Preclinical Evaluation of Antimicrobial Compounds: An Investigation of Natural Products, Natural Product Derivates, and Synthetic Compounds

The increased proliferation of antimicrobial resistance is a major public health challenge of our time.

Effective treatment of resistant infections requires the development of new antibacterial drugs with yet unexploited mechanisms of action, a task where natural products have historically been a convenient starting point. In this thesis, various synthetics, natural product derivatives, and natural products were screened for antibacterial and antibiofilm activity using broth microdilution and crystal violet methods.

For active compounds, potential targets, mechanisms of action, and early pharmacokinetic properties were investigated.

First, a group of seven synthetic analogues of the breitfussin natural products were investigated in paper I. Two of the tested compounds inhibited the growth of Gram-positive bacteria in their planktonic state at concentrations at or below 50 µM. One of the active compounds inhibited the formation of Staphylococcus epidermidis biofilm at sub-growth-inhibitory, low micromolar concentrations. Initial mechanistic studies revealed low impact on cell surface hydrophobicity and early-stage adhesion to polystyrene.

Next, in paper II, forty-two new synthetic molecules based on an N-arylsulfonyl pyridinyl- or indolyl hydrazone framework were investigated for antimicrobial activity. Twelve molecules were found to inhibit Gram-positive bacteria at concentrations as low as 1.25 µM. Using a Bacillus subtilis biosensor assay, cell envelope stress and interference with fatty acid synthesis type II were identified as likely mechanisms of action. A luciferin-based cell membrane integrity assay demonstrated that selected compounds increased bacterial membrane permeability, supporting the envelope stress hypothesis.

Through addition of 0.1 % Tween® 20 to the growth medium, bacterial growth was rescued, supporting the fatty acid synthesis inhibition hypothesis. The three most active compounds showed between 1.6 and >40 times higher activity towards bacterial cells over eukaryotic cells and high metabolic stability.

Finally, in paper III, the natural product ianthelline with known antibacterial activity was isolated from Porifera indet. extract and investigated for potential targets and mechanisms of action. To this end, proteins affected directly or indirectly by Escherichia coli exposure to ianthelline were investigated using a proteome integral solubility alteration whole cell thermal proteomics method combined with expression proteomics and mass spectrometry using data-independent acquisition. Ianthelline induced cell membrane permeability and likely interfered with transcriptomic or translational mechanisms, but further investigation is needed.