Dieter Häbich

Identification and Characterization of the First Class of Potent Bacterial Acetyl-CoA Carboxylase Inhibitors with Antibacterial Activity

The multisubunit acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis, is broadly conserved among bacteria. Its rate-limiting role in formation of fatty acids makes this enzyme an attractive target for the design of novel broad-spectrum antibacterials. However, no potent inhibitors have been discovered so far. This report describes the identification and characterization of highly potent bacterial acetyl-CoA carboxylase inhibitors with antibacterial activity for the first time. We demonstrate that pseudopeptide pyrrolidine dione antibiotics such as moiramide B inhibit the Escherichia coli enzyme at nanomolar concentrations. Moiramide B targets the carboxyltransferase reaction of this enzyme with a competitive inhibition pattern versus malonyl-CoA (Ki value = 5 nm). Inhibition at nanomolar concentrations of the pyrrolidine diones is also demonstrated using recombinantly expressed carboxyltransferases from other bacterial species (Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa). We isolated pyrrolidine dione-resistant strains of E. coli, S. aureus, and Bacillus subtilis, which contain mutations within the carboxyltransferase subunits AccA or AccD. We demonstrate that such mutations confer resistance to pyrrolidine diones. Inhibition values (IC50) of >100 μm regarding an eukaryotic acetyl-CoA carboxylase from rat liver indicate high selectivity of pyrrolidine diones for the bacterial multisubunit enzyme. The natural product moiramide B and synthetic analogues show broad-spectrum antibacterial activity. The knowledge of the target and the availability of facile assays using carboxyltransferases from different pathogens will enable evaluation of the antibacterial potential of the pyrrolidine diones as a promising antibacterial compound class acting via a novel mode of action.

Specific and Potent Inhibition of NAD+-dependent DNA Ligase by Pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gramnegative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 μm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low μg/ml range, whereas eukaryotic cells were not affected up to 60 μg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.

Platensimycin, a New Antibiotic and “Superbug Challenger” from Nature

Multi-resistant “superbugs” have become a physician’s nightmare, particularly in hospitals, where antibiotics are heavily used. Bacterial infections increasingly evade standard treatment as resistance to existing antibiotics is spreading throughout the world. Reports of escalating treatment costs and—often fatal—therapy failures are on the rise. Of particular concern are infections by Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae (PRSP). Regardless of their historic titles, they all have acquired resistance to multiple antibiotic classes. As bacteria can replicate in less than half an hour, they are able to swiftly mutate and outsmart the antibiotic pressure by clever mechanisms that quickly spread through their microbial populations and help select for resistant organisms. Evolving resistance calls for new, effective, and safe antibacterial drugs without cross-resistance to antibiotics in clinical use. Only the persistent discovery and development of new antibiotics will guarantee future therapy.

Medicinal chemistry optimization of acyldepsipeptides of the enopeptin class antibiotics.

