Metallacarborane Derivatives Effective against Pseudomonas aeruginosa and Yersinia enterocolitica

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen that has become a nosocomial health problem worldwide. The pathogen has multiple drug removal and virulence secretion systems, is resistant to many antibiotics, and there is no commercial vaccine against it. Yersinia pestis is a zoonotic pathogen that is on the Select Agents list. The bacterium is the deadliest pathogen known to humans and antibiotic-resistant strains are appearing naturally. There is no commercial vaccine against the pathogen, either. In the current work, novel compounds based on metallacarborane cage were studied on strains of Pseudomonas aeruginosa and a Yersinia pestis substitute, Yersinia enterocolitica. The representative compounds had IC₅₀ values below 10 µM against Y. enterocolitica and values of 20-50 μM against P. aeruginosa. Artificial generation of compound-resistant Y. enterocolitica suggested a common mechanism for drug resistance, the first reported in the literature, and suggested N-linked metallacarboranes as impervious to cellular mechanisms of resistance generation. SEM analysis of the compound-resistant strains showed that the compounds had a predominantly bacteriostatic effect and blocked bacterial cell division in Y. enterocolitica. The compounds could be a starting point towards novel anti-Yersinia drugs and the strategy presented here proposes a mechanism to bypass any future drug resistance in bacteria.

Keywords: COSAN; Pseudomonas aeruginosa; Yersinia enterocolitica; antibacterials; boron clusters; metallacarboranes; resistance generation.

Figure 1

Screening strategy of small compounds library against P. aeruginosa and Y. enterocolitica. A diversity set of the complete library was screened on LB agar plates and then in a liquid medium as described under Materials and Methods.

Figure 2

Growth inhibition of Y. enterocolitica by metallacarboranes. (A) Bacterial growth inhibitors selected from a 20 μM screen against Y. enterocolitica. Only compounds showing at least 50% growth inhibition are shown. (B) Bacteria were grown in 96-well flat-bottom plates at 37 °C in the presence of 200 µM compounds for 24 h. The growth was followed by measurement of OD600 absorbance as described under Materials and Methods. Measurements at 8 h post-inoculation are shown. Error bars correspond to the SEM value from 3–5 measurements. DMSO, dimethyl sulfoxide; LB, Luria-Bertani broth.

Scheme 1

The synthetic scheme used to generate the top compounds from the screening strategy. Detailed synthetic procedures and analysis of compounds are provided under the Materials and Methods section.

Figure 3

Scanning electron microscopy of drug-resistant Y. enterocolitica strain 2080. The SEM was performed on bacteria passaged at least nine times in the presence of 200 μM compound 1 (B), 7 (C,D), or 3 (E,F), and deposited on polished silicon chips as described in Materials and Methods. Bacteria grown without targeting compounds displayed normal morphology (A). After exposure to compound 1 for nine passages, there were only fragments of organic debris visible (B). In the presence of compounds 7 and 3, there were multiple bacteria observed with apparently normal morphology. Scale bars: (A) and (B) 500 nm, (C) and (E) 2.5 μm, (D) and (F) 1 μm.

Figure 4

Toxicity of selected compounds on zebrafish (Danio rerio) larvae. Fish larvae were grown in 6-well plates in the presence of compounds for up to 5 days and the morphological deformities were analyzed from images as described under Materials and Methods. Representative photographs are shown. S, scoliosis; TA, tail autophagy; PE, pericardial edema.

Figure 5

Mortality rates of selected compounds on zebrafish (Danio rerio) larvae. The population size (n = 10) from Figure 4 was followed up to day 5 as described under Materials and Methods. Representative graphs are shown.