Structure and Function of a Dehydrating Condensation Domain in Nonribosomal Peptide Biosynthesis.

Dehydroamino acids are important structural motifs and biosynthetic intermediates for natural products. Many bioactive natural products of nonribosomal origin contain dehydroamino acids; however, the biosynthesis of dehydroamino acids in most nonribosomal peptides is not well understood. Here, we provide biochemical and bioinformatic evidence in support of the role of a unique class of condensation domains in dehydration (CmodAA). We also obtain the crystal structure of a CmodAA domain, which is part of the nonribosomal peptide synthetase AmbE in the biosynthesis of the antibiotic methoxyvinylglycine. Biochemical analysis reveals that AmbE-CmodAA modifies a peptide substrate that is attached to the donor carrier protein. Mutational studies of AmbE-CmodAA identify several key residues for activity, including four residues that are mostly conserved in the CmodAA subfamily. Alanine mutation of these conserved residues either significantly increases or decreases AmbE activity. AmbE exhibits a dimeric conformation, which is uncommon and could enable transfer of an intermediate between different protomers. Our discovery highlights a central dehydrating function for CmodAA domains that unifies dehydroamino acid biosynthesis in diverse nonribosomal peptide pathways. Our work also begins to shed light on the mechanism of CmodAA domains. Understanding CmodAA domain function may facilitate identification of new natural products that contain dehydroamino acids and enable engineering of dehydroamino acids into nonribosomal peptides.

Biosynthesis of fluopsin C, a copper-containing antibiotic from Pseudomonas aeruginosa.

Metal-binding natural products contribute to metal acquisition and bacterial virulence, but their roles in metal stress response are underexplored. We show that a five-enzyme pathway in Pseudomonas aeruginosa synthesizes a small-molecule copper complex, fluopsin C, in response to elevated copper concentrations. Fluopsin C is a broad-spectrum antibiotic that contains a copper ion chelated by two minimal thiohydroxamates. Biosynthesis of the thiohydroxamate begins with cysteine and requires two lyases, two iron-dependent enzymes, and a methyltransferase. The iron-dependent enzymes remove the carboxyl group and the α carbon from cysteine through decarboxylation, N-hydroxylation, and methylene excision. Conservation of the pathway in P. aeruginosa and other bacteria suggests a common role for fluopsin C in the copper stress response, which involves fusing copper into an antibiotic against other microbes.

Exceedingly Small Gadolinium Oxide Nanoparticles with Remarkable Relaxivities for Magnetic Resonance Imaging of Tumors.

Gd chelates have occupied most of the market of magnetic resonance imaging (MRI) contrast agents for decades. However, there have been some problems (nephrotoxicity, non-specificity, and low r1 ) that limit their applications. Herein, a wet-chemical method is proposed for facile synthesis of poly(acrylic acid) (PAA) stabilized exceedingly small gadolinium oxide nanoparticles (ES-GON-PAA) with an excellent water dispersibility and a size smaller than 2.0 nm, which is a powerful T1 -weighted MRI contrast agent for diagnosis of diseases due to its remarkable relaxivities (r1 = 70.2 ± 1.8 mM-1 s-1 , and r2 /r1 = 1.02 ± 0.03, at 1.5 T). The r1 is much higher and the r2 /r1 is lower than that of the commercial Gd chelates and reported gadolinium oxide nanoparticles (GONs). Further ES-GON-PAA is developed with conjugation of RGD2 (RGD dimer) (i.e., ES-GON-PAA@RGD2) for T1 -weighted MRI of tumors that overexpress RGD receptors (i.e., integrin αv ß3 ). The maximum signal enhancement (ΔSNR) for T1 -weighted MRI of tumors reaches up to 372 ± 56% at 2 h post-injection of ES-GON-PAA@RGD2, which is much higher than commercial Gd-chelates (<80%). Due to the high biocompatibility and high tumor accumulation, ES-GON-PAA@RGD2 with remarkable relaxivities is a promising and powerful T1 -weighted MRI contrast agent.

Physicochemical characterization of ferumoxytol, heparin and protamine nanocomplexes for improved magnetic labeling of stem cells.

Stem cell-based therapies have become a major focus in regenerative medicine and to treat diseases. A straightforward approach combining three drugs, heparin (H), protamine (P) with ferumoxytol (F) in the form of nanocomplexes (NCs) effectively labeled stem cells for cellular MRI. We report on the physicochemical characteristics for optimizing the H, P, and F components in different ratios, and mixing sequences, producing NCs that varied in hydrodynamic size. NC size depended on the order in which drugs were mixed in media. Electron microscopy of HPF or FHP showed that F was located on the surface of spheroidal shaped HP complexes. Human stem cells incubated with FHP NCs resulted in a significantly greater iron concentration per cell compared to that found in HPF NCs with the same concentration of F. These results indicate that FHP could be useful for labeling stem cells in translational studies in the clinic.

