Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics.

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6')-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

Signal Propagation in the ATPase Domain of Mycobacterium tuberculosis DNA Gyrase from Dynamical-Nonequilibrium Molecular Dynamics Simulations.

DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.

Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Ligand-Based Virtual Screening for Discovery of Indole Derivatives as Potent DNA Gyrase ATPase Inhibitors Active against Mycobacterium tuberculosis and Hit Validation by Biological Assays.

Mycobacterium tuberculosis is the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosis DNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosis GyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosis DNA gyrase ATPase inhibitors. IC50 values of compounds 8, 11, and 14 (0.26, 0.56, and 0.08 µM, respectively) for inhibition of M. tuberculosis DNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosis H37Ra are 25.0, 3.1, and 6.2 µg/mL, respectively, superior to novobiocin (MIC > 100.0 µg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14 to M. tuberculosis GyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosis DNA gyrase ATPase activity in vitro and growth of M. tuberculosis bacteria.

The Ableist stare: an interdisciplinary, narrative-driven exploration of staring at disabled bodies.

In this paper, I explore a phenomenon those with visible disabilities are all too familiar with: being stared at for their disabled bodies. Drawing on the interrelated fields of psychology, narrative, autoethnography and philosophy, I argue that staring at disabled bodies morally harms disabled people. This moral harm arises from the fact that not only does staring at disabled people fundamentally treat them as means to ends in which they cannot share, and thus, violates the Kantian formula of humanity, but also because this staring results in further, consequential harms for disabled people as well. In elaborating on these consequential harms, I draw largely on the works of disability ethicists Rosemarie Garland-Thomson and Elizabeth Barnes and argue that staring at disabled people contributes to the hermeneutical injustice disabled people face in their largely ableist world. Having identified these harms, I then explore the ameliorative potential of elevating disability narrative (with various disability narratives largely leading the discussion, including my own), drawing on Hilden Lindemann's Damaged Identities, Narrative Repair, and hope to call attention to the ways in which our broader structurally ableist world contributes to disabled people being stared at for their bodies in such harmful fashion.

Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 ß-Lactamase.

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-ß-lactamase (SBL) responsible for extensive ß-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate ß-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent ß-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25-1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different ß-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

Discovering Quantum Phase Transitions with Fermionic Neural Networks.

Deep neural networks have been very successful as highly accurate wave function Ansätze for variational Monte Carlo calculations of molecular ground states. We present an extension of one such Ansatz, FermiNet, to calculations of the ground states of periodic Hamiltonians, and study the homogeneous electron gas. FermiNet calculations of the ground-state energies of small electron gas systems are in excellent agreement with previous initiator full configuration interaction quantum Monte Carlo and diffusion Monte Carlo calculations. We investigate the spin-polarized homogeneous electron gas and demonstrate that the same neural network architecture is capable of accurately representing both the delocalized Fermi liquid state and the localized Wigner crystal state. The network converges on the translationally invariant ground state at high density and spontaneously breaks the symmetry to produce the crystalline ground state at low density, despite being given no a priori knowledge that a phase transition exists.

Bioisosteric Design Identifies Inhibitors of Mycobacterium tuberculosis DNA Gyrase ATPase Activity.

Mutations in DNA gyrase confer resistance to fluoroquinolones, second-line antibiotics for Mycobacterium tuberculosis infections. Identification of new agents that inhibit M. tuberculosis DNA gyrase ATPase activity is one strategy to overcome this. Here, bioisosteric designs using known inhibitors as templates were employed to define novel inhibitors of M. tuberculosis DNA gyrase ATPase activity. This yielded the modified compound R3-13 with improved drug-likeness compared to the template inhibitor that acted as a promising ATPase inhibitor against M. tuberculosis DNA gyrase. Utilization of compound R3-13 as a virtual screening template, supported by subsequent biological assays, identified seven further M. tuberculosis DNA gyrase ATPase inhibitors with IC50 values in the range of 0.42-3.59 µM. The most active compound 1 showed an IC50 value of 0.42 µM, 3-fold better than the comparator ATPase inhibitor novobiocin (1.27 µM). Compound 1 showed noncytotoxicity to Caco-2 cells at concentrations up to 76-fold higher than its IC50 value. Molecular dynamics simulations followed by decomposition energy calculations identified that compound 1 occupies the binding pocket utilized by the adenosine group of the ATP analogue AMPPNP in the M. tuberculosis DNA gyrase GyrB subunit. The most prominent contribution to the binding of compound 1 to M. tuberculosis GyrB subunit is made by residue Asp79, which forms two hydrogen bonds with the OH group of this compound and also participates in the binding of AMPPNP. Compound 1 represents a potential new scaffold for further exploration and optimization as a M. tuberculosis DNA gyrase ATPase inhibitor and candidate anti-tuberculosis agent.

