RESUMO
A 2-naphthol derivative 2 corresponding to the aromatic ring moiety of neocarzinostatin chromophore was found to degrade proteins under photo-irradiation with long-wavelength UV light without any additives under neutral conditions. Structure-activity relationship studies of the derivative revealed that methylation of the hydroxyl group at the C2 position of 2 significantly suppressed its photodegradation ability. Furthermore, a purpose-designed synthetic tumor-related biomarker, a H2 O2 -activatable photosensitizer 8 possessing a H2 O2 -responsive arylboronic ester moiety conjugated to the hydroxyl group at the C2 position of 2, showed significantly lower photodegradation ability compared to 2. However, release of the 2 from 8 by reaction with H2 O2 regenerated the photodegradation ability. Compound 8 exhibited selective photo-cytotoxicity against high H2 O2 -expressing cancer cells upon irradiation with long-wavelength UV light.
Assuntos
Naftóis , Proteínas , Zinostatina/análogos & derivados , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/química , Camundongos , Naftóis/química , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/toxicidade , Proteínas/efeitos dos fármacos , Proteínas/efeitos da radiação , Zinostatina/química , Zinostatina/toxicidadeRESUMO
Neocarzinostatin (NCS) consists of an enediyne chromophore and an apoprotein (NCP). Lidamycin (LDM) is composed of another active enediyne chromophore (AE) and an acidic protein (LDP). Although the structures of NCP and LDP are very similar, LDM has been shown to have an increased tumor-suppressive activity than that of NCS. The aim of this study was to construct a chimeric protein (CMP) that consists of both the terminus residue of NCP and an LDP pocket-forming residue that can bind AE. This CMP will have a structure similar to NCS and an antitumor activity similar to LDM. The assembling efficiency of LDP, CMP, and NCP was 73.9, 1.5, and 1.1%, respectively. The cytotoxicity was consistent with their assembling efficiency of AE in proteins. When CMP-AE and NCP-AE were administered at equivalent AE doses of LDM, the inhibition rate of CMP-AE was the same as LDM and significantly higher than that of NCP-AE. Our study implied that the binding activity between LDP and AE was very specific. The terminus residue of LDP could affect the specifically binding activity. The pocket-forming residue could confer a protective function to the chromophore. Further investigation of its bioactivity might serve as a new drug design strategy and drug-delivery carrier in targeted cancer therapy.
Assuntos
Aminoglicosídeos/química , Antineoplásicos/química , Apoproteínas/química , Enedi-Inos/química , Proteínas Recombinantes de Fusão/química , Zinostatina/química , Aminoglicosídeos/genética , Animais , Antineoplásicos/farmacologia , Apoproteínas/genética , Apoproteínas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Neocarzinostatin (NCS) a potent DNA-damaging, anti-tumor toxin extracted from Streptomyces carzinostaticus that recognizes double-stranded DNA bulge and induces DNA damage. 2 Fluoro (2F) Modified EpCAM RNA aptamer is a 23-mer that targets EpCAM protein, expressed on the surface of epithelial tumor cells. Understanding the interaction between NCS and the ligand is important for carrying out the targeted tumor therapy. In this study, we have investigated the biophysical interactions between NCS and 2-fluro Modified EpCAM RNA aptamer using Circular Dichroism (CD) and Infra-Red (IR) spectroscopy. The aromatic amino acid residues spanning the ß sheets of NCS are found to participate in intermolecular interactions with 2 F Modified EpCAM RNA aptamer. In-silico modeling and simulation studies corroborate with CD spectra data. Furthermore, it reinforces the involvement of C and D1 strand of NCS in intermolecular interactions with EpCAM RNA aptamer. This the first report on interactions involved in the stabilization of NCS-EpCAM aptamer complex and will aid in the development of therapeutic modalities towards targeted cancer therapy.
