Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

Fecal Carriage and Molecular Characterization of Carbapenemase-Producing Enterobacterales in the Pediatric Population in Qatar.

Whole-genome sequencing was used to characterize carbapenemase-producing Enterobacterales (CPE) strains recovered from rectal screening swab samples obtained from children at a tertiary-care pediatric hospital in Qatar during a 3-year period. A total of 72 CPE isolates recovered from 61 fecal carriers were characterized. Escherichia coli (47 isolates [65.3%]) and Klebsiella pneumoniae (22 isolates [30.6%]) were the most common species identified. High levels of genetic diversity were observed for both species. These 72 isolates produced 78 carbapenemases, characterized as either NDM-type (41 enzymes [52.6%]) or OXA-48-type (37 enzymes [47.4%]). NDM-5 (24 enzymes [30.8%]), NDM-1 (15 enzymes [19.2%]), and OXA-181 (15 enzymes [19.2%]) were the most common variants detected within each type. Twenty-three NDM producers exhibited difficult-to-treat resistance, compared with only 2 of the OXA-48 producers. Multiple comorbidities were identified in 88.5% of the patients, whereas recent travel history to countries in which CPE are endemic was documented for 57.4% of the patients. All 9 blaOXA-48-type-gene-containing E. coli sequence type 38 (ST38) strains were isolated from patients without international travel history. The mean quarterly incidence of fecal carriage decreased more than 6-fold after the implementation of coronavirus disease 2019 (COVID-19)-related international travel restrictions in Qatar in mid-March 2020. Our data suggest that NDM-type and OXA-48-type carbapenemases expressed by a large diversity of E. coli and K. pneumoniae genotypes are largely dominant in the pediatric population of Qatar. Although our data indicate successful local expansion of E. coli ST38 strains harboring blaOXA-244 genes, at least within health care settings, blaOXA-48-type and blaNDM-type genes appear to have been mainly introduced sporadically by asymptomatic carriers who visited or received health care in some nearby countries in which the genes are endemic. IMPORTANCE To the best of our knowledge, this is the first study addressing the molecular characteristics of CPE in a pediatric population in Qatar using whole-genome sequencing. Since several countries in the Arabian Peninsula share relatively similar demographic patterns and international links, it is plausible that the molecular characteristics of CPE in children, at least in the middle and eastern parts of the region, are similar to those observed in our study.

Novel N-Substituted 3-Aryl-4-(diethoxyphosphoryl)azetidin-2-ones as Antibiotic Enhancers and Antiviral Agents in Search for a Successful Treatment of Complex Infections.

A novel series of N-substituted cis- and trans-3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones were synthesized by the Kinugasa reaction of N-methyl- or N-benzyl-(diethyoxyphosphoryl)nitrone and selected aryl alkynes. Stereochemistry of diastereoisomeric adducts was established based on vicinal H3-H4 coupling constants in azetidin-2-one ring. All the obtained azetidin-2-ones were evaluated for the antiviral activity against a broad range of DNA and RNA viruses. Azetidin-2-one trans-11f showed moderate inhibitory activity against human coronavirus (229E) with EC50 = 45 µM. The other isomer cis-11f was active against influenza A virus H1N1 subtype (EC50 = 12 µM by visual CPE score; EC50 = 8.3 µM by TMS score; MCC > 100 µM, CC50 = 39.9 µM). Several azetidin-2-ones 10 and 11 were tested for their cytostatic activity toward nine cancerous cell lines and several of them appeared slightly active for Capan-1, Hap1 and HCT-116 cells values of IC50 in the range 14.5-97.9 µM. Compound trans-11f was identified as adjuvant of oxacillin with significant ability to enhance the efficacy of this antibiotic toward the highly resistant S. aureus strain HEMSA 5. Docking and molecular dynamics simulations showed that enantiomer (3R,4S)-11f can be responsible for the promising activity due to the potency in displacing oxacillin at ß-lactamase, thus protecting the antibiotic from undesirable biotransformation.

Molecular basis of the beta-lactamase protein using comparative modelling, drug screening and molecular dynamics studies to understand the resistance of ß-lactam antibiotics.

Beta-lactamase (ampC) in general causes the onset of antibiotic resistance in pathogenic bacteria against the ß-lactam antibiotics. Morganella morganii which belongs to the Proteae tribe of the Enterobacteriaceae family is a Gram-negative bacillus. Gram-negative bacteria are the key problematic agents among the human population in overexpressing resistance against ß-lactam antibiotics. These ß-lactam antibiotics being experimentally well studied still lack the key information and mechanism for their resistance. The structural information of the ampC protein is unknown and poorly studied; hence, it is the need of the hour to find effective inhibitors against it. In our study, the prediction of the three-dimensional structure of ampC protein from Morganella morganii was performed using a comparative modelling approach. The predicted structure was energetically stabilized and functional conformations were mapped through 100-ns molecular dynamics simulation runs. Also, Ramachandran plot shows the model to be stereo-chemically stable with most residues found under core allowed regions. Drug screening with several experimentally tested inhibitors was then confirmed to check the activity against ampC protein using an AutoDock tool. The results suggested OncoglabrinolC molecule as the best inhibitor (out of 21 drug molecules) with a binding affinity of - 11.44 kcal/mol. Anti-bacterial/anti-parasitic inhibitors have not only been used against bacterial infections, but later reports have also shown them to work against deadly viruses such as SARS-CoV2. This key structural and inhibitory information is certain to help in the discovery of specific and potent substitute therapeutic drugs and the development of experimental procedures against human infection.