RESUMO
Changes in DNA methylation patterning have been reported to be a key hallmark of aged human skin. The altered DNA methylation patterns are correlated with deregulated gene expression and impaired tissue functionality, leading to the well-known skin aging phenotype. Searching for small molecules, which correct the aged methylation pattern therefore represents a novel and attractive strategy for the identification of anti-aging compounds. DNMT1 maintains epigenetic information by copying methylation patterns from the parental (methylated) strand to the newly synthesized strand after DNA replication. We hypothesized that a modest inhibition of this process promotes the restoration of the ground-state epigenetic pattern, thereby inducing rejuvenating effects. In this study, we screened a library of 1800 natural substances and 640 FDA-approved drugs and identified the well-known antioxidant and anti-inflammatory molecule dihydromyricetin (DHM) as an inhibitor of the DNA methyltransferase DNMT1. DHM is the active ingredient of several plants with medicinal use and showed robust inhibition of DNMT1 in biochemical assays. We also analyzed the effect of DHM in cultivated keratinocytes by array-based methylation profiling and observed a moderate, but significant global hypomethylation effect upon treatment. To further characterize DHM-induced methylation changes, we used published DNA methylation clocks and newly established age predictors to demonstrate that the DHM-induced methylation change is associated with a reduction in the biological age of the cells. Further studies also revealed re-activation of age-dependently hypermethylated and silenced genes in vivo and a reduction in age-dependent epidermal thinning in a 3-dimensional skin model. Our findings thus establish DHM as an epigenetic inhibitor with rejuvenating effects for aged human skin.
RESUMO
DNA methyltransferases (DNMTs) represent promising targets for the development of unique anticancer drugs. However, all DNMT inhibitors currently in clinical use are nonselective cytosine analogs with significant cytotoxic side-effects. Several natural products, covering diverse chemical classes, have indicated DNMT inhibitory activity, but these effects have yet to be systematically evaluated. In this study, we provide experimental data suggesting that two of the most prominent natural products associated with DNA methylation inhibition, (-)-epigallocathechin-3-gallate (EGCG) and curcumin, have little or no pharmacologically relevant inhibitory activity. We therefore conducted a virtual screen of a large database of natural products with a validated homology model of the catalytic domain of DNMT1. The virtual screening focused on a lead-like subset of the natural products docked with DNMT1, using three docking programs, following a multistep docking approach. Prior to docking, the lead-like subset was characterized in terms of chemical space coverage and scaffold content. Consensus hits with high predicted docking affinity for DNMT1 by all three docking programs were identified. One hit showed DNMT1 inhibitory activity in a previous study. The virtual screening hits were located within the biological-relevant chemical space of drugs, and represent potential unique DNMT inhibitors of natural origin. Validation of these virtual screening hits is warranted.
Assuntos
Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Simulação por Computador , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Produtos Biológicos/química , Catequina/análogos & derivados , Catequina/metabolismo , Catequina/farmacologia , Biologia Computacional , Curcumina/metabolismo , Curcumina/farmacologia , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/química , Células HCT116 , Células HEK293 , Células HL-60 , Humanos , Modelos Moleculares , Interface Usuário-ComputadorRESUMO
The DNA methyltransferase (DNMT) enzyme family consists of four members with diverse functions and represents one of the most promising targets for the development of novel anticancer drugs. However, the standard drugs for DNMT inhibition are non-selective cytosine analogues with considerable cytotoxic side-effects that have been developed several decades ago. In this work, we conducted a virtual screening of more than 65,000 lead-like compounds selected from the National Cancer Institute collection using a multistep docking approach with a previously validated homology model of the catalytic domain of human DNMT1. Experimental evaluation of top-ranked molecules led to the discovery of novel small molecule DNMT1 inhibitors. Virtual screening hits were further evaluated for DNMT3B inhibition revealing several compounds with selectivity towards DNMT1. These are the first small molecules reported with biochemical selectivity towards an individual DNMT enzyme capable of binding in the same pocket as the native substrate cytosine, and are promising candidates for further rational optimization and development as anticancer drugs. The availability of enzyme-selective inhibitors will also be of great significance for understanding the role of individual DNMT enzymes in epigenetic regulation.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Clonagem Molecular , Cristalografia por Raios X , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/isolamento & purificação , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Relação Estrutura-Atividade , DNA Metiltransferase 3BRESUMO
