RESUMO
Kempopeptin C, a novel chlorinated analogue of kempopeptin B, was discovered from a marine cyanobacterium collected from Kemp Channel in Florida. The structure was elucidated using NMR spectroscopy and mass spectrometry (MS). The presence of the basic Lys residue adjacent to the N-terminus of the 3-amino-6-hydroxy-2-piperidone (Ahp) moiety contributed to its selectivity towards trypsin and related proteases. The antiproteolytic activity of kempopeptin C was evaluated against trypsin, plasmin and matriptase and found to inhibit these enzymes with IC50 values of 0.19, 0.36 and 0.28 µM, respectively. Due to the significance of these proteases in cancer progression and metastasis, as well as their functional redundancy with respect to targeting overlapping substrates, we examined the effect of kempopeptin C on the downstream cellular substrates of matriptase: CDCP1 and desmoglein-2 (Dsg-2). Kempopeptin C was shown to inhibit the cleavage of both substrates in vitro. Additionally, kempopeptin C reduced the cleavage of CDCP1 in MDA-MB-231 cells up to 10 µM. The functional relevance of targeting matriptase and related proteases was investigated by assessing the effect of kempopeptin C on the migration of breast cancer cells. Kempopeptin C inhibited the migration of the invasive MDA-MB-231 cells by 37 and 60% at 10 and 20 µM, respectively.
Assuntos
Antineoplásicos/farmacologia , Organismos Aquáticos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral/efeitos dos fármacos , Cianobactérias/química , Depsipeptídeos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Animais , Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Depsipeptídeos/uso terapêutico , Feminino , Humanos , Inibidores de Serina Proteinase/uso terapêuticoRESUMO
Largazole is a potent and class I-selective histone deacetylase (HDAC) inhibitor purified from marine cyanobacteria and was demonstrated to possess antitumor activity. Largazole employs a unique prodrug strategy, via a thioester moiety, to liberate the bioactive species largazole thiol. Here we report alternate prodrug strategies to modulate the pharmacokinetic and pharmacodynamics profiles of new largazole-based compounds. The in vitro effects of largazole analogues on cancer cell proliferation and enzymatic activities of purified HDACs were comparable to the natural product. However, in vitro and in vivo histone hyperacetylation in HCT116 cells and implanted tumors, respectively, showed differences, particularly in the onset of action and oral bioavailability. These results indicate that, by employing a different approach to disguise the "warhead" moiety, the functional consequence of these prodrugs can be significantly modulated. Our data corroborate the role of the pharmacokinetic properties of this class of compounds to elicit the desired and timely functional response.
RESUMO
Largazole is a potent class I selective histone deacetylase (HDAC) inhibitor. The majority of largazole analogues to date have modified the thiazole-thiazoline and the warhead moiety. In order to elucidate class I-specific structure-activity relationships, a series of analogues with modifications in the valine or the linker region were prepared and evaluated for their class I isoform selectivity. The inhibition profile showed that the C2 position of largazole has an optimal steric requirement for efficient HDAC inhibition and that substitution of the trans-alkene in the linker with an aromatic group results in complete loss of activity. This data will aid the design of class I isoform selective HDAC inhibitors.
Assuntos
Depsipeptídeos/farmacologia , Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Tiazóis/farmacologia , Depsipeptídeos/síntese química , Depsipeptídeos/química , Relação Dose-Resposta a Droga , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
A highly sensitive and specific LC-MS/MS method for the quantitation of largazole thiol, the active species of the marine-derived preclinical histone deacetylase inhibitor, largazole (prodrug), was developed and validated. Largazole thiol was extracted with ethyl acetate from human or rat plasma along with the internal standard, harmine. Samples were separated on an Onyx Monolithic C18 column by a stepwise gradient elution with 0.1% formic acid in methanol and 0.1% aqueous formic acid employing multiple reaction monitoring (MRM) detection. Linear calibration curves were obtained in the range of 12.5-400 ng/mL with 200 µL of human plasma. The overall intra-day precision was from 3.87% to 12.6%, and the inter-day precision was from 7.12% to 9.8%. The accuracy at low, medium and high concentrations ranged from 101.55% to 105.84%. Plasma protein bindings of largazole thiol in human and rat plasma as determined by an ultrafiltration method were 90.13% and 77.14%, respectively. Plasma drug concentrations were measured by this LC-MS/MS method. The pharmacokinetics of largazole thiol in rats was studied following i.v. administration at 10 mg/kg and found to follow a two-compartment model. Largazole thiol was rapidly eliminated from systemic circulation within 2 h. The established LC-MS/MS method is suitable for the analysis of largazole thiol in human plasma, as well.
