RESUMO
Small molecules that bind in the minor groove of DNA are in clinical use as antibiotics and antitumor drugs. Two members of this class of molecules, netropsin and chromomycin, are shown here to displace DNA from the nucleosome and promote transfer of the histone octamer to an acceptor protein. The effects of these groove-binding molecules are exploited to address an outstanding problem in the mechanism of the RSC chromatin remodeling complex. RSC and other remodeling complexes are DNA translocases, acting near the center of the nucleosomal DNA, but translocation is apparently impossible because DNA cannot slide across the histone surface in the nucleosome. Netropsin and chromomycin promote the release of DNA from the histone surface, enhance the formation of a RSC-nucleosome complex, and synergize with RSC in chromatin remodeling. These findings are in keeping with an involvement of bulge translocation in chromatin remodeling.
Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Histonas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Montagem e Desmontagem da Cromatina , Relevância Clínica , Netropsina/metabolismo , DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , CromatinaRESUMO
The structural analysis of guest molecules in rationally designed and self-assembling DNA crystals has proven an elusive goal since its conception. Oligonucleotide frameworks provide an especially attractive route toward studying DNA-binding molecules by using three-dimensional lattices with defined sequence and structure. In this work, we site-specifically position a suite of minor groove binding molecules, and solve their structures via X-ray crystallography as a proof-of-principle toward scaffolding larger guest species. Two crystal motifs were used to precisely immobilize the molecules DAPI, Hoechst, and netropsin at defined positions in the lattice, allowing us to control occupancy within the crystal. We also solved the structure of a three-ring imidazole-pyrrole-pyrrole polyamide molecule, which sequence-specifically packs in an antiparallel dimeric arrangement within the minor groove. Finally, we engineered a crystal designed to position both netropsin and the polyamide at two distinct locations within the same lattice. Our work elucidates the design principles for the spatial arrangement of functional guests within lattices and opens new potential opportunities for the use of DNA crystals to display and structurally characterize small molecules, peptides, and ultimately proteins of unknown structure.
Assuntos
Netropsina , Nylons , Netropsina/química , DNA/química , Oligonucleotídeos , Pirróis/química , Conformação de Ácido NucleicoRESUMO
Immobilization of DNA to surfaces offers a convenient means of screening the binding affinity and selectivity of potential small-molecule therapeutic candidates. Unfortunately, most surface-sensitive methods for detecting these binding interactions are not informative of the molecular structure, information that is valuable for understanding the non-covalent interactions that stabilize binding. In this work, we report a method to meet this challenge by employing confocal Raman microscopy to quantify the association of a minor-groove-binding antimicrobial peptide, netropsin, to duplex DNA hairpin sequences immobilized on the interior surfaces of porous silica particles. To assess binding selectivity, particles functionalized with different sequences of DNA were equilibrated with solutions of 100 nM netropsin, and selective association was detected based on the presence of netropsin Raman scattering in the particles. The selectivity study revealed that netropsin binds to sequences of duplex DNA having AT-rich recognition regions. To quantify binding affinities, these AT-rich DNA sequences were equilibrated with a range of netropsin solution concentrations (1 to 100 nM). Raman scattering intensities of netropsin versus solution concentration were well described by single-binding-site Langmuir isotherms with nanomolar dissociation constants, in agreement with previous isothermal calorimetry and surface plasmon resonance results. Target sequence binding was accompanied with changes in netropsin and DNA vibrational modes consistent with the hydrogen bonding between the amide groups of netropsin and adenine and thymine bases in the DNA minor groove. The binding of netropsin to a control sequence lacking the AT-rich recognition region exhibited an affinity nearly 4 orders of magnitude weaker than found for the target sequences. The Raman spectrum of netropsin interacting with this control sequence showed broad pyrrole and amide mode vibrations at frequencies similar to a free solution, revealing less constrained conformations compared with the specific binding interactions observed with AT-rich sequences.