Constrained analogues of procaine were synthesized, and their inhibiting activity against DNMT1 was tested. Among them, the most potent compound, derivative 3b, was also able to induce a recognizable demethylation of chromosomal satellite repeats in HL60 human myeloid leukemia cells and thus represents a lead compound for the development of a novel class of non-nucleoside DNMT1 inhibitors.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Oxazóis/farmacologia , Procaína/análogos & derivados , Procaína/farmacologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Peso Molecular , Oxazóis/síntese química , Oxazóis/química , Procaína/química , Proteínas Recombinantes/antagonistas & inibidores , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy. More recently, due to the advantages they offer for gene transfer, several AAV serotypes have gained increasing interest. However, monoclonal antibodies for the characterization and quantitation of vectors derived from different serotypes are at present not available. Serotype-specific monoclonal antibodies (mAbs) against the capsids of the serotypes 1/6, 4 and 5 are described. These antibodies, designated as ADK1a and b, ADK4 or ADK5a and b, reacted specifically with the indicated serotype capsids in cell lysates. ADK 1a and b cross-reacted with its highly related AAV6 serotype, but not with the other serotypes tested. The new antibodies recognized exclusively assembled capsids and neither free nor denatured capsid proteins as shown by fractionation experiments. In immunofluorescence experiments, the mAbs stained only distinct intranuclear foci in cells expressing the capsid protein. The development of capture ELISAs for quantitation of AAV1 and 6, AAV4 or AAV5 capsids illustrates that these novel monoclonal antibodies provide valuable tools for characterization of vector stocks.
Assuntos
Anticorpos Monoclonais/imunologia , Dependovirus/genética , Ensaio de Imunoadsorção Enzimática/métodos , Capsídeo/metabolismo , Linhagem Celular , Reações Cruzadas , Dependovirus/classificação , Imunofluorescência , Humanos , Plasmídeos , SorotipagemRESUMO
The patent presents 140 purine and 7-azapurine derivatives as potent inhibitors of DOT1L histone methyltransferase that might be useful in the treatment of leukemia with MLL rearrangements. It is becoming more and more evident that the deregulation of chromatin modifiers such as DOT1L plays a critical role in tumorigenesis. As yet, the number of pharmaceutical agents targeting chromatin modifiers is still limited. The market for such compounds has been estimated to be potentially as large as one third of all cancer patients. Overall, the prospective of a targeted product (i.e., a drug targeting a commonly affected chromatin modifier) is very promising, and exponentially growing investments into this market are anticipated.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Leucemia/tratamento farmacológico , Metiltransferases/antagonistas & inibidores , Terapia de Alvo Molecular , Purinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Controle de Medicamentos e Entorpecentes , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase , Humanos , Leucemia/enzimologia , Leucemia/genética , Leucemia/patologia , Metiltransferases/metabolismo , Estrutura Molecular , Patentes como Assunto , Purinas/química , Purinas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs.
Assuntos
Proliferação de Células , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Interferência de RNA , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Immunoblotting , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Metiltransferase 3BRESUMO
A series of Δ(2)-isoxazoline constrained analogues of procaine/procainamide (7a-k and 8a-k) were prepared and their inhibitory activity against DNA methyltransferase 1 (DNMT1) was tested. Among them, derivative 7b is far more potent in vitro (IC(50) = 150 µM) than other non-nucleoside inhibitors and also exhibits a strong and dose-dependent antiproliferative effect against HCT116 human colon carcinoma cells. The binding mode of 7b with the enzyme was also investigated by means of a simple competition assay as well as of docking simulations conducted using the recently published crystallographic structure of human DNMT1. On the basis of the findings, we assessed that the mode of inhibition of 7b is consistent with a competition with the cofactor and propose it as a novel lead compound for the development of non-nucleoside DNMT inhibitors.
