Potential Transition State Analogue Inhibitors for the Penicillin-Binding Proteins

Abstract

Penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis. The development of new PBP inhibitors is a potentially viable strategy for developing new antibacterial agents. Several potential transition state analogue inhibitors for the PBPs were synthesized, including peptide chloromethyl ketones, trifluoromethyl ketones, aldehydes, and boronic acids. These agents were characterized chemically, stereochemically, and as inhibitors of a set of low molecular mass PBPs: Escherichia coli (EC) PBP 5, Neisseria gonorrhoeae (NG) PBP 3, and NG PBP 4. A peptide boronic acid was the most effective PBP inhibitor in the series, with a preference observed for a d-boroAla-based over an l-boroAla-based inhibitor, as expected given that physiological PBP substrates are based on d-Ala at the cleavage site. The lowest K(I) of 370 nM was obtained for NG PBP 3 inhibition by Boc-l-Lys(Cbz)-d-boroAla (10b). Competitive inhibition was observed for this enzyme-inhibitor pair, as expected for an active site-directed inhibitor. For the three PBPs included in this study, an inverse correlation was observed between the values for log K(I) with 10b and the values for log(k(cat)/K(m)) for activity against the analogous substrate, and K(m)/K(I) ratios were 90, 1900, and 9600 for NG PBP 4, EC PBP 5, and NG PBP 3, respectively. These results demonstrate that peptide boronic acids can be effective transition state analogue inhibitors for the PBPs and provide a basis for the use of these agents as probes of PBP structure, function, and mechanism, as well as a possible basis for the development of new PBP-targeted antibacterial agents.

Crystal Structure of Escherichia coli Penicillin-Binding Protein 5 Bound to a Tripeptide Boronic Acid Inhibitor: A Role for Ser-110 in Deacylation

“…Mechanism-based inhibitors of PBPs are of interest as probes of the essential features of substrate binding and catalytic mechanism in these enzymes, as well as for their potential to be developed into new classes of antibacterial agents. Recently, peptide boronic acids were demonstrated to be effective inhibitors of several PBPs (31), and this study presents the first structural description of a PBP-peptide boronic acid complex.…”

“…This substrate concentration is well below the K m of this substrate for E. coli PBP 5 (subsaturating conditions) (15). Peptide boronic acids were previously demonstrated to be competitive inhibitors for the PBPs (31). Under the conditions of enzyme (PBP) inhibition at subsaturating substrate concentrations ([S] < K m ), the following equation will apply:…”

Resistance to ?-lactam antibiotics

“…Finally, given the therapeutic success of b-lactams, the search is now on for novel agents that also target PBPs (e.g. [386][387][388][389]). …”

Boronic Acid Transition State Inhibitors Active against KPC and Other Class A β-Lactamases: Structure-Activity Relationships as a Guide to Inhibitor Design

“…Of late, great interest has been focused on inhibition of common serine ␤-lactamases (9). Attention to novel BLIs bearing an electrophilic center (phosphonates, aldehydes, trifluoromethylketones, and boronic acids) that can covalently modify the nucleophilic catalytic serine is conceptually advancing our understanding in this field (10).…”