Assuntos
Antígenos de Neoplasias/química , Antígenos de Neoplasias/ultraestrutura , Aptâmeros de Nucleotídeos/química , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/ultraestrutura , Modelos Químicos , Simulação de Acoplamento Molecular , Zinostatina/química , Sítios de Ligação , Citotoxinas , Molécula de Adesão da Célula Epitelial , Conformação Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas/métodosRESUMO
The enhanced permeability and retention (EPR) of nanoparticles in tumors has long stood as one of the fundamental principles of cancer drug delivery, holding the promise of safe, simple and effective therapy. By allowing particles preferential access to tumors by virtue of size and longevity in circulation, EPR provided a neat rationale for the trend toward nano-sized drug carriers. Following the discovery of the phenomenon by Maeda in the mid-1980s, this rationale appeared to be well justified by the flood of evidence from preclinical studies and by the clinical success of Doxil. Clinical outcomes from nano-sized drug delivery systems, however, have indicated that EPR is not as reliable as previously thought. Drug carriers generally fail to provide superior efficacy to free drug systems when tested in clinical trials. A closer look reveals that EPR-dependent drug delivery is complicated by high tumor interstitial fluid pressure (IFP), irregular vascular distribution, and poor blood flow inside tumors. Furthermore, the animal tumor models used to study EPR differ from clinical tumors in several key aspects that seem to make EPR more pronounced than in human patients. On the basis of this evidence, we believe that EPR should only be invoked on a case-by-case basis, when clinical evidence suggests the tumor type is susceptible.
Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/química , Nanopartículas , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/história , Portadores de Fármacos/história , História do Século XX , Humanos , Anidridos Maleicos/administração & dosagem , Anidridos Maleicos/química , Anidridos Maleicos/história , Nanopartículas/química , Neoplasias/patologia , Permeabilidade , Poliestirenos/administração & dosagem , Poliestirenos/química , Poliestirenos/história , Zinostatina/administração & dosagem , Zinostatina/análogos & derivados , Zinostatina/química , Zinostatina/históriaRESUMO
In the field of pharmaceuticals there is an increasing need for new delivery systems to overcome the issues of solubility, penetration, toxicity and drug resistance. One of the possible strategies is to use biocarriers such as proteins to encourage the cell-penetration of drugs. In this paper, the use of the apo-protein neocarzinostatin (apo-NCS) as a carrier-protein for two Cu(II) glycocomplexes, previously characterized, and Cu(II) ions was investigated. Its interaction with the metallic compounds was analyzed using microcalorimetry. The dissociation constants were shown to be in the micromolar range. The Cu(II) glycocomplexes, in absence of apo-NCS, were found to be cytotoxic in the U937 and HT29 cell lines whereas the corresponding glycoligands showed no toxicity. The leukemic cell line (U937) seems to be more sensitive to glycocomplexes than the colon cancer cell line (HT29). Interestingly, apo-NCS was shown to increase systematically the antiproliferative activity by a factor of 2 and 3 for Cu(II) glycocomplexes and Cu(II) respectively. The antiproliferative activity detected was not related to proteasome inhibition. This result stresses the importance of new molecular tools for the delivery of Cu(II) to tumor cells using non-covalent association with carriers proteins.
Assuntos
Antineoplásicos/química , Proteínas de Bactérias/química , Complexos de Coordenação/química , Cobre/química , Portadores de Fármacos/química , Zinostatina/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoproteínas/química , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Glicolipídeos/química , Células HT29 , Humanos , Concentração Inibidora 50RESUMO
Neocarzinostatin is an antibiotic chromoprotein produced by Streptomyces carzinostaticus. Its enediyne-containing chromophore exhibits high DNA cleavage activity and belongs to one of the most potent categories of antitumor agents. The labile chromophore is readily inactivated by environmental thiols including the most abundant glutathione. How the microorganism preserves the secreted antibiotic and at the same time is immune to its toxicity are of interest. Site-directed mutagenesis studies of the neocarzinostatin protein have shown that residues D33 and D99 play primary and secondary roles, respectively, in preserving neocarzinostatin from acidic glutathione whereas D79 and other residues around the opening of the binding cleft have an insignificant effect. Biothiol analyses revealed that cells of S. carzinostaticus produced no glutathione, but instead neutral mycothiol, which is known to serve functions analogous to glutathione. Mycothiol was the only neutral-charged thiol produced by the organism; all other identified biothiols carried at least partial negative charges. When the bacteria were cultured under conditions that stimulated the biosynthesis of neocarzinostatin, the yield of mycothiol increased significantly, which suggests mycothiol-dependent cellular detoxification. Treating neocarzinostatin samples with the cell extract that retained active sulfhydryls led to efficient drug inactivation, which indicates that mycothiol is allowed to approach the protein-bound chromophore. The anionic side-chains of D33 and D99 in the neocarzinostatin protein played two critical roles in a single thiol-screening operation: Preserving the antibiotic for defense and survival by rejecting the ubiquitous glutathione through charge-charge repulsion in the outer-cell environment and detoxifying the toxin in the inner-cell body for self-resistance by accepting the cell-produced neutral mycothiol.
