RESUMO
Histone deacetylases (HDACs) are a class of enzymes that control chromatin state and influence cell fate. We evaluated the chromatin accessibility and transcriptome dynamics of zinc-containing HDACs during cell differentiation in vitro coupled with chemical perturbation to identify the role of HDACs in mesendoderm cell fate specification. Single-cell RNA sequencing analyses of HDAC expression during human pluripotent stem cell (hPSC) differentiation in vitro and mouse gastrulation in vivo reveal a unique association of HDAC1 and -3 with mesendoderm gene programs during exit from pluripotency. Functional perturbation with small molecules reveals that inhibition of HDAC1 and -3, but not HDAC2, induces mesoderm while impeding endoderm and early cardiac progenitor specification. These data identify unique biological functions of the structurally homologous enzymes HDAC1-3 in influencing hPSC differentiation from pluripotency toward mesendodermal and cardiac progenitor populations.
Assuntos
Endoderma , Histona Desacetilases , Células-Tronco Pluripotentes , Animais , Diferenciação Celular/genética , Cromatina/metabolismo , Endoderma/citologia , Endoderma/enzimologia , Endoderma/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/enzimologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Malaria is caused by infection with Plasmodium parasites and results in significant health and economic impacts. Malaria eradication is hampered by parasite resistance to current drugs and the lack of a widely effective vaccine. Compounds that target epigenetic regulatory proteins, such as histone deacetylases (HDACs), may lead to new therapeutic agents with a different mechanism of action, thereby avoiding resistance mechanisms to current antimalarial drugs. The anticancer HDAC inhibitor AR-42, as its racemate (rac-AR-42), and 36 analogues were investigated for in vitro activity against P. falciparum. Rac-AR-42 and selected compounds were assessed for cytotoxicity against human cells, histone hyperacetylation, human HDAC1 inhibition and oral activity in a murine malaria model. Rac-AR-42 was tested for ex vivo asexual and in vitro exoerythrocytic stage activity against P. berghei murine malaria parasites. Rac-AR-42 and 13 achiral analogues were potent inhibitors of asexual intraerythrocytic stage P. falciparum 3D7 growth in vitro (IC50 5-50 nM), with four of these compounds having >50-fold selectivity for P. falciparum versus human cells (selectivity index 56-118). Rac-AR-42 induced in situ hyperacetylation of P. falciparum histone H4, consistent with PfHDAC(s) inhibition. Furthermore, rac-AR-42 potently inhibited P. berghei infected erythrocyte growth ex vivo (IC50 40 nM) and P. berghei exoerythrocytic forms in hepatocytes (IC50 1 nM). Oral administration of rac-AR-42 and two achiral analogues inhibited P. berghei growth in mice, with rac-AR-42 (50 mg/kg/day single dose for four days) curing all infections. These findings demonstrate curative properties for HDAC inhibitors in the oral treatment of experimental mouse malaria.
Assuntos
Antimaláricos , Malária , Parasitos , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Malária/tratamento farmacológico , Camundongos , Plasmodium berghei , Plasmodium falciparumRESUMO
The zinc-containing histone deacetylase enzyme HDAC7 is emerging as an important regulator of immunometabolism and cancer. Here, we exploit a cavity in HDAC7, filled by Tyr303 in HDAC1, to derive new inhibitors. Phenacetyl hydroxamates and 2-phenylbenzoyl hydroxamates bind to Zn2+ and are 50-2700-fold more selective inhibitors of HDAC7 than HDAC1. Phenylbenzoyl hydroxamates are 30-70-fold more potent HDAC7 inhibitors than phenacetyl hydroxamates, which is attributed to the benzoyl aromatic group interacting with Phe679 and Phe738. Phthalimide capping groups, including a saccharin analogue, decrease rotational freedom and provide hydrogen bond acceptor carbonyl/sulfonamide oxygens that increase inhibitor potency, liver microsome stability, solubility, and cell activity. Despite being the most potent HDAC7 inhibitors to date, they are not selective among class IIa enzymes. These strategies may help to produce tools for interrogating HDAC7 biology related to its catalytic site.