Assuntos
Netropsina , Análise Espectral Raman , Sequência de Bases , Netropsina/química , Netropsina/metabolismo , Conformação de Ácido Nucleico , DNA/química , Sítios de Ligação , AntibacterianosRESUMO
The production of specialized metabolites by Streptomyces bacteria is usually temporally regulated. This regulation is complex and frequently involves both global and pathway-specific mechanisms. Streptomyces ambofaciens ATCC23877 produces several specialized metabolites, including spiramycins, stambomycins, kinamycins and congocidine. The production of the first three molecules has been shown to be controlled by one or several cluster-situated transcriptional regulators. However, nothing is known regarding the regulation of congocidine biosynthesis. Congocidine (netropsin) belongs to the family of pyrrolamide metabolites, which also includes distamycin and anthelvencins. Most pyrrolamides bind into the minor groove of DNA, specifically in A/T-rich regions, which gives them numerous biological activities, such as antimicrobial and antitumoral activities. We previously reported the characterization of the pyrrolamide biosynthetic gene clusters of congocidine (cgc) in S. ambofaciens ATCC23877, distamycin (dst) in Streptomyces netropsis DSM40846, and anthelvencins (ant) in Streptomyces venezuelae ATCC14583. The three gene clusters contain a gene encoding a putative transcriptional regulator, cgc1, dst1, and ant1, respectively. Cgc1, Dst1, and Ant1 present a high percentage of amino acid sequence similarity. We demonstrate here that Cgc1, an atypical orphan response regulator, activates the transcription of all cgc genes in the stationary phase of S. ambofaciens growth. We also show that the cgc cluster is constituted of eight main transcriptional units. Finally, we show that congocidine induces the expression of the transcriptional regulator Cgc1 and of the operon containing the resistance genes (cgc20 and cgc21, coding for an ABC transporter), and propose a model for the transcriptional regulation of the cgc gene cluster. IMPORTANCE Understanding the mechanisms of regulation of specialized metabolite production can have important implications both at the level of specialized metabolism study (expression of silent gene clusters) and at the biotechnological level (increase of the production of a metabolite of interest). We report here a study on the regulation of the biosynthesis of a metabolite from the pyrrolamide family, congocidine. We show that congocidine biosynthesis and resistance are controlled by Cgc1, a cluster-situated regulator. As the gene clusters directing the biosynthesis of the pyrrolamides distamycin and anthelvencin encode a homolog of Cgc1, our findings may be relevant for the biosynthesis of other pyrrolamides. In addition, our results reveal a new type of feed-forward induction mechanism, in which congocidine induces its own biosynthesis through the induction of the transcription of cgc1.
Assuntos
Regulação Bacteriana da Expressão Gênica , Netropsina , Streptomyces , Distamicinas , Genes Bacterianos , Família Multigênica , Netropsina/biossíntese , Streptomyces/genética , Streptomyces/metabolismoRESUMO
Optical DNA mapping (ODM) allows visualization of long-range sequence information along single DNA molecules. The data can for example be used for detecting long range structural variations, for aiding DNA sequence assembly of complex genomes and for mapping epigenetic marks and DNA damage across the genome. ODM traditionally utilizes sequence specific marks based on nicking enzymes, combined with a DNA stain, YOYO-1, for detection of the DNA contour. Here we use a competitive binding approach, based on YOYO-1 and netropsin, which highlights the contour of the DNA molecules, while simultaneously creating a continuous sequence specific pattern, based on the AT/GC variation along the detected molecule. We demonstrate and validate competitive-binding-based ODM using bacterial artificial chromosomes (BACs) derived from the human genome and then turn to DNA extracted from white blood cells. We generalize our findings with in-silico simulations that show that we can map a vast majority of the human genome. Finally, we demonstrate the possibility of combining competitive binding with enzymatic labeling by mapping DNA damage sites induced by the cytotoxic drug etoposide to the human genome. Overall, we demonstrate that competitive-binding-based ODM has the potential to be used both as a standalone assay for studies of the human genome, as well as in combination with enzymatic approaches, some of which are already commercialized.