Gadolinium MRI contrast agents based on triazine dendrimers: relaxivity and in vivo pharmacokinetics.

Four gadolinium (Gd)-based macromolecular contrast agents, G3-(Gd-DOTA)(24), G5-(Gd-DOTA)(96), G3-(Gd-DTPA)(24), and G5-(Gd-DTPA)(96), were prepared that varied in the size of dendrimer (generation three and five), the type of chelate group (DTPA or DOTA), and the theoretical number of metalated chelates (24 and 96). Synthesis relied on a dichlorotriazine derivatized with a DOTA or DTPA ligand that was incorporated into the dendrimer and ultimately metalated with Gd ions. Paramagnetic characteristics and in vivo pharmacokinetics of all four contrast agents were investigated. The DOTA-containing agents, G3-(Gd-DOTA)(24) and G5-(Gd-DOTA)(96), demonstrated exceptionally high r1 relaxivity values at off-peak magnetic fields. Additionally, G5-(Gd-DOTA)(96) showed increased r1 relaxivity in serum compared to that in PBS, which was consistent with in vivo images. While G3-(Gd-DOTA)(24) and G3-(Gd-DTPA)(24) were rapidly excreted into the urine, G5-(Gd-DOTA)(96) and G5-(Gd-DTPA)(96) did not clear as quickly through the kidneys. Molecular simulation of the DOTA-containing dendrimers suggests that a majority of the metalated ligands are accessible to water. These triazine dendrimer-based MRI contrast agents exhibit several promising features such as high in vivo r1 relaxivity, desirable pharmacokinetics, and well-defined structure.

Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging.

We report on a new straightforward magnetic cell-labeling approach that combines three US Food and Drug Administration (FDA)-approved drugs--ferumoxytol, heparin and protamine--in serum-free medium to form self-assembling nanocomplexes that effectively label cells for in vivo magnetic resonance imaging (MRI). We observed that the ferumoxytol-heparin-protamine (HPF) nanocomplexes were stable in serum-free cell culture medium. HPF nanocomplexes show a threefold increase in T2 relaxivity compared to ferumoxytol. Electron microscopy showed internalized HPF in endosomes, which we confirmed by Prussian blue staining of labeled cells. There was no long-term effect or toxicity on cellular physiology or function of HPF-labeled hematopoietic stem cells, bone marrow stromal cells, neural stem cells or T cells when compared to controls. In vivo MRI detected 1,000 HPF-labeled cells implanted in rat brains. This HPF labeling method should facilitate the monitoring by MRI of infused or implanted cells in clinical trials.

Polyaspartic acid coated manganese oxide nanoparticles for efficient liver MRI.

We report in this communication a simple, facile surface modification strategy to transfer hydrophobic manganese oxide nanoparticles (MONPs) into water by using polyaspartic acid (PASP). We systematically investigated the effect of the size of PASP-MONPs on MRI of normal liver and found that the particles with a core size of 10 nm exhibited greater enhancement than those with larger core sizes.

Functional MnO nanoclusters for efficient siRNA delivery.

A non-viral gene delivery nanovehicle based on Alkyl-PEI2k capped MnO nanoclusters was synthesized via a simple, facile method and used for efficient siRNA delivery and magnetic resonance imaging.

Dendrimer-based MRI contrast agents: the effects of PEGylation on relaxivity and pharmacokinetics.

Polyethylene glycol (PEG) surface modification can make nanomaterials highly hydrophilic, reducing their sequestration in the reticuloendothelial system. In this study, polyamidoamine (PAMAM) dendrimers bearing gadolinium (Gd) chelates were PEGylated with different PEG-chain lengths, and the effects on paramagnetic and pharmacokinetic properties were evaluated. Specifically, Gd chelate-bearing PAMAM dendrimers (generations 4 and 5; G4 and G5) were conjugated with two different PEG chains (2 kDa and 5 kDa; 2k and 5k). Long PEG chains (5k) on the smaller (G4) dendrimer resulted in reduced relaxivity compared to non-PEGylated dendrimers, whereas short PEG chains (2k) on a larger (G5) dendrimer produced relaxivities comparable to non-PEGylated G4 dendrimers. The relaxivity of all PEGylated or lysine-conjugated dendrimers increased at higher temperature, whereas that of intact G4 Gd-PAMAM dendrimer decreased. All PEGylated dendrimers had minimal liver and kidney uptake and remained in circulation for at least 1 hour. Thus, surface-PEGylated Gd-PAMAM dendrimers showed decreased plasma clearance and prolonged retention in the blood pool. Shorter PEG, higher generation conjugates led to higher relaxivity. FROM THE CLINICAL EDITOR: In this study, polyamidoamine dendrimers bearing gadolinium (Gd) chelates were PEGylated with different PEG-chain lengths, and the effects on paramagnetic and pharmacokinetic properties were evaluated.