Assuntos
Benzamidas/farmacologia , Benzenoacetamidas/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Benzamidas/síntese química , Benzamidas/metabolismo , Benzenoacetamidas/síntese química , Benzenoacetamidas/metabolismo , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/farmacologia , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Células THP-1RESUMO
BACKGROUND & AIMS: Chronic bowel inflammation increases the risk of colon cancer; colitis-associated cancer (CAC). Thiopurine treatments are associated with a reduction in dysplasia and CAC in inflammatory bowel disease (IBD). Abnormal Wnt/ß-catenin signalling is characteristic of >90% of colorectal cancers. Immunosuppression by thiopurines is via Rac1 GTPase, which also affects Wnt/ß-catenin signalling. Autophagy is implicated in colonic tumors, and topical delivery of the thiopurine thioguanine (TG) is known to alleviate colitis and augment autophagy. This study investigated the effects of TG in a murine model of CAC and potential mechanisms. METHODS: Colonic dysplasia was induced by exposure to azoxymethane (AOM) and dextran sodium sulfate (DSS) in wild-type (WT) mice and mice harboring intestinal epithelial cell-specific deletion of autophagy related 7 gene (Atg7ΔIEC). TG or vehicle was administered intrarectally, and the effect on tumor burden and ß-catenin activity was assessed. The mechanisms of action of TG were investigated in vitro and in vivo. RESULTS: TG ameliorated DSS colitis in wild-type but not Atg7ΔIEC mice, demonstrating that anti-inflammatory effects of locally delivered TG are autophagy-dependent. However, TG inhibited CAC in both wild-type and Atg7ΔIEC mice. This was associated with decreased ß-catenin activation/nuclear translocation demonstrating that TG's inhibition of tumorigenesis occurred independently of anti-inflammatory and pro-autophagic actions. These results were confirmed in cell lines, and the dependency on Rac1 GTPase was demonstrated by siRNA knockdown and overexpression of constitutively active Rac1. CONCLUSIONS: Our findings provide evidence for a new mechanism that could be exploited to improve CAC chemoprophylactic approaches.
Assuntos
Neoplasias Associadas a Colite/prevenção & controle , Colite/tratamento farmacológico , Tioguanina/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Administração Retal , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Células CACO-2 , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Neoplasias Associadas a Colite/imunologia , Neoplasias Associadas a Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Mercaptopurina/farmacologia , Mercaptopurina/uso terapêutico , Camundongos , Camundongos Transgênicos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Tioguanina/uso terapêutico , beta Catenina/análise , beta Catenina/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
AR-42 is an orally active inhibitor of histone deacetylases (HDACs) in clinical trials for multiple myeloma, leukemia, and lymphoma. It has few hydrogen bond donors and acceptors but is a chiral 2-arylbutyrate and potentially prone to racemization. We report achiral AR-42 analogues incorporating a cycloalkyl group linked via a quaternary carbon atom, with up to 40-fold increased potency against human class I HDACs (e.g., JT86, IC50 0.7 nM, HDAC1), 25-fold increased cytotoxicity against five human cancer cell lines, and up to 70-fold less toxicity in normal human cells. JT86 was ninefold more potent than racAR-42 in promoting accumulation of acetylated histone H4 in MM96L melanoma cells. Molecular modeling and structure-activity relationships support binding to HDAC1 with tetrahydropyran acting as a hydrophobic shield from water at the enzyme surface. Such potent inhibitors of class I HDACs may show benefits in diseases (cancers, parasitic infections, inflammatory conditions) where AR-42 is active.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/química , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Desenho de Fármacos , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Simulação de Acoplamento Molecular , Fenilbutiratos , Relação Estrutura-AtividadeRESUMO
Breast cancer brain metastases remain largely incurable. Although several mouse models have been developed to investigate the genes and mechanisms regulating breast cancer brain metastasis, these models often lack clinical relevance since they require the use of immunocompromised mice and/or are poorly metastatic to brain from the mammary gland. We describe the development and characterisation of an aggressive brain metastatic variant of the 4T1 syngeneic model (4T1Br4) that spontaneously metastasises to multiple organs, but is selectively more metastatic to the brain from the mammary gland than parental 4T1 tumours. As seen by immunohistochemistry, 4T1Br4 tumours and brain metastases display a triple-negative phenotype, consistent with the high propensity of this breast cancer subtype to spread to brain. In vitro assays indicate that 4T1Br4 cells have an enhanced ability to adhere to or migrate across a brain-derived endothelial monolayer and greater invasive response to brain-derived soluble factors compared to 4T1 cells. These properties are likely to contribute to the brain selectivity of 4T1Br4 tumours. Expression profiling and gene set enrichment analyses demonstrate the clinical relevance of the 4T1Br4 model at the transcriptomic level. Pathway analyses implicate tumour-intrinsic immune regulation and vascular interactions in successful brain colonisation, revealing potential therapeutic targets. Evaluation of two histone deacetylase inhibitors, SB939 and 1179.4b, shows partial efficacy against 4T1Br4 metastasis to brain and other sites in vivo, and potent radio-sensitising properties in vitro The 4T1Br4 model provides a clinically relevant tool for mechanistic studies and to evaluate novel therapies against brain metastasis.This article has an associated First Person interview with Soo-Hyun Kim, joint first author of the paper.