Interaction of Zn(II)bleomycin-A2 and Zn(II)peplomycin with a DNA hairpin containing the 5'-GT-3' binding site in comparison with the 5'-GC-3' binding site studied by NMR spectroscopy.

Bleomycins are a group of glycopeptide antibiotics synthesized by Streptomyces verticillus that are widely used for the treatment of various neoplastic diseases. These antibiotics have the ability to chelate a metal center, mainly Fe(II), and cause site-specific DNA cleavage. Bleomycins are differentiated by their C-terminal regions. Although this antibiotic family is a successful course of treatment for some types of cancers, it is known to cause pulmonary fibrosis. Previous studies have identified that bleomycin-related pulmonary toxicity is linked to the C-terminal region of these drugs. This region has been shown to closely interact with DNA. We examined the binding of Zn(II)peplomycin and Zn(II)bleomycin-A2 to a DNA hairpin of sequence 5'-CCAGTATTTTTACTGG-3', containing the binding site 5'-GT-3', and compared the results with those obtained from our studies of the same MBLMs bound to a DNA hairpin containing the binding site 5'-GC-3'. We provide evidence that the DNA base sequence has a strong impact in the final structure of the drug-target complex.

NMR study of the effects of some bleomycin C-termini on the structure of a DNA hairpin with the 5'-GC-3' binding site.

The antibiotics known as bleomycins constitute a family of natural products clinically employed for the treatment of a wide spectrum of cancers. The drug acts as an antitumor agent by virtue of the ability of a metal complex of the antibiotic to cleave DNA. Bleomycins are differentiated by their C-terminal regions. Previous structural studies involving metal-bleomycin-DNA triads have allowed the identification of the bithiazole-(C-terminus substituent) segment in this molecule as the one that most closely interacts with DNA. Three different modes of binding of metallo-bleomycins to DNA (partial or total intercalation of the bithiazole unit between DNA bases, or binding to the minor groove) have been proposed in the literature. The therapeutic use of bleomycin is frequently associated with the development of pulmonary fibrosis. The severity of this side effect has been attributed to the C-terminus of the antibiotic by some researchers. The degree of pulmonary toxicity of bleomycin-A2 and -A5, were found to be higher than those of bleomycin-B2 and peplomycin. Since the introduction of Blenoxane to clinical medicine in 1972, attempts have been made at modifying the basic bleomycin structure at the C-terminus to improve its therapeutic index. However, the pharmacological and toxicological importance of particular C-termini on bleomycin remains unclear. The present study was designed to determine the effect of Zn(II)bleomycin-A2, -A5, -B2, and Zn(II)peplomycin on the structure of a DNA hairpin containing the 5'-GC-3' binding site. We provide evidence that different Zn(II)bleomycins affect the structure of the tested DNA segment in different fashions.

Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c-di-GMP-dependent regulation.

Elevated levels of the second messenger c-di-GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food-borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface-bound. Secreted carbohydrates represent exclusively cell-wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a ß-1,4-linked N-acetylmannosamine chain decorated with terminal α-1,6-linked galactose. All genes of the pssA-E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS-specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS-mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c-di-GMP-dependent EPS production.

Novel curcumin derivative CNB-001 mitigates obesity-associated insulin resistance.

Type 2 diabetes is growing at epidemic proportions, and pharmacological interventions are being actively sought. This study examined the effect of a novel neuroprotective curcuminoid, CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl)vinyl)-2-methoxy-phenol], on glucose intolerance and insulin signaling in high-fat diet (HFD)-fed mice. C57BL6 mice (5-6 weeks old) were randomly assigned to receive either a HFD (45% fat) or a low-fat diet (LFD, 10% fat) for 24 weeks, together with CNB-001 (40 mg/kg i.p. per day). Glucose tolerance test revealed that the area under the curve of postchallenge glucose concentration was elevated on HF-feeding, which was attenuated by CNB-001. CNB-001 attenuated body weight gain, serum triglycerides, and IL-6, and augmented insulin signaling [elevated phosphoprotein kinase B (p-Akt), and phosphoinsulin receptor (p-IR)ß, lowered endoplasmic reticulum (ER) stress, protein-tyrosine phosphatase 1B (PTP1B)] and glucose uptake in gastrocnemius muscle of HFD-fed mice. Respiratory quotient, measured using a metabolic chamber, was elevated in HFD-fed mice, which was unaltered by CNB-001, although CNB-001 treatment resulted in higher energy expenditure. In cultured myotubes, CNB-001 reversed palmitate-induced impairment of insulin signaling and glucose uptake. Docking studies suggest a potential interaction between CNB-001 and PTP1B. Taken together, CNB-001 alleviates obesity-induced glucose intolerance and represents a potential candidate for further development as an antidiabetic agent.

T1BT* structural study of an anti-plasmodial peptide through NMR and molecular dynamics.