Assuntos
Antibacterianos/química , Enedi-Inos/química , Streptomyces/química , Compostos de Sulfidrila/análise , Zinostatina/química , Antibacterianos/metabolismo , Cisteína/metabolismo , Enedi-Inos/metabolismo , Glutationa/química , Glutationa/metabolismo , Glicopeptídeos/metabolismo , Inositol/metabolismo , Estrutura Molecular , Streptomyces/metabolismo , Compostos de Sulfidrila/química , Zinostatina/biossíntese , Zinostatina/metabolismoRESUMO
Neocarzinostatin (NCS), a potent mutagen and carcinogen, consists of an enediyne prodrug and a protein carrier. It has a unique double role in that it intercalates into DNA and imposes radical-mediated damage after thiol activation. Here we employed NCS as a probe to examine the DNA-protection capability of caffeine, one of common dietary phytochemicals with potential cancer-chemopreventive activity. NCS at the nanomolar concentration range could induce significant single- and double-strand lesions in DNA, but up to 75 ± 5% of such lesions were found to be efficiently inhibited by caffeine. The percentage of inhibition was caffeine-concentration dependent, but was not sensitive to the DNA-lesion types. The well-characterized activation reactions of NCS allowed us to explore the effect of caffeine on the enediyne-generated radicals. Postactivation analyses by chromatographic and mass spectroscopic methods identified a caffeine-quenched enediyne-radical adduct, but the yield was too small to fully account for the large inhibition effect on DNA lesions. The affinity between NCS chromophore and DNA was characterized by a fluorescence-based kinetic method. The drug-DNA intercalation was hampered by caffeine, and the caffeine-induced increases in DNA-drug dissociation constant was caffeine-concentration dependent, suggesting importance of binding affinity in the protection mechanism. Caffeine has been shown to be both an effective free radical scavenger and an intercalation inhibitor. Our results demonstrated that caffeine ingeniously protected DNA against the enediyne-induced damages mainly by inhibiting DNA intercalation beforehand. The direct scavenging of the DNA-bound NCS free radicals by caffeine played only a minor role.
Assuntos
Antibióticos Antineoplásicos/química , Cafeína/química , Sondas de DNA/química , DNA/química , Zinostatina/química , Sequestradores de Radicais Livres/química , Radicais Livres/química , Substâncias Intercalantes/química , Cinética , Mutagênicos/química , Zinostatina/análogos & derivadosRESUMO
The antibiotic neocarzinostatin comprises a carrier protein with a well-defined cavity for accommodating an active enediyne chromophore. The protein has two disulfides, one (Cys(37)-Cys(47)) lies on the cavity bottom and the other (Cys(88)-Cys(93)) in a constrained short loop. When the chromophore is not bound to the protein, a thiol-induced cycloaromatization of the enediyne into a tetrahydroindacene derivative is responsible for the potent antitumor activity. When it is protein-bound, the protein diverts the cycloaromatization pathway to form a distinct hydroxyisochromene-type product. How the protein directs the enediyne chemistry is an interesting puzzle, and various suggestions have been proposed in the past. We screened more than fifty thiols and manipulated conditions to locate reaction features and search for factors that could influence the protein directing strength. Thiol- and oxygen-concentration-dependence studies suggested that disulfides, which maintain the steric rigidity of the protein, could play a key role in diverting the cycloaromatization pathway. For direct proofs, we made mutations at each of the two disulfides by replacing sulfur atoms with oxygen. Circular dichroism and two-dimensional NMR spectroscopy studies suggested that the mutations changed neither the protein conformation nor the ligand interactions. Analyses of the thiol-induced cycloaromatization revealed that rupture of Cys(37)-Cys(47) made the protein almost completely lose its chemical directing ability, whereas rupture of Cys(88)-Cys(93) had only a minor influence. The results demonstrated that the steric rigidity of the binding cavity, but not necessary the whole protein, played an important role in the protein-directed mechanism.