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Genes Neoplásicos , Inibidores de Histona Desacetilases/uso terapêutico , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Invasividade Neoplásica , Fenótipo , Tolerância a Radiação/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Peptides and proteins are not orally bioavailable in mammals, although a few peptides are intestinally absorbed in small amounts. Polypeptides are generally too large and polar to passively diffuse through lipid membranes, while most known active transport mechanisms facilitate cell uptake of only very small peptides. Systematic evaluations of peptides with molecular weights above 500 Da are needed to identify parameters that influence oral bioavailability. Here we describe 125 cyclic peptides containing four to thirty-seven amino acids that are orally absorbed by mammals. Cyclization minimizes degradation in the gut, blood, and tissues by removing cleavable N- and C-termini and by shielding components from metabolic enzymes. Cyclization also folds peptides into bioactive conformations that determine exposure of polar atoms to solvation by water and lipids and therefore can influence oral bioavailability. Key chemical properties thought to influence oral absorption and bioavailability are analyzed, including molecular weight, octanol-water partitioning, hydrogen bond donors/acceptors, rotatable bonds, and polar surface area. The cyclic peptides violated to different degrees all of the limits traditionally considered to be important for oral bioavailability of drug-like small molecules, although fewer hydrogen bond donors and reduced flexibility generally favored oral absorption.
Assuntos
Absorção Fisico-Química , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Humanos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismoRESUMO
Histone deacetylase inhibitors (HDACi) are being investigated for the control of various human parasites. Here we investigate their potential as insecticides for the control of a major ecto-parasite of sheep, the Australian sheep blowfly, Lucilia cuprina. We assessed the ability of HDACi from various chemical classes to inhibit the development of blowfly larvae in vitro, and to inhibit HDAC activity in nuclear protein extracts prepared from blowfly eggs. The HDACi prodrug romidepsin, a cyclic depsipeptide that forms a thiolate, was the most potent inhibitor of larval growth, with equivalent or greater potency than three commercial blowfly insecticides. Other HDACi with potent activity were hydroxamic acids (trichostatin, CUDC-907, AR-42), a thioester (KD5170), a disulphide (Psammaplin A), and a cyclic tetrapeptide bearing a ketone (apicidin). On the other hand, no insecticidal activity was observed for certain other hydroxamic acids, fatty acids, and the sesquiterpene lactone parthenolide. The structural diversity of the 31 hydroxamic acids examined here revealed some structural requirements for insecticidal activity; for example, among compounds with flexible linear zinc-binding extensions, greater potency was observed in the presence of branched capping groups that likely make multiple interactions with the blowfly HDAC enzymes. The insecticidal activity correlated with inhibition of HDAC activity in blowfly nuclear protein extracts, indicating that the toxicity was most likely due to inhibition of HDAC enzymes in the blowfly larvae. The inhibitor potencies against blowfly larvae are different from inhibition of human HDACs, suggesting some selectivity for human over blowfly HDACs, and a potential for developing compounds with the inverse selectivity. In summary, these novel findings support blowfly HDAC enzymes as new targets for blowfly control, and point to development of HDAC inhibitors as a promising new class of insecticides.
Assuntos
Dípteros/efeitos dos fármacos , Dípteros/enzimologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Inseticidas/farmacologia , Animais , Austrália , Depsipeptídeos/farmacologia , Dípteros/crescimento & desenvolvimento , Concentração Inibidora 50 , Resistência a Inseticidas , Inseticidas/química , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Carneiro Doméstico/parasitologiaRESUMO
Cyclic peptides and macrocycles have the potential to be membrane permeable and orally bioavailable, despite often not complying with the "rule of five" used in medicinal chemistry to guide the discovery of oral drugs. Here we compare solvent-dependent three-dimensional structures of three cyclic hexapeptides containing d-amino acids, prolines, and intramolecular hydrogen bonds. Conformational rigidity rather than flexibility resulted in higher membrane permeability, metabolic stability and oral bioavailability, consistent with less polar surface exposure to solvent and a reduced entropy penalty for transition between polar and nonpolar environments.
