued. Trans-2-phenylcyclopropylamine (2-PCPA)-based inhibitors featuring substitutions on the amino group have emerged, with sub-micromolar affinities toward LSD1 and high selectivities over monoamine oxidases (MAOs). We synthesized two N-alkylated 2-PCPA-based LSD1 inhibitors, S2116 and S2157, based on the previously developed S2101. S2116 and S2157 exhibited enhanced potency for LSD1 by 2.0- to 2.6-fold, as compared with S2101. In addition, they exhibited improved selectivity over MAOs. Structural analyses of LSD1 co-crystallized with S2101, S2116, S2157, or another N-alkylated inhibitor (FCPA-MPE) confirmed that the N-substituents enhance the potency of a 2-PCPA-based inhibitor of LSD1, without constituting the adduct formed with FAD.

Tranylcypromine-Based LSD1 Inhibitors: Structure-Activity Relationships, Antiproliferative Effects in Leukemia, and Gene Target Modulation.

LSD1 is a lysine demethylase highly involved in initiation and development of cancer. To design highly effective covalent inhibitors, a strategy is to fill its large catalytic cleft by designing tranylcypromine (TCP) analogs decorated with long, hindered substituents. We prepared three series of TCP analogs, carrying aroyl- and arylacetylamino (1 a-h), Z-amino acylamino (2 a-o), or double-substituted benzamide (3 a-n) residues at the C4 or C3 position of the phenyl ring. Further fragments obtained by chemical manipulation applied on the TCP scaffold (compounds 4 a-i) were also prepared. When tested against LSD1, most of 1 and 3 exhibited IC50 values in the low nanomolar range, with 1 e and 3 a,d,f,g being also the most selective respect to monoamine oxidases. In MV4-11 AML and NB4 APL cells compounds 3 were the most potent, displaying up to sub-micromolar cell growth inhibition against both cell lines (3 a) or against NB4 cells (3 c). The most potent compounds in cellular assays were also able to induce the expression of LSD1 target genes, such as GFI-1b, ITGAM, and KCTD12, as functional read-out for LSD1 inhibition. Mouse and human intrinsic clearance data highlighted the high metabolic stability of compounds 3 a, 3 d and 3 g. Further studies will be performed on the new compounds 3 a and 3 c to assess their anticancer potential in different cancer contexts.

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.

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.

Tying up tranylcypromine: Novel selective histone lysine specific demethylase 1 (LSD1) inhibitors.

Aberrant expression of lysine specific histone demethylase 1 (LSD1) has been increasingly associated with numerous cancer cells and several proof-of-concept studies are strongly suggestive of its potential as a druggable target. Tranylcypromine (TCP) is an antidepressant originally known to target the monoamine oxidases A and B (MAO-A and MAO-B), which are structurally related to LSD1. A number of TCP derivatives have been identified as potent LSD1 inhibitors, with a handful of them currently being tested in clinical trials. However, thus far the majority of structure-activity relationship studies reported on these TCP derivatives have been mostly limited to the racemates. In this study, we present the SAR data for a novel series of conformationally-restricted TCP-based LSD1 inhibitors, both in their racemic and enantiomerically pure forms. Compounds 18b and 19b were identified as the most potent LSD1 inhibitors within this series, possessing excellent selectivity (>10,000-fold) against MAO-A and MAO-B. These compounds activated CD86 expression on the human MV4-11 AML cells following 10 days of exposure, accompanied with the apparent cytotoxicity. Taken together, these findings are consistent with the pharmacological inhibition of LSD1 and further provide structural insights on the binding modes of these TCP derivatives and their enantiomers at the LSD1.

Design and synthesis of tranylcypromine derivatives as novel LSD1/HDACs dual inhibitors for cancer treatment.

Lysine specific demethylase 1 (LSD1) and Histone deacetylases (HDACs) are promising drug targets for cancers. Recent studies reveal an important functional interplay between LSD1 and HDACs, and there is evidence for the synergistic effect of combined LSD1 and HDAC inhibitors on cancers. Therefore, development of inhibitors targeting both LSD1 and HDACs might be a promising strategy for epigenetic therapy of cancers. We report herein the synthesis of a series of tranylcypromine derivatives as LSD1/HDACs dual inhibitors. Most compounds showed potent LSD1 and HDACs inhibitory activity, especially compound 7 displayed the most potent inhibitory activity against HDAC1 and HDAC2 with IC50 of 15 nM and 23 nM, as well as potent inhibition against LSD1 with IC50 of 1.20 µM. Compound 7 demonstrated stronger anti-proliferative activities than SAHA with IC50 values ranging from 0.81 to 4.28 µM against MGC-803, MCF-7, SW-620 and A-549 human cancer cell lines. Further mechanistic studies showed that compound 7 treatment in MGC-803 cells dose-dependently increased cellular H3K4 and H3K9 methylation, as well as H3 acetylation, decreased the mitochondrial membrane potential and induced remarkable apoptosis. Docking studies showed that compound 7 can be well docked into the active binding sites of LSD1 and HDAC2. This finding highlights the potential for the development of LSD1/HDACs dual inhibitors as novel anticancer drugs.

