Screening and structure-based design of novel ligands for FabF and KPC-2 : first steps to counteract an antibiotic crisis
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Abstract
The number of infections with multidrug resitant bacteria is constantly
increasing and thus, the need for new antibiotics is becoming more urgent.
The World Health Organisation (WHO) recently published a list of 12 bacteria
for which there is a critical, high or medium need for new antibiotics.
In particular, Gram-negative bacteria like Pseudomonas aeruginosa are associated
with a broad spectrum of infections. Attractive targets for drug design
are the enzymes involved in cell membrane and wall synthesis.
In this thesis, the elongation condensing enzyme FabF of the fatty acid synthesis
pathway was chosen as target for further studies. Fatty acids are
essential for the formation of the inner and outer cellmembrane of Gramnegative
bacteria. Using virtual screening, 2 compounds sharing a thiazolidine-
4-carboxylic acid scaffold binding non-covalently to FabF were identified.
Binding affinities of 278 uM and 321 uM were determined using biolayer
interferometry. In addition, 1 compound binding covalently was identified
using MALDI mass spectrometry. Further investigations of the active
site regarding the binding mode of the natural product inhibitor platensimycin
were carried out by introducing the point mutation for T271A using
site directed mutagenesis and solving the structure of FabF T271A. A
fragment library of 651 compounds was screened using biolayer interferometry
(BLI). Due to a limited amount of publications using BLI in fragment
screens, this gave new insights in the assessment of fragment binding
profiles and responses. The false-positive rate was lowered by screening
with FabF wild type with a closed binding site and FabF C164Q with an
open binding site. Finally, 16 FabF C164Q specific hits were identified (hit
rate: 2.5%) out of 67 hits found in the primary screening (10.3%).
Another approach of targeting the cell wall synthesis of bacteria is the inhibition
of the glycopeptide transpeptidase by beta-lactame antibiotics. However,
this approach is limited due to the defence mechanism of bacteria,
the production of beta-lactamases, which can inactivate beta-lactame antibiotics
through hydrolysis. The carbapenemase KPC-2 was chosen as target enzyme
for further studies. Using virtual screening, 10 ligands of KPC-2 were
identified, the most potent with a KI of 30 uM. In attempts to improve the
binding affinity of this hit compound an amide linker was replaced by a
sulfonamide in order to change the geometry of the ligand and to vary the
attached group. This substitution targeted the side chains corresponding to
amino acids Asn 132, Asn 170 and Leu 166. Finally, it was shown that the
sulfonamide linker is not suitable for improving the binding affinity.