Inspiration

Nothing in pipeline? - The problem of modern antibiotic research

Worldwide globalisation and a rising world population creates a new problem for our society: Pathogens acquire mutations and different resistance factors leaving us with less and less effective antimicrobials. The World Health Organisation (WHO) considers the most problematic organisms to be multi-resistant Mycobacterium tuberculosis, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and many others (Geneva: World Health Organization 2017).

Besides fighting antibiotic misuse by raising awareness and health education to decrease the number of new resistant bacteria, there is a necessity for new antibacterial substances and therapies. This lies within the responsibility of universities and companies. In contrast, every year, the FDA approves less and less new antibacterial compounds. This is due to the fact, that research in the field of new antibiotics for pharmaceutical companies is not attractive: New substances are usually only used as reserve antibiotics for patients suffering from infections with highly resistant pathogens and therefore provides in its patented period only a low budget income.

Most new antibiotic substances are found in new bacterial strains, while (bio-)chemically modified compounds are rather rare. At the same time, it gets harder to identify new potent candidates. We need new strategies to produce effective compounds that are specifically engineered to fight resistant pathogens and new methods to achieve these modifications. This is where antibiotic classes, that provide potency in theory, but fail in compliance and practical application, come into play.

Introducing the aminocoumarins: potent antibiotics with little relevance in clinical therapy

Aminocoumarins are one of many substance classes with almost no use in the antibacterial therapy. So far, three aminocoumarins are known: Novobiocin, Clorobiocin and Coumermycin A1. (Heide 2014) Furthermore, the structurally different Simocyclinone D8 has been described as a fourth aminocoumarin (Schimana et al. 2000).

All aminocoumarins produced by different streptomyces species have an aminocoumarin (Figure 3 green) and a deoxyribose (Figure 3 blue) moiety in common. Aminocoumarins irreversibly bind to and inhibit the B subunit of the bacterial gyrase and therefore prevent the cell from successful mitosis (Lawson and Stevenson 2012). The human topoisomerase II is a known off-target, which can cause toxicity when highly dosed. Resistances are mediated by unspecific multidrug exporters (MDR) or mutations in the gyrase binding pockets. The latter are only found in aminocoumarin producing Streptomyces strains or bacteria that were forced to mutate by selection under low dose aminocoumarin application (Fujimoto-Nakamura et al. 2005).
Disadvantages that prevent aminocoumarins from more frequent clinical use include path of administration (i.v. only), a narrow efficacy spectrum (no gram-negative bacteria are affected), unfavorable pharmacokinetics caused by a low water solubility or irritations at the injection spot caused by toxicity due to a slow drug distribution in the blood (Grayson et al 2010). So far, Novobiocin is the only aminocoumarin approved by the FDA for antibiotic therapy. Lately, studies have found beneficial results for cancer therapies with topoisomerase II inhibitors in combination with Novobiocin.

Engineering the warhead: the ß-lactam motive as an activator in resistant bacteria

Our way to a new aminocoumarin antibiotic with an activation mechanism started with the question, which of the known aminocoumarins our substance should be based on. We decided to work with clorobiocin, as it is not used as antibiotic in its current form and provides perfect circumstances for different chemical modifications.
A basic aminocoumarin provides three sites open to (bio-)chemical modifications without loss of activity: While R1 allows little modification, as this part of the molecule is synthesised first (further involved enzymes wouldn't recognize their new substrate) and R2 can only be substituted with small chemical groups to retain the antibacterial properties, R3 provides the biggest variety in possible substrates. For an effective gyrase inhibition a nonpolar, hydrophobic moiety at R3 is preferred. Novobiocin and clorobiocin carry an isoprenyl moiety at this position. Therefore, we chose a modification at R3, that lowers the hydrophobic affinity in inactive state but keeps its linear, hydrophobic character after activation. R3 can be modified by knocking out genes in the antibiotic gene cluster that are involved in the synthesis of the para-hydroxy-benzoic acid derivative (Figure 3 red). Afterwards, chemically produced substances are fed to the producing Streptomyces which can then be used by the corresponding amide synthase to connect it with the residual molecule.
Many multiresistant pathogens acquired different resistances for many antibiotic classes. Frequently these pathogens integrated a kind of ß-lactamase into their genome because of the high frequency of ß-lactam motives in many different antibiotic classes. That's why we decided to use a ß-lactam ring as warhead for our compound: Without cleavage by a ß-lactamase, the aminocoumarin provides a slightly hydrophilic and bigger moiety then an isoprenyl at R3, not allowing an effective binding in the target's gyrase binding pocket, while after cleavage the opened ß-lactam ring mimics a hydrophobic N-isobutyl moiety leading to an effective inhibition (Figure 5). These considerations lead us to our new aminocoumarin Troiacin.

Troiacin: Structural advancement leads to confined efficacy

Troiacin integrates many of our considerations listed above with our new semi-biosynthetic synthesis. Troiacin has a ß-lactam ring structure similar to the carbapenem ß-lactam allowing an effective cleavage by carbapenem resistant pathogens. While the expected pathogen spectrum remains the same (mainly gram-positive bacteria including S. aureus, Actinobacteria, and M. tuberculosis), we propose that Troiacin has a decreased off-target effect by decreased binding affinity to the human topoisomerase II. Furthermore, it is better soluble in water then clorobiocin leading to a better properties in pharmacokinetics, lower toxicity reactions at the infusion position and a better distribution in the patient's body. Besides, the need of a lower antibiotic dose reduces the risks of off-target effects in the human topoisomerase II even further. Therefore, Troiacin can be used for a positive antibiotic selection in carbapenem resistant bacteria.