Pyrazolopyrimidines Establish MurC as a Vulnerable Target in Pseudomonas aeruginosa and Escherichia coli

Abstract

The bacterial peptidoglycan biosynthesis pathway provides multiple targets for antibacterials, as proven by the clinical success of β-lactam and glycopeptide classes of antibiotics. The Mur ligases play an essential role in the biosynthesis of the peptidoglycan building block, N-acetyl-muramic acid-pentapeptide. MurC, the first of four Mur ligases, ligates l-alanine to UDP-N-acetylmuramic acid, initiating the synthesis of pentapeptide precursor. Therefore, inhibiting the MurC enzyme should result in bacterial cell death. Herein, we report a novel class of pyrazolopyrimidines with subnanomolar potency against both Escherichia coli and Pseudomonas aeruginosa MurC enzymes, which demonstrates a concomitant bactericidal activity against efflux-deficient strains. Radio-labeled precursor incorporation showed these compounds selectively inhibited peptidoglycan biosynthesis, and genetic studies confirmed the target of pyrazolopyrimidines to be MurC. In the presence of permeability enhancers such as colistin, pyrazolopyrimidines exhibited low micromolar MIC against the wild-type bacteria, thereby, indicating permeability and efflux as major challenges for this chemical series. Our studies provide biochemical and genetic evidence to support the essentiality of MurC and serve to validate the attractiveness of target for antibacterial discovery.

Novel Antibacterial Targets and Compounds Revealed by a High-Throughput Cell Wall Reporter Assay

“…In order to confirm the results of the AmpC reporter assay, compounds 1 and 2 were assayed for inhibition of the incorporation of cellular pathway-specific radioactive precursors (23). Both were found to inhibit the incorporation of [ 3 H]diaminopimelic acid, which is a component of the E. coli peptidoglycan, indicating that the activity of these compounds is related to the inhibition of cell wall biogenesis ( Table 2).…”

“…Inhibition of cell wall, fatty acid, DNA, RNA, and protein biosynthesis processes was measured as previously described, using an E. coli W3110 ⌬tolC ⌬lysA strain (23).…”

The biology of Mur ligases as an antibacterial target

“…Recently, a new class of pyrazolopyrimidine inhibitors with sub‐nanomolar potency against E. coli and Pseudomonas aeruginosa MurC enzymes have been reported. These compounds have been shown to inhibit PG biosynthesis in vivo due to the targeting of MurC (Hameed et al ., ). These studies suggest that the Mur ligases perform rate‐limiting steps and confirm their validity as antibacterial targets.…”

“…These studies suggest that the Mur ligases perform rate-limiting steps and confirm their validity as antibacterial targets. However, the antibacterial activities of these Mur inhibitors were observed only when the bacterial cells were permeabilized (Baum et al, 2007;2009;Hameed et al, 2014).…”

Trends and Exceptions of Physical Properties on Antibacterial Activity for Gram-Positive and Gram-Negative Pathogens

“…Three examples of Hit-to-Lead programs which illustrate some of the extremes of physical property changes over the course of an antibacterial drug discovery program are highlighted below and help to reinforce the conclusions by Manchester et al These examples further illustrate that simply relying on confines of physical property space may be helpful, but not sufficient to have whole cell activity against Gram-negative pathogens. Figure 19(a) highlights, the progression of clogD vs. time in the MurC program, which utilized a P.aeruginosa enzyme as the biochemical assay 30. In this depiction, the X-axis contains the consecutive compounds which were registered for the project and the vertical scale is the clogD of each compound.…”