Assuntos
Depsipeptídeos/farmacocinética , Inibidores de Histona Desacetilases/farmacocinética , Tiazóis/farmacocinética , Animais , Cromatografia Líquida de Alta Pressão , Meia-Vida , Humanos , Indicadores e Reagentes , Masculino , Espectrometria de Massas , Plasma/química , Ligação Proteica , Controle de Qualidade , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
We discovered new structural diversity to a prevalent, yet medicinally underappreciated, cyanobacterial protease inhibitor scaffold and undertook comprehensive protease profiling to reveal potent and selective elastase inhibition. Structure-activity relationship (SAR) studies and X-ray cocrystal structure analysis allowed a detailed assessment of critical and tunable structural elements. To realize the therapeutic potential of these cyclodepsipeptides, we probed the cellular effects of a novel and representative family member, symplostatin 5 (1), which attenuated the downstream cellular effects of elastase in an epithelial lung airway model system, alleviating clinical hallmarks of chronic pulmonary diseases such as cell death, cell detachment, and inflammation. This compound attenuated the effects of elastase on receptor activation, proteolytic processing of the adhesion protein ICAM-1, NF-κB activation, and transcriptomic changes, including the expression of pro-inflammatory cytokines IL1A, IL1B, and IL8. Compound 1 exhibited activity comparable to the clinically approved elastase inhibitor sivelestat in short-term assays and demonstrated superior sustained activity in longer-term assays.
Assuntos
Anti-Inflamatórios/farmacologia , Brônquios/efeitos dos fármacos , Cianobactérias/química , Inibidores Enzimáticos/farmacologia , Elastase Pancreática/antagonistas & inibidores , Brônquios/citologia , Linhagem Celular , Cristalografia por Raios X , Inibidores Enzimáticos/isolamento & purificação , Células Epiteliais/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Inspired by marine cyanobacterial natural products, we synthesized modified peptides with a central statine-core unit, characteristic for aspartic protease inhibition. A series of tasiamide B analogues inhibited BACE1, a therapeutic target in Alzheimer's disease. We probed the stereospecificity of target engagement and determined additional structure-activity relationships with respect to BACE1 and related aspartic proteases, cathepsins D and E. We cocrystallized selected inhibitors with BACE1 to reveal the structural basis for the activity. Hybrid molecules that combine features of tasiamide B and an isophthalic acid moiety-containing sulfonamide showed nanomolar cellular activity. Compounds were screened in a series of rigorous complementary cell-based assays. We measured secreted APP ectodomain (sAPPß), membrane bound carboxyl terminal fragment (CTF), levels of ß-amyloid (Aß) peptides and selectivity for ß-secretase (BACE1) over γ-secretase. Prioritized compounds showed reasonable stability in vitro and in vivo, and our most potent inhibitor showed efficacy in reducing Aß levels in the rodent brain.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Proteases/química , Cianobactérias/química , Peptídeos/química , Peptídeos/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Desenho de Fármacos , Modelos Moleculares , Sondas Moleculares , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
Small molecules that modulate histone acetylation by targeting key enzymes mediating this posttranslational modification - histone acetyltransferases and histone deacetylases - are validated chemotherapeutic agents for the treatment of cancer. This area of research has seen a rapid increase in interest in the past decade, with the structurally diverse natural products-derived compounds at its forefront. These secondary metabolites from various biological sources target this epigenetic modification through distinct mechanisms of enzyme regulation by utilizing a diverse array of pharmacophores. We review the discovery of these compounds and discuss their modes of inhibition together with their downstream biological effects.
Assuntos
Produtos Biológicos/farmacologia , Descoberta de Drogas , Histonas/metabolismo , Acetilação , Produtos Biológicos/química , Processamento de Proteína Pós-TraducionalRESUMO
Cytotoxicity-directed purification of a Symploca cf. hydnoides sample from Cetti Bay, Guam, afforded seven new cyclic depsipeptides, veraguamides A-G (1-7), together with the known compound dolastatin 16. The planar structures of 1-7 were elucidated using NMR and MS experiments, while enantioselective HPLC and Mosher's analysis of acid and base hydrolysates, respectively, were utilized to assign the absolute configurations of the stereocenters. Veraguamides A-G (1-7) are characterized by the presence of an invariant proline residue, multiple N-methylated amino acids, an α-hydroxy acid, and a C8-polyketide-derived ß-hydroxy acid moiety with a characteristic terminus as either an alkynyl bromide, alkyne, or vinyl group. These compounds and a semisynthetic analogue (8) showed moderate to weak cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cell lines. Preliminary structure-activity relationship analysis identified several sensitive positions in the veraguamide scaffold that affect the cytotoxic activity of this compound class. Dolastatin 16 showed only weak cytotoxic activity on both cell lines tested. The complete stereostructure of dolastatin 16 was proposed for the first time through degradation followed by a combination of advanced Marfey's analysis and modified Mosher's analysis using phenylglycine methyl ester as a chiral anisotropic reagent.