Preparation, characterization and in vivo assessment of Gd-albumin and Gd-dendrimer conjugates as intravascular contrast-enhancing agents for MRI.

We report in vivo and in vitro MRI properties of six gadolinium-dendrimer and gadolinium-albumin conjugates of derivatized acyclic diethylenetriamine-N,N',N',N″, N″-pentaacetic acid (1B4M) and macrocyclic 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (C-DOTA). The three albumin-based agents have comparable protein to chelate ratios (1:16-18) as well as molar relaxivity (8.8-10.4 mM(-1) s(-1)). The three dendrimer based agents have blood clearance half-lives ranging from 17 to 66 min while that of the three albumin-based agents are comparable to one another (40-47 min). The dynamic image obtained from use of the albumin conjugate based on the macrocycle (C-DOTA) showed a higher contrast compared to the remaining two albumin based agents. Our conclusion from all of the results is that the macrocyclic-based (DOTA) agents are more suitable than the acyclic-based (1B4M) agent for in vivo use based on their MRI properties combined with the kinetic inertness property associated with the more stable Gd(III) DOTA complex.

Poly(amidoamine) dendrimer based MRI contrast agents exhibiting enhanced relaxivities derived via metal preligation techniques.

This report presents the preparation and characterization of three [Gd-C-DOTA](-1)-dendrimer assemblies by way of analysis, NMRD spectroscopy, and photon correlation spectroscopy (PCS). The metal-ligand chelates were preformed in alcohol media prior to conjugation to generation 4, 5, and 6 PAMAM dendrimers. The dendrimer-based agents were purified by Sephadex G-25 column chromatography. The combustion analysis, SE-HPLC, and UV-vis data indicated chelate to dendrimer ratios of 28:1, 61:1 and 115:1, respectively. Molar relaxivity measured at pH 7.4, 22 degrees C, and 3 T (29.6, 49.8, and 89.1 mM(-1) s(-1)) indicated the viability of conjugates as MRI contrast agents. 1/T(1) NMRD profiles were measured at 23 degrees C and indicated that at 22 MHz the 1/T(1) reached a plateau at 60, 85, and 140 mM(-1) s(-1) for the generation 4, 5, and 6 dendrimer conjugates, respectively. The PCS data showed the respective sizes of 5.2, 6.5, and 7.8 nm for G-4, 5, and 6 conjugates.

New nanosized biocompatible MR contrast agents based on lysine-dendri-graft macromolecules.

Paramagnetic nanomaterials for use as magnetic resonance imaging (MRI) contrast agents have higher relaxivity than conventional low molecular weight MRI agents. However, the biocompatibility and pharmacokinetics of such nanomaterials will strongly affect the likelihood of clinical approval. We synthesized MRI contrast agents based on biocompatible lysine-dendri-grafts: Gd-BzDTPA-lysineG2 and Gd-BzDTPA-lysineG3. The relaxivity of Gd-BzDTPA-lysineG2 and Gd-BzDTPA-lysineG3 increased with sample temperature, while the relaxivity of Gd-BzDTPA-PAMAMG4 decreased with increasing sample temperature. The increase in relaxivity with increasing temperature may be attributed to accessibility of water to the internal Gd chelates with lysine-dendri-grafts, which does not occur with PAMAM dendrimers. Gd-BzDTPA-lysineG3 had a long intravascular half-life but were largely excreted by the kidneys. Therefore, Gd-BzDTPA-lysineG3 enhanced the blood vessels for longer periods than Gd-BzDTPA-PAMAMG4, but was still excreted through the kidney. Because of their biocompatibility, desirable magneto-physical characteristics and favorable pharmacokinetics, which are derived from different interior structures rather than the physical size, lysine-dendri-graft MR contrast agents may be feasible for clinical use.

Synthesis and evaluation of novel macrocyclic and acyclic ligands as contrast enhancement agents for magnetic resonance imaging.