Assuntos
Peptídeos Cíclicos/metabolismo , Administração Oral , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Disponibilidade Biológica , Dicroísmo Circular , Entropia , Meia-Vida , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Masculino , Microssomos Hepáticos , Modelos Moleculares , Azeite de Oliva/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Permeabilidade , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Solventes/químicaRESUMO
Protease activated receptor 2 (PAR2) is an unusual G-protein coupled receptor (GPCR) involved in inflammation and metabolism. It is activated through cleavage of its N-terminus by proteases. The new N-terminus functions as a tethered ligand that folds back and intramolecularly activates PAR2, initiating multiple downstream signaling pathways. The only compounds reported to date to inhibit PAR2 activation are of moderate potency. Three structural models for PAR2 have been constructed based on sequence homology with known crystal structures for bovine rhodopsin, human ORL-1 (also called nociceptin/orphanin FQ receptor), and human PAR1. The three PAR2 model structures were compared and used to predict potential interactions with ligands. Virtual screening for ligands using the Chembridge database, and either ORL-1 or PAR1 derived PAR2 models led to identification of eight new small molecule PAR2 antagonists (IC50 10-100 µM). Notably, the most potent compound 1 (IC50 11 µM) was derived from the less homologous template protein, human ORL-1. The results suggest that virtual screening against multiple homology models of the same GPCR can produce structurally diverse antagonists and that this may be desirable even when some models have less sequence homology with the target protein.
Assuntos
Descoberta de Drogas/métodos , Simulação de Acoplamento Molecular , Receptor PAR-2/antagonistas & inibidores , Receptor PAR-2/química , Homologia de Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Membrana Celular/metabolismo , Bases de Dados de Proteínas , Avaliação Pré-Clínica de Medicamentos , Células HT29 , Humanos , Ligantes , Estrutura Terciária de Proteína , Receptor PAR-2/metabolismoRESUMO
Accurately predicting relative binding affinities and biological potencies for ligands that interact with proteins remains a significant challenge for computational chemists. Most evaluations of docking and scoring algorithms have focused on enhancing ligand affinity for a protein by optimizing docking poses and enrichment factors during virtual screening. However, there is still relatively limited information on the accuracy of commercially available docking and scoring software programs for correctly predicting binding affinities and biological activities of structurally related inhibitors of different enzyme classes. Presented here is a comparative evaluation of eight molecular docking programs (Autodock Vina, Fitted, FlexX, Fred, Glide, GOLD, LibDock, MolDock) using sixteen docking and scoring functions to predict the rank-order activity of different ligand series for six pharmacologically important protein and enzyme targets (Factor Xa, Cdk2 kinase, Aurora A kinase, COX-2, pla2g2a, ß Estrogen receptor). Use of Fitted gave an excellent correlation (Pearson 0.86, Spearman 0.91) between predicted and experimental binding only for Cdk2 kinase inhibitors. FlexX and GOLDScore produced good correlations (Pearson>0.6) for hydrophilic targets such as Factor Xa, Cdk2 kinase and Aurora A kinase. By contrast, pla2g2a and COX-2 emerged as difficult targets for scoring functions to predict ligand activities. Although possessing a high hydrophobicity in its binding site, ß Estrogen receptor produced reasonable correlations using LibDock (Pearson 0.75, Spearman 0.68). These findings can assist medicinal chemists to better match scoring functions with ligand-target systems for hit-to-lead optimization using computer-aided drug design approaches.
Assuntos
Simulação de Acoplamento Molecular , Proteínas/química , Algoritmos , Domínio Catalítico , Humanos , Interações Hidrofóbicas e Hidrofílicas , LigantesRESUMO
Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.
RESUMO
The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation.
