Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-β-lactamases (IMP-1 and IMP-6) Associated with a Single Amino Acid Substitution

Abstract

Gram-negative bacteria producing metallo-β-lactamases (MBLs) have become a considerable threat to public health. MBLs including the IMP, VIM, and NDM types are Zn(II) enzymes that hydrolyze the β-lactam ring present in a broad range of antibiotics, such as N-benzylpenicillin, meropenem, and imipenem. Among IMPs, IMP-1 and IMP-6 differ in a single amino acid substitution at position 262, where serine in IMP-1 is replaced by glycine in IMP-6, conferring a change in substrate specificity. To investigate how this mutation influences enzyme function, we examined lactamase inhibition by thiol compounds. Ethyl 3-mercaptopropionate acted as a competitive inhibitor of IMP-1, but a noncompetitive inhibitor of IMP-6. A comparison of the crystal structures previously reported for IMP-1 (PDB code: 5EV6) and IMP-6 (PDB code: 6LVJ) revealed a hydrogen bond between the side chain of Ser262 and Cys221 in IMP-1 but the absence of hydrogen bond in IMP-6, which affects the Zn2 coordination sphere in its active site. We investigated the demetallation rates of IMP-1 and IMP-6 in the presence of chelating agent ethylenediaminetetraacetic acid (EDTA) and found that the demetallation reactions had fast and slow phases with a first-order rate constant (k fast = 1.76 h −1 , k slow = 0.108 h −1 for IMP-1, and k fast = 14.0 h −1 and k slow = 1.66 h −1 for IMP-6). The difference in the flexibility of the Zn2 coordination sphere between IMP-1 and IMP-6 may influence the demetallation rate, the catalytic efficiency against β-lactam antibiotics, and the inhibitory effect of thiol compounds.

Hydroxyhexylitaconic acids as potent IMP-type metallo-β-lactamase inhibitors for controlling carbapenem resistance in Enterobacterales

“…The bacterial strains used in this study, MBL-producing E. coli transformants and Enterobacterales , have been listed in our previous reports ( 11 , 45 ). The expression and purification of IMP-1, IMP-6, NDM-1, VIM-2, and SMB-1 were performed according to previously described methods ( 11 , 45 , 46 ).…”

Drug Discovery in the Field of β-Lactams: An Academic Perspective

“…Unfortunately, resistance to β-lactam-BLI combinations has been identified both in the laboratory and clinical settings, including against avibactam and vaborbactam, even before their approval by the FDA [25][26][27]. Recent studies have demonstrated antibiotic resistance against combination therapies involving the latest BLIs and against new β-lactams, such as durlobactam [28], relebactam [29,30], zidebactam [31], tazobactam [32], taniborbactam [33], thiol-containing BLIs under development [34], and cefiderocol [35]. The resistance mechanisms include upregulation of efflux, mutations in the β-lactam target PBPs, and expression of β-lactamases and mutants less susceptible to the specific BLI, such as KPC-109 [36], NDM-9 [33], IMP-6 [34], and CMY-178 [37].…”

“…Recent studies have demonstrated antibiotic resistance against combination therapies involving the latest BLIs and against new β-lactams, such as durlobactam [28], relebactam [29,30], zidebactam [31], tazobactam [32], taniborbactam [33], thiol-containing BLIs under development [34], and cefiderocol [35]. The resistance mechanisms include upregulation of efflux, mutations in the β-lactam target PBPs, and expression of β-lactamases and mutants less susceptible to the specific BLI, such as KPC-109 [36], NDM-9 [33], IMP-6 [34], and CMY-178 [37]. These resistance mutants highlight the need for the antimicrobial field to constantly explore novel inhibitor chemotypes to counter future resistance.…”