Assuntos
Proteínas de Transporte/química , Cisteína/química , Enedi-Inos/química , Zinostatina/química , Antibióticos Antineoplásicos/química , Proteínas de Transporte/metabolismo , Cisteína/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Compostos de Sulfidrila/químicaRESUMO
The nine-membered enediyne class has drawn extensive interest because of extremely high antitumor potency and intricate interactions with its carrier protein. While the drug-induced DNA cleavage reactions have been mostly elucidated, the critical release-transport process of the labile enediyne molecule in cellular environment remained obscure. Using neocarzinostatin chromoprotein as a model, we demonstrated a lipid bilayer-assisted release mechanism. The in vitro enediyne release rate under aqueous conditions was found to be too slow to account for its efficient DNA cleavage action. Via the presence of lipid bilayers, chaotropic agents, or organic solvents, we found the release was substantially enhanced. The increased rate was linearly dependent on the lipid bilayer concentration and the dielectric value of the binary organic solvent mixtures. While lipid bilayers provided a low surrounding dielectricity to assist in drug release, there were no major conformational changes in the apo and holo forms of the carrier protein. In addition, the lifespan of the released enediyne chromophore was markedly extended through partitioning of the chromophore in the hydrophobic bilayer phase, and the lipid bilayer-stabilized enediyne chromophore significantly enhanced DNA cleavage in vitro. Collectively, we depicted how a lipid bilayer membrane efficiently enhanced dissociation of the enediyne chromophore through a hydrophobic sensing release mechanism and then acted as a protector of the released enediyne molecule until its delivery to the target DNA. The proposed membrane-assisted antibiotic release-transport model might signify a new dimension to our understanding of the modus operandi of the antitumor enediyne drugs.
Assuntos
Antibióticos Antineoplásicos/química , Enedi-Inos/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Zinostatina/química , Antibióticos Antineoplásicos/metabolismo , Clivagem do DNA , Enedi-Inos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Espectrometria de Fluorescência , Zinostatina/metabolismoRESUMO
Neocarzinostatin (NCS) is an antitumor chromophore carrier protein with many therapeutic applications. To characterize its binding and release mechanism, we have carried out molecular dynamics (MD) simulations for apo-NCS and holo-NCS. Although the beta-sheet regions of the protein exhibit restricted motion, large atomic fluctuations were observed in the loop regions, especially loop 99-104. This loop undergoes a "door-opening" motion that can facilitate chromophore binding and release. Calculated NMR order parameters confirm the simulated loop flexibility. We also provide a proposed explanation for the release rate difference for two mutants F78L and F78A through our simulation. The binding site structures of holo-NCS were also validated by chemical shift perturbations. Based on these results, a new binding and release mechanism for the NCS chromophore is proposed.
Assuntos
Antineoplásicos/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Zinostatina/análogos & derivados , Zinostatina/químicaRESUMO
BACKGROUND: Neocarzinostatin is a potent antitumor drug consisting of an enediyne chromophore and a protein carrier. METHODS: We characterized an intermediate in the equilibrium unfolding pathway of aponeocarzinostatin, using a variety of biophysical techniques including 1-anilino-8-napthalene sulfonate binding studies, size-exclusion fast protein liquid chromatography, intrinsic tryptophan fluorescence, circular dichroism, and 1H-15N heteronuclear single quantum coherence spectroscopy. RESULTS: The partially unfolded protein is in molten globule-like state, in which approximately 60% and approximately 20% tertiary and secondary structure is disrupted respectively. Despite lacking a fully coordinated tertiary structure for assembling a functional binding cleft, the protein in molten globule-like state is still able to fully protect the labile chromophore. Titration of chromophore leads the partially denatured apoprotein to fold into its native state. CONCLUSION: These findings bring insight into conserving mechanism of neocarzinostatin under harsh environment, where even the partially denatured apoprotein exhibits protective effect, confirming the superiority of the drug carrier.