Novel chelates PIP-DTPA, AZEP-DTPA, NETA, NPTA, and PIP-DOTA were synthesized and evaluated as potential magnetic resonance imaging (MRI) contrast enhancement agents. The T1 and T2 relaxivities of their corresponding Gd(III) complexes are reported. At clinically relevant field strengths, the relaxivities of the complexes are comparable to that of the clinically used contrast agents Gd(DTPA) and Gd(DOTA). The serum stability of the 153Gd-labeled complexes, Gd(PIP-DTPA), Gd(AZEP-DTPA), Gd(PIP-DOTA), Gd(NETA), and Gd(NPTA), was assessed by measuring the release of 153Gd from the complexes. 153Gd(NETA), 153Gd(PIP-DTPA), and 153Gd(PIP-DOTA) were found to be stable in human serum for up to 14 days without any measurable loss of radioactivity. Significant release of 153Gd was observed with the 153Gd(III) radiolabled NPTA. In vivo biodistribution of the153Gd-labeled complexes was performed to evaluate their in vivo stability. While Gd(AZEP-DTPA) and Gd(NPTA) were found to be unstable in vivo, Gd(NETA), Gd(PIP-DTPA), and Gd(PIP-DOTA) were excreted without dissociation. These results suggest that the Gd(III) complexes of the novel chelates NETA, PIP-DTPA, and PIP-DOTA possess potential as MRI contrast enhancement agents. In particular, the piperidine backboned chelates Gd(PIP-DTPA) and Gd(PIP-DOTA) displayed reduced kidney retention as compared to the nonspecific MRI contrast agent Gd(DOTA) at all time points, although the observed effects were relatively small at 0.5 h postinjection. Incorporation of the lipophilic piperidine ring appears to confer a moderate effect on the liver uptake of these two chelates.

Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents.

PURPOSE: To label mammalian and stem cells by combining commercially available transfection agents (TAs) with superparamagnetic iron oxide (SPIO) magnetic resonance (MR) imaging contrast agents. MATERIALS AND METHODS: Three TAs were incubated with ferumoxides and MION-46L in cell culture medium at various concentrations. Human mesenchymal stem cells, mouse lymphocytes, rat oligodendrocyte progenitor CG-4 cells, and human cervical carcinoma cells were incubated 2-48 hours with 25 microg of iron per milliliter of combined TAs and SPIO. Cellular labeling was evaluated with T2 relaxometry, MR imaging of labeled cell suspensions, and Prussian blue staining for iron assessment. Proliferation and viability of mesenchymal stem cells and human cervical carcinoma cells labeled with a combination of TAs and ferumoxides were evaluated. RESULTS: When ferumoxides-TA or MION-46L-TA was used, intracytoplasmic particles stained with Prussian blue stain were detected for all cell lines with a labeling efficiency of nearly 100%. Limited or no uptake was observed for cells incubated with ferumoxides or MION-46L alone. For TA-SPIO-labeled cells, MR images and relaxometry findings showed a 50%-90% decrease in signal intensity and a more than 40-fold increase in T2s. Cell viability varied from 103.7% +/- 9 to 123.0% +/- 9 compared with control cell viability at 9 days, and cell proliferation was not affected by endosomal incorporation of SPIO nanoparticles. Iron concentrations varied with ferumoxides-TA combinations and cells with a maximum of 30.1 pg +/- 3.7 of iron per cell for labeled mesenchymal stem cells. CONCLUSION: Magnetic labeling of mammalian cells with use of ferumoxides and TAs is possible and may enable cellular MR imaging and tracking in experimental and clinical settings.

Test of Electron Delocalization Effects on Water-Proton Spin-Lattice Relaxation by Bromination of [Tetrakis(4-sulfonatopheny)porphine]manganese.

The potential value of electron spin delocalization as a means for substantially increasing the ability of a paramagnetic metal complex to induce nuclear spin relaxation of water protons has been examined by covalent attachment of bromine atoms in the beta-pyrrole positions of the [5,10,15,20-tetrakis(4-sulfonatophenyl)-21H,23H-porphine]manganese complexes[Mn(III)TPPS](3)(-) and [Mn(II)TPPS].(4)(-) The water-proton spin-lattice relaxivities are reported as a function of magnetic field strength for the brominated and nonbrominated metalloporphyrins over the range of magnetic field strengths corresponding to proton Larmor frequencies between 0.01 and 30 MHz. The brominated metalloporphyrins increase the water-proton relaxativities compared to the nonbrominated metalloporphrins, and, at low magnetic field strengths, the brominated [Mn(II)TPPS](4)(-) complex rivals the efficiency of the hexaaquomanganese(II) ion. Attempts to fit the experimental data to theories for paramagnetic relaxation, which are based on the point-dipole approximation, result in distances between the paramagnetic center and the water proton that are unreasonably short based on published structural data. The excess relaxivity implies that the point-dipole approximation may be inappropriate for these porphyrin systems and electron spin delocalization may provide a significant contribution to nuclear spin relaxation that may be fruitfully exploited in construction of contrast agents for magnetic resonance imaging.