Assuntos
Benzoxazóis/química , Mapeamento Cromossômico/métodos , DNA/química , Genoma Humano , Netropsina/química , Compostos de Quinolínio/química , Análise de Sequência de DNA/métodos , Antineoplásicos Fitogênicos/farmacologia , Sítios de Ligação , Ligação Competitiva , Cromossomos Artificiais Bacterianos/química , DNA/genética , Etoposídeo/farmacologia , Corantes Fluorescentes/química , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Imagem Individual de Molécula/métodosRESUMO
A-tracts are sequences of repeated adenine bases that, under the proper conditions, are capable of mediating DNA curvature. A-tracts occur naturally in the regulatory regions of many organisms, yet their biological functions are not fully understood. Orienting multiple A-tracts together constructively or destructively in a phase has the potential to create different shapes in the DNA helix axis. One means of detecting these molecular shape differences is from altered DNA mobilities measured using electrophoresis. The small molecule netropsin binds the minor groove of DNA, particularly at AT-rich sequences including A-tracts. Here, we systematically test the hypothesis that netropsin binding eliminates the curvature of A-tracts by measuring the electrophoretic mobilities of seven 98-base pair DNA samples containing different numbers and arrangements of centrally located A-tracts under varying conditions with netropsin. We find that netropsin binding eliminates the mobility difference between the DNA fragments with different A-tract arrangements in a concentration-dependent manner. This work provides evidence for the straightening of A-tracts upon netropsin binding and illustrates an artificial approach to re-sculpt DNA shape.
Assuntos
Antibacterianos/química , DNA/química , Eletroforese/métodos , Netropsina/química , Conformação de Ácido Nucleico , Sequência de Bases , Humanos , Estrutura Molecular , Homologia de SequênciaRESUMO
The recognition of specific DNA sequences in processes such as transcription is associated with a cooperative binding of proteins. Some transcription regulation mechanisms involve additional proteins that can influence the binding cooperativity by acting as corepressors or coactivators. In a conditional cooperativity mechanism, the same protein can induce binding cooperativity at one concentration and inhibit it at another. Here, we use calorimetric (ITC) and spectroscopic (UV, CD) experiments to show that such conditional cooperativity can also be achieved by the small DNA-directed oligopeptides distamycin and netropsin. Using a global thermodynamic analysis of the observed binding and (un)folding processes, we calculate the phase diagrams for this system, which show that distamycin binding cooperativity is more pronounced at lower temperatures and can be first induced and then reduced by increasing the netropsin or/and Na+ ion concentration. A molecular interpretation of this phenomenon is suggested.
Assuntos
DNA/metabolismo , Oligopeptídeos/metabolismo , Distamicinas/metabolismo , Netropsina/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Sódio/metabolismo , Termodinâmica , Transcrição Gênica/genéticaRESUMO
Covering: 1990 to 2019 Many medicinally-relevant compounds are derived from non-ribosomal peptide synthetase (NRPS) products. Type I NRPSs are organized into large modular complexes, while type II NRPS systems contain standalone or minimal domains that often encompass specialized tailoring enzymes that produce bioactive metabolites. Protein-protein interactions and communication between the type II biosynthetic machinery and various downstream pathways are critical for efficient metabolite production. Importantly, the architecture of type II NRPS proteins makes them ideal targets for combinatorial biosynthesis and metabolic engineering. Future investigations exploring the molecular basis or protein-protein recognition in type II NRPS pathways will guide these engineering efforts. In this review, we consolidate the broad range of NRPS systems containing type II proteins and focus on structural investigations, enzymatic mechanisms, and protein-protein interactions important to unraveling pathways that produce unique metabolites, including dehydrogenated prolines, substituted benzoic acids, substituted amino acids, and cyclopropanes.
