Elucidating the Molecular Basis of Avibactam‐Mediated Inhibition of Class A β‐Lactamases

Abstract

Disseminating antibiotic resistance rendered by bacteria against the widely used β−lactam antibiotics is a serious concern in the public health care. Development of inhibitors for drug-resistant β−lactamase enzymes is vital to combat this rapidly escalating problem. Recently, the Food and Drug Administration has approved a non-βlactam inhibitor called avibactam for the treatment of complicated intra-abdominal and urinary tract infections caused by drug-resistant Gram-negative bacteria. This work sheds light on the molecular origin of the inhibitory effect of avibactam against drugresistant CTX-M variant of Class-A β-lactamase. Especially, we probed the structural evolution, dynamical features and energetics along the acylation and the deacylation reaction pathways through reliable enhanced sampling molecular dynamics methods and free energy calculations. We scrutinize the roles of active site residues, the nature of the carbonyl linkage formed in the inhibitor-enzyme covalent intermediate and other structural features of the inhibitor molecule. While unraveling the reasons behind the inhibition of all the deacylation routes, this study explains various experimental 1 structural and kinetics data, and paves the way to design new inhibitors based on the β-lactam framework. 1 Introduction β-Lactam antibiotics are antibacterials that are routinely used to inhibit the bacterial cellwall synthesizing enzymes known as penicillin binding proteins (PBPs). It is now well established that their clinical efficacy is progressively deteriorating due to the emergence of drug-resistance in bacteria, primarily associated with their expression of β-lactamases. 1,2 These enzymes hydrolyze β-lactam antibiotics in an efficient manner, preventing the drug molecules to react with PBPs. Four major classes of β-lactamases have been identified: A, B, C and D. 3 Among them, classes A, C and D β-lactamases use their active site serine residue for the catalysis, while class B β-lactamase employs either single zinc ion, or two zinc ions. Here, we focus on class-A serine β-lactamases (ABL). This particular class of β-lactamase includes extended-spectrum β-lactamases (such as SHV, TEM and CTX-M)and serine carbapenemases (for e.g. KPC), that are largely responsible for major outbreaks of antibiotic resistance. 1,4,5 Mechanistically, the inactivation of β-lactam antibiotics by A, C and D β-lactamases occurs through two distinct chemical steps. In the first step (viz. acylation), the βlactam drug reacts with the active site serine, resulting in the formation of a covalent drug-enzyme intermediate, which undergoes further hydrolysis (deacylation) in the next step and dissociates from the active site; see Figure 1. To restore the efficacy of βlactam antibiotics, they are often prescribed in combination with slowly deacylating βlactamase inhibitors. Amoxicillin-clavulanate, ticarcillin-clavulanate, ampicillin-sulbactam and piperacillin-tazobactam are some generally prescribed combinations of antibiotics and inhibitors. 6,7 These β-lactamase inhibitor...

Achieving an Order of Magnitude Speedup in Hybrid-Functional- and Plane-Wave-Based Ab Initio Molecular Dynamics: Applications to Proton-Transfer Reactions in Enzymes and in Solution

“…They inhibit the bacterial-cell-wall-synthesizing enzymes known as penicillin binding proteins (PBPs). , However, the clinical efficacy of these life-saving drugs progressively deteriorates due to the emergence of drug resistance in bacteria, primarily associated with the expression of β-lactamases. − These enzymes hydrolyze β-lactam antibiotics in an efficient manner, preventing the drug molecules to react with PBPs. In the past, our group contributed to the understanding of the mechanism of hydrolysis of different classes of β-lactamases. ,− Here, we focus on class C serine β-lactamases that are majorly responsible for hospital-acquired infections. It was found that the protonation states of the active-site residues Lys 67 and Tyr 150 of class C β-lactamase play a crucial role in determining the hydrolysis mechanism. ,, In the Michaelis complex of cephalothin and the enzyme, two protonation states are to be considered: K + Y – and KY .…”

Boosting the conformational sampling by combining replica exchange with solute tempering and well‐sliced metadynamics

“…This method is suited to study systems with broad, flat, and unbound free energy landscapes. This method has been applied to study various problems 59–64 and has also been extended to deal with high‐dimensional CV‐space 47 . By combining REST2 with WSMTD, we hope to boost the sampling of hidden transverse coordinates while exploring the relevant CV space.…”

Inhibition Mechanism of Class D β-Lactamases by Avibactam

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“…It uses an umbrella sampling bias for a controlled sampling of a CV along which a broad and unbound basin is expected, whereas the orthogonal coordinates are sampled using a well-tempered metadynamics bias and high temperature. , In conventional metadynamics, the negative sum of the total augmented bias potentials gives a good estimation of the underlying free energy, while for TASS, the same is obtained from a reweighted distribution of CVs, as described in ref . It is worth mentioning that TASS and metadynamics were successfully used in many of our previous studies for modeling chemical reactions. ,,− …”