Michael Kurz

Targeting DnaN for tuberculosis therapy using novel griselimycins

New for old—TB drug development Tuberculosis (TB) is a global health threat for which there is only lengthy drug treatment. Patients need to consume multiple tablets over several months and frequently fail to complete their treatment. Consequently, drug-resistant strains of the pathogen have emerged, which add to the threat. Kling et al. revisited a natural product called griselimycin, extracted from the same organism that produced the prototype anti-TB drug, streptomycin. Unmodified griselimycin has poor pharmacological properties. However, one synthetic derivative had improved oral uptake and penetrated cells of the immune system that harbor the TB mycobacterium. In combination with other drugs, the griselimycin derivative showed high potency in mice with TB. Science, this issue p. 1106 A griselimycin-derived drug that blocks the DNA polymerase sliding clamp is a potent anti-tuberculosis lead. The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.

Identification of the bile acid-binding site of the ileal lipid-binding protein by photoaffinity labeling, matrix-assisted laser desorption ionization-mass spectrometry, and NMR structure.

The ileal lipid-binding protein (ILBP) is the only physiologically relevant bile acid-binding protein in the cytosol of ileocytes. To identify the bile acid-binding site(s) of ILBP, recombinant rabbit ILBP photolabeled with 3-azi- and 7-azi-derivatives of cholyltaurine was analyzed by a combination of enzymatic fragmentation, gel electrophoresis, and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. The attachment site of the 3-position of cholyltaurine was localized to the amino acid triplet His100-Thr101-Ser102using the photoreactive 3,3-azo-derivative of cholyltaurine. With the corresponding 7,7-azo-derivative, the attachment point of the 7-position could be localized to the C-terminal part (position 112–128) as well as to the N-terminal part suggesting more than one binding site for bile acids. By chemical modification and NMR structure of ILBP, arginine residue 122 was identified as the probable contact point for the negatively charged side chain of cholyltaurine. Consequently, bile acids bind to ILBP with the steroid nucleus deep inside the protein cavity and the negatively charged side chain near the entry portal. The combination of photoaffinity labeling, enzymatic fragmentation, MALDI-mass spectrometry, and NMR structure was successfully used to determine the topology of bile acid binding to ILBP.

Insights into the bile acid transportation system: the human ileal lipid-binding protein-cholyltaurine complex and its comparison with homologous structures.

Bile acids are generated in vivo from cholesterol in the liver, and they undergo an enterohepatic circulation involving the small intestine, liver, and kidney. To understand the molecular mechanism of this transportation, it is essential to gain insight into the three‐dimensional (3D) structures of proteins involved in the bile acid recycling in free and complexed form and to compare them with homologous members of this protein family. Here we report the solution structure of the human ileal lipid‐binding protein (ILBP) in free form and in complex with cholyltaurine. Both structures are compared with a previously published structure of the porcine ILBP‐cholylglycine complex and with related lipid‐binding proteins. Protein structures were determined in solution by using two‐dimensional (2D)‐ and 3D‐homo and heteronuclear NMR techniques, leading to an almost complete resonance assignment and a significant number of distance constraints for distance geometry and restrained molecular dynamics simulations. The identification of several intermolecular distance constraints unambiguously determines the cholyltaurine‐binding site. The bile acid is deeply buried within ILBP with its flexible side‐chain situated close to the fatty acid portal as entry region into the inner ILBP core. This binding mode differs significantly from the orientation of cholylglycine in porcine ILBP. A detailed analysis using the GRID/CPCA strategy reveals differences in favorable interactions between protein‐binding sites and potential ligands. This characterization will allow for the rational design of potential inhibitors for this relevant system. Proteins 2003;50:312–328.

Isolation and Structural Elucidation of Armeniaspirols A–C: Potent Antibiotics against Gram‐Positive Pathogens

In an antibiotic lead discovery program, the known strain Streptomyces armeniacus DSM19369 has been found to produce three new natural products when cultivated on a malt-containing medium. The challenging structural elucidation of the isolated compounds was achieved by using three independent methods, that is, chemical degradation followed by NMR spectroscopy, a computer-assisted structure prediction algorithm, and X-ray crystallography. The compounds, named armeniaspirolu2005A-C (2-4), exhibit a compact, hitherto unprecedented chlorinated spiro[4.4]non-8-ene scaffold. Labeling experiments with [1-(13)C] acetate, [1,2-(13)C2] acetate, and [U-(13)C] proline suggest a biosynthesis through a rare two-chain mechanism. Armeniaspirols displayed moderate to high in vitro activities against gram-positive pathogens such as methicillin-resistant S. aureus (MRSA) or vancomycin resistant E. faecium (VRE). As analogue 2 was active in vivo in an MRSA sepsis model, and showed no development of resistance in a serial passaging experiment, it represents a new antibiotic lead structure.