Assuntos
Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Ácido Benzoico/química , Ácido Benzoico/metabolismo , Ciclopropanos/química , Ciclopropanos/metabolismo , Hidroxilação , Lactamas/metabolismo , Macrolídeos/metabolismo , Netropsina/biossíntese , Peptídeo Sintases/genética , Prolina/metabolismo , Mapas de Interação de Proteínas , Pirróis/química , Pirróis/metabolismo , Tiazóis/metabolismo , Tionas/metabolismoRESUMO
Netropsin is one of the first ligands to be discovered that selectively binds to the minor groove of DNA and is actively used as a scaffold for developing potential anticancer and antibiotic agents. The mechanism by which netropsin binds to hairpin DNA remains controversial with two competing mechanisms having been proposed. In one mechanism, netropsin binding induces a hairpin-to-duplex DNA transition. Alternatively, netropsin binds in two thermodynamically different modes at a single duplexed AATT site. Here, results from native mass spectrometry (MS) with nanoscale ion emitters indicate that netropsin can simultaneously and sequentially bind to both hairpin and duplex DNA. Duplex DNA was not detected using conventional MS with larger emitters because nanoscale emitters significantly reduce the extent of salt adduction to ligand-DNA complex ions, including in the presence of relatively high concentrations of nonvolatile salts. Based on native MS and polyacrylamide gel electrophoresis results, the abundances of hairpin and duplex DNA are unaffected by the addition of netropsin. By native MS, the binding affinities for five ligand-DNA and DNA-DNA interactions can be rapidly obtained simultaneously. This research indicates a "simultaneous binding mechanism" for the interactions of netropsin with DNA.
Assuntos
DNA/metabolismo , Netropsina/metabolismo , DNA/genética , Eletroforese em Gel de Poliacrilamida , Sequências Repetidas Invertidas , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray/métodos , Streptomyces/químicaRESUMO
Alchemically derived free energies are artifacted when the perturbed moiety has a nonzero net charge. The source of the artifacts lies in the effective treatment of the electrostatic interactions within and between the perturbed atoms and remaining (partial) charges in the simulated system. To treat the electrostatic interactions effectively, lattice-summation (LS) methods or cutoff schemes in combination with a reaction-field contribution are usually employed. Both methods render the charging component of the calculated free energies sensitive to essential parameters of the system like the cutoff radius or the box side lengths. Here, we discuss the results of three previously published studies of ligand binding. These studies presented estimates of binding free energies that were artifacted due to the charged nature of the ligands. We show that the size of the artifacts can be efficiently calculated and raw simulation data can be corrected. We compare the corrected results with experimental estimates and nonartifacted estimates from path-sampling methods. Although the employed correction scheme involves computationally demanding continuum-electrostatics calculations, we show that the correction estimate can be deduced from a small sample of configurations rather than from the entire ensemble. This observation makes the calculations of correction terms feasible for complex biological systems. To show the general applicability of the proposed procedure, we also present results where the correction scheme was used to correct independent free energies obtained from simulations employing a cutoff scheme or LS electrostatics. In this work, we give practical guidelines on how to apply the appropriate corrections easily.
Assuntos
Eletricidade Estática , Artefatos , Sítios de Ligação , DNA/química , Distamicinas/química , Ligantes , Simulação de Dinâmica Molecular , Netropsina/química , Solventes/química , Termodinâmica , Inibidores da Tripsina/químicaRESUMO
Overexpression of phosphopantetheinyl transferase (PPtase)-encoding genes sfp and svp in the marine-derived Verrucosispora sp. SCSIO 40062 led to the production of two new aminofuran monomers, proximicin F (1) and proximicin G (3) and a new dimer diproximicin A (2), along with two known compounds, proximicins B (4) and C (5). Their structures were unambiguously elucidated on the basis of detailed NMR spectroscopic analysis and high-resolution electrospray ionization mass spectrometry (HRESIMS) data. Proximicin B (4) showed moderate antibacterial activities against Staphylococcus aureus, methicillin-resistant S. aureus, and Bacillus subtilis.
Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Micromonosporaceae/química , Netropsina/análogos & derivados , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Netropsina/química , Netropsina/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Staphylococcus aureus/químicaRESUMO
Conioidine A (1), isolated in 1993 with unknown relative and absolute configuration, was suggested to be a DNA-binding compound by an indirect technique. Four stereoisomers of conioidine A have been synthesized from d- and l-proline, and the natural product has been identified as possessing (4R,6R) absolute configuration. Binding of the conioidine diastereomers to calf thymus DNA (CT DNA) and human serum albumin (HSA) has been investigated by fluorescence spectroscopy and isothermal titration calorimetry (ITC). All stereoisomers display at least an order of magnitude weaker binding to DNA than the control compound netropsin; however, a strong association with HSA was observed for the (4R,6S) stereoisomer.
