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.

The vasoactive cyclic 11-amino acid peptide urotensin II (U-II) has recently been discovered as the endogenous ligand of the orphan G-protein-coupled receptor GPR14. As U-II might be involved in the regulation of cardiovascular homeostasis and pathology, a nonpeptidic GPR14/U-II antagonist is of considerable basic and therapeutic interest. We have performed structure-activity relationship studies on U-II by investigating 25 peptide analogues to mobilize intracellular calcium in GPR14-transfected CHO cells, demonstrating that only the side chains of the residues Trp-7, Lys-8, and Tyr-9 are required for receptor recognition and activation. The solution structure of U-II derived by nuclear magnetic resonance has served as a structural template for a three-dimensional three point pharmacophore query for the virtual screening of the Aventis compound repository for nonpeptidic U-II receptor antagonists. Highly active lead compounds of six different scaffold classes could be identified, antagonizing the biological activity of U-II in vitro. The most potent compound identified by the virtual screening approach, 1-(3-carbamimidoyl-benzyl)-4-methyl-1H-indole-2-carboxylic acid (naphthalen-1-ylmethyl)amide, reveals an IC(50) of 400 nM in a functional fluorometric imaging plate reader assay and constitutes a promising lead.

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Dual activation of the glucagon-like peptide 1 (GLP-1) and glucagon receptor has the potential to lead to a novel therapy principle for the treatment of diabesity. Here, we report a series of novel peptides with dual activity on these receptors that were discovered by rational design. On the basis of sequence analysis and structure-based design, structural elements of glucagon were engineered into the selective GLP-1 receptor agonist exendin-4, resulting in hybrid peptides with potent dual GLP-1/glucagon receptor activity. Detailed structure-activity relationship data are shown. Further modifications with unnatural and modified amino acids resulted in novel metabolically stable peptides that demonstrated a significant dose-dependent decrease in blood glucose in chronic studies in diabetic db/db mice and reduced body weight in diet-induced obese (DIO) mice. Structural analysis by NMR spectroscopy confirmed that the peptides maintain an exendin-4-like structure with its characteristic tryptophan-cage fold motif that is responsible for favorable chemical and physical stability.

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.

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.

Urotensin II (U-II; cyclo [5][6][7][8][9][10] [H-Glu-Thr-Pro-Asp-Cys-Phe-TrpLys-Tyr-Cys-Val-OH]) is a potent vasoconstrictor in mammals, and it is postulated that it plays a central role in cardiovascular homeostasis. Thus, we initiated a structure-to-function analysis of this peptide characterized by a N-terminal tail and a cyclic core formed through a disulfide bridging. A total of 41 analogs focusing on these characteristics were developed and evaluated using a binding assay on membranes from a stable HEK-293 cell line containing the human or rat U-II receptor, a functional assay for Ca 2ϩ mobilization on transiently transfected CHO-K1 cells with the human or rat U-II receptor, and a rat thoracic aorta bioassay. At first, the focus was applied on peptide compounds containing exocyclic modifications. From this series, it appeared that only valine-11 played a significant role although it is not an essential amino acid. Similarly, endocyclic and ring transformations of hU-II were also studied. In most cases, a detrimental effect on affinity and biological activity was observed. However, two compounds, [Tyr 6 ]hU-II and [Phe 9 ]hU-II, retained affinity and activity. So far, our binding, functional, and pharmacological data clearly demonstrated the minor contribution of the N-terminal segment and the essential role of the cyclic structure. More particularly, three residues within the loop, i.e., Trp-7, Lys-8, and Tyr-9, are required for receptor recognition and activation. This three-pole feature, kept by the disulfide bond in a correct spatial arrangement, appears as the key pharmacophore for the U-II receptor.