Isolation and structure elucidation of vancoresmycin—a new antibiotic from Amycolatopsis sp. ST 101170

A new antibiotic, active against Gram-positive bacteria, has been isolated from the fermentation broth of the actinomycete Amycolatopsis sp. ST 101170. The structure of the compound named vancoresmycin, a tetramic acid derivative with a highly oxygenated long alkyl chain, was elucidated by extensive spectroscopic studies and derivatization.

Structure and biosynthesis of xenoamicins from entomopathogenic Xenorhabdus.

During the search for novel natural products from entomopathogenic Xenorhabdus doucetiae DSM17909 and X. mauleonii DSM17908 novel peptides named xenoamicins were identified in addition to the already known antibiotics xenocoumacin and xenorhabdin. Xenoamicins are acylated tridecadepsipeptides consisting of mainly hydrophobic amino acids. The main derivative xenoamicin A (1) was isolated from X. mauleonii DSM17908, and its structure elucidated by detailed 1D and 2D NMR experiments. Detailed MS experiments, also in combination with labeling experiments, confirmed the determined structure and allowed structure elucidation of additional derivatives. Moreover, the xenoamicin biosynthesis gene cluster was identified and analyzed in X. doucetiae DSM17909, and its participation in xenoamicin biosynthesis was confirmed by mutagenesis. Advanced Marfeys analysis of 1 showed that the absolute configuration of the amino acids is in agreement with the predicted stereochemistry deduced from the nonribosomal peptide synthetase XabABCD. Biological testing revealed activity of 1 against Plasmodium falciparum and other neglected tropical diseases but no antibacterial activity.

Fabclavines : bioactive peptide-polyketide-polyamino hybrids from Xenorhabdus

The structure of the fabclavines—unique mixtures of nonribosomally derived peptide–polyketide hybrids connected to an unusual polyamino moiety—has been solved by detailed NMR and MS methods. These compounds have been identified in two different entomopathogenic Xenorhabdus strains, thereby leading also to the identification of the fabclavine biosynthesis gene cluster. Detailed analysis of these clusters and initial mutagenesis experiments allowed the prediction of a biosynthesis pathway in which the polyamino moiety is derived from an unusual type of fatty acid synthase that is normally involved in formation of polyunsaturated fatty acids. As fabclavines show broad‐spectrum activity against bacteria, fungi, and other eukaryotic cells, they might act as “protection factors” against all kinds of food competitors during the complex life cycle of Xenorhabdus, its nematode host, and their insect prey.

Antiparasitic Chaiyaphumines from Entomopathogenic Xenorhabdus sp. PB61.4

A new class of four depsipentapeptides called chaiyaphumines A-D (1-4) was isolated from Xenorhabdus sp. PB61.4. Their structures were elucidated by detailed 1D and 2D NMR experiments and by a Marfeys analysis following flash hydrolysis of the peptide. Verification of the structure was achieved by three-dimensional modeling using NOE-derived distance constraints, molecular dynamics, and energy minimization. Chaiyaphumine A (1) showed good activity against Plasmodium falciparum (IC50 of 0.61 μM), the causative agent of malaria, and was active against other protozoal tropical disease causing agents.

Structure determination of the bioactive depsipeptide xenobactin from Xenorhabdus sp. PB30.3

A new hexadepsipeptide called xenobactin (1) was isolated from Xenorhabdus sp. PB30.3. Structure elucidation was performed after isolation by extensive 1D- and 2D-NMR experiments. To determine the absolute configuration of the amino acids, modifications of the advanced Marfeys method were applied avoiding racemization and additionally allowing the stereochemical assignment of tryptophan. Moreover, the three dimensional structure was modeled by ROE derived constraints and molecular dynamics runs. The major conformation was verified by comparison of the modeled and experimentally predicted hydrogen bonds. Moreover, bioactivity testing of 1 revealed good antiprotozoal activity against Plasmodium falciparum and a specific antibiotic activity against the Gram positive bacterium Micrococcus luteus, whereas no cytotoxicity could be observed.

CHEMICAL MODIFICATION OF THE GE 2270A MACROCYCLE (MDL 62,879)

Abstract Controlled acid hydrolysis of compound 2 resulted in the selective opening of the macrocycle while in basic condition a retro-aldol reaction occurred at the level of phenylserine thiazole. Compound 2 can be easily prepared from the natural antibiotic MDL 62,879. All the compounds prepared resulted less active than MDL 62,879.