Monoclonal Antibody Therapy against Acinetobacter baumannii.

Extremely drug-resistant (XDR) Acinetobacter baumannii is a notorious and frequently encountered pathogen demanding novel therapeutic interventions. An initial monoclonal antibody (MAb), C8, raised against A. baumannii capsule, proved a highly effective treatment against a minority of clinical isolates. To overcome this limitation, we broadened coverage by developing a second antibody for use in a combination regimen. We sought to develop an additional anti-A. baumannii MAb through hybridoma technology by immunizing mice with sublethal inocula of virulent, XDR clinical isolates not bound by MAb C8. We identified a new antibacterial MAb, 65, which bound to strains in a pattern distinct from and complementary to that of MAb C8. MAb 65 enhanced macrophage opsonophagocytosis of targeted strains and markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia murine models of A. baumannii infection. MAb 65 was also synergistic with colistin, substantially enhancing protection compared to monotherapy. Treatment with MAb 65 significantly reduced blood bacterial density, ameliorated cytokine production (interleukin-1ß [IL-1ß], IL-6, IL-10, and tumor necrosis factor), and sepsis biomarkers. We describe a novel MAb targeting A. baumannii that broadens immunotherapeutic strain coverage, is highly potent and effective, and synergistically improves outcomes in combination with antibiotics.

Role of Synonymous Mutations in the Evolution of TEM ß-Lactamase Genes.

Nonsynonymous mutations are well documented in TEM ß-lactamases. The resulting amino acid changes often alter the conferred phenotype from broad spectrum (2b) conferred by TEM-1 to extended spectrum (2be), inhibitor resistant (2br), or both extended spectrum and inhibitor resistant (2ber). The encoding blaTEM genes also deviate in numerous synonymous mutations, which are not well understood. blaTEM-3 (2be), blaTEM-33 (2br), and blaTEM-109 (2ber) were studied in comparison to blaTEM-1blaTEM-33 was chosen for more detailed studies because it deviates from blaTEM-1 by a single nonsynonymous mutation and three additional synonymous mutations. Genes encoding the enzymes with only nonsynonymous or all (including synonymous) mutations plus all permutations between blaTEM-1 and blaTEM-33 were expressed in Escherichia coli cells. In disc diffusion assays, genes encoding TEM-3, TEM-33, and TEM-109 with all synonymous mutations resulted in higher resistance levels than genes without synonymous mutations. Disc diffusion assays with the 16 genes carrying all possible nucleotide change combinations between blaTEM-1 and blaTEM-33 indicated different susceptibilities for different variants. Nucleotide BLAST searches did not identify genes without synonymous mutations but did identify some without nonsynonymous mutations. Energies of possible secondary mRNA structures calculated with mfold are generally higher with synonymous mutations, suggesting that their role could be to destabilize the mRNA and facilitate its unfolding for efficient translation. In summary, our data indicate that transition from blaTEM-1 to other variant genes by simply acquiring the nonsynonymous mutations is not favored. Instead, synonymous mutations seem to support the transition to other variant genes with nonsynonymous mutations leading to different phenotypes.

Virtual Screening and Experimental Testing of B1 Metallo-ß-lactamase Inhibitors.

The global rise of metallo-ß-lactamases (MBLs) is problematic due to their ability to inactivate most ß-lactam antibiotics. MBL inhibitors that could be coadministered with and restore the efficacy of ß-lactams are highly sought after. In this study, we employ virtual screening of candidate MBL inhibitors without thiols or carboxylates to avoid off-target effects using the Avalanche software package, followed by experimental validation of the selected compounds. As target enzymes, we chose the clinically relevant B1 MBLs NDM-1, IMP-1, and VIM-2. Among 32 compounds selected from an approximately 1.5 million compound library, 6 exhibited IC50 values less than 40 µM against NDM-1 and/or IMP-1. The most potent inhibitors of NDM-1, IMP-1, and VIM-2 had IC50 values of 19 ± 2, 14 ± 1, and 50 ± 20 µM, respectively. While chemically diverse, the most potent inhibitors all contain combinations of hydroxyl, ketone, ester, amide, or sulfonyl groups. Docking studies suggest that these electron-dense moieties are involved in Zn(II) coordination and interaction with protein residues. These novel scaffolds could serve as the basis for further development of MBL inhibitors. A procedure for renaming NDM-1 residues to conform to the class B ß-lactamase (BBL) numbering scheme is also included.

