Phenylboronic Acid Derivatives as Validated Leads Active in Clinical Strains Overexpressing KPC‐2: A Step against Bacterial Resistance

Abstract

The emergence and dissemination of multidrug resistant (MDR) pathogens resistant to nearly all available antibiotics poses a significant threat in clinical therapy. Among them, Klebsiella pneumoniae clinical isolates overexpressing KPC-2 carbapenemase are the most worrisome, extending bacterial resistance to last-resort carbapenems. In this study, we investigate the molecular recognition requirements in the KPC-2 active site by small phenylboronic acid derivatives. Four new phenylboronic acid derivatives were designed and tested against KPC-2. For the most active, despite their simple chemical structures, nanomolar affinity was achieved. The new derivatives restored susceptibility to meropenem in clinical strains overexpressing KPC-2. Moreover, no cytotoxicity was detected in cell-viability assays, which further validated the designed leads. Two crystallographic binary complexes of the best inhibitors binding KPC-2 were obtained at high resolution. Kinetic descriptions of slow binding, time-dependent inhibition, and interaction geometries in KPC-2 were fully investigated. This study will ultimately lead toward the optimization and development of more-effective KPC-2 inhibitors.

In Silico Identification and Experimental Validation of Novel KPC-2 β-lactamase Inhibitors

“…Candidates were predicted to orient the anionic side of their moiety in the carboxylic acid binding site of KPC-2, delimited by Ser130, Thr235 and Thr237 and present in all serine-based Beta-lactamases. In the above mentioned site, in fact, binds the C(3)4’ carboxylate of β-lactams antibiotics as well as the sulfate group of avibactam and the carboxylic group of other known BLs inhibitors [12,32,37,38,39].…”

“…The three additional strains harbored the blaKPC-2 gene and were resistant to meropenem (Table 4). [32] Noteworthy none of the tested compounds had intrinsic antibiotic activity (MIC >256 ug/mL), against the employed strains, included the susceptible one. The results show that in most cases the compounds were not able to reverse antibiotic resistance and did not showed synergism with meropenem.…”

“…The bla KPC-2 gene was kindly provided by Prof. Sergei Vakulenko (University of Notre Dame du Lac, Indiana, USA) and cloned as already reported [32] and transformed into competent E.coli BL21 (DE3) cells for protein expression. 50 mL of Tryptic Soy Broth (TSB) (50 mg/L kanamycin) were inoculated with fresh colonies and grown at 37°C.…”

“…The protein was conveniently purified in a single step using a Macro-Prep High S resin and eluted using sodium acetate 50 mM pH 5.0 and a sodium chloride (NaCl) linear gradient from 100 to 500 mM. The purified protein was dialyzed overnight in sodium phosphate buffer 50 mM, pH 7.0 [32,33].…”

Discovery of [1,2,4]Triazole Derivatives as New Metallo-β-Lactamase Inhibitors

“…The IC 50 values for the compounds (with 10 different concentrations in threefold dilution) were further determined. All determinations were tested in triplicate [12,20,38]. The IC 50 values were obtained from the plot of activity versus inhibitor concentration by using the GraphPad Prism software.…”