echinomycin and thiocoraline

Echinomycin is the prototypical bisintercalator, a molecule that binds to DNA by inserting two planar chromophores between the base-pairs of duplex DNA, placing its cyclic depsipeptide backbone in the minor groove. As such, it has been the focus of an extensive number of investigations into its biological activity, nucleic acid binding and, to some extent, its structure-activity relationships. However, echinomycin is also the parent member of an extended family of natural products that interact with DNA by a similar mechanism of bisintercalation. The structural variety in these compounds leads to changes in sequence selectivity and and biological activity, particularly as anti-tumour and anti-viral agents. One of the more recently identified marine natural products that is moving close to clinical development is thiocoraline, and it therefore seems timely to review the various bisintercalator natural products.

Topics: Biological Products; Depsipeptides; DNA; Echinomycin; Intercalating Agents; Peptides; Peptides, Cyclic; Quinolines; Quinoxalines

Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.

Topics: Adenosine Monophosphate; Depsipeptides; Echinomycin; Kinetics; Methylation; Methyltransferases; Microsporidia; Peptide Biosynthesis, Nucleic Acid-Independent; Peptide Synthases; Protein Domains; Streptomyces; Sulfhydryl Compounds

A sensitive high-performance liquid chromatography-tandem mass spectrometry assay for thiocoraline, an anti-tumor depsipeptide, in mouse plasma is described. Echinomycin, a quinoxaline peptide, was used as an internal standard. Thiocoraline was recovered from the mouse plasma using protein precipitation with acetonitrile and followed by solid-phase extraction of the supernatant. The mobile phase consisted of methanol (0.1% formic acid)-water (0.1% formic acid) (90:10, v/v). The analytical column was a YMC C(18). The standard curve was linear from 0.1 to 50 ng/ml (R(2)>0.99). The lower limit of quantitation was 0.1 ng/ml. The assay was specific based on the multiple reaction monitoring transitions at m/z 1157-->215 and m/z 1101-->243 for thiocoraline and the internal standard, echinomycin, respectively. The mean intra- and inter-day assay accuracies remained below 5 and 12%, respectively, for all calibration standards and quality control (QC) samples. The intra- and inter-day assay precisions were less than 11.4 and 9.5% for all QC levels, respectively. The utility of the assay was demonstrated by a pharmacokinetic study of i.v. (bolus) thiocoraline on CD-1 mice. Thiocoraline was stable in mouse plasma in an ice-water bath for 6 h and for three freeze-thaw cycles. The reconstituted thiocoraline after extraction and drying sample process was stable in the autosampler for over 24 h. The assay was able to quantify thiocoraline in plasma up to 48 h following dose. Pharmacokinetic analysis showed that thiocoraline has distinct pharmacokinetic profiling when dosed in different formulation solutions. The assay is currently used to measure thiocoraline plasma concentrations in support of a project to develop a suitable formulation with a desirable pharmacokinetic profile.

Topics: Animals; Depsipeptides; Echinomycin; Female; Male; Mass Spectrometry; Mice; Peptides; Quality Control; Reproducibility of Results; Sensitivity and Specificity

Full details of the total syntheses of thiocoraline (1) and BE-22179 (2), C(2) symmetric bicyclic octadepsipeptides possessing two pendant 3-hydroxyquinoline chromophores, are described in which their relative and absolute stereochemistry were established. Key elements of the approach include the late-stage introduction of the chromophore, symmetrical tetrapeptide coupling, macrocyclization of the 26-membered octadepsipeptide conducted at the single secondary amide site following disulfide formation, and a convergent assemblage of the tetradepsipeptide with introduction of the labile thiol ester linkage in the final coupling reaction under near racemization free conditions. By virtue of the late-stage introduction of the chromophore and despite the challenges this imposes on the synthesis, this approach provides ready access to a range of key chromophore analogues. Thiocoraline and BE-22179 were shown to bind to DNA by high-affinity bisintercalation analogous to echinomycin, but with little or no perceptible sequence selectivity. Both 1 and 2 were found to exhibit exceptional cytotoxic activity (IC(50) = 200 and 400 pM, respectively, L1210 cell line) comparable to echinomycin and one analogue, which bears the luzopeptin chromophore, was also found to be a potent cytotoxic agent.

Topics: Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Cattle; Cell Survival; Depsipeptides; DNA; Echinomycin; HIV-1; Inhibitory Concentration 50; Mice; Peptides; Reverse Transcriptase Inhibitors; Tumor Cells, Cultured