Azolylthioacetamides as a potent scaffold for the development of metallo-ß-lactamase inhibitors.

In an effort to develop new inhibitors of metallo-ß-lactamases (MßLs), twenty-eight azolylthioacetamides were synthesized and assayed against MßLs. The obtained benzimidazolyl and benzioxazolyl substituted 1-19 specifically inhibited the enzyme ImiS, and 10 was found to be the most potent inhibitor of ImiS with an IC50 value of 15 nM. The nitrobenzimidazolyl substituted 20-28 specifically inhibited NDM-1, with 27 being the most potent inhibitor with an IC50 value of 170 nM. Further studies with 10, 11, and 27 revealed a mixed inhibition mode with competitive and uncompetitive inhibition constants in a similar range as the IC50 values. These inhibitors resulted in a 2-4-fold decrease in imipenem MIC values using E. coli cells producing ImiS or NDM-1. While the source of uncompetitive (possibly allosteric) inhibition remains unclear, docking studies indicate that 10 and 11 may interact orthosterically with Zn2 in the active site of CphA, while 27 could bridge the two Zn(II) ions in the active site of NDM-1 via its nitro group.

Optimization of amino acid thioesters as inhibitors of metallo-ß-lactamase L1.

The emergence of antibiotic resistance caused by metallo-ß-lactamases (MßLs) is a global public health problem. Recently, we found amino acid thioesters to be a highly promising scaffold for inhibitors of the MßL L1. In order to optimize this series of inhibitors, nine new amino acid thioesters were developed by modifying the substituents on the N-terminus of the thioesters and the groups representing the amino acid side chain. Biological activity assays indicate that all of them are very potent inhibitors of L1 with an IC50 value range of 20-600nM, lower than those of most of the previously reported inhibitors of this scaffold. Analysis of structure-activity relationship reveals that big hydrophobic substituents on the N-terminus and a methionine amino acid side chain improves inhibitory activity of the thioesters. All these inhibitors are able to restore antibacterial activity of a ß-lactam antibiotic against Escherichia coli BL21(DE3) cells producing L1 to that against E. coli cells lacking a ß-lactamase. Docking studies reveal that a large N-terminal hydrophobic group results in a slightly different binding mode than smaller hydrophobic groups at the same position.

Functionalizing the γ-Position of α-Diazo-ß-ketoesters.

Although α-diazo-ß-ketoesters are synthetically versatile intermediates, methodology for introducing this functionality into complex molecules is still limited, most frequently involving a carboxylic acid precursor, which is then activated and transformed into a ß-ketoester, with the diazo group being subsequently added with a diazo transfer reagent. While introducing this highly functional moiety in a convergent one step process would be ideal, such an objective is limited by the relatively few studies which address functionalization of the α-diazo-ß-ketoester at the γ-position. In the present investigation, we evaluate strategies, both new and established, for functionalizing α-diazo-ß-ketoesters, particularly with regard to generating compounds prospectively useful in the synthesis of C1-substituted carbapenems. We report the first δ-aldehydo-α-diazo-ß-ketoester as well as a method for its oxidation to the corresponding methyl ester, and the formation of a new substituted pyrazole under basic conditions.

Meropenem and chromacef intermediates observed in IMP-25 metallo-ß-lactamase-catalyzed hydrolysis.

Metallo-ß-lactamases inactivate most ß-lactam antibacterials, and much attention has been paid to their catalytic mechanism. One issue of controversy has been whether ß-lactam hydrolysis generally proceeds through an anionic intermediate bound to the active-site Zn(II) ions or not. The formation of an intermediate has not been shown conclusively in imipenemase (IMP) enzymes to date. Here, we provide evidence that intermediates are formed during the hydrolysis of meropenem and chromacef catalyzed by the variant IMP-25 and, to a lesser degree, IMP-1.