Assuntos
Oligopeptídeos/farmacocinética , Peptídeos Cíclicos/farmacocinética , Administração Oral , Sequência de Aminoácidos , Disponibilidade Biológica , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/química , Conformação ProteicaRESUMO
Histone deacetylase (HDAC) enzymes posttranslationally modify lysines on histone and nonhistone proteins and play crucial roles in epigenetic regulation and other important cellular processes. HDAC inhibitors (e.g., suberoylanilide hydroxamic acid [SAHA; also known as vorinostat]) are used clinically to treat some cancers and are under investigation for use against many other diseases. Development of new HDAC inhibitors for noncancer indications has the potential to be accelerated by piggybacking onto cancer studies, as several HDAC inhibitors have undergone or are undergoing clinical trials. One such compound, SB939, is a new orally active hydroxamate-based HDAC inhibitor with an improved pharmacokinetic profile compared to that of SAHA. In this study, the in vitro and in vivo antiplasmodial activities of SB939 were investigated. SB939 was found to be a potent inhibitor of the growth of Plasmodium falciparum asexual-stage parasites in vitro (50% inhibitory concentration [IC(50)], 100 to 200 nM), causing hyperacetylation of parasite histone and nonhistone proteins. In combination with the aspartic protease inhibitor lopinavir, SB939 displayed additive activity. SB939 also potently inhibited the in vitro growth of exoerythrocytic-stage Plasmodium parasites in liver cells (IC(50), ~150 nM), suggesting that inhibitor targeting to multiple malaria parasite life cycle stages may be possible. In an experimental in vivo murine model of cerebral malaria, orally administered SB939 significantly inhibited P. berghei ANKA parasite growth, preventing development of cerebral malaria-like symptoms. These results identify SB939 as a potent new antimalarial HDAC inhibitor and underscore the potential of investigating next-generation anticancer HDAC inhibitors as prospective new drug leads for treatment of malaria.
Assuntos
Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Malária/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cérebro/parasitologia , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidadeRESUMO
Transformation of proteins and peptides to fibrillar aggregates rich in ß sheets underlies many diseases, but mechanistic details of these structural transitions are poorly understood. To simulate aggregation, four equivalents of a water-soluble, α-helical (65 %) amphipathic peptide (AEQLLQEAEQLLQEL) were assembled in parallel on an oxazole-containing macrocyclic scaffold. The resulting 4α-helix bundle is monomeric and even more α helical (85 %), but it is also unstable at pHâ 4 and undergoes concentration-dependent conversion to ß-sheet aggregates and amyloid fibrils. Fibrils twist and grow with time, remaining flexible like rope (>1â µm long, 5-50â nm wide) with multiple strings (2â nm), before ageing to matted fibers. At pHâ 7 the fibrils revert back to soluble monomeric 4α-helix bundles. During αâß folding we were able to detect soluble 3(10) helices in solution by using 2D-NMR, CD and FTIR spectroscopy. This intermediate satisfies the need for peptide elongation, from the compressed α helix to the fully extended ß strand/sheet, and is driven here by 3(10) -helix aggregation triggered in this case by template-promoted helical bundling and by hydrogen-bonding glutamic acid side chains. A mechanism involving αâα(4) â(3(10) )(4) â(3(10) )(n) â(ß)(n) âm(ß)(n) equilibria is plausible for this peptide and also for peptides lacking hydrogen-bonding side chains, with unfavourable equilibria slowing the αâß conversion.
Assuntos
Amiloide/química , Oligopeptídeos/síntese química , Sequência de Aminoácidos , Amiloide/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/química , Oxazóis/química , Estrutura Terciária de ProteínaRESUMO
Histone deacetylase inhibitors with anticancer or anti-inflammatory activity bind to Class I or Class I and II HDAC enzymes. Here we compare selectivity of inhibitors of a Class II HDAC enzyme (HDAC6) and find one that retains high selectivity in macrophages.
