The clinical effectiveness of the important β-lactam class of antibiotics is under threat by the emergence of resistance, mostly due to the production of acquired serine- (SBL) and metallo-β-lactamase (MBL) enzymes. To address this resistance issue, multiple β-lactam/β-lactamase inhibitor combinations have been successfully introduced into the clinic over the past several decades. However, all of those combinations contain SBL inhibitors and, as yet, there are no MBL inhibitors in clinical use. Consequently, there exists an unaddressed yet growing healthcare problem due to the rise in recent years of highly resistant strains which produce New Delhi metallo (NDM)-type metallo-carbapenemases. Previously, we reported the characterization of an advanced MBL inhibitor lead compound, ANT431. Herein, we discuss the completion of a lead optimization campaign culminating in the discovery of the preclinical candidate ANT2681, a potent NDM inhibitor with strong potential for clinical development.

Bacterial resistance has become a worldwide concern after the emergence of metallo-β-lactamases (MBLs). They represent one of the major mechanisms of bacterial resistance against beta-lactam antibiotics. Among MBLs, New Delhi metallo-β-lactamase-1 NDM-1, the most prevalent type, is extremely efficient in inactivating nearly all-available antibiotics including last resort carbapenems. No inhibitors for NDM-1 are currently available in therapy, making the spread of NDM-1 producing bacterial strains a serious menace. With this perspective, we performed a structure-based screening of a commercially available library using FLAPdock and identified several, non-β-lactam derivatives as promising candidates active against NDM-1. The binding affinities of the highest scoring hits were measured revealing, for some of them, low micromolar affinity toward NDM-1. For the best inhibitors, efficacy against resistant bacterial strains overexpressing NDM-1 was validated, confirming their favorable synergistic effect in combination with the carbapenem Meropenem.

The diazabicyclooctanes (DBOs) are a class of serine β-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of β-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of β-lactam antibiotics against carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO “critical priority pathogen” producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure–activity relationship, leading to the discovery of a novel DBO, ANT3310, which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.

To fight the increasingly worrying bacterial resistance to antibiotics, the discovery and development of new therapeutics is urgently needed. Here, we report on a new series of 1,2,4-triazole-3-thione compounds as inhibitors of metallo-β-lactamases (MBLs), which represent major resistance determinants to β-lactams, and especially carbapenems, in Gram-negative bacteria. These molecules are stable analogs of 4-amino-1,2,4-triazole-derived Schiff bases, where the hydrazone-like bond has been reduced (hydrazine series) or the 4-amino group has been acylated (hydrazide series); the synthesis and physicochemical properties thereof are described. The inhibitory potency was determined on the most clinically relevant acquired MBLs (IMP-, VIM-, and NDM-types subclass B1 MBLs). When compared with the previously reported hydrazone series, hydrazine but not hydrazide analogs showed similarly potent inhibitory activity on VIM-type enzymes, especially VIM-2 and VIM-4, with Ki values in the micromolar to submicromolar range. One of these showed broad-spectrum inhibition as it also significantly inhibited VIM-1 and NDM-1. Restoration of β-lactam activity in microbiological assays was observed for one selected compound. Finally, the binding to the VIM-2 active site was evaluated by isothermal titration calorimetry and a modeling study explored the effect of the linker structure on the mode of binding with this MBL.

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1 I35T and NDM-1 I35S enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. The k cat , K m , and k cat /K m values calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, the k cat /K m of NDM-1 I35S for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1 I35T and NDM-1 I35S enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of ␣-helix content in the mutants. The NDM-1 metallo-␤-lactamase (MBL) was first described in a urinary Klebsiella pneumoniae isolate recovered from a Swedish patient who traveled to New Delhi and who had received medical care in India (1). This is the most recent MBL to have widely spread around the world among enterobacterial strains, Pseudomonas aeruginosa (2), Acinetobacter baumannii (3), Morganella morganii (4), Alcaligenes faecalis, Vibrio cholerae, and Stenotrophomonas maltophilia (5, 6). NDM-1-producing bacteria have been recovered from many infection sites as hospital-acquired and community-acquired infections but also in environmental samples (7). Since its finding, 12 NDM variants have been identified (http://www.lahey.org/Studies/). The factor that has influenced the wide geographic spread of bla NDM-1 gene is its localization on complex plasmids that mediate the transfer of this resistant determinant under the selective pressure of antibiotic therapy (8, 9). Metallo-␤-lactamases show a broad-spectrum substrate profile; they are resistant to classical ␤-lactamase inhibitors and hydrolyze carbapenems very efficiently. MBLs require one or two zinc ions to catalyze the hydrolysis of ␤-lactams. It is commonly suggested that the zinc ion acts as a Lewis acid to stabilize the transient tetrahedral intermediate formed by the nucleophilic attachment of a hydroxide ion to the carboxyl group of the ␤-lactam ring (10). NDM-1 enzyme shows a great ability to hydrolyze all ␤-lactam antibiotics (11). Several crystal structures of NDM-1 have been solved, and the enzyme displays the typical ␣␤/␤␣ fold of MBLs (12,13,14). NDM-1 belongs to subclass B1, and it contains a binuclear Zn active site surrounded by several loops responsible for substrate binding and specificity (13). Several studies pointed out the attention focused on the L3 loop, which could be involved in the hydrophobic contacts with substrates through the presence of an aromatic residue. Moreover, its great flexibility makes the loop able to drive substrate into the active site. The role of the L3 loop has been studied in such subclass B1 MBLs as IMP-1 and VIM-2 (15), but no experimental data are available for the NDM-1 enzyme. In comparison to the IMP and VIM variants, NDM-1 has a longer N terminus which forms two extra strands that pack on the L3 loop through an isoleucin...