Assuntos
Antineoplásicos/administração & dosagem , Apoproteínas/química , Portadores de Fármacos/química , Enedi-Inos/administração & dosagem , Zinostatina/química , Antineoplásicos/química , Sítios de Ligação , Dicroísmo Circular , Enedi-Inos/química , Guanidina/farmacologia , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide-free NCS variants suitable for a variety of purposes, including intracellular applications.The chromoprotein neocarzinostatin (NCS) has been intensively studied for its antitumour properties. It has recently been redesigned as a potential drug-carrying scaffold. A potential limit of this protein scaffold, especially for intracellular applications, is the presence of disulfide bonds. The objective of this work was to create a disulfide-free NCS-derived scaffold. A generic targeted approach was developed by using directed evolution methods. As a starting point we used a previously engineered NCS variant in which a hapten binding site had been created. A library was then generated in which cysteine Cys88 and Cys93 and neighbouring residues were randomly substituted. Variants that preserved the hapten binding function were selected by phage display and further screened by colony filtration methods. Several sequences with common features emerged from this process. The corresponding proteins were expressed, purified and their biophysical properties characterised. How these selected sequences rescued folding ability and stability of the disulfide-free protein was carefully examined by using calorimetry and the results were interpreted with molecular simulation techniques.
Assuntos
Antineoplásicos/química , Dissulfetos/química , Zinostatina/química , Sequência de Aminoácidos , Antineoplásicos/metabolismo , Sequência de Bases , Simulação por Computador , Cristalografia por Raios X , Evolução Molecular Direcionada , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Engenharia de Proteínas , Testosterona/química , Termodinâmica , Zinostatina/metabolismoRESUMO
Neocarzinostatin, a clinical anticancer drug, is the archetypal member of the chromoprotein family of enediyne antitumor antibiotics that are composed of a nonprotein chromophore and an apoprotein. The neocarzinostatin chromophore consists of a nine-membered enediyne core, a deoxyaminosugar, and a naphthoic acid moiety. We have previously cloned and sequenced the neocarzinostatin biosynthetic gene cluster and proposed that the biosynthesis of the naphthoic acid moiety and its incorporation into the neocarzinostatin chromophore are catalyzed by five enzymes NcsB, NcsB1, NcsB2, NcsB3, and NcsB4. Here we report the biochemical characterization of NcsB1, unveiling that: (i) NcsB1 is an S-adenosyl-L-methionine-dependent O-methyltransferase; (ii) NcsB1 catalyzes regiospecific methylation at the 7-hydroxy group of its native substrate, 2,7-dihydroxy-5-methyl-1-naphthoic acid; (iii) NcsB1 also recognizes other dihydroxynaphthoic acids as substrates and catalyzes regiospecific O-methylation; and (iv) the carboxylate and its ortho-hydroxy groups of the substrate appear to be crucial for NcsB1 substrate recognition and binding, and O-methylation takes place only at the free hydroxy group of these dihydroxynaphthoic acids. These findings establish that NcsB1 catalyzes the third step in the biosynthesis of the naphthoic acid moiety of the neocarzinostatin chromophore and further support the early proposal for the biosynthesis of the naphthoic acid and its incorporation into the neocarzinostatin chromophore with free naphthoic acids serving as intermediates. NcsB1 represents another opportunity that can now be exploited to produce novel neocarzinostatin analogs by engineering neocarzinostatin biosynthesis or applying directed biosynthesis strategies.