Enhancement by pyrazolones of colistin efficacy against mcr-1-expressing E. coli: an in silico and in vitro investigation.

Owing to the emergence of antibiotic resistance, the polymyxin colistin has been recently revived to treat acute, multidrug-resistant Gram-negative bacterial infections. Positively charged colistin binds to negatively charged lipids and damages the outer membrane of Gram-negative bacteria. However, the MCR-1 protein, encoded by the mobile colistin resistance (mcr) gene, is involved in bacterial colistin resistance by catalysing phosphoethanolamine (PEA) transfer onto lipid A, neutralising its negative charge, and thereby reducing its interaction with colistin. Our preliminary results showed that treatment with a reference pyrazolone compound significantly reduced colistin minimal inhibitory concentrations in Escherichia coli expressing mcr-1 mediated colistin resistance (Hanpaibool et al. in ACS Omega, 2023). A docking-MD combination was used in an ensemble-based docking approach to identify further pyrazolone compounds as candidate MCR-1 inhibitors. Docking simulations revealed that 13/28 of the pyrazolone compounds tested are predicted to have lower binding free energies than the reference compound. Four of these were chosen for in vitro testing, with the results demonstrating that all the compounds tested could lower colistin MICs in an E. coli strain carrying the mcr-1 gene. Docking of pyrazolones into the MCR-1 active site reveals residues that are implicated in ligand-protein interactions, particularly E246, T285, H395, H466, and H478, which are located in the MCR-1 active site and which participate in interactions with MCR-1 in ≥ 8/10 of the lowest energy complexes. This study establishes pyrazolone-induced colistin susceptibility in E. coli carrying the mcr-1 gene, providing a method for the development of novel treatments against colistin-resistant bacteria.

Postoperative Delirium Severity and Recovery Correlate With Electroencephalogram Spectral Features.

BACKGROUND: Delirium is an acute syndrome characterized by inattention, disorganized thinking, and an altered level of consciousness. A reliable biomarker for tracking delirium does not exist, but oscillations in the electroencephalogram (EEG) could address this need. We evaluated whether the frequencies of EEG oscillations are associated with delirium onset, severity, and recovery in the postoperative period. METHODS: Twenty-six adults enrolled in the Electroencephalography Guidance of Anesthesia to Alleviate Geriatric Syndromes (ENGAGES; ClinicalTrials.gov NCT02241655) study underwent major surgery requiring general anesthesia, and provided longitudinal postoperative EEG recordings for this prespecified substudy. The presence and severity of delirium were evaluated with the confusion assessment method (CAM) or the CAM-intensive care unit. EEG data obtained during awake eyes-open and eyes-closed states yielded relative power in the delta (1-4 Hz), theta (4-8 Hz), and alpha (8-13 Hz) bands. Discriminability for delirium presence was evaluated with c-statistics. To account for correlation among repeated measures within patients, mixed-effects models were generated to assess relationships between: (1) delirium severity and EEG relative power (ordinal), and (2) EEG relative power and time (linear). Slopes of ordinal and linear mixed-effects models are reported as the change in delirium severity score/change in EEG relative power, and the change in EEG relative power/time (days), respectively. Bonferroni correction was applied to confidence intervals (CIs) to account for multiple comparisons. RESULTS: Occipital alpha relative power during eyes-closed states offered moderate discriminability (c-statistic, 0.75; 98% CI, 0.58-0.87), varying inversely with delirium severity (slope, -0.67; 98% CI, -1.36 to -0.01; P = .01) and with severity of inattention (slope, -1.44; 98% CI, -2.30 to -0.58; P = .002). Occipital theta relative power during eyes-open states correlated directly with severity of delirium (slope, 1.28; 98% CI, 0.12-2.44; P = .007), inattention (slope, 2.00; 98% CI, 0.48-3.54; P = .01), and disorganized thinking (slope, 3.15; 98% CI, 0.66-5.65; P = .01). Corresponding frontal EEG measures recapitulated these relationships to varying degrees. Severity of altered level of consciousness correlated with frontal theta relative power during eyes-open states (slope, 11.52; 98% CI, 6.33-16.71; P < .001). Frontal theta relative power during eyes-open states correlated inversely with time (slope, -0.05; 98% CI, -0.12 to -0.04; P = .002). CONCLUSIONS: Presence, severity, and core features of postoperative delirium covary with spectral features of the EEG. The cost and accessibility of EEG facilitate the translation of these findings to future mechanistic and interventional trials.

Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance.

Class A serine ß-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize ß-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent ß-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), for example, cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show that KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than altered KM values. High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω, 240, and 270 loops, which border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω loop, adopts two distinct conformations in KPC-2, either pointing "in" or "out" of the active site; with only the "in" form compatible with deacylation. The "out" conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate.

A multiscale approach to predict the binding mode of metallo beta-lactamase inhibitors.

Antibiotic resistance is a major threat to global public health. ß-lactamases, which catalyze breakdown of ß-lactam antibiotics, are a principal cause. Metallo ß-lactamases (MBLs) represent a particular challenge because they hydrolyze almost all ß-lactams and to date no MBL inhibitor has been approved for clinical use. Molecular simulations can aid drug discovery, for example, predicting inhibitor complexes, but empirical molecular mechanics (MM) methods often perform poorly for metalloproteins. Here we present a multiscale approach to model thiol inhibitor binding to IMP-1, a clinically important MBL containing two catalytic zinc ions, and predict the binding mode of a 2-mercaptomethyl thiazolidine (MMTZ) inhibitor. Inhibitors were first docked into the IMP-1 active site, testing different docking programs and scoring functions on multiple crystal structures. Complexes were then subjected to molecular dynamics (MD) simulations and subsequently refined through QM/MM optimization with a density functional theory (DFT) method, B3LYP/6-31G(d), increasing the accuracy of the method with successive steps. This workflow was tested on two IMP-1:MMTZ complexes, for which it reproduced crystallographically observed binding, and applied to predict the binding mode of a third MMTZ inhibitor for which a complex structure was crystallographically intractable. We also tested a 12-6-4 nonbonded interaction model in MD simulations and optimization with a SCC-DFTB QM/MM approach. The results show the limitations of empirical models for treating these systems and indicate the need for higher level calculations, for example, DFT/MM, for reliable structural predictions. This study demonstrates a reliable computational pipeline that can be applied to inhibitor design for MBLs and other zinc-metalloenzyme systems.

Inhibition of Mycobacterium tuberculosis InhA by 3-nitropropanoic acid.

3-Nitropropanoic acid (3NP), a bioactive fungal natural product, was previously demonstrated to inhibit growth of Mycobacterium tuberculosis. Here we demonstrate that 3NP inhibits the 2-trans-enoyl-acyl carrier protein reductase (InhA) from Mycobacterium tuberculosis with an IC50 value of 71 µM, and present the crystal structure of the ternary InhA-NAD+ -3NP complex. The complex contains the InhA substrate-binding loop in an ordered, open conformation with Tyr158, a catalytically important residue whose orientation defines different InhA substrate/inhibitor complex conformations, in the "out" position. 3NP occupies a hydrophobic binding site adjacent to the NAD+ cofactor and close to that utilized by the diphenyl ether triclosan, but binds predominantly via electrostatic and water-mediated hydrogen-bonding interactions with the protein backbone and NAD+ cofactor. The identified mode of 3NP binding provides opportunities to improve inhibitory activity toward InhA.

Identification of Potent DNA Gyrase Inhibitors Active against Mycobacterium tuberculosis.

Mycobacterium tuberculosis DNA gyrase manipulates the DNA topology using controlled breakage and religation of DNA driven by ATP hydrolysis. DNA gyrase has been validated as the enzyme target of fluoroquinolones (FQs), second-line antibiotics used for the treatment of multidrug-resistant tuberculosis. Mutations around the DNA gyrase DNA-binding site result in the emergence of FQ resistance in M. tuberculosis; inhibition of DNA gyrase ATPase activity is one strategy to overcome this. Here, virtual screening, subsequently validated by biological assays, was applied to select candidate inhibitors of the M. tuberculosis DNA gyrase ATPase activity from the Specs compound library (www.specs.net). Thirty compounds were identified and selected as hits for in vitro biological assays, of which two compounds, G24 and G26, inhibited the growth of M. tuberculosis H37Rv with a minimal inhibitory concentration of 12.5 µg/mL. The two compounds inhibited DNA gyrase ATPase activity with IC50 values of 2.69 and 2.46 µM, respectively, suggesting this to be the likely basis of their antitubercular activity. Models of complexes of compounds G24 and G26 bound to the M. tuberculosis DNA gyrase ATP-binding site, generated by molecular dynamics simulations followed by pharmacophore mapping analysis, showed hydrophobic interactions of inhibitor hydrophobic headgroups and electrostatic and hydrogen bond interactions of the polar tails, which are likely to be important for their inhibition. Decreasing compound lipophilicity by increasing the polarity of these tails then presents a likely route to improving the solubility and activity. Thus, compounds G24 and G26 provide attractive starting templates for the optimization of antitubercular agents that act by targeting DNA gyrase.