Assuntos
Pirrolidinas/química , Pirrolidinas/síntese química , Alcaloides de Solanáceas/química , Alcaloides de Solanáceas/síntese química , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Calorimetria , Dicroísmo Circular , DNA/química , Etídio , Simulação de Acoplamento Molecular , Estrutura Molecular , Netropsina/química , Netropsina/metabolismo , Prolina/química , Albumina Sérica Humana/química , Espectrometria de Fluorescência , EstereoisomerismoRESUMO
A stable intense resistance called "nonhost resistance" generates a complete multiple-gene resistance against plant pathogenic species that are not pathogens of pea such as the bean pathogen, Fusarium solani f. sp. phaseoli (Fsph). Chitosan is a natural nonhost resistance response gene activator of defense responses in peas. Chitosan may share with cancer-treatment compounds, netropsin and some anti-cancer drugs, a DNA minor groove target in plant host tissue. The chitosan heptamer and netropsin have the appropriate size and charge to reside in the DNA minor groove. The localization of a percentage of administered radio-labeled chitosan in the nucleus of plant tissue in vivo indicates its potential to transport to site(s) within the nuclear chromatin (1,2). Other minor groove-localizing compounds administered to pea tissue activate the same secondary plant pathway that terminates in the production of the anti-fungal isoflavonoid, pisatin an indicator of the generated resistance response. Some DNA minor groove compounds also induce defense genes designated as "pathogenesis-related" (PR) genes. Hypothetically, DNA targeting components alter host DNA in a manner enabling the transcription of defense genes previously silenced or minimally expressed. Defense-response-elicitors can directly (a) target host DNA at the site of transcription or (b) act by a series of cascading events beginning at the cell membrane and indirectly influence transcription. A single defense response, pisatin induction, induced by chitosan and compounds with known DNA minor groove attachment potential was followed herein. A hypothesis is formulated suggesting that this DNA target may be accountable for a portion of the defense response generated in nonhost resistance.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Quitosana/farmacologia , Substâncias Intercalantes/farmacologia , Netropsina/farmacologia , Pisum sativum/genética , Doenças das Plantas/genética , Pterocarpanos/farmacologia , Antineoplásicos Fitogênicos/química , Benzimidazóis/química , Benzimidazóis/farmacologia , Quitosana/química , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Cromomicinas/química , Cromomicinas/farmacologia , DNA de Plantas/genética , DNA de Plantas/metabolismo , Resistência à Doença/genética , Fusarium/crescimento & desenvolvimento , Fusarium/patogenicidade , Regulação da Expressão Gênica de Plantas , Proteínas HMGA/genética , Proteínas HMGA/metabolismo , Substâncias Intercalantes/química , Netropsina/química , Pisum sativum/imunologia , Pisum sativum/metabolismo , Pisum sativum/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pterocarpanos/química , Transcrição GênicaRESUMO
Studies on DNA-ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA-ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA-ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro-validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off-target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of drug-like molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.