BACKGROUND: T1BT* is a peptide construct containing the T1 and B epitopes located in the 5' minor repeat and the 3' major repeat of the central repeat region of the Plasmodium falciparum circumsporozoite protein (CSP), respectively, and the universal T* epitope located in the C-terminus of the same protein. This peptide construct, with B = (NANP)3, has been found to elicit antisporozoite antibodies and gamma-interferon-screening T-cell responses in inbred strains of mice and in outbred nonhuman primates. On the other hand, NMR and CD spectroscopies have identified the peptide B' = (NPNA)3 as the structural unit of the major repeat in the CSP, rather than the more commonly quoted NANP. With the goal of assessing the structural impact of the NPNA cadence on a proven anti-plasmodial peptide, the solution structures of T1BT* and T1B'T* were determined in this work. METHODS: NMR spectroscopy and molecular dynamics calculations were used to determine the solution structures of T1BT* and T1B'T*. These structures were compared to determine the main differences and similarities between them. RESULTS: Both peptides exhibit radically different structures, with the T1B'T* showing strong helical tendencies. NMR and CD data, in conjunction with molecular modelling, provide additional information about the topologies of T1BT* and T1B'T*. Knowing the peptide structures required to elicit the proper immunogenic response can help in the design of more effective, conformationally defined malaria vaccine candidates. If peptides derived from the CSP are required to have helical structures to interact efficiently with their corresponding antibodies, a vaccine based on the T1B'T* construct should show higher efficiency as a pre-erythrocyte vaccine that would prevent infection of hepatocytes by sporozoites.

Structural changes of Zn(II)bleomycin complexes when bound to DNA hairpins containing the 5'-GT-3' and 5'-GC-3' binding sites studied through NMR spectroscopy.

Bleomycins are antitumor antibiotics that can chelate a metal center and cause site-specific DNA cleavage at 5'-Gpyrimidine-3' regions of DNA. These antibiotics are successful in the treatment of various cancers, but are known to cause pulmonary fibrosis to patients under bleomycin regimes. Substantial research has resulted in the development of over 300 bleomycin analogs, aiming to improve the therapeutic index of the drug. Previous studies have proposed that the lung toxicity caused by bleomycin is related to the C-terminal regions of these drugs, which have been shown to closely interact with DNA in metal-bleomycin-DNA complexes. Some of the research studying metallo-bleomycin-DNA interactions have suggested three different binding modes of the metal form of the drug to DNA, including total and/or partial intercalation, and minor groove binding. However, there is still lack of consensus regarding this matter, and solid conclusions on the subject have not yet been established. Previously we investigated the diverse levels of disruption caused to DNA hairpins containing 5'-GC-3' and 5'-GT-3' binding sites, which are consequence of the binding of bleomycins with different C-termini. The results of these investigation indicate that both the DNA-binding site and the bleomycin C-termini have an impact on the final conformations of drug and target. The present study focuses on the structural alterations exhibited by Zn(II)bleomycin-A2, -B2, -A5 and Zn(II)peplomycin upon binding to DNA hairpins containing 5'-GC-3' and 5'-GT-3' binding sites. Evidence that each Zn(II)bleomycin is structurally affected depending on both its C-terminus and the DNA-binding site present in the hairpin is provided.

Effects of enzymatically inactive recombinant botulinum neurotoxin type A at the mouse neuromuscular junctions.

Botulinum neurotoxin A (BoNT/A) is used clinically to treat several neurological and metabolic diseases. However, the mechanisms that underlie the clinical use of the toxin remain still to be elusive. BoNT/A inhibits acetylcholine (ACh) release at the motor nerve terminals (MNT) and causes neuroparalysis. The toxic effects of BoNT/A at the MNT occur in sub-pico molar range, and it is invaluable to determine the half-life and the persistence of catalytic activity of the toxin to develop therapeutics against BoNT/A intoxication. However, the use of extremely low concentrations of BoNT/A in cellular, or animal models due to high toxicity makes it difficult to determine new cellular mechanisms and binding or interacting partners of BoNT/A. In order to address this, a catalytically deactivated, non-toxic version of BoNT/A, designated as DrBoNT/A, was characterized. DrBoNT/A lacks endoprotease activity (SNAP-25 cleavage) at concentrations as high as 46,875-fold, compared to wild-type BoNT/A. Unlike BoNT/A injection (3.2 pg), injection of the recombinant product (150 ng or 3.2 pg) into mouse hind limbs failed to cause neuroparalysis as exhibited by the lack of inhibition of toe spread reflex (ability of the mouse to spread its hindlimb toes), and inhibit ACh release at the MNT. The in vitro experiments also demonstrate that DrBoNT/A uptake (at concentrations equivalent to BoNT/A), internalization and localization at the MNT remained unaltered. In addition, modeling studies support that DrBoNT/A lacked the zinc binding ability, and the ability to directly participate in the hydrolysis of SNAP-25 substrate. Collectively, we demonstrate that DrBoNT/A is non-toxic to the MNT and can be used as a surrogate tool to understand the mechanism by which BoNT/A modulates signal transduction mechanisms.