Elucidating the Role of Residue 67 in IMP-Type Metallo-ß-Lactamase Evolution.

Antibiotic resistance in bacteria is ever changing and adapting, as once-novel ß-lactam antibiotics are losing their efficacy, primarily due to the production of ß-lactamases. Metallo-ß-lactamases (MBLs) efficiently inactivate a broad range of ß-lactam antibiotics, including carbapenems, and are often coexpressed with other antibacterial resistance factors. The rapid dissemination of MBLs and lack of novel antibacterials pose an imminent threat to global health. In an effort to better counter these resistance-conferring ß-lactamases, an investigation of their natural evolution and resulting substrate specificity was employed. In this study, we elucidated the effects of different amino acid substitutions at position 67 in IMP-type MBLs on the ability to hydrolyze and confer resistance to a range of ß-lactam antibiotics. Wild-type ß-lactamases IMP-1 and IMP-10 and mutants IMP-1-V67A and IMP-1-V67I were characterized biophysically and biochemically, and MICs for Escherichia coli cells expressing these enzymes were determined. We found that all variants exhibited catalytic efficiencies (kcat/Km) equal to or higher than that of IMP-1 against all tested ß-lactams except penicillins, against which IMP-1 and IMP-1-V67I showed the highest kcat/Km values. The substrate-specific effects of the different amino acid substitutions at position 67 are discussed in light of their side chain structures and possible interactions with the substrates. Docking calculations were employed to investigate interactions between different side chains and an inhibitor used as a ß-lactam surrogate. The differences in binding affinities determined experimentally and computationally seem to be governed by hydrophobic interactions between residue 67 and the inhibitor and, by inference, the ß-lactam substrates.

Triethysilyl Enol Ethers in the Synthesis of Carbapenem Precursors.

A diastereoselective process for the formation of intermediates suitable for the preparation of C1 substituted carbapenems was developed. The process is readily scalable and does not involve organometallics or strong bases such as LDA.

Molecular Mechanisms and the Significance of Synonymous Mutations.

Synonymous mutations result from the degeneracy of the genetic code. Most amino acids are encoded by two or more codons, and mutations that change a codon to another synonymous codon do not change the amino acid in the gene product. Historically, such mutations have been considered silent because they were assumed to have no to very little impact. However, research in the last few decades has produced several examples where synonymous mutations play important roles. These include optimizing expression by enhancing translation initiation and accelerating or decelerating translation elongation via codon usage and mRNA secondary structures, stabilizing mRNA molecules and preventing their breakdown before translation, and faulty protein folding or increased degradation due to enhanced ubiquitination and suboptimal secretion of proteins into the appropriate cell compartments. Some consequences of synonymous mutations, such as mRNA stability, can lead to different outcomes in prokaryotes and eukaryotes. Despite these examples, the significance of synonymous mutations in evolution and in causing disease in comparison to nonsynonymous mutations that do change amino acid residues in proteins remains controversial. Whether the molecular mechanisms described by which synonymous mutations affect organisms can be generalized remains poorly understood and warrants future research in this area.

Biochemical characterization of IMP-30, a metallo-ß-lactamase with enhanced activity toward ceftazidime.

IMP-type enzymes constitute a clinically important family of metallo-ß-lactamases that has grown dramatically in the past decade to its current 45 known members. Here, we report the biochemical characterization of IMP-30 in comparison to IMP-1, from which it deviates by a single E59K mutation. Kinetics, MIC assays, docking, and molecular dynamics simulations support a scenario in which Lys59 interacts with the ceftazidime R1 group, resulting in increased water access and enhanced turnover and MIC of ceftazidime.

New ß-phospholactam as a carbapenem transition state analog: Synthesis of a broad-spectrum inhibitor of metallo-ß-lactamases.