Virtual Screening Identifies Novel and Potent Inhibitors of Mycobacterium tuberculosis PknB with Antibacterial Activity.

Mycobacterium tuberculosis protein kinase B (PknB) is essential to mycobacterial growth and has received considerable attention as an attractive target for novel anti-tuberculosis drug development. Here, virtual screening, validated by biological assays, was applied to select candidate inhibitors of M. tuberculosis PknB from the Specs compound library (www.specs.net). Fifteen compounds were identified as hits and selected for in vitro biological assays, of which three indoles (2, AE-848/42799159; 4, AH-262/34335013; 10, AP-124/40904362) inhibited growth of M. tuberculosis H37Rv with minimal inhibitory concentrations of 6.2, 12.5, and 6.2 µg/mL, respectively. Two compounds, 2 and 10, inhibited M. tuberculosis PknB activity in vitro, with IC50 values of 14.4 and 12.1 µM, respectively, suggesting this to be the likely basis of their anti-tubercular activity. In contrast, compound 4 displayed anti-tuberculosis activity against M. tuberculosis H37Rv but showed no inhibition of PknB activity (IC50 > 128 µM). We hypothesize that hydrolysis of its ethyl ester to a carboxylate moiety generates an active species that inhibits other M. tuberculosis enzymes. Molecular dynamics simulations of modeled complexes of compounds 2, 4, and 10 bound to M. tuberculosis PknB indicated that compound 4 has a lower affinity for M. tuberculosis PknB than compounds 2 and 10, as evidenced by higher calculated binding free energies, consistent with experiment. Compounds 2 and 10 therefore represent candidate inhibitors of M. tuberculosis PknB that provide attractive starting templates for optimization as anti-tubercular agents.

Cladobotric Acids: Metabolites from Cultures of Cladobotryum sp., Semisynthetic Analogues and Antibacterial Activity.

Three new polyketide-derived natural products, cladobotric acids G-I (1-3), and six known metabolites (4, 5, 8-11) were isolated from fermentation of the fungus Cladobotryum sp. grown on rice. Their structures were elucidated by extensive spectroscopic methods. Two metabolites, cladobotric acid A (4) and pyrenulic acid A (10), were converted to a series of new products (12-20) by semisynthesis. The antibacterial activities of all these compounds were investigated against the Gram-positive pathogen Staphylococcus aureus including methicillin-susceptible (MSSA), methicillin-resistant and vancomycin-intermediate (MRSA/VISA), and heterogeneous vancomycin-intermediate (hVISA) strains. Results of these antibacterial assays revealed structural features of the unsaturated decalins important for biological activity.

Tumour treating fields therapy for glioblastoma: current advances and future directions.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults and continues to portend poor survival, despite multimodal treatment using surgery and chemoradiotherapy. The addition of tumour-treating fields (TTFields)-an approach in which alternating electrical fields exert biophysical force on charged and polarisable molecules known as dipoles-to standard therapy, has been shown to extend survival for patients with newly diagnosed GBM, recurrent GBM and mesothelioma, leading to the clinical approval of this approach by the FDA. TTFields represent a non-invasive anticancer modality consisting of low-intensity (1-3 V/cm), intermediate-frequency (100-300 kHz), alternating electric fields delivered via cutaneous transducer arrays configured to provide optimal tumour-site coverage. Although TTFields were initially demonstrated to inhibit cancer cell proliferation by interfering with mitotic apparatus, it is becoming increasingly clear that TTFields show a broad mechanism of action by disrupting a multitude of biological processes, including DNA repair, cell permeability and immunological responses, to elicit therapeutic effects. This review describes advances in our current understanding of the mechanisms by which TTFields mediate anticancer effects. Additionally, we summarise the landscape of TTFields clinical trials across various cancers and consider how emerging preclinical data might inform future clinical applications for TTFields.

