The emergence and spread of carbapenem-resistant Gram-negative pathogens is a global public health problem. The acquisition of metallo-β-lactamases (MBLs) such as NDM-1 is a principle contributor to the emergence of carbapenem-resistant Gram-negative pathogens that threatens the use of penicillin, cephalosporin, and carbapenem antibiotics to treat infections. So far a clinical inhibitor of MBLs that could reverse resistance and re-sensitize resistant Gram-negative pathogens to carbapenems does not exist. Here we have identified a fungal natural product, aspergillomarasmine A (AMA) that is a rapid and potent inhibitor of the NDM-1 enzyme and another clinically relevant MBL, VIM-2. AMA also fully restored the activity of meropenem against Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. possessing either VIM or NDM-type alleles. In mice infected with NDM-1-expressing Klebsiella pneumoniae, AMA efficiently restored meropenem activity, demonstrating that a combination of AMA and a carbapenem antibiotic has therapeutic potential to address the clinical challenge of MBL positive carbapenem-resistant Gram-negative pathogens.

The β-lactam antibiotics have long been a cornerstone for the treatment of bacterial disease. Recently, a readily transferable antibiotic resistance factor called the New Delhi metallo-β-lactamase-1 (NDM-1) has been found to confer enteric bacteria resistance to nearly all β-lactams, including the heralded carbapenems, posing a serious threat to human health. The crystal structure of NDM-1 bound to meropenem shows for the first time the molecular details of how carbapenem antibiotics are recognized by dizinc-containing metallo-β-lactamases. Additionally, product complex structures of hydrolyzed benzylpenicillin-, methicillin-, and oxacillin-bound NDM-1 have been solved to 1.8, 1.2, and 1.2 Å, respectively, and represent the highest-resolution structural data for any metallo-β-lactamase reported to date. Finally, we present the crystal structure of NDM-1 bound to the potent competitive inhibitor l-captopril, which reveals a unique binding mechanism. An analysis of the NDM-1 active site in these structures reveals key features important for the informed design of novel inhibitors of NDM-1 and other metallo-β-lactamases.

b-Lactams are the most commonly prescribed class of antibiotics and have had an enormous impact on human health. Thus, it is disquieting that an enzyme called New Delhi metallo-b-lactamase-1 (NDM-1) can confer Enterobacteriaceae with nearly complete resistance to all b-lactam antibiotics including the carbapenams. We have determined the crystal structure of Klebsiella pneumoniae apo-NDM-1 to 2.1-Å resolution. From the structure, we see that NDM-1 has an expansive active site with a unique electrostatic profile, which we propose leads to a broader substrate specificity. In addition, NDM-1 undergoes important conformational changes upon substrate binding. These changes have not been previously observed in metallo-b-lactamase enzymes and may have a direct influence on substrate recognition and catalysis.

Emerging β-lactamase-mediated resistance is threatening the clinical utility of the single most prominent class of antibacterial agents used in medicine, the β-lactams. The diazabicyclooctane avibactam is able to inhibit a wider range of serine β-lactamases than has been previously observed with β-lactamase inhibitors such as the widely prescribed clavulanic acid. However, despite its broad-spectrum activity, variable levels of inhibition have been observed for molecular class D β-lactamases. In order to better understand the molecular basis and spectrum of inhibition by avibactam, we provide structural and mechanistic analysis of the compound in complex with important class A and D serine β-lactamases. Herein, we reveal the 1.7- and 2.0-Å-resolution crystal structures of avibactam covalently bound to class D β-lactamases OXA-10 and OXA-48. Furthermore, a kinetic analysis of key active-site mutants for class A β-lactamase CTX-M-15 allows us to propose a validated mechanism for avibactam-mediated β-lactamase inhibition including a unique role for S130, which acts as a general base. This study provides molecular insights that will aid in the design and development of avibactam-based chemotherapeutic agents effective against emerging drug-resistant microorganisms.

O-GlcNAc hydrolase, OGA, removes O-linked N-acetylglucosamine (O-GlcNAc) from myriad nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments we determined the 3-D structure of human OGA, revealing an unusual helix exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors having clinical value.

Edited by F. Peter GuengerichIn Escherichia coli, the peptidoglycan cell wall is synthesized by bifunctional penicillin-binding proteins such as PBP1b that have both transpeptidase and transglycosylase activities. The PBP1b transpeptidase domain is a major target of ␤-lactams, and therefore it is important to attain a detailed understanding of its inhibition. The peptidoglycan glycosyltransferase domain of PBP1b is also considered an excellent antibiotic target yet is not exploited by any clinically approved antibacterials. Herein, we adapt a pyrophosphate sensor assay to monitor PBP1b-catalyzed glycosyltransfer and present an improved crystallographic model for inhibition of the PBP1b glycosyltransferase domain by the potent substrate analog moenomycin. We elucidate the structure of a previously disordered region in the glycosyltransferase active site and discuss its implications with regards to peptidoglycan polymerization. Furthermore, we solve the crystal structures of E. coli PBP1b bound to multiple different ␤-lactams in the transpeptidase active site and complement these data with gel-based competition assays to provide a detailed structural understanding of its inhibition. Taken together, these biochemical and structural data allow us to propose new insights into inhibition of both enzymatic domains in PBP1b.

Background: Coding region determinant-binding protein (CRD-BP) interacts physically with oncogenic mRNAs. Results: Point mutation in the K-homology (KH) domains of CRD-BP abolishes its RNA-binding ability. Conclusion: Two KH domains of CRD-BP are required for efficient binding to oncogenic mRNAs and for granule formation in zebrafish embryos. Significance: Learning how the KH domains interact with mRNAs is crucial for understanding oncogenic function of CRD-BP.

Avibactam is a diazabicyclooctane β-lactamase inhibitor possessing outstanding but incomplete efficacy against multidrug-resistant Gram-negative pathogens in combination with β-lactam antibiotics. Significant pharmaceutical investment in generating derivatives of avibactam warrants a thorough characterization of their activity. We show here through structural and kinetic analysis that select diazabicyclooctane derivatives display effective but varied inhibition of two clinically important β-lactamases (CTX-M-15 and OXA-48). Furthermore, these derivatives exhibit considerable antimicrobial activity (MIC ≤ 2 μg/mL) against clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Enterobacter spp. Imaging of cell phenotype along with structural and biochemical experiments unambiguously demonstrate that this activity, in E. coli, is a result of targeting penicillin-binding protein 2. Our results suggest that structure-activity relationship studies for the purpose of drug discovery must consider both β-lactamases and penicillin-binding proteins as targets. We believe that this approach will yield next-generation combination or monotherapies with an expanded spectrum of activity against currently untreatable Gram-negative pathogens.