Assuntos
Anti-Infecciosos/farmacologia , DNA/metabolismo , Naftalenos/farmacologia , Netropsina/farmacologia , Anti-Infecciosos/química , Pareamento de Bases/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/química , Descoberta de Drogas , Humanos , Ligantes , Naftalenos/química , Netropsina/química , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação de Ácido Nucleico/efeitos dos fármacosRESUMO
Trichomonas vaginalis (T. vaginalis) is a common sexually transmitted infection, affecting the urogenital tract. Trichomoniasis is customarily treated with metronidazole (MTZ). MTZ is known to cause undesirable side effects and there is several reports on MTZ resistant T. vaginalis. Thus, the present study aimed to in-vitro evaluate the activity of DNA minor groove binder drug ''Netropsin dihydrochloride'' against metronidazole-sensitive T. vaginalis isolates (G and U isolates) and resistant T. vaginalis isolate (ATCC50138) (R isolate). Netropsin was tested at concentrations ranging from 3.5 to 200 µg/ml. It showed effectiveness against all isolates with MLC of 12.5 µg/ml for G and U isolates and of 25 µg/ml for R isolate. Cytotoxicity assay of isolates exposed to the respective MLC of netropsin for 42 h showed a highly significant reduction in the death percentage of MCDK cell line as compared to the effect elicited by drug free controls. The hemolytic activity was evaluated by hemolytic assay and by monitoring the interaction of T. vaginalis isolates with human erythrocytes by inverted microscopy and scanning electron microscopy. The hemolytic assay showed (0%) hemolysis of RBCs incubated with T. vaginalis isolates treated with the corresponding MLC of netropsin for 24 h. Scanning electron microscopy revealed cytoskeletal deformities of netropsin treated isolates. Taken together, these observations suggest that netropsin is a promising therapy for T. vaginalis infection affecting its viability, virulence, cytopathogenic and hemolytic activity with a mechanism of action that might overcome T. vaginalis resistance to metronidazole.
Assuntos
Antibacterianos/farmacologia , Netropsina/farmacologia , Vaginite por Trichomonas/tratamento farmacológico , Trichomonas vaginalis/efeitos dos fármacos , Animais , Antibacterianos/uso terapêutico , Cães , Resistência a Medicamentos , Feminino , Hemólise/imunologia , Humanos , Células Madin Darby de Rim Canino , Metronidazol/farmacologia , Metronidazol/uso terapêutico , Netropsina/uso terapêutico , Testes de Sensibilidade Parasitária , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/imunologia , Trichomonas vaginalis/isolamento & purificação , Trichomonas vaginalis/patogenicidade , Trofozoítos/efeitos dos fármacos , Trofozoítos/imunologia , Vagina/parasitologiaRESUMO
The small molecules that bind to DNA minor groove are considered as potential therapeutic agents to fight against many human diseases. They induce cell death by interfering with transcription, replication and progression of cell cycle. Herein, we report the synthesis of imidazopyridine-3-amines using sulfated ceria catalyst by employing Groebkee-Blackburne-Bienayme reaction. We evaluated the possible antiproliferative and antimycobacterial activity against A549 cells and Mycobacterium tuberculosis, respectively. Among the tested compounds, N-tert-butyl-2-(2-butyl-4-chloro-1H-imidazol-5-yl)-5,7-dimethylimidazo[1,2-a]pyridin-3-amine (4g) was identified as cytotoxic heterocycle and antimycobacterial agent. Molecular docking studies of the imidazopyridine derivatives revealed the consistent positioning in the minor groove with a tight shape fit between receptor and ligands. Therefore, we speculate that new imidazopyridines induce their pharmacological effect by targeting the minor groove of DNA.
Assuntos
Antituberculosos/síntese química , Cério/química , DNA/química , Imidazóis/química , Piridinas/química , Células A549 , Antituberculosos/química , Antituberculosos/farmacologia , Sítios de Ligação , Catálise , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ciclização , DNA/metabolismo , Humanos , Imidazóis/síntese química , Imidazóis/farmacologia , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Netropsina/química , Netropsina/metabolismo , Conformação de Ácido Nucleico , Piridinas/síntese química , Piridinas/farmacologia , Relação Estrutura-Atividade , Sulfatos/químicaRESUMO
HMGA2 is an important chromatin factor that interacts with DNA via three AT-hook domains, thereby regulating chromatin architecture and transcription during embryonic and fetal development. The protein is absent from differentiated somatic cells, but aberrantly re-expressed in most aggressive human neoplasias where it is causally linked to cell transformation and metastasis. DNA-binding also enables HMGA2 to protect cancer cells from DNA-damaging agents. HMGA2 therefore is considered to be a prime drug target for many aggressive malignancies. Here, we have developed a broadly applicable cell-based reporter system which can identify HMGA2 antagonists targeting functionally important protein domains, as validated with the known AT-hook competitor netropsin. In addition, high-throughput screening can uncover functional links between HMGA2 and cellular factors important for cell transformation. This is demonstrated with the discovery that HMGA2 potentiates the clinically important topoisomerase I inhibitor irinotecan/SN-38 in trapping the enzyme in covalent DNA-complexes, thereby attenuating transcription.