Development of (4-Cyanophenyl)glycine Derivatives as Reversible Inhibitors of Lysine Specific Demethylase 1.

Inhibition of lysine specific demethylase 1 (LSD1) has been shown to induce the differentiation of leukemia stem cells in acute myeloid leukemia (AML). Irreversible inhibitors developed from the nonspecific inhibitor tranylcypromine have entered clinical trials; however, the development of effective reversible inhibitors has proved more challenging. Herein, we describe our efforts to identify reversible inhibitors of LSD1 from a high throughput screen and subsequent in silico modeling approaches. From a single hit (12) validated by biochemical and biophysical assays, we describe our efforts to develop acyclic scaffold-hops from GSK-690 (1). A further scaffold modification to a (4-cyanophenyl)glycinamide (e.g., 29a) led to the development of compound 32, with a Kd value of 32 nM and an EC50 value of 0.67 µM in a surrogate cellular biomarker assay. Moreover, this derivative does not display the same level of hERG liability as observed with 1 and represents a promising lead for further development.

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.

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.

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.

Irreversible LSD1 Inhibitors: Application of Tranylcypromine and Its Derivatives in Cancer Treatment.

Due to the increasing costs and time consuming for new drug discovery, a large number of pharmaceutical firms have chosen to modify the existing drug molecules for repositioning candidates with new or improved properties, especially those with severe adverse effects, thereby accelerating the drug discovery process. Such strategy has witnessed its success with several examples reported. As the first identified histone lysine specific demethylase, lysine specific demethylase 1 (LSD1) is classified as a member of monoamine oxidase (MAO) superfamily, and specifically removes mono- and dimethylated histone 3 lysine 4 (H3K4) and H3 lysine 9 (H3K9). It has been reported that LSD1 and its downstream targets are involved in cancer cell growth and metastasis. Meanwhile, it is overexpressed in a variety of tumor cells. Inactivating LSD1 specifically inhibits tumor progression and metastasis. Hence, LSD1 inhibition may represent a new and promising direction in anti-cancer drug discovery. Based on the structure and cofactor of LSD1, some clinical applied MAO inhibitors have been identified as LSD1 inactivators. Among them, tranylcypromine presented the most potency against LSD1 and its derivatives were further developed by medicinal chemists in order to develop potent and selective LSD1 inhibitors. Currently, a number of tranylcypromine based LSD1 inhibitors have been developed and two of them, ORY-1001 and GSK2879552, are in clinical trials for cancer treatment. This review highlights recent advances in the repurposing of tranylcypromine and its derivatives as irreversible LSD1 inhibitors for cancer treatment, which are conventionally used for the treatment of depression.

Discovery of a Novel Inhibitor of Histone Lysine-Specific Demethylase 1A (KDM1A/LSD1) as Orally Active Antitumor Agent.

We report the stereoselective synthesis and biological activity of a novel series of tranylcypromine (TCPA) derivatives (14a-k, 15, 16), potent inhibitors of KDM1A. The new compounds strongly inhibit the clonogenic potential of acute leukemia cell lines. In particular three molecules (14d, 14e, and 14g) showing selectivity versus MAO A and remarkably inhibiting colony formation in THP-1 human leukemia cells, were assessed in mouse for their preliminary pharmacokinetic. 14d and 14e were further tested in vivo in a murine acute promyelocytic leukemia model, resulting 14d the most effective. Its two enantiomers were synthesized: the (1S,2R) enantiomer 15 showed higher activity than its (1R,2S) analogue 16, in both biochemical and cellular assays. Compound 15 exhibited in vivo efficacy after oral administration, determining a 62% increased survival in mouse leukemia model with evidence of KDM1A inhibition. The biological profile of compound 15 supports its further investigation as a cancer therapeutic.