Structural role of K224 in taniborbactam inhibition of NDM-1

Abstract

Taniborbactam (TAN; VNRX-5133) is a novel bicyclic boronic acid β-lactamase inhibitor (BLI) being developed in combination with cefepime (FEP). TAN inhibits both serine and some metallo-β-lactamases. Previously, the substitution R228L in VIM-24 was shown to increase activity against oxyimino-cephalosporins like FEP and ceftazidime (CAZ). We hypothesized that substitutions at K224, the homologous position in NDM-1, could impact FEP/TAN resistance. To evaluate this, a library of codon-optimized NDM K224X clones for minimum inhibitory concentration (MIC) measurements was constructed; steady-state kinetics and molecular docking simulations were next performed. Surprisingly, our investigation revealed that the addition of TAN restored FEP susceptibility only for NDM-1, as the MICs for the other 19 K224X variants remained comparable to those of FEP alone. Moreover, compared to NDM-1, all K224X variants displayed significantly lower MICs for imipenem, tebipenem, and cefiderocol (32-, 133-, and 33-fold lower, respectively). In contrast, susceptibility to CAZ was mostly unaffected. Kinetic assays with the K224I variant, the only variant with hydrolytic activity to FEP comparable to NDM-1, confirmed that the inhibitory capacity of TAN was modestly compromised (IC₅₀ 0.01 µM vs 0.14 µM for NDM-1). Lastly, structural modeling and docking simulations of TAN in NDM-1 and in the K224I variant revealed that the hydrogen bond between TAN's carboxylate with K224 is essential for the productive binding of TAN to the NDM-1 active site. In addition to the report of NDM-9 (E149K) as FEP/TAN resistant, this study demonstrates the fundamental role of single amino acid substitutions in the inhibition of NDM-1 by TAN.

Keywords: K224; NDM-1; metallo-β-lactamase; taniborbactam; β-lactamase inhibitor.

FIG 1

General catalysis mechanism of B1-metallo-β-lactamases. The resting unliganded B1 MBL contains a dizinc-bridging hydroxide ion within the hydrogen bonding distance of the side chain of Asp120 (E). Substrates bind with their β-lactam carbonyl oxygen coordinating to Zn1 and the conserved carboxylate coordinates to Zn2 as well as a backbone amide nitrogen provided by a structurally conserved K/R residue (ES). Upon the nucleophilic attack performed by the zinc-bound hydroxide, a high-energy tetrahedral intermediate (HE-TI) is formed, which quickly transitions to form the anionic intermediate (AI) after the cleavage of the C-N bond in the β-lactam ring. The negative charge is stabilized by a strong interaction with Zn2 and is delocalized within the structure of the hydrolyzed antibiotic. In the last step, protonation of the N atom (the rate-limiting step) and the addition of a water molecule complete the product and restore the enzyme for another cycle of catalysis (EP).

FIG 2

Structures of β-lactams used in this study and taniborbactam (VNRX-5133).

FIG 3

Minimum inhibitory concentrations (MICs) of different β-lactams against E. coli DH10B strains harboring the pHSG298 bla_NDM-1(K224X) gene. The heat map represents the doubling dilution changes in the MIC values of the K224X variants compared with that of the NDM-1 wild type. The results shown are the mode of three biological replicates. FEP, cefepime; CAZ, ceftazidime; IMI, imipenem; TBPM, tebipenem; FDC, cefiderocol; WT, wild type.

FIG 4

Minimum inhibitory concentrations (MICs) of different β-lactams with or without EDTA against E. coli DH10B strains harboring the pHSG298 bla_NDM-1(K224X) gene. The heat map represents the doubling dilution changes in the MIC values with EDTA compared with those without EDTA. MIC fold change calculations were conducted by converting “>” to the next higher dilution (e.g., >512 µg/mL was converted to 1,024 µg/mL), and “≤” was assumed to be the same value (e.g., ≤0.125 µg/mL was converted to 0.125 µg/mL). The results shown are the mode of three biological replicates. Abbreviations: FEP, cefepime; CAZ, ceftazidime; IMI, imipenem; WT, wild type.

FIG 5

Expression of the bla_{NDM K224X} library in E. coli DH10B cells. The steady-state expression of the K224X library in E. coli DH10B was analyzed by immunoblotting, as described in Material and Methods. * NDM-1 K224I, ** NDM-1 K224R.

Fig 6

Productive binding of taniborbactam to NDM-1 is mediated by K224.Energy-minimized models of NDM-1 wild-type (A) and NDM K224I (B and C) in complex with taniborbactam. Notice that in (A) taniborbactam is anchored by hydrogen-bonds from(K224 and E149, and the boron (B) atom is properly positioned for nucleophilic attack. In contrast, in (B and C) taniborbactam loses the interaction with K224 and E149, resulting in a non-productive binding that impairs inhibition. C atoms of the protein are shown in cyan, C atoms of taniborbactam in orange, O atoms in red, N atoms in blue, S atoms in yellow and B atom in pink. Zn(II) ions are shown as grey spheres. Taniborbactam-protein interactions are depicted in dash lines.