Alberto Plaza

Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting

Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.

Celebesides A-C and Theopapuamides B-D, Depsipeptides from an Indonesian Sponge that Inhibit HIV-1 Entry

Six new depsipeptides belonging to two different structural classes, termed celebesides A-C and theopapuamides B-D, have been isolated from the marine sponge Siliquariaspongia mirabilis. Their structures were determined using extensive 2D NMR and ESI-MS/MS techniques. Celebesides are unusual cyclic depsipeptides that comprise a polyketide moiety and five amino acid residues, including an uncommon 3-carbamoyl threonine, and a phosphoserine residue in celebesides A and B. Theopapuamides B-D are undecapeptides with an N-terminal fatty acid moiety containing two previously unreported amino acids, 3-acetamido-2-aminopropanoic acid and 4-amino-2,3-dihydroxy-5-methylhexanoic acid. The relative configuration of the polyketide moiety in celebesides was resolved by J-based analysis and quantum mechanical calculations, the results of which were self-consistent. Celebeside A neutralized HIV-1 in a single-round infectivity assay with an IC(50) value of 1.9 +/- 0.4 microg/mL while the nonphosphorylated analog celebeside C was inactive at concentrations as high as 50 microg/mL. Theopapuamides A-C showed cytotoxicity against human colon carcinoma (HCT-116) cells with IC(50) values between 2.1 and 4.0 microg/mL and exhibited strong antifungal activity against wildtype and amphotericin B-resistant strains of Candida albicans at loads of 1-5 microg/disk.

In Vivo Evidence for a Prodrug Activation Mechanism during Colibactin Maturation

Releasing the cytopath: We have identified an N-myristoyl-D-asparagine (1) as the free N-terminal prodrug scaffold in cytopathogenic Escherichia coli strains expressing the colibactin gene cluster. Colibactin is released in vivo upon cleavage of precolibactin. We provide for the first time in vivo evidence of the prodrug-like release mechanism of colibactin.

Myxoprincomide: A Natural Product from Myxococcus xanthus Discovered by Comprehensive Analysis of the Secondary Metabolome

Bacteria are important sources of therapeutically relevant natural products. Early established bacterial producers include the streptomycetes, pseudomonads, and bacilli, but the list has recently expanded to include further sources, such as the myxobacteria. Access to whole genome sequences has indicated that among bacteria producing natural products through the action of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) the number of known compounds is remarkably lower than the genetic capacity of the organism for secondary-metabolite biosynthesis. 6] Thus, identification of new compound classes and their assignment to biosynthetic gene clusters is a crucial step in the discovery of novel natural products. Here, we report the discovery and structural elucidation of myxoprincomide (1), a novel NRPS/PKS natural product from Myxococcus xanthus DK1622, by combining methods of targeted mutagenesis, liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), and statistical data evaluation (Figure 1). Myxoprincomide (1), a linear peptide bearing some unusual residues, is produced by the NRPS/PKS biosynthetic machinery encoded by the mxp (MXAN_3779) gene locus. Moreover, by correlating additional very-low-abundance natural products to biosynthetic pathways in DK1622 we demonstrate how a comprehensive “metabolome-mining” approach can complement genomemining strategies in the discovery of secondary metabolites. The myxobacterial strain DK1622, considered a model organism for the study of myxobacterial social motility and multicellular differentiation, was not recognized as a producer of secondary metabolites until its genome was sequenced. However, interest in resolving its secondary metabolome has increased since bioinformatic analysis revealed it to contain 18 biosynthetic gene clusters encoding NRPS, PKS, and NRPS/PKS hybrid systems. To date only five compound classes derived from NRPS and PKS biosynthetic machineries have been characterized and correlated to their gene clusters in DK1622 (see Figure 1 in the Supporting Information). Intriguingly, evaluation at the transcriptomic and proteomic levels asserts that the remaining 13 unassigned pathways are active under standard conditions for the cultivation of DK1622. 15] We reasoned that the low abundance of the corresponding compounds may have previously prevented their detection, and thus set out to find these compounds by utilizing advanced analytical methods. The available DK1622 genome sequence facilitated the construction of a targeted mutant library including knockouts of every secondary-metabolite biosynthetic gene cluster (Table 1 in the Supporting Information). As the process of finding the possibly subtle differences between wild-type and mutant secondary-metabolite profiles by manual comparison of LC-MS data is frequently tedious, error-prone, and low in sensitivity, we sought to implement statistical tools in order to expedite our LC-MS data evaluation. In preparation for the comprehensive statistical analysis, mutant and wildtype strains were grown in small-scale fermentation in quadruplicate, replicate extracts were analyzed by LCHRMS, and data were pretreated by using a compoundfinding algorithm, resulting in the definition of > 1000 molecular features per sample. In order to identify molecular features specifically missing in culture extracts from DK1622 mutant strains, we applied principal-component analysis (PCA) to the preprocessed LC-MS datasets [*] M. Sc. N. S. Cortina, Dr. D. Krug, Dr. A. Plaza, Dipl.-Chem. O. Revermann, Prof. Dr. R. M ller Abteilung Mikrobielle Naturstoffe, Helmholtz-Institut f r Pharmazeutische Forschung Saarland (HIPS), Helmholtz-Zentrum f r Infektionsforschung and Institut f r Pharmazeutische Biotechnologie, Universit t des Saarlandes Campus, Geb ude C2.3, 66123 Saarbr cken (Deutschland) E-mail: rom@helmholtz-hzi.de Homepage: http://www.helmholtz-hzi.de/hips [] These authors contributed equally to this work.