Assuntos
Anti-Inflamatórios/química , Antineoplásicos/química , Inibidores de Histona Desacetilases/química , Animais , Linhagem Celular , Histona Desacetilase 1/efeitos dos fármacos , Desacetilase 6 de Histona , Histona Desacetilases/efeitos dos fármacos , Humanos , Macrófagos/enzimologia , Camundongos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Malaria is the most lethal parasite-mediated tropical infectious disease, killing 1-2 million people each year. An emerging drug target is the enzyme Plasmodium falciparum histone deacetylase 1 (PfHDAC1). We report 26 compounds designed to bind the zinc and exterior surface around the entrance to the active site of PfHDAC1, 16 displaying potent in vitro antimalarial activity (IC(50)<100 nM) against P. falciparum. Selected compounds were shown to cause hyperacetylation of P. falciparum histones and be >10-fold more cytotoxic towards P. falciparum than a normal human cell type (NFF). Twenty-two inhibitors feature cinnamic acid derivatives or non-steroidal anti-inflammatory drugs (NSAIDs) as HDAC-binding components. A homology model of PfHDAC1 enzyme gives new insights to interactions likely made by some of these inhibitors. Results support PfHDAC1 as a promising new antimalarial drug target.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antimaláricos/química , Cinamatos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Plasmodium falciparum/enzimologia , Acetilação/efeitos dos fármacos , Antimaláricos/farmacologia , Sítios de Ligação , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases/química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Plasmodium falciparum/efeitos dos fármacos , Ligação ProteicaRESUMO
Chemotherapy in the last century was characterized by cytotoxic drugs that did not discriminate between cancerous and normal cell types and were consequently accompanied by toxic side effects that were often dose limiting. The ability of differentiating agents to selectively kill cancer cells or transform them to a nonproliferating or normal phenotype could lead to cell- and tissue-specific drugs without the side effects of current cancer chemotherapeutics. This may be possible for a new generation of histone deacetylase inhibitors derived from amino acids. Structure-activity relationships are now reported for 43 compounds derived from 2-aminosuberic acid that kill a range of cancer cells, 26 being potent cytotoxins against MM96L melanoma cells (IC50 20 nM-1 microM), while 17 were between 5- and 60-fold more selective in killing MM96L melanoma cells versus normal (neonatal foreskin fibroblasts, NFF) cells. This represents a 10- to 100-fold increase in potency and up to a 10-fold higher selectivity over previously reported compounds derived from cysteine (J. Med. Chem. 2004, 47, 2984). Selectivity is also an underestimate, because the normal cells, NFF, are rarely all killed by the drugs that also induce selective blockade of the cell cycle for normal but not cancer cells. Selected compounds were tested against a panel of human cancer cell lines (melanomas, prostate, breast, ovarian, cervical, lung, and colon) and found to be both selective and potent cytotoxins (IC50 20 nM-1 microM). Compounds in this class typically inhibit human histone deacetylases, as evidenced by hyperacetylation of histones in both normal and cancer cells, induce expression of p21, and differentiate surviving cancer cells to a nonproliferating phenotype. These compounds may be valuable leads for the development of new chemotherapeutic agents.
Assuntos
Aminoácidos Dicarboxílicos/síntese química , Antineoplásicos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Acetilação , Aminoácidos Dicarboxílicos/química , Aminoácidos Dicarboxílicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores de Histona Desacetilases , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Concentração Inibidora 50 , Relação Estrutura-AtividadeRESUMO
A major chemical challenge is the structural mimicry of discontinuous protein surfaces brought into close proximity through polypeptide folding. We report the design, synthesis, and solution structure of a highly functionalized saddle-shaped macrocyclic scaffold, constrained by oxazoles and thiazoles, supporting two short peptide loops projecting orthogonally from the same face of the scaffold. This structural mimetic of two interhelical loops of cytochrome b(562) illustrates a promising approach to structurally mimicking discontinuous loops of proteins.
Assuntos
Grupo dos Citocromos b/química , Proteínas de Escherichia coli/química , Compostos Macrocíclicos/química , Oxazóis/química , Tiazóis/química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Grupo dos Citocromos b/metabolismo , Proteínas de Escherichia coli/metabolismo , Compostos Macrocíclicos/metabolismo , Modelos Moleculares , Estrutura Quaternária de Proteína , Soluções , Propriedades de SuperfícieRESUMO
[reaction: see text] Cyclopropylmethyltrimethylstannanes undergo electrophilic cyclopropane cleavage in chloroform with simple inorganic electrophiles (H(+), SO(2), I(2)) in a homologous reaction to the S(E)' cleavage of allylic stannanes. The sigma-sigma conjugation between the carbon-tin bond and cyclopropane orbitals observed spectroscopically in the parent cyclopropylmethyltrimethylstannane is responsible for a rate enhancement of ca. 10(2) toward iodinolysis, relative to comparable alkyl stannanes. This acceleration is considerably less, however, than the ca. 10(9)-fold rate enhancement provided by the corresponding sigma-pi conjugation in allylic stannanes. Methanol-tin coordination appears to reduce the activating influence of the metal, promoting methyl cleavage over cyclopropane fission with acid and iodine. Decreased sigma-sigma conjugation can also explain the decreased reactivity of cyclopropyltriphenylstannane compared with its trimethyltin counterpart. Cyclopropylmethylstannanes do not undergo the synthetically useful addition of aldehydes under conditions that facilitate the corresponding reaction of allylic stannanes.