Optimization and Mechanistic Characterization of Pyridopyrimidine Inhibitors of Bacterial Biotin Carboxylase

A major challenge for new antibiotic
discovery is predicting the
physicochemical properties that enable small molecules to permeate
Gram-negative bacterial membranes. We have applied physicochemical
lessons from previous work to redesign and improve the antibacterial
potency of pyridopyrimidine inhibitors of biotin carboxylase (BC)
by up to 64-fold and 16-fold against Escherichia coli and Pseudomonas aeruginosa, respectively.
Antibacterial and enzyme potency assessments in the presence of an
outer membrane-permeabilizing agent or in efflux-compromised strains
indicate that penetration and efflux properties of many redesigned
BC inhibitors could be improved to various extents. Spontaneous resistance
to the improved pyridopyrimidine inhibitors in P. aeruginosa occurs at very low frequencies between 10–8 and
10–9. However, resistant isolates had alarmingly
high minimum inhibitory concentration shifts (16- to >128-fold)
compared
to the parent strain. Whole-genome sequencing of resistant isolates
revealed that either BC target mutations or efflux pump overexpression
can lead to the development of high-level resistance.