New boronate drugs and evolving NDM-mediated beta-lactam resistance

ABSTRACT Taniborbactam and xeruborbactam are dual serine-/metallo-beta-lactamase inhibitors (BLIs) based on a cyclic boronic acid pharmacophore that undergo clinical development. Recent report demonstrated that New Delhi metallo-beta-lactamase (NDM)-9 (differs from NDM-1 by a single amino acid substitution, E152K, evolved to overcome Zn (II) deprivation) is resistant to inhibition by taniborbactam constituting pre-existing taniborbactam resistance mechanism. Using microbiological and biochemical experiments, we show that xeruborbactam is capable of inhibiting NDM-9 and propose the structural basis for differences between two BLIs.

R ecent studies suggest that NDM [metallo-beta-lactamase (MBL) of the New Delhi group] variants are evolving to overcome Zn (II) deprivation, a condition that can be elicited by the immune system in response to infection (1). One of these variants is NDM-9 that differs from NDM-1 by a single amino acid substitution, E152K (2), and has been reported in several species of Enterobacterales worldwide (3). Recent report (4) demonstrated that NDM-9 is resistant to inhibition by taniborbactam, a dual serine-(SBL)/metallo-beta-lactamase inhibitor (BLI) based on a cyclic boronic acid pharma cophore (5) that has completed Phase 3 clinical development in combination with cefepime (NCT03840148). With their study, the authors raised awareness of differences in sensitivity of MBLs to new inhibitors, including pre-existing resistance to cefepime-tani borbactam. This finding is of concern as it demonstrates that NDM-9 has an advantage either in the presence or in the absence of selective pressure from cefepime-taniborbac tam.
Xeruborbactam is another cyclic boronate dual SBL/MBL BLI (6) (Fig. 1). Xeruborbac tam has completed Phase 1 studies (NCT04380207, NCT04578873) administered by the IV or oral route (as a prodrug form). In these studies, xeruborbactam was found to be safe and well tolerated at exposures that exceeded non-clinical PK-PD targets (7)(8)(9). The spectrum of beta-lactamase inhibition by xeruborbactam is the broadest among marketed BLIs and those in clinical development (10). Compared to taniborbactam, xeruborbactam has a broader MBL inhibition spectrum, which includes MBLs of the IMP-type that are not inhibited by taniborbactam (11,12). The objective of our study was to investigate whether NDM-9 is susceptible to inhibition by xeruborbactam.
Four clinical isolates of Klebsiella pneumoniae and one isolate of Escherichia coli producing NDM-9 (and other beta-lactamases, Table 1) were included in this study; NDM-1, NDM-4, NDM-5, NDM-6, and NDM-7-producing isolates were added for compari son. Nine out of 13 isolates used in this study were collected by IHMA (International Health Management Associates, Schaumburg, IL) or JMI (Jones Microbiology Institute, North Liberty, IA) as a part of various worldwide surveillance studies. KP1280 (0106), (KP1297 (0143), EC1100 (0128), and EC1104 (0151) were obtained from the CDC & FDA Antibiotic Resistance Isolate Bank. Isolates were tested for antimicrobial susceptibility using the broth microdilution methodology per Clinical and Laboratory Standards Institute M07 (2018) guidelines (13). The MICs of meropenem and cefepime in combination with xeruborbactam (fixed 4 µg/mL or 8 µg/mL) and taniborbactam (fixed 4 µg/mL) were determined using this panel. Xerubor bactam at both concentrations enhanced in vitro potency of meropenem and cefepime against all the NDM-producing strains including those producing NDM-9 (Table 1).