In an effort to test whether a transition state analog is an inhibitor of the metallo-ß-lactamases, a phospholactam analog of carbapenem has been synthesized and characterized. The phospholactam 1 proved to be a weak, time-dependent inhibitor of IMP-1 (70%), CcrA (70%), L1 (70%), NDM-1 (53%), and Bla2 (94%) at an inhibitor concentration of 100µM. The phospholactam 1 activated ImiS and BcII at the same concentration. Docking studies were used to explain binding and to offer suggestions for modifications to the phospholactam scaffold to improve binding affinities.

Exploring the conformational and reactive dynamics of biomolecules in solution using an extended version of the glycine reactive force field.

In order to describe possible reaction mechanisms involving amino acids, and the evolution of the protonation state of amino acid side chains in solution, a reactive force field (ReaxFF-based description) for peptide and protein simulations has been developed as an expansion of the previously reported glycine parameters. This expansion consists of adding to the training set more than five hundred molecular systems, including all the amino acids and some short peptide structures, which have been investigated by means of quantum mechanical calculations. The performance of this ReaxFF protein force field on a relatively short time scale (500 ps) is validated by comparison with classical non-reactive simulations and experimental data of well characterized test cases, comprising capped amino acids, peptides, and small proteins, and reaction mechanisms connected to the pharmaceutical sector. A good agreement of ReaxFF predicted conformations and kinetics with reference data is obtained.

Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a Qnr protein from a gram-negative bacterium.

Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for gram-negative Qnr proteins. The AhQnr crystal structure, the first for any gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A 'tower' and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in gram-negative bacteria.

Strategies to Name Metallo-ß-Lactamases and Number Their Amino Acid Residues.

Metallo-ß-lactamases (MBLs), also known as class B ß-lactamases (BBLs), are Zn(II)-containing enzymes able to inactivate a broad range of ß-lactams, the most commonly used antibiotics, including life-saving carbapenems. They have been known for about six decades, yet they have only gained much attention as a clinical problem for about three decades. The naming conventions of these enzymes have changed over time and followed various strategies, sometimes leading to confusion. We are summarizing the naming strategies of the currently known MBLs. These enzymes are quite diverse on the amino acid sequence level but structurally similar. Problems trying to describe conserved residues, such as Zn(II) ligands and other catalytically important residues, which have different numbers in different sequences, have led to the establishment of a standard numbering scheme for BBLs. While well intended, the standard numbering scheme is not trivial and has not been applied consistently. We revisit this standard numbering scheme and suggest some strategies for how its implementation could be made more accessible to researchers. Standard numbering facilitates the comparison of different enzymes as well as their interaction with novel antibiotics and BBL inhibitors.

A self-reported inhibitor of metallo-carbapenemases for reversing carbapenem resistance.

Metallo-carbapenemases-mediated carbapenem-resistant Enterobacterales (CREs) has been acknowledged as "urgent threat" by the World Health Organization. The discovery of new strategies that block metallo-carbapenemases activity to reverse carbapenem resistance is an urgent need. In this study, a coumarin copper complex containing a PEG linker and glucose ligand, GluC-Cu, was used to reverse carbapenem resistance. Interestingly, it could effectively inhibit metallo-carbapenemases (NDM-1, IMP-1 and ImiS) with an IC50 value in the range of 0.23-1.21 µM, and simultaneously release the green fluorescence signal (GluC), therefore exhibiting self-reported inhibition performance. The inhibition mechanism of oxidizing Zn(II) thiolate site of NDM-1 from Cu2+ to Cu+ was verified by fluorescence assay, HR-MS, and XPS. Moreover, GluC-Cu in combination with meropenem showed excellent synergistic antibacterial effect to effectively combat E. coli expressing metallo-carbapenemases in vitro and in a mice infection model. This bifunctional metallo-carbapenemases inhibitor provides a novel chemical tool to overcome carbapenem resistance.

A dual covalent binder for labelling and inhibiting serine and metallo-carbapenemases.

