Berthold Hinzen

Dysregulation of bacterial proteolytic machinery by a new class of antibiotics.

Here we show that a new class of antibiotics—acyldepsipeptides—has antibacterial activity against Gram-positive bacteria in vitro and in several rodent models of bacterial infection. The acyldepsipeptides are active against isolates that are resistant to antibiotics in clinical application, implying a new target, which we identify as ClpP, the core unit of a major bacterial protease complex. ClpP is usually tightly regulated and strictly requires a member of the family of Clp-ATPases and often further accessory proteins for proteolytic activation. Binding of acyldepsipeptides to ClpP eliminates these safeguards. The acyldepsipeptide-activated ClpP core is capable of proteolytic degradation in the absence of the regulatory Clp-ATPases. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death.

Improving the hit-to-lead process: data-driven assessment of drug-like and lead-like screening hits

Drug-like and lead-like hits derived from HTS campaigns provide good starting points for lead optimization. However, too strong emphasis on potency as hit-selection parameter might hamper the success of such projects. A detailed absorption, distribution, metabolism, excretion and toxicology (ADME-Tox) profiling is needed to help identify hits with a minimum number of (known) liabilities. This is particularly true for drug-like hits. Herein, we describe how to break down large numbers of screening hits and we provide a comprehensive overview of the strengths and weaknesses for each structural class. The overall profile (e.g. ligand efficiency, selectivity and ADME-Tox) is the distinctive feature that will define the priority for follow-up.

In Silico ADMET Traffic Lights as a Tool for the Prioritization of HTS Hits

The need for in silico characterization of HTS hit structures as part of a data‐driven hit‐selection process is demonstrated. A solution is described in the form of an in silico ADMET traffic light and PhysChem scoring system. This has been extensively validated with in‐house data at Bayer, published data, and a collection of launched small‐molecule oral drugs.

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.

Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease.

The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram‐positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl‐leucyl‐leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP‐activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP‐dependent protein degradation.