Assuntos
Antineoplásicos/síntese química , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Isoxazóis/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Ligação Competitiva , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/química , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Isoxazóis/química , Isoxazóis/farmacologia , Modelos Moleculares , Ligação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , S-Adenosilmetionina/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Enzymes involved in the epigenetic regulation of the genome represent promising starting points for therapeutic intervention by small molecules, and DNA methyltransferases (DNMT) are emerging targets for the development of a new class of cancer therapeutics. In this work, we present nanaomycin A, initially identified by a virtual screening for inhibitors against DNMT1, as a compound inducing antiproliferative effects in three different tumor cell lines originating from different tissues. Nanaomycin A treatment reduced the global methylation levels in all three cell lines and reactivated transcription of the RASSF1A tumor suppressor gene. In biochemical assays, nanaomycin A revealed selectivity toward DNMT3B. To the best of our knowledge, this is the first DNMT3B-selective inhibitor identified to induce genomic demethylation. Our study thus establishes the possibility of selectively inhibiting individual DNMT enzymes.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Genes Supressores de Tumor/efeitos dos fármacos , Naftoquinonas/farmacologia , Neoplasias/genética , Ativação Transcricional/efeitos dos fármacos , Antineoplásicos/farmacologia , Metilação de DNA/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células HCT116 , Células HL-60 , Células HeLa , Humanos , Concentração Inibidora 50 , Modelos Biológicos , Neoplasias/patologia , Especificidade por Substrato , Células Tumorais Cultivadas , DNA Metiltransferase 3BRESUMO
Aberrant DNA methylation patterns, including hypermethylation of tumor suppressor genes, have been described in many human cancers. These epigenetic mutations can be reversed by DNA methyltransferase inhibitors, which provide novel opportunities for cancer therapy. Clinical concepts for epigenetic therapies are currently being developed by using azanucleosides for the treatment of leukemias and other tumors. These trials will greatly benefit from the inclusion of molecular markers for monitoring epigenetic changes in patients and for maximizing biologic responses. In addition, novel inhibitors need to be developed that result in a direct and specific inhibition of DNA methyltransferase activity. Several recent developments indicate that rational design of small molecule DNA methyltransferase inhibitors is feasible and that this approach can result in the establishment of novel drug candidates. The use of novel DNA methyltransferase inhibitors in clinical trials that allow monitoring of drug-induced DNA methylation changes should provide the foundation for improved epigenetic cancer therapies.
Assuntos
Metilases de Modificação do DNA/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Azacitidina/análogos & derivados , Azacitidina/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Decitabina , Epigênese Genética/efeitos dos fármacos , Humanos , Neoplasias/genéticaRESUMO
Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy in many preclinical studies, as well as in clinical trials. However, only a few approaches have used recombinant AAV (rAAV) to deliver vaccine antigens. We generated an rAAV encoding the major capsid protein L1 (L1h) from the human papillomavirus type 16 (HPV16), aiming to develop a prophylactic vaccine against HPV16 infections, which are the major cause of cervical cancer in women worldwide. A single dose of rAAV5 L1h administered intranasally was sufficient to induce high titers of L1-specific serum antibodies, as well as mucosal antibodies in vaginal washes. Seroconversion was maintained for at least 1 year. In addition, a cellular immune response was still detectable 60 weeks after immunization. Furthermore, lyophilized rAAV5 L1h successfully evoked a systemic and mucosal immune response in mice. These data clearly show the efficacy of a single-dose intranasal immunization against HPV16 based on the recombinant rAAV5L1h vector without the need of an adjuvant.
Assuntos
Proteínas do Capsídeo/imunologia , Dependovirus/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Vacinas Virais/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Feminino , Células HeLa , Humanos , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Baço/citologia , Baço/imunologia , Vacinação , Vagina/imunologiaRESUMO
Infections by human papillomaviruses (HPV) are the major cause of uterine cancer in women worldwide. Aiming to develop a combined prophylactic and therapeutic vaccine we have previously demonstrated immunogenicity of chimeric virus-like particles consisting of a C-terminally truncated HPV 16 L1 capsid protein fused to an E7 portion. Here we show that genetic vaccination with a corresponding DNA was inefficient in the induction of a L1-specific prophylactic immune response. DNA immunization with C-terminally truncated HPV 16 L1 genes of different lengths revealed that only short deletions (L1(1-498)) were tolerated for eliciting a humoral immune response against viral capsids. This correlates with the observation that the C-terminal sequences are critical for nuclear localization, capsomere and capsid assembly. However, only the ability of L1 protein to form capsomeres or capsids showed a direct influence on the outcome of the immune response. C-terminal insertion of 60 amino acids of E7 was tolerated in fusion constructs, whereas insertion of full-length E7(1-98) or shuffled E7 (149 aa) completely abolished the humoral immune response. The L1(1-498)/E7(1-60) fusion construct not only induced L1-specific antibodies but also L1- and E7-specific CTL responses after DNA vaccination.