Possible structural role of the disaccharide unit in Fe-bleomycin before and after oxygen activation.

Our previous investigation of the solution structure of Fe(II)-bleomycin pointed toward the carbamoyl group in the mannose moiety or a water molecule as possible alternative axial ligands to the metal center in this metallo-bleomycin. The possibility of a solvent molecule occupying the apical position trans to the primary amine has not been ruled out yet. In order to explore this possibility even further, the coordination chemistry of azide-bound Fe(II)-bleomycin was investigated with the use of NMR applied to paramagnetic molecules. Fe(II)- and apo-bleomycin were also re-visited. Comparison of the NMR results for both Fe(II)-bound molecules obtained in the present study strongly suggests that the carbamoyl oxygen is ligated to Fe(II), and it is released from coordination upon azide binding. This event is suggested based on the diminished paramagnetic character exhibited by the carbohydrate moiety in Fe(II)-azide-bleomycin when compared with its parent metal complex. A possible structural role for the glucopyranose fragment, which changes throughout the process that starts with metallo-bleomycin formation and ends with DNA binding, is discussed. The study of the coordination of azide by Fe(II)-bleomycin through NMR has not been reported previously. Unlike magnetic CD data, NMR offers a residue-by-residue account of the possible structural changes that take place in Fe(II)-bleomycin after azide binding.

NMR study of peplomycin in aqueous solution. Assignment of resonances by means of two-dimensional spectroscopy.

¹H-NMR spectra of peplomycin (PEP) recorded at 400 and, for the first time, 900 MHz at 2 °C were examined. All the spin systems in the PEP molecule were identified through 2D NMR spectroscopy. The use of NMR spectroscopy allowed the unambiguous assignment of 62 protons, generating 47 non-exchangeable and 15 exchangeable signals. The analysis of the signals observed in 2D-NOE spectra indicates that PEP exhibits an extended conformation at 2 °C. A comparison between the solution conformation of apo-PEP and the solution structure of HOO-Co(III)-PEP indicates that the overall structure of apo-PEP is extended in solution, but exhibiting a conformation of the bithiazole (B)-sulfonium (S) unit similar to that of HOO-Co(III)-PEP. The present investigation represents the initial stage of an NMR study of the solution conformation and dynamics of PEP, its derivatives, its metal complexes and the interactions of metallo-PEPs with their target DNA.

Structural study of copper(I)-bleomycin.

Previous NMR studies on Cu(I)-bleomycin have suggested that this adduct has a geometry distinct from Fe(II)BLM. The coordination chemistry of this bleomycin derivative has been investigated through the extension of the NMR data reported previously, and the use of molecular dynamics calculations. The data collected from the NMR experiments support the coordination to the metal center of the primary and secondary amines in beta-aminoalanine and the pyrimidine ring. The detection in the NMR spectra of the signal derived from the amide hydrogen in beta-hydroxyhistidine indicates that this amide is protonated in Cu(I)-bleomycin, precluding participation of the pyrimidinyl carboxamide nitrogen in the coordination of Cu(I), as previously reported. Three-dimensional solution structures compatible with the NMR data have been assayed for Cu(I)-bleomycin for the first time by way of molecular dynamics calculations, and two models showing four and five coordination have been found to be those that better fit the experimental data. In both models the primary amine in beta-aminoalanine is coordinated such that it is located on the same side, with respect to the coordination cage, as the peptide linker fragment. This result seems important for the favored models to be compatible with either their possible oxidation to become one of the reported structures for Cu(II)BLM, or their transformation into Fe(II) adducts able to cause DNA damage.

Molecular modeling of the three-dimensional structure of Fe(II)-bleomycin: are the Co(II) and Fe(II) adducts isostructural?

The molecular modeling of Co(II)-bleomycin previously performed by us through NMR and molecular dynamics indicates that the most favorable structure for this complex is six-coordinate, with the secondary amine in beta-aminoalanine, the N5 and N1 nitrogens in the pyrimidine and imidazole rings, respectively, and the amide nitrogen in beta-hydroxyhistidine as equatorial ligands. The primary amine and either the carbamoyl group or a solvent molecule are proposed to occupy the axial sites. In this report, the results of the molecular modeling of Fe(II)-bleomycin are presented. The NMR data for the ferrous derivative of the drug have already been reported by us, and were used here to generate the necessary restraints for this modeling work. For Co(II)-bleomycin, two new models exhibiting N-carbamoyl ligation to the metal centers were also assayed and compared with the ones previously examined. The results of this investigation on Fe(II)- and Co(II)-bleomycin are most consistent with a six-coordinate structure with five endogenous N-donors and a solvent molecule or the carbamoyl group as the sixth ligand. Comparisons of the best Co(II)- and Fe(II)-bleomycin models with the NMR-generated structures for some relevant metallo-BLMs favor the model with only endogenous ligands and N-carbamoyl ligation as the structure probably held in solution by both Co(II)- and Fe(II)-bleomycin.