Mutremdamide A and koshikamides C-H, peptide inhibitors of HIV-1 entry from different Theonella species.

A new sulfated cyclic depsipeptide, termed mutremdamide A, and six new highly N-methylated peptides, termed koshikamides C-H, were isolated from different deep-water specimens of Theonella swinhoei and Theonella cupola. Their structures were determined using extensive 2D NMR, ESI, or CDESI and QTOF-MS/MS experiments and absolute configurations established by quantum mechanical calculations, advanced Marfeys method, and chiral HPLC. Mutremdamide A displays a rare 2-amino-3-(2-hydroxyphenyl)propanoic acid and a new N(delta)-carbamoyl-beta-sulfated asparagine. Koshikamides C-E are linear undecapeptides, and koshikamides F-H are 17-residue depsipeptides containing a 10-residue macrolactone. Koshikamides F and G differ from B and H in part by the presence of the conjugated unit 2-(3-amino-5-oxopyrrolidin-2-ylidene)propanoic acid. Cyclic koshikamides F and H inhibited HIV-1 entry at low micromolar concentrations while their linear counterparts were inactive. The Theonella collections studied here are distinguished by co-occurrence of mutremdamide A, koshikamides, and theonellamides, the combination of which appears to define a new Theonella chemotype that can be found in deeper waters.

Motualevic Acids A−F, Antimicrobial Acids from the Sponge Siliquariaspongia sp.

Seven new antibacterials, motualevic acids A-F (1-6) and (4E)-(R)-antazirine (7), have been isolated from the marine sponge Siliquariaspongia sp. and their structures elucidated by spectroscopic methods. Motualevic acids A-D are the first glycyl conjugates of the omega-brominated lipid (E)-14,14-dibromotetradeca-2,13-dienoic acid, and motualevic acid F is the first long-chain 2H-azirine 2-carboxylic acid to be found in nature. Carboxylic acid-containing compounds 1 and 6 inhibit the growth of Staphylococcus aureus and methicillin-resistant S. aureus at 1.2-10.9 microg/mL.

New unusual pregnane glycosides with antiproliferative activity from Solenostemma argel

Seven new 15-keto pregnane glycosides, namely Stemmosides E--K, were isolated from Solenostemma argel. Stemmosides E--J are characterized by the occurrence of an uncommon 14 beta proton configuration while stemmosides E and F possess in addition a rare enolic function in C-16. On the other hand, stemmosides G-J display an unusual C-17 alpha side chain. Their structures were established by ESI-MS and NMR experiments. Moreover, the effect of these compounds on the VEGF-induced in Kaposis sarcoma cell proliferation was tested. Results indicated that all the compounds reduced the cell proliferation in a dose dependent manner.

Combining in silico and biophysical methods for the development of Pseudomonas aeruginosa quorum sensing inhibitors: an alternative approach for structure-based drug design.

The present work deals with the optimization of an inhibitor of PqsD, an enzyme essential for Pseudomonas aeruginosa quorum sensing apparatus. Molecular docking studies, supported by biophysical methods (surface plasmon resonance, isothermal titration calorimetry, saturation transfer difference NMR), were used to illuminate the binding mode of the 5-aryl-ureidothiophene-2-carboxylic acids. Enabled to make profound predictions, structure-based optimization led to increased inhibitory potency. Finally a covalent inhibitor was obtained. Binding to the active site was confirmed by LC-ESI-MS and MALDI-TOF-MS experiments. Following this rational approach, potent PqsD inhibitors were efficiently developed within a short period of time. This example shows that a combination and careful application of in silico and biophysical methods represents a powerful complement to cocrystallography.

Mirabilin, an Antitumor Macrolide Lactam from the Marine Sponge Siliquariaspongia mirabilis

A new highly unsaturated macrolide lactam, termed mirabilin ( 1), was isolated from the aqueous extract of the marine sponge Siliquariaspongia mirabilis. Mirabilin is characterized by the presence of a 35-membered macrolide lactam ring bearing a pentadiene conjugated system and a tetrasubstituted tetrahydropyran ring. A linear polyketide moiety is attached to the macrocyclic ring through an amide linkage. The structure of mirabilin was determined using extensive 2D NMR and ESIMS and tandem MS techniques. Mirabilin inhibits the growth of the tumor cell line HCT-116 with an IC 50 value of 0.27 +/- 0.09 microM and is noncytotoxic to several other cell lines.

Discovery and Synthesis of Namalide Reveals a New Anabaenopeptin Scaffold and Peptidase Inhibitor

The discovery, structure elucidation, and solid-phase synthesis of namalide, a marine natural product, are described. Namalide is a cyclic tetrapeptide; its macrocycle is formed by only three amino acids, with an exocyclic ureido phenylalanine moiety at its C-terminus. The absolute configuration of namalide was established, and analogs were generated through Fmoc-based solid phase peptide synthesis. We found that only natural namalide and not its analogs containing l-Lys or l-allo-Ile inhibited carboxypeptidase A at submicromolar concentrations. In parallel, an inverse virtual screening approach aimed at identifying protein targets of namalide selected carboxypeptidase A as the third highest scoring hit. Namalide represents a new anabaenopeptin-type scaffold, and its protease inhibitory activity demonstrates that the 13-membered macrolactam can exhibit similar activity as the more common hexapeptides.