The therapy of life-threatening infections is significantly weakened by the global spread of antibiotic resistance. Among Gram-positive bacteria, the development of resistance of staphylococci, streptococci, and enterococci is of particular concern. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of complicated nosocomial infections, and its prevalence in hospitals has increased during the last decade. Furthermore, the frequent reliance on vancomycin as a last line of defense has led to a high level of vancomycin-resistant nosocomial isolates. Streptococcus pneumoniae is an important community-acquired respiratory tract pathogen, and a leading cause of morbidity and mortality. More than 30% of US isolates are penicillin-resistant (penicillin-resistant S. pneumoniae, PRSP) and many of these strains are also resistant towards a large number of other antibiotics. Novel antibacterial agents with unprecedented mechanisms of action, which are devoid of pre-existing cross-resistances, are therefore very necessary. Most of the currently marketed antibiotic classes originate from the secondary metabolism of bacteria or fungi, which emphasizes that natural products are a valuable source of novel antibacterial agents. Thus, depsipeptides of the enopeptin family are of interest. In 1982, the isolation of depsipeptide antibiotics A54556A and B (Scheme 1, 1 and 2, respectively), from a mixture of eight individual depsipeptidic factors (A– H), produced by aerobic fermentation of Streptomyces hawaiiensis (NRRL 15010) was described. Enopeptin A (3) and B (4) (Scheme 1) were isolated in 1991 from a culture broth of Streptomyces sp. RK-1051, found in a soil sample from Tsuruoka City, Japan. The enopeptin structure consists of a lactone core composed of five S-configured amino acids and a lipophilic acylated phenylalanine side chain attached to a serine nitrogen. This macrocyclic, peptidic structure made these acyldepsipeptides interesting candidates for total synthesis. The level of interest was further increased by mode of action studies with B. subtilis, demonstrating impaired bacterial cell division and induction of filamentation as the underlying causes of antibacterial activity (Figure 1). Applying reversed genomics technologies, Brçtz-Oesterhelt and co-workers were able to demonstrate that the lead structure 1 acts by binding to caseine lytic protease (ClpP), the core unit of a major bacterial–protease complex. In order to protect the bacterial cell from the destructive power of this universal protease, ClpP is tightly regulated and requires a ClpATPase, and often other accessory proteins for activation. Binding of 1 to ClpP eliminates the requirement of Clp-ATPases and other regulatory factors for proteolytic degradation. Therefore, uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death. With respect to drug discovery, natural acyldepsipeptides 1 and 2 displayed only limited in vitro activity against the Grampositive pathogen S. aureus (Table 1) and Gram-negative bacteria were not susceptible. Furthermore, 1 and 2 were not effective in standard mouse models of lethal bacterial infection, and their physicochemical and pharmacokinetic profile was dominated by poor aqueous solubility and high clearance. Thus, 1 and 2 were not favorable candidates for drug development. Finally, natural enopeptin acyldepsipeptide antibiotics 1 and 2 are challenging lead structures from a chemical and synthetic viewpoint. Several functional groups limited their stability: (a) the lactone core was readily hydrolyzed in basic and acidic aqueous media; (b) the acylated serine hydroxy group eliminated readily under non-aqueous basic conditions; (c) the conjugated triene was sensitive to temperature and light (cyclization and aromatization reactions) ; and (d) solubility was not sufficient for parenteral application. However, the novel target and the absence of cross-resistance to established antibiotics strongly encouraged the initiation of a medicinal chemistry program, the objective of which was to improve these deficiencies using a thorough understanding of the lead conformation, based on x-ray structure analysis of the synthetic congener 5 (Figure 2). Crystallization of 5 from toluene gave a solvate with two toluene molecules, whereas solvent free crystals were obtained from aqueous acetonitrile. In the case of the solvate crystal, a Scheme 1. Natural enopeptin depsipeptide antibiotics.

Total Synthesis and Initial Structure―Activity Relationships of Longicatenamycin A

Natural products have provided the majority of lead structures for marketed antibacterials. In addition, they are biological guide principles to new therapies. Nevertheless, numerous “old” classes of antibiotics such as the longicatenamycins have never been explored by chemical postevolution. Longicatenamycin A is the first defined longicatenamycin congener that has been totally synthesized and tested in pure form. This venture required the de novo syntheses of the non‐proteinogenic amino acids (2S,3R)‐β‐hydroxyglutamic acid (HyGlu), 5‐chloro‐D‐tryptophan (D‐ClTrp), and (S)‐2‐amino‐6‐methylheptanoic acid (hhLeu). In the key step, the sensitive HyGlu building block was coupled as a pentafluorophenyl active ester to the unprotected H‐D‐ClTrp‐Glu‐hhLeu‐D‐Val‐D‐(Cbz)Orn‐OH fragment. This first total synthesis of longicatenamycin A provided new congeners of the natural product (deacetyllongicatenamycin, dechlorolongicatenamycin, and longicatenamycin‐A‐amide).

Improved synthesis of antibacterial 3-substituted 6-anilinouracils.

3-Substituted 6-anilinouracils, presently the most promising class of inhibitors of the bacterial DNA polymerase in Gram-positive bacteria, have been prepared by a general and straightforward three-step procedure starting from a readily available 1-benzyloxymethyl-protected derivative of 6-chlorouracil.