The continuous emergence of multi-drug resistant pathogens co-expressing serine and metallo-carbapenemases seriously threatens the efficacy of carbapenem. Here, we report the first SeCN-derived dual inhibitor of serine and metallo-carbapenemases with IC50 values ranging from 0.0038 to 1.27 µg mL-1. The inhibitor was shown to form covalent bonds with Cys221 of NDM-1 and Ser70 of KPC-2, respectively, achieving selective labelling and cross-class inhibition for carbapenemases. Our results provide a potential strategy to develop clinically useful dual inhibitors targeting serine and metallo-carbapenemases to combat superbugs.

The sequence-activity relationship between metallo-ß-lactamases IMP-1, IMP-6, and IMP-25 suggests an evolutionary adaptation to meropenem exposure.

Metallo-ß-lactamases are important determinants of antibacterial resistance. In this study, we investigate the sequence-activity relationship between the closely related enzymes IMP-1, IMP-6, and IMP-25. While IMP-1 is the more efficient enzyme across the overall spectrum of tested ß-lactam antibacterial agents, IMP-6 and IMP-25 seem to have evolved to specifically inactivate the newer carbapenem meropenem. Molecular modeling indicates that the G235S mutation distinguishing IMP-25 from IMP-1 and IMP-6 may affect enzyme activity via Asn233.

Systematic analysis of metallo-ß-lactamases using an automated database.

Metallo-ß-lactamases (MBLs) are enzymes that hydrolyze ß-lactam antibiotics, resulting in bacterial resistance to these drugs. These proteins have caused concerns due to their facile transference, broad substrate spectra, and the absence of clinically useful inhibitors. To facilitate the classification, nomenclature, and analysis of MBLs, an automated database system was developed, the Metallo-ß-Lactamase Engineering Database (MBLED) (http://www.mbled.uni-stuttgart.de). It contains information on MBLs retrieved from the NCBI peptide database while strictly following the nomenclature by Jacoby and Bush (http://www.lahey.org/Studies/) and the generally accepted class B ß-lactamase (BBL) standard numbering scheme for MBLs. The database comprises 597 MBL protein sequences and enables systematic analyses of these sequences. A systematic analysis employing the database resulted in the generation of mutation profiles of assigned IMP- and VIM-type MBLs, the identification of five MBL protein entries from the NCBI peptide database that were inconsistent with the Jacoby and Bush nomenclature, and the identification of 15 new IMP candidates and 9 new VIM candidates. Furthermore, the database was used to identify residues with high mutation frequencies and variability (mutation hot spots) that were unexpectedly distant from the active site located in the ßß sandwich: positions 208 and 266 in the IMP family and positions 215 and 258 in the VIM family. We expect that the MBLED will be a valuable tool for systematically cataloguing and analyzing the increasing number of MBLs being reported.

Polymorphisms in common antihypertensive targets: Pharmacogenomic implications for the treatment of cardiovascular disease.

The idea of personalized medicine came to fruition with sequencing the human genome; however, aside from a few cases, the genetic revolution has yet to materialize. Cardiovascular diseases are the leading cause of death globally, and hypertension is a common prelude to nearly all cardiovascular diseases. Thus, hypertension is an ideal candidate disease to apply tenants of personalized medicine to lessen cardiovascular disease. Herein is a survey that visually depicts the polymorphisms in the top eight antihypertensive targets. Although there are numerous genome-wide association studies regarding cardiovascular disease, few studies look at the effects of receptor polymorphisms on drug treatment. With 17,000+ polymorphisms in the combined target proteins examined, it is expected that some of the clinical variability in the treatment of hypertension is due to polymorphisms in the drug targets. Recent advances in techniques and technology, such as high throughput examination of single mutations, structure prediction, computational power for modeling, and CRISPR models of point mutations, allow for a relatively rapid and comprehensive examination of the effects of known and future polymorphisms on drug affinity and effects. As hypertension is easy to measure and has a plethora of clinically viable ligands, hypertension makes an excellent disease to study pharmacogenomics in the lab and the clinic. If the promises of personalized medicine are to materialize, a concerted effort to examine the effects polymorphisms have on drugs is required. A clinician with the knowledge of a patient's genotype can then prescribe drugs that are optimal for treating that specific patient.