RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Tranilcipromina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Relação Estrutura-Atividade , Tranilcipromina/síntese química , Tranilcipromina/químicaRESUMO
Lysine-specific demethylase 1 (LSD1 or KDM1A) is a FAD-dependent enzyme that acts as a transcription corepressor or coactivator by regulating the methylation status of histone H3 lysines K4 and K9, respectively. KDM1A represents an attractive target for cancer therapy. While, in the past, the main medicinal chemistry strategy toward KDM1A inhibition was based on the optimization of ligands that irreversibly bind the FAD cofactor within the enzyme catalytic site, we and others have also identified reversible inhibitors. Herein we reported the discovery of 5-imidazolylthieno[3,2-b]pyrroles, a new series of KDM1A inhibitors endowed with picomolar inhibitory potency, active in cells and efficacious after oral administration in murine leukemia models.
RESUMO
A number of mono- and bis-quaternary ammonium salts, containing edrophonium-like and coumarin moieties tethered by an appropriate linker, proved to be highly potent and selective dual binding site acetylcholinesterase inhibitors with good selectivity over butyrylcholinesterase. Homobivalent bis-quaternary inhibitors 11 and 12, differing by only one methylene unit in the linker, were the most potent and selective inhibitors exhibiting a sub-nanomolar affinity (IC(50)=0.49 and 0.17 nM, respectively) and a high butyryl-/acetylcholinesterase affinity ratio (SI=1465 and 4165, respectively). The corresponding hetero-bivalent coumarinic inhibitors 13 and 14 were also endowed with excellent inhibitory potency but a lower AChE selectivity (IC(50)=2.1 and 1.0 nM, and SI=505 and 708, respectively). Docking simulations enabled clear interpretation of the structure-affinity relationships and detection of key binding interactions at the primary and peripheral AChE binding sites.
Assuntos
Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Edrofônio/química , Edrofônio/farmacologia , Acetilcolinesterase/metabolismo , Animais , Butirilcolinesterase/metabolismo , Bovinos , Simulação por Computador , Desenho de Fármacos , Cavalos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Histone lysine demethylases (KDMs) are well-recognized targets in oncology drug discovery. They function at the post-translation level controlling chromatin conformation and gene transcription. KDM1A is a flavin adenine dinucleotide-dependent amine oxidase, overexpressed in several tumor types, including acute myeloid leukemia, neuroblastoma and non-small-cell lung cancer. Among the many known monoamine oxidase inhibitors screened for KDM1A inhibition, tranylcypromine emerged as a moderately active hit, which irreversibly binds to the flavin adenine dinucleotide cofactor. MATERIAL & METHODS: The KDM1A inhibitors 5a-w were synthesized and tested in vitro and in vivo. The biochemical potency was determined, modulation of target in cells was demonstrated on KDM1A-dependent genes and the anti-clonogenic activity was performed in murine acute promyelocytic Leukemia (APL) blasts. An in vivo efficacy experiment was conducted using an established murine promyelocytic leukemia model. RESULTS: We report a new series of tranylcypromine derivatives substituted on the cyclopropyl moiety, endowed with high potency in both biochemical and cellular assays. CONCLUSION: The most interesting derivative (5a) significantly improved survival rate after oral administration in a murine model of promyelocitic leukemia.
Assuntos
Antineoplásicos/síntese química , Histona Desmetilases/antagonistas & inibidores , Leucemia Promielocítica Aguda/tratamento farmacológico , Tranilcipromina/análogos & derivados , Tranilcipromina/síntese química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Leucemia Promielocítica Aguda/patologia , Camundongos , Relação Estrutura-Atividade , Tranilcipromina/farmacocinética , Tranilcipromina/farmacologiaRESUMO
The balance of methylation levels at histone H3 lysine 4 (H3K4) is regulated by KDM1A (LSD1). KDM1A is overexpressed in several tumor types, thus representing an emerging target for the development of novel cancer therapeutics. We have previously described ( Part 1, DOI 10.1021.acs.jmedchem.6b01018 ) the identification of thieno[3,2-b]pyrrole-5-carboxamides as novel reversible inhibitors of KDM1A, whose preliminary exploration resulted in compound 2 with biochemical IC50 = 160 nM. We now report the structure-guided optimization of this chemical series based on multiple ligand/KDM1A-CoRest cocrystal structures, which led to several extremely potent inhibitors. In particular, compounds 46, 49, and 50 showed single-digit nanomolar IC50 values for in vitro inhibition of KDM1A, with high selectivity in secondary assays. In THP-1 cells, these compounds transcriptionally affected the expression of genes regulated by KDM1A such as CD14, CD11b, and CD86. Moreover, 49 and 50 showed a remarkable anticlonogenic cell growth effect on MLL-AF9 human leukemia cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Lisina/química , Pirróis/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Histona Desmetilases , Humanos , Concentração Inibidora 50 , Pirróis/química , Relação Estrutura-AtividadeRESUMO