Antimicrobial Resistance Conferred by OXA-48 ß-Lactamases: Towards a Detailed Mechanistic Understanding.

OXA-48-type ß-lactamases are now routinely encountered in bacterial infections caused by carbapenem-resistant Enterobacterales These enzymes are of high and growing clinical significance due to the importance of carbapenems in treatment of health care-associated infections by Gram-negative bacteria, the wide and increasing dissemination of OXA-48 enzymes on plasmids, and the challenges posed by their detection. OXA-48 confers resistance to penicillin (which is efficiently hydrolyzed) and carbapenem antibiotics (which is more slowly broken down). In addition to the parent enzyme, a growing array of variants of OXA-48 is now emerging. The spectrum of activity of these variants varies, with some hydrolyzing expanded-spectrum oxyimino-cephalosporins. The growth in importance and diversity of the OXA-48 group has motivated increasing numbers of studies that aim to elucidate the relationship between structure and specificity and establish the mechanistic basis for ß-lactam turnover in this enzyme family. In this review, we collate recently published structural, kinetic, and mechanistic information on the interactions between clinically relevant ß-lactam antibiotics and inhibitors and OXA-48 ß-lactamases. Collectively, these studies are starting to form a detailed picture of the underlying bases for the differences in ß-lactam specificity between OXA-48 variants and the consequent differences in resistance phenotype. We focus specifically on aspects of carbapenemase and cephalosporinase activities of OXA-48 ß-lactamases and discuss ß-lactamase inhibitor development in this context. Throughout the review, we also outline key open research questions for future investigation.

Catalytic mechanism of the colistin resistance protein MCR-1.

The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyses phosphoethanolamine transfer onto bacterial lipid A, making bacteria resistant to colistin, a last-resort antibiotic. Mechanistic understanding of this process remains incomplete. Here, we investigate possible catalytic pathways using DFT and ab initio calculations on cluster models and identify a complete two-step reaction mechanism. The first step, formation of a covalent phosphointermediate via transfer of phosphoethanolamine from a membrane phospholipid donor to the acceptor Thr285, is rate-limiting and proceeds with a single Zn2+ ion. The second step, transfer of the phosphoethanolamine group to lipid A, requires an additional Zn2+. The calculations suggest the involvement of the Zn2+ orbitals directly in the reaction is limited, with the second Zn2+ acting to bind incoming lipid A and direct phosphoethanolamine addition. The new level of mechanistic detail obtained here, which distinguishes these enzymes from other phosphotransferases, will aid in the development of inhibitors specific to MCR-1 and related bacterial phosphoethanolamine transferases.

Crystallography and QM/MM Simulations Identify Preferential Binding of Hydrolyzed Carbapenem and Penem Antibiotics to the L1 Metallo-ß-Lactamase in the Imine Form.

Widespread bacterial resistance to carbapenem antibiotics is an increasing global health concern. Resistance has emerged due to carbapenem-hydrolyzing enzymes, including metallo-ß-lactamases (MßLs), but despite their prevalence and clinical importance, MßL mechanisms are still not fully understood. Carbapenem hydrolysis by MßLs can yield alternative product tautomers with the potential to access different binding modes. Here, we show that a combined approach employing crystallography and quantum mechanics/molecular mechanics (QM/MM) simulations allow tautomer assignment in MßL:hydrolyzed antibiotic complexes. Molecular simulations also examine (meta)stable species of alternative protonation and tautomeric states, providing mechanistic insights into ß-lactam hydrolysis. We report the crystal structure of the hydrolyzed carbapenem ertapenem bound to the L1 MßL from Stenotrophomonas maltophilia and model alternative tautomeric and protonation states of both hydrolyzed ertapenem and faropenem (a related penem antibiotic), which display different binding modes with L1. We show how the structures of both complexed ß-lactams are best described as the (2S)-imine tautomer with the carboxylate formed after ß-lactam ring cleavage deprotonated. Simulations show that enamine tautomer complexes are significantly less stable (e.g., showing partial loss of interactions with the L1 binuclear zinc center) and not consistent with experimental data. Strong interactions of Tyr32 and one zinc ion (Zn1) with ertapenem prevent a C6 group rotation, explaining the different binding modes of the two ß-lactams. Our findings establish the relative stability of different hydrolyzed (carba)penem forms in the L1 active site and identify interactions important to stable complex formation, information that should assist inhibitor design for this important antibiotic resistance determinant.