Assuntos
Antibacterianos/biossíntese , Antineoplásicos/metabolismo , Metiltransferases/metabolismo , Naftalenossulfonatos/metabolismo , Zinostatina/biossíntese , Antibacterianos/química , Antineoplásicos/química , Catálise , Clonagem Molecular , Expressão Gênica , Metilação , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Estrutura Molecular , Especificidade por Substrato , Zinostatina/químicaRESUMO
The enediynes, unified by their unique molecular architecture and mode of action, represent some of the most potent anticancer drugs ever discovered. The biosynthesis of the enediyne core has been predicted to be initiated by a polyketide synthase (PKS) that is distinct from all known PKSs. Characterization of the enediyne PKS involved in C-1027 (SgcE) and neocarzinostatin (NcsE) biosynthesis has now revealed that (i) the PKSs contain a central acyl carrier protein domain and C-terminal phosphopantetheinyl transferase domain; (ii) the PKSs are functional in heterologous hosts, and coexpression with an enediyne thioesterase gene produces the first isolable compound, 1,3,5,7,9,11,13-pentadecaheptaene, in enediyne core biosynthesis; and (iii) the findings for SgcE and NcsE are likely shared among all nine-membered enediynes, thereby supporting a common mechanism to initiate enediyne biosynthesis.
Assuntos
Proteína de Transporte de Acila/química , Antibióticos Antineoplásicos/biossíntese , Enedi-Inos/metabolismo , Polienos/química , Policetídeo Sintases/química , Proteína de Transporte de Acila/genética , Sequência de Aminoácidos , Aminoglicosídeos/biossíntese , Aminoglicosídeos/química , Sítios de Ligação , Biologia Computacional , Enedi-Inos/química , Dados de Sequência Molecular , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/química , Policetídeo Sintases/genética , Estrutura Terciária de Proteína , Zinostatina/biossíntese , Zinostatina/químicaRESUMO
The enediyne chromoproteins are a class of potent antitumour antibiotics comprising a 1:1 complex of a protein and a noncovalently bound chromophore. The protein is required to protect and transport the highly labile chromophore, which acts as the cytotoxic component by reacting with DNA leading to strand cleavage. A derivative of the best-studied member of this class, neocarzinostatin (NCS), is currently in use as a chemotherapeutic in Japan. The application of the chromoproteins as therapeutics along with their unique mode of action has prompted widespread interest in this area. Notable developments include the discovery of non-natural ligands for the apoproteins and the observation that multiple binding modes are available for these ligands in the binding site. Mutation studies on the apoproteins have revealed much about their stability and variability, and the application of an in vitro evolution method has conferred new binding specificity for unrelated ligands. These investigations hold great promise for the application of the apoproteins for drug-delivery, transport and stabilisation systems.
Assuntos
Antibacterianos/química , Proteínas/química , Zinostatina/química , Ligantes , Modelos Moleculares , Sondas Moleculares , Mutação , Proteínas/genéticaRESUMO
The enediyne antitumor antibiotic chromoproteins are very potent in causing DNA damages. During the drug delivery time course, the stability of the carrier protein becomes an important concern. To simulate conceivably offensive environment in biological contexts, such as cell membrane, we studied structural endurance of aponeocarzinostatin against several denaturants by circular dichroism and nuclear magnetic resonance spectroscopy. For comparison, we also examined proteins known to be stable and similar in size to aponeocarzinostatin. The results highlight the unusual structural stability of aponeocarzinostatin against chemical denaturants, suggesting the potential of aponeocarzinostatin as an inherently superior carrier in drug delivery systems.
Assuntos
Apoproteínas/química , Portadores de Fármacos/química , Desnaturação Proteica , Zinostatina/química , Apoproteínas/farmacologia , Dicroísmo Circular , Portadores de Fármacos/farmacologia , Espectroscopia de Ressonância Magnética , Compostos Orgânicos/química , Conformação Proteica/efeitos dos fármacos , Zinostatina/farmacologiaRESUMO
The biosynthetic gene cluster for the enediyne antitumor antibiotic neocarzinostatin (NCS) was localized to 130 kb continuous DNA from Streptomyces carzinostaticus ATCC15944 and confirmed by gene inactivation. DNA sequence analysis of 92 kb of the cloned region revealed 68 open reading frames (ORFs), 47 of which were determined to constitute the NCS cluster. Sequence analysis of the genes within the NCS cluster suggested dNDP-D-mannose as a precursor for the deoxy aminosugar, revealed two distinct type I polyketide synthases (PKSs), and supported a convergent model for NCS chromophore biosynthesis from the deoxy aminosugar, naphthoic acid, and enediyne core building blocks. These findings shed light into deoxysugar biosynthesis, further support the iterative type I PKS paradigm for enediyne core biosynthesis, and unveil a mechanism for microbial polycyclic aromatic polyketide biosynthesis by an iterative type I PKS.
