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Carbapenem antibiotics are the last resort for control of severe infectious diseases, bloodstream infections, and pneumonia caused by Gram-negative bacteria, including Enterobacteriaceae. However, carbapenem-resistant Enterobacteriaceae (CRE) strains have spread globally and are a critical concern in clinical settings because CRE infections are recognized as a leading cause of increased mortality among hospitalized patients. Most CRE produce certain kinds of serine carbapenemases (e.g., KPC- and GES-type β-lactamases) or metallo-β-lactamases (MBLs), which can hydrolyze carbapenems. Although effective MBL inhibitors are expected to restore carbapenem efficacy against MBL-producing CRE, no MBL inhibitor is currently clinically available. Here, we synthesized 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), which is a potent inhibitor of MBLs. SPC is a remarkable lead compound for clinically useful MBL inhibitors and can potentially provide a considerable benefit to patients receiving treatment for lethal infectious diseases caused by MBL-producing CRE., Production of metallo-β-lactamases (MBLs), which hydrolyze carbapenems, is a cause of carbapenem resistance in Enterobacteriaceae. Development of effective inhibitors for MBLs is one approach to restore carbapenem efficacy in carbapenem-resistant Enterobacteriaceae (CRE). We report here that sulfamoyl heteroarylcarboxylic acids (SHCs) can competitively inhibit the globally spreading and clinically relevant MBLs (i.e., IMP-, NDM-, and VIM-type MBLs) at nanomolar to micromolar orders of magnitude. Addition of SHCs restored meropenem efficacy against 17/19 IMP-type and 7/14 NDM-type MBL-producing Enterobacteriaceae to satisfactory clinical levels. SHCs were also effective against IMP-type MBL-producing Acinetobacter spp. and engineered Escherichia coli strains overproducing individual minor MBLs (i.e., TMB-2, SPM-1, DIM-1, SIM-1, and KHM-1). However, SHCs were less effective against MBL-producing Pseudomonas aeruginosa. Combination therapy with meropenem and SHCs successfully cured mice infected with IMP-1-producing E. coli and dually NDM-1/VIM-1-producing Klebsiella pneumoniae clinical isolates. X-ray crystallographic analyses revealed the inhibition mode of SHCs against MBLs; the sulfamoyl group of SHCs coordinated to two zinc ions, and the carboxylate group coordinated to one zinc ion and bound to positively charged amino acids Lys224/Arg228 conserved in MBLs. Preclinical testing revealed that the SHCs showed low toxicity in cell lines and mice and high stability in human liver microsomes. Our results indicate that SHCs are promising lead compounds for inhibitors of MBLs to combat MBL-producing CRE.

Metallo-β-lactamases (MBLs) confer resistance to carbapenems, and their increasing global prevalence is a growing clinical concern. To elucidate the mechanisms by which these enzymes recognize and hydrolyze carbapenems, we solved 1.4 to 1.6 Å crystal structures of SMB-1 ( S erratia m etallo- β -lactamase 1), a subclass B3 MBL, bound to hydrolyzed carbapenems (doripenem, meropenem, and imipenem). In these structures, SMB-1 interacts mainly with the carbapenem core structure via elements in the active site, including a zinc ion (Zn-2), Q157[113] (where the position in the SMB-1 sequence is in brackets after the BBL number), S221[175], and T223[177]. There is less contact with the carbapenem R2 side chains, strongly indicating that SMB-1 primarily recognizes the carbapenem core structure. This is the first report describing how a subclass B3 MBL recognizes carbapenems. We also solved the crystal structure of SMB-1 in complex with the approved drugs captopril, an inhibitor of the angiotensin-converting enzyme, and 2-mercaptoethanesulfonate, a chemoprotectant. These drugs are inhibitors of SMB-1 with K i values of 8.9 and 184 μM, respectively. Like carbapenems, these inhibitors interact with Q157[113] and T223[177] and their thiol groups coordinate the zinc ions in the active site. Taken together, the data indicate that Q157[113], S221[175], T223[177], and the two zinc ions in the active site are key targets in the design of SMB-1 inhibitors with enhanced affinity. The structural data provide a solid foundation for the development of effective inhibitors that would overcome the carbapenem resistance of MBL-producing multidrug-resistant microbes.

A novel subclass B3 metallo-β-lactamase (MBL), SMB-1, recently identified from a Serratia marcescens clinical isolate, showed a higher hydrolytic activity against a wide range of β-lactams than did the other subclass B3 MBLs, i.e., BJP-1 and FEZ-1, from environmental bacteria. To identify the mechanism underlying the differences in substrate specificity among the subclass B3 MBLs, we determined the structure of SMB-1, using 1.6-Å diffraction data. Consequently, we found that SMB-1 reserves a space in the active site to accommodate β-lactam, even with a bulky R1 side chain, due to a loss of amino acid residues corresponding to F31 and L226 of BJP-1, which protrude into the active site to prevent β-lactam from binding. The protein also possesses a unique amino acid residue, Q157, which probably plays a role in recognition of β-lactams via the hydrogen bond interaction, which is missing in BJP-1 and FEZ-1, whose K m values for β-lactams are particularly high. In addition, we determined the mercaptoacetate (MCR)-complexed SMB-1 structure and revealed the mode of its inhibition by MCR: the thiolate group bridges to two zinc ions (Zn1 and Zn2). One of the carboxylate oxygen atoms of MCR makes contact with Zn2 and Ser221, and the other makes contact with T223 and a water molecule. Our results demonstrate the possibility that MCR could be a potent inhibitor for subclass B3 MBLs and that the screening technique using MCR as an inhibitor would be effective for detecting subclass B3 MBL producers.