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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - AND (Antibacterial (Nano)medicines Development)

Teaser

Summary

The discovery of antibiotics in the 1950s saved millions of lives from once deadly diseases and opened possibilities for medical interventions such as organ transplants or cancer treatment, that would not be possible due to risk of life-threatening infections. However, the overuse and misuse of antibiotics has promoted the development and spread of antimicrobial resistance (AMR), including antibiotic resistance. The growing problem of AMR threatens health security, and damages trade and economies.
The evolution of resistant strains is a natural phenomenon that happens when microorganisms are exposed to antimicrobial drugs, and resistant traits are transferred between microorganisms through horizontal gene transfer mechanisms. Efforts to reduce the inappropriate use of antibiotics in humans and animals, improve infection control strategies and promote international cooperation will help to reduce the spread of AMR. However, research on new prevention and therapeutic strategies for bacterial infections is also urgently needed to address the lack of new drugs entering the market in the past 30 years. This was the focus of the research performed within the project â€œAntibacterial (Nano)medicines Developmentâ€, AND-659121.
The specific objectives of the project were to identify a lead-candidate for the development of a novel broad-spectrum antibacterial (nano)medicine by improving the pharmacological properties and Gram-negative membrane permeability of previously identified bacterial histidine kinase inhibitors (HKIs). Histidine kinases (HKs) are a component of the bacterial two-component systems (TCSs), major signalling systems in bacteria.

Work performed

From the beginning of the project more potent HKIs were identified via computer-aided medicinal chemistry approaches. The latest HKIs are bactericidal against Gram- positive pathogens with MICs sometimes as low as 0.2 Âµg/ml, which is 40 times improvement compared to the hit compound we started with. The susceptible Gram positive pathogens include S. aureus, vancomycin-resistant Enterococcus faecium, Staphylococcus pseudintermedius, multi-drug resistant pathogen of dogs and cats, and the zoonotic pathogen S. suis. The newly discovered HKIs show moderate activity in vitro against Mycobacterium marinum, a fish pathogen used as model organism in anti-tuberculosis drug discovery. Furthermore, the HKIs are active against some pathogenic Gram-negative pathogens, including the zoonotic pathogen P. multocida and E. coli mutants which are more susceptible to current antibiotics.
The HKIs are not toxic to G. mellonella larvae at a single dose of 40 mg/kg. Noteworthy, some of the HKIs showed very low or no detectable mutation rates, indicating their low potential for resistance development.
By using nanoparticles we showed that the HKIs are active against Gram negative bacteria such as E. coli and S. marcescens provided they pass the outer membrane. We have also demonstrated that the used nanotool does not interfere with the immune function of immune cells in vitro and is not cytotoxic. In fact, the HKIs delivered via the nanotool were less toxic to mammalian cells in vitro than the HKIs alone.
Altogether, the identified HKIs have a great potential in the fight against the so-called ESKAPE pathogens and important zoonotic pathogens.
Based on the results obtained so far, we have rationally designed next generation putative HKIs and will confirm their activity once they are synthesized (expected soon after the end of the project).
Ideally during the hit-to-lead optimization of candidates for antibacterial medicines one would like to focus on hits with demonstrated in vivo safety and efficacy. However, testing the in vivo toxicity and efficacy with mammalian in vivo models is time- and resource-consuming and raises ethical issues. For this, we chose to use G. mellonella larvae to assess the toxicity and the efficacy of the HKIs in vivo. Furthermore, we validated G. mellonella as a model to study the virulence of the important zoonotic pathogen S. suis.
The obtained results have been disseminated to the scientific community via publications (1 review and 3 research papers), poster presentations at 2 international conferences and 1 international summer school and short talks at national meetings of the NCOH-AMR. Additionally, a patent application is in progress to protect intellectual property around these inhibitors and their therapeutic use against bacterial infections.

Final results

The performed research has a very high potential to impact on society by (i) providing candidates for novel antibacterial medicines with low potential for resistance development or adverse effects, the current technology readiness level (TLR) is between TRL3 and 4 (experimental proof of concept, and technology validated in the lab) [1], (ii) validating the safety of a nanotool for broadening the spectrum of antibacterial medicines to Gram negatives [2], and (iii) validating a model for the rapid assessment of the virulence of the emerging zoonotic pathogen S. suis and therapies against S. suis [3]. In the longer-term (8-12 years after the end of the project) this could have a social benefit reducing deaths due to drug resistant infections which are currently estimated at 50,000 per annum in Europe and predicted to increase dramatically in the coming decades.
The identification of improved HKIs with potential for development as novel antibacterials for animal and human medicine has led to the generation of intellectual property which will bring economic benefits to society if they are successful in clinical trials.
The validated insect in vivo infection model will potentially reduce the number of pigs and mice used in vivo infection studies with S. suis by for example allowing rapid preliminary assessment of the virulence of newly isolated strains and rapid preliminary assessment of the efficacy of therapies against S. suis. Thus, contributing to replacement, reduction, and refinement of the use of animal models (3Rs).
The national and international collaborations within the project have also strengthened academic research networks, aligned resources, attracted interest from industry and funding from national and international organizations. The ER is a member of a JPIAMR international working group (2017) and as a co-applicant has secured funding for the further development of the HKIs from ZonMw (2017-2021). Her research has also been supported by a travel grant and a short-term research grant from the Dutch Cystic Fibrosis Foundation and from FEBS, respectively.

1. Velikova N, Fulle S, Manso AS, Mechkarska M, Finn P, Conlon JM, Oggioni MR, Wells JM, Marina A: Putative histidine kinase inhibitors with antibacterial effect against multi-drug resistant clinical isolates identified by in vitro and in silico screens. Scientific reports 2016, 6:26085.
2. Velikova N, Mas N, Miguel-Romero L, Aguado LP, Stolte E, Zaccaria E, Cao R, Taverne N, Murguia JR, Martinez-Manez R et al: Broadening the antibacterial spectrum of histidine kinase autophosphorylation inhibitors via the use of epsilon-poly-L-lysine capped mesoporous silica-based nanoparticles. Nanomedicine : nanotechnology, biology, and medicine 2016.
3. Velikova N, Kavanagh K, Wells JM: Evaluation of Galleria mellonella larvae for studying the virulence of Streptococcus suis. BMC microbiology 2016, 16(1):291.