Avibactam is a β-lactamase inhibitor that is in clinical development, combined with β-lactam partners, for the treatment of bacterial infections comprising Gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a β-lactam core but maintains the capacity to covalently acylate its β-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the offrate for deacylation. The deacylation off-rate was 0.045 min −1 , which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant β-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among β-lactamase inhibitors.antibacterial | drug discovery | enzymology T here is an urgent need for new antibacterial agents that are active against drug-resistant bacteria. In particular, some Gram-negative pathogens have accumulated enough resistance mechanisms to render them virtually untreatable by modern antibacterial chemotherapy (1, 2). A mainstay for treatment of Gram-negative infections is the β-lactam classes of drugs. The most common form of resistance to β-lactam antibiotics is the expression of various β-lactamase enzymes capable of hydrolyzing the β-lactam ring of β-lactam drugs, rendering them ineffective. As new β-lactams have been introduced into clinical use, a changing landscape of β-lactamases has been selected and disseminated. Presently, over 1,000 β-lactamases have been documented comprising several structural classes and a wide range of substrate promiscuities and catalytic efficiencies (3, 4).In efforts to restore the efficacy of β-lactam antibiotics, β-lactamases have also been targeted with a variety of inhibitors (5, 6). The three inhibitors approved for clinical use are clavulanic acid, tazobactam, and sulbactam, all of which contain a β-lactam core. A challenge for the development of broad-spectrum β-lactamase inhibitors is the mechanistic diversity in β-lactamase enzymes, with the largest distinction being between the enzyme classes that use a serine residue as the nucleophilic species and the metallo-β-lactamases, which directly activate water for hydrolysis (7). A shared mechanistic feature of the marketed β-lactam-based inhibitors is their reaction with the serine enzymes to form a covalent acylenzyme intermediate. On ring opening, the acyl-enzyme intermediate can undergo additional rearrangements or be released through hydrolysis to regenerate the active β-lactamase enzyme (8). Originally designed to combat class A serine β-lactamase enzymes such as TEM-1, the clinical use of β-lactam-based inhibitors has been diminished by the emergence of enzymes against which they are ineffective. Despite intense investigation by pharmaceutical companies, no new β-lactamas...

The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.

Oxazolidinones represent a new and promising class of antibacterial agents. Current research in this area is mainly concentrated on improving the safety profile and the antibacterial spectrum. Many oxazolidinones, including linezolid (marketed as Zyvox), are inhibitors of monoamine oxidase A (MAO-A), which presents an undesired side effect. Recently, it was found that the 1,2,3-triazole is a good replacement for the conventional acetamide functionality found in oxazolidinones. We now disclose the finding that 1,2,3-triazoles bearing a substituent like methyl, small substituted methyl, bromo, or a linear (sp-hybridized) group at the 4 position (compounds such as 5, 16, 19, and 21) are good antibacterials with reduced or no activity, within the detection limit of the assay, against MAO-A. The results are especially promising for the development of oxazolidinones with an improved safety profile. The MAO-A SAR can be rationalized on the basis of docking studies to a MAO-A/MAO-B homology model.