Assuntos
Camptotecina/análogos & derivados , DNA Topoisomerases Tipo I/fisiologia , Proteína HMGA2/fisiologia , Inibidores da Topoisomerase I/farmacologia , Sequência de Aminoácidos , Camptotecina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Estabilidade Enzimática , Genes Reporter , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Irinotecano , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Netropsina/farmacologia , Regiões Promotoras Genéticas , Transcrição Gênica/efeitos dos fármacos , Ativação TranscricionalRESUMO
DNA is the target of chemical compounds (drugs, pollutants, photosensitizers, etc.), which bind through non-covalent interactions. Depending on their structure and their chemical properties, DNA binders can associate to the minor or to the major groove of double-stranded DNA. They can also intercalate between two adjacent base pairs, or even replace one or two base pairs within the DNA double helix. The subsequent biological effects are strongly dependent on the architecture of the binding motif. Discriminating between the different binding patterns is of paramount importance to predict and rationalize the effect of a given compound on DNA. The structural characterization of DNA complexes remains, however, cumbersome at the experimental level. In this contribution, we employed all-atom molecular dynamics simulations to determine the standard binding free energy of DNA with netropsin, a well-characterized antiviral and antimicrobial drug, which associates to the minor groove of double-stranded DNA. To overcome the sampling limitations of classical molecular dynamics simulations, which cannot capture the large change in configurational entropy that accompanies binding, we resort to a series of potentials of mean force calculations involving a set of geometrical restraints acting on collective variables.
Assuntos
DNA/química , Modelos Moleculares , Netropsina/química , Conformação de Ácido Nucleico , Conformação Proteica , Algoritmos , Sítios de Ligação , DNA/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Netropsina/metabolismo , Relação Estrutura-AtividadeRESUMO
DNA-binding and RNA-binding proteins are usually considered 'undruggable' partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein-nucleic acids interactions based on protein-DNA or protein-RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2-DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2-DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2-DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Ensaio de Imunoadsorção Enzimática/métodos , Ensaios de Triagem em Larga Escala/métodos , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Animais , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína HMGA2/antagonistas & inibidores , Proteína HMGA2/metabolismo , Camundongos , Netropsina/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismoRESUMO
Microbes belonging to the genus Verrucosispora possess significant chemical diversity and biological properties. They have attracted the interests of many researchers and are becoming promising resources in the marine natural product research field. A bioassay-guided isolation from the crude extract of Verrucosispora sp. strain MS100047, isolated from sediments collected from the South China Sea, has led to the identification of a new salicylic derivative, glycerol 1-hydroxy-2,5-dimethyl benzoate (1), along with three known compounds, brevianamide F (2), abyssomicin B (3), and proximicin B (4). Compound 1 showed selective activity against methicillin-resistant Staphylococcus aureus (MRSA) with a minimum inhibitory concentration (MIC) value of 12.5 µg/mL. Brevianamide F (2), which was isolated from actinomycete for the first time, showed a good anti-BCG activity with a MIC value of 12.5 µg/mL that has not been reported previously in literatures. Proximicin B (4) showed significant anti-MRSA (MIC = 3.125 µg/mL), anti-BCG (MIC = 6.25 µg/mL), and anti-tuberculosis (TB) (MIC = 25 µg/mL) activities. This is the first report on the anti-tubercular activities of proximicins. In addition, Verrucosispora sp. strain MS100047 was found to harbor 18 putative secondary metabolite gene clusters based on genomic sequence analysis. These include the biosynthetic loci encoding polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) consistent with abyssomicins and proximicins, respectively. The biosynthetic pathways of these isolated compounds have been proposed. These results indicate that MS100047 possesses a great potential as a source of active secondary metabolites.