Assuntos
Genes Bacterianos , Genes Sintéticos/genética , Família Multigênica/fisiologia , Policetídeo Sintases/biossíntese , Streptomyces/enzimologia , Streptomyces/genética , Zinostatina/biossíntese , Clonagem Molecular , Ordem dos Genes , Policetídeo Sintases/química , Policetídeo Sintases/genética , Análise de Sequência de DNA/métodos , Streptomyces/isolamento & purificação , Zinostatina/químicaRESUMO
Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.
Assuntos
Zinostatina/química , Cristalografia por Raios X , Enedi-Inos , Espectroscopia de Ressonância Magnética , Estrutura Secundária de Proteína , Soluções , Zinostatina/análogos & derivadosRESUMO
Neocarzinostatin (NCS) is an antitumor antibiotic comprising a 1:1 protein-chromophore complex and exhibits cytotoxic action through DNA cleavage via H-abstraction. Cytotoxic activity resides with the chromophore 1 alone, while the protein (apoNCS) protects and transports labile 1. The naphthoate portion (2) of NCS chromophore (1) is important for binding to apoNCS and DNA intercalation. In this paper we describe our attempts to use apoNCS to improve the hydrolytic stability of novel bifunctional DNA alkylating agents. The nitrogen mustards, melphalan and chlorambucil, were both conjugated to 2, and the biological activities of these conjugates were assessed. Chlorambucil did not benefit from conjugation. The melphalan conjugate (6) formed covalent DNA adducts at guanine bases and exhibited greater in vitro cytotoxic activity than unmodified melphalan. Fluorescence and NMR spectroscopy showed that 6 binds to apoNCS. Binding to apoNCS-protected 6 reduced the extent of hydrolysis of the conjugate. This novel approach demonstrates for the first time that an enediyne apo-protein can be used to improve the stability of substances that are of potential interest in cancer chemotherapy.
Assuntos
Desenho de Fármacos , Compostos de Mostarda Nitrogenada/química , Compostos de Mostarda Nitrogenada/síntese química , Zinostatina/química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação , Clorambucila/química , Clorambucila/farmacologia , Adutos de DNA/química , Adutos de DNA/metabolismo , Hidrólise , Espectroscopia de Ressonância Magnética , Melfalan/química , Melfalan/farmacologia , Modelos Moleculares , Estrutura Molecular , Compostos de Mostarda Nitrogenada/metabolismo , Compostos de Mostarda Nitrogenada/farmacologia , Estrutura Terciária de Proteína , Taq Polimerase/antagonistas & inibidores , Taq Polimerase/metabolismoRESUMO
Neocarzinostatin (NCS) is a small "all beta" protein displaying the same overall fold as immunoglobulins. This protein possesses a well-defined hydrophobic core and two loops structurally equivalent to the CDR1 and CDR3 of immunoglobulins. NCS is the most studied member of the enediynechromoprotein family, and is clinically used as an antitumoral agent. NCS has promise as a drug delivery vehicle if new binding specificities could be conferred on its protein scaffold. Previous studies have shown that the binding specificity of the crevasse can be extended to compounds completely unrelated to the natural enediyne chromophore family. We show here that it is possible to introduce new interaction capacities to obtain a protein useful for drug targeting by modifying the immunoglobulin CDR-like loops. We transferred the CDR3 of the VHH chain of camel antilysozyme immunoglobulin to the equivalent site in the corresponding loop of neocarzinostatin. We then evaluated the stability of the resulting structure and its affinity for lysozyme. The engineered NCS-CDR3 presents a structure similar to that of the wild-type NCS, and is stable and efficiently produced. ELISA, ITC, and SPR measurements demonstrated that the new NCS-CDR3 specifically bound lysozyme.