Assuntos
Antineoplásicos/isolamento & purificação , Cianobactérias/química , Depsipeptídeos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/farmacologia , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Guam , Células HT29 , Células HeLa , Humanos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Relação Estrutura-AtividadeRESUMO
A Phormidium spp. collection from Key West, Florida, afforded caylobolide B (1), an analogue of the known macrolactone caylobolide A, previously isolated from a Lyngbya majuscula collection from the Bahamas. The planar structure of 1 was determined using NMR and MS experiments. The relative configuration for subunits C7-C9 and C25-C29 was assigned using Kishi's Universal NMR Database. Caylobolide B (1) displayed cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells with IC(50) values of 4.5 and 12.2 µM, respectively.
Assuntos
Antineoplásicos/isolamento & purificação , Cianobactérias/química , Lactonas/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/farmacologia , Bahamas , Depsipeptídeos/química , Depsipeptídeos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Florida , Células HT29 , Células HeLa , Humanos , Concentração Inibidora 50 , Lactonas/química , Lactonas/farmacologia , Biologia Marinha , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Collections of the marine cyanobacterium Lyngbya bouillonii from shallow patch reefs in Apra Harbor, Guam, afforded three hitherto undescribed analogues of the glycosidic macrolide lyngbyaloside, namely, 2-epi-lyngbyaloside (1) and the regioisomeric 18E- and 18Z-lyngbyalosides C (2 and 3). Concurrently we discovered two new analogues of the cytoskeletal actin-disrupting lyngbyabellins, 27-deoxylyngbyabellin A (4) and lyngbyabellin J (5), a novel macrolide of the laingolide family, laingolide B (6), and a linear modified peptide, lyngbyapeptin D (7), along with known lyngbyabellins A and B, lyngbyapeptin A, and lyngbyaloside. The structures of 1-7 were elucidated by a combination of NMR spectroscopic and mass spectrometric analysis. Compounds 1-6 were either brominated (1-3) or chlorinated (4-6), consistent with halogenation being a hallmark of many marine natural products. All extracts derived from these L. bouillonii collections were highly cytotoxic due to the presence of apratoxin A or apratoxin C. Compounds 1-5 showed weak to moderate cytotoxicity to HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Cianobactérias/química , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Hidrocarbonetos Halogenados/isolamento & purificação , Hidrocarbonetos Halogenados/farmacologia , Toxinas de Lyngbya/química , Macrolídeos/isolamento & purificação , Macrolídeos/farmacologia , Antineoplásicos/química , Depsipeptídeos/química , Ensaios de Seleção de Medicamentos Antitumorais , Guam , Células HT29 , Células HeLa , Humanos , Hidrocarbonetos Halogenados/química , Macrolídeos/química , Biologia Marinha , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , EstereoisomerismoRESUMO
Histone deacetylases (HDACs) are validated targets for anticancer therapy as attested by the approval of suberoylanilide hydroxamic acid (SAHA) and romidepsin (FK228) for treating cutaneous T cell lymphoma. We recently described the bioassay-guided isolation, structure determination, synthesis, and target identification of largazole, a marine-derived antiproliferative natural product that is a prodrug that releases a potent HDAC inhibitor, largazole thiol. Here, we characterize the anticancer activity of largazole by using in vitro and in vivo cancer models. Screening against the National Cancer Institute's 60 cell lines revealed that largazole is particularly active against several colon cancer cell types. Consequently, we tested largazole, along with several synthetic analogs, for HDAC inhibition in human HCT116 colon cancer cells. Enzyme inhibition strongly correlated with the growth inhibitory effects, and differential activity of largazole analogs was rationalized by molecular docking to an HDAC1 homology model. Comparative genomewide transcript profiling revealed a close overlap of genes that are regulated by largazole, FK228, and SAHA. Several of these genes can be related to largazole's ability to induce cell cycle arrest and apoptosis. Stability studies suggested reasonable bioavailability of the active species, largazole thiol. We established that largazole inhibits HDACs in tumor tissue in vivo by using a human HCT116 xenograft mouse model. Largazole strongly stimulated histone hyperacetylation in the tumor, showed efficacy in inhibiting tumor growth, and induced apoptosis in the tumor. This effect probably is mediated by the modulation of levels of cell cycle regulators, antagonism of the AKT pathway through insulin receptor substrate 1 down-regulation, and reduction of epidermal growth factor receptor levels.