Lysine specific demethylase 1 KDM1A (LSD1) regulates histone methylation and it is increasingly recognized as a potential therapeutic target in oncology. We report on a high-throughput screening campaign performed on KDM1A/CoREST, using a time-resolved fluorescence resonance energy transfer (TR-FRET) technology, to identify reversible inhibitors. The screening led to 115 hits for which we determined biochemical IC50, thus identifying four chemical series. After data analysis, we have prioritized the chemical series of N-phenyl-4H-thieno[3, 2-b]pyrrole-5-carboxamide for which we obtained X-ray structures of the most potent hit (compound 19, IC50 = 2.9 µM) in complex with the enzyme. Initial expansion of this chemical class, both modifying core structure and decorating benzamide moiety, was directed toward the definition of the moieties responsible for the interaction with the enzyme. Preliminary optimization led to compound 90, which inhibited the enzyme with a submicromolar IC50 (0.162 µM), capable of inhibiting the target in cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Pirróis/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Pirróis/química , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Histona Desmetilases/antagonistas & inibidores , Leucemia Promielocítica Aguda/tratamento farmacológico , Tranilcipromina/química , Tranilcipromina/uso terapêutico , Administração Oral , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Histona Desmetilases/metabolismo , Humanos , Leucemia Promielocítica Aguda/metabolismo , Relação Estrutura-Atividade , Tranilcipromina/administração & dosagem , Tranilcipromina/farmacologiaRESUMO
Histone demethylase KDM1A (also known as LSD1) has become an attractive therapeutic target for the treatment of cancer as well as other disorders such as viral infections. We report on the synthesis of compounds derived from the expansion of tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. These compounds, which are substituted on the cyclopropyl core moiety, were evaluated for their ability to inhibit KDM1A in vitro as well as to function in cells by modulating the expression of Gfi-1b, a well recognized KDM1A target gene. The molecules were all found to covalently inhibit KDM1A and to become increasingly selective against human monoamine oxidases MAO A and MAO B through the introduction of bulkier substituents on the cyclopropylamine ring. Structural and biochemical analysis of selected trans isomers showed that the two stereoisomers are endowed with similar inhibitory activities against KDM1A, but form different covalent adducts with the FAD co-enzyme.
Assuntos
Ciclopropanos/farmacologia , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Monoaminoxidase/metabolismo , Cristalografia por Raios X , Ciclopropanos/síntese química , Ciclopropanos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Histona Desmetilases/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
In this account, we report the development of a series of substituted cinnamic anilides that represents a novel class of mitochondrial permeability transition pore (mPTP) inhibitors. Initial class expansion led to the establishment of the basic structural requirements for activity and to the identification of derivatives with inhibitory potency higher than that of the standard inhibitor cyclosporine-A (CsA). These compounds can inhibit mPTP opening in response to several stimuli including calcium overload, oxidative stress, and thiol cross-linkers. The activity of the cinnamic anilide mPTP inhibitors turned out to be additive with that of CsA, suggesting for these inhibitors a molecular target different from cyclophylin-D. In vitro and in vivo data are presented for (E)-3-(4-fluoro-3-hydroxy-phenyl)-N-naphthalen-1-yl-acrylamide 22, one of the most interesting compounds in this series, able to attenuate opening of the mPTP and limit reperfusion injury in a rabbit model of acute myocardial infarction.
Assuntos
1-Naftilamina/análogos & derivados , Acrilamidas/química , Anilidas/química , Cinamatos/química , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , 1-Naftilamina/síntese química , 1-Naftilamina/química , 1-Naftilamina/farmacologia , Acrilamidas/síntese química , Acrilamidas/farmacologia , Anilidas/síntese química , Anilidas/farmacologia , Animais , Cálcio/metabolismo , Cinamatos/síntese química , Cinamatos/farmacologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Coelhos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The histone deacetylases (HDACs) are able to regulate gene expression, and histone deacetylase inhibitors (HDACi) emerged as a new class of agents in the treatment of cancer as well as other human disorders such as neurodegenerative diseases. In the present investigation, we report on the synthesis and biological evaluation of compounds derived from the expansion of a HDAC inhibitor scaffold having N-hydroxy-3-phenyl-2-propenamide and N-hydroxy-3-(pyridin-2-yl)-2-propenamide as core structures and containing a phenyloxopropenyl moiety, either unsubstituted or substituted by a 4-methylpiperazin-1-yl or 4-methylpiperazin-1-ylmethyl group. The compounds were evaluated for their ability to inhibit nuclear HDACs, as well as for their in vitro antiproliferative activity. Moreover, their metabolic stability in microsomes and aqueous solubility were studied and selected compounds were further characterized by in vivo pharmacokinetic experiments. These compounds showed a remarkable stability in vivo, compared to hydroxamic acid HDAC inhibitors that have already entered clinical trials. The representative compound 30b showed in vivo antitumor activity in a human colon carcinoma xenograft model.