Meropenem MIC values were reduced by xeruborbactam (fixed 8 µg/mL) from 16 to >64 µg/mL to ≤0.06 µg/mL for 9 out of 13 tested isolates, including three of five NDM-9 producing strains. Meropenem-xeruborbactam (8 µg/mL) MIC values for the remaining four strains (two with NDM-9-and two with NDM-1) ranged from 1 to 8 µg/mL. These four strains carried various mutations affecting expression or functionality of the major porins OmpF/OmpK35 and/or OmpF/OmpK36 that have previously been demonstrated to restrict meropenem and xeruborbactam entry (14) and have been associated with the increase in meropenem-xeruborbactam MIC (15) ( Table 1). No such mutations were present in the strains with meropenem-xeruborbactam (8 µg/mL) MIC values of ≤0.06 µg/mL. Meropenem-xeruborbactam and cefepime-xeruborbactam MIC values were highly correlated: same strains that had low meropenem-xeruborbactam (8 µg/mL) MIC values also had low cefepime-xeruborbactam (8 µg/mL) values (≤0.06 µg/mL, reduced from 32 to >64 for the cefepime alone), including NDM-9-producing strains and vice versa. The only exceptions were the NDM-1 or NDM-5-producing strains of E. coli, EC100 and EC1104, respectively, with the low meropenem-xeruborbactam (8 µg/mL) and an increased cefepime-xeruborbactam (8 µg/mL) MIC values. These strains carried four amino acids, YRIN, insertion in PBP3 associated with a decreased susceptibility of cephalosporins and monobactams but not of meropenem (16,17). Of note, these strains were also resistant to cefepime-taniborbactam (MIC value of 32-64 µg/mL) consistent with recent reports (18,19). Based on microbiological results, we conclude that xerubor bactam inhibits all the tested NDM variants, including the NDM-9 variant, and that increased MIC values of xeruborbactam combinations are due to the presence of nonbeta-lactamase-mediated resistance mechanisms, including porin and/or PBP mutations. As expected, taniborbactam did not enhance activity of either meropenem or cefepime against any of the NDM-9-producing strains but demonstrated enhancement of potency of these antibiotics against isolates producing other NDM variants.
Inhibition of NDM-9 by xeruborbactam was also demonstrated in biochemical experiments. We determined IC 50 of xeruborbactam (and taniborbactam) inhibition of imipenem hydrolysis in cell lysates prepared from either NDM-1 (KP1297) or NDM-9 (KP1671) producing strains. Bacterial lysates were prepared as previously described (20); imipenem was used at a final concentration of 100 µL. Absorbance profiles at 295 nm (as a result of imipenem cleavage) were recorded every 10 s for 30 min using Tecan plate reader. Initial reaction rates were calculated in OD/min and used to generate dose response curves vs BLI concentration. IC 50 values of BLI effect on imipenem cleavage were calculated by fitting the resulting curves in "dose-response-inhibition, variable slope (four parameters)" equation using Prizm software. The IC 50 s of xeruborbactam inhibition of imipenem hydrolysis by NDM-1 (0.77 ± 0.12 µM) or NDM-9 (1.2 ± 0.1 µM) were essentially the same. Taniborbactam inhibited NDM-1-meditaed hydrolysis (IC 50 = 0.24 ± 0.052 µM) but not NDM-9-mediated hydrolysis (IC 50 >60 µM).
Available crystallography data (6, 12) provide a clear explanation for why the NDM-9 variant that differs from NDM-1 by a single E152K substitution is resistant to the inhibition by taniborbactam but remains susceptible to inhibition by xeruborbactam. In NDM-1, the negatively charged glutamic acid (E152) (corresponds to E149 in VIM-2) forms a salt bridge with the positively charged amine of the taniborbactam sidechain (Fig. 2). In NDM-9, E152 is substituted by a positively charged lysine, E152L. This E to K substitution turns the previously favorable interaction between taniborbactam and the enzyme at this position into repulsion, which explains resistance to taniborbactam inhibition. In contrast, the xeruborbactam molecule doesn't rely on this interaction and is predicted to not be affected by mutations in these positions.
In conclusion, we have shown that xeruborbactam retains high potency against NDM-9 as well as against other NDM variants (e.g., NDM-4 and NDM-6) that also evolved to withstand Zn (II) deprivation (1). This finding highlights the differences between boronic-acid-based dual spectrum BLIs and underscores the importance of further development of xeruborbactam. In NDM-9, the negatively charged glutamic acid (E152) is changed to a positively charged lysine (E152K). The E to K substitution observed in NDM-9 or the VIM mutant [generated by Le Terrier et al. (4)] turns the previously favorable interaction between taniborbactam and the enzyme at this position into repulsion, which explains resistance to taniborbactam inhibition. In contrast, xeruborbactam molecule doesn't rely on this interaction at E149 in VIM-2 (panel B) or E152 in NDM-1 (panel C) and is predicted to not be affected by mutations in these positions.