Profiling interactions of vaborbactam with metallo-β-lactamases

β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two mechanistic groups: the serine β-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo β-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both β-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-β-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-β-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20–100 fold below that by which it inhibits its current clinical targets, the Class A serine β-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of β-lactamases and justify further work on the development of boronates as broad-spectrum β-lactamase inhibitors

Bicyclic Boronate VNRX-5133 Inhibits Metallo- and Serine-β-Lactamases

The bicyclic boronate VNRX-5133 (taniborbactam) is a new type of β-lactamase inhibitor in clinical development. We report that VNRX-5133 inhibits serine-β-lactamases (SBLs) and some clinically important metallo-β-lactamases (MBLs), including NDM-1 and VIM-1/2. VNRX-5133 activity against IMP-1 and tested B2/B3 MBLs was lower/not observed. Crystallography reveals how VNRX-5133 binds to the class D SBL OXA-10 and MBL NDM-1. The crystallographic results highlight the ability of bicyclic boronates to inhibit SBLs and MBLs via binding of a tetrahedral (sp3) boron species. The structures imply conserved binding of the bicyclic core with SBLs/MBLs. With NDM-1, by crystallography, we observed an unanticipated VNRX-5133 binding mode involving cyclization of its acylamino oxygen onto the boron of the bicyclic core. Different side-chain binding modes for bicyclic boronates for SBLs and MBLs imply scope for side-chain optimization. The results further support the "high-energy-intermediate" analogue approach for broad-spectrum β-lactamase inhibitor development and highlight the ability of boron inhibitors to interchange between different hybridization states/binding modes

Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential beta-lactamase stable beta-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.Peer reviewe

Investigating multiple factors affecting the successful infection and transmission of arboviruses

Arboviruses are arthropod-borne viruses that cycle between a vertebrate host and an arthropod vector. This disparate life cycle involves adapting to two alternate animals and the differing tissues and systems within them. A range of factors influence the ability of a mosquito to transmit a virus, including internal factors such as mosquito immunity, the mosquito microbiota and the mutation rate of the virus itself, and external factors such as exposure to agricultural chemicals and warming global temperatures. I looked at several factors affecting the establishment of infection and transmission of arboviruses. Three low-fidelity mutants of Venezuelan Equine Encephalitis Virus (VEEV), which produce a higher mutation rate than the wild-type virus, were previously identified as unable to successfully infect mosquitoes. To understand why these low-fidelity mutants were attenuated, I serially passaged wildtype VEEV and the three low-fidelity mutants in a new cell culture model designed to replicate bottlenecks within arbovirus transmission. This would enable us to understand why the low-fidelity mutants were attenuated in previous in vivo mosquito and mouse infections. Furthermore, we wanted to see if the low-fidelity nature changed the RNAi response, a key antiviral immune response within mosquitoes. We looked to see if the RNAi response was different for TC83_3X (a low-fidelity mutant) compared to the parent TC83 VEEV-vaccine strain using in vitro cell culture experiments and RNA sequencing. Finally, I wanted to understand the impact of external factors on vector competence, given many factors will act on wild mosquito populations. I determined if two agricultural chemicals, glyphosate and triticonazole, or exposure to the insect-specific virus Negevirus (NEGV), altered the vector competence of Aedes aegypti for Chikungunya virus (CHIKV) or Zika virus (ZIKV). To summarise the results, we found that the previously seen attenuation of the three low-fidelity VEEV variants is likely due to their inability to traverse bottlenecks associated with mosquito infection, and that the RNAi response did not differ between parent TC83 and low-fidelity TC83_3X. Both Glyphosate and Triticonazole slightly reduced the vector competence of A. aegypti for both CHIKV and ZIKV whilst exposure to NEGV increased the percentage of the mosquito population infected with both viruses. However, NEGV exposure reduced the percentage of infected mosquitoes with dissemination, which may subsequently reduce the likelihood of transmission due to lower likelihood of infectious virus reaching mosquito salivary glands. Our work covered a range of internal and external factors and identified their influence on vector competence. This has greater implications for our understanding of the establishment of arboviral infection and their transmission

In vitro efficacy of Imipenem-Relebactam and Cefepime-AAI101 against a global collection of ESBL-positive and carbapenemase-producing Enterobacteriaceae

Objectives: In vitro evaluation of the potential clinical efficacy of the novel β-lactam/β-lactamase-inhibitor combinations including imipenem-relebactam (IPM-REL) and cefepime-AAI101 (enmetazobactam) (FEP-AAI) against contemporary multidrug-resistant Enterobacteriaceae. Methods: Agar-based MIC screening against MDR-Enterobacteriaceae (n=264) was used to evaluate the in vitro efficacy of IPM-REL and FEP-AAI, to compare the results with established combinations, and to investigate alternative β-lactam partners for REL and AAI. The inhibition activities of REL, AAI and the comparators avibactam (AVI) and tazobactam, against isolated recombinant β-lactamases covering representatives from all four Ambler classes of β-lactamases were tested using a fluorescence-based assay. Results: Using recombinant proteins, all four inhibitors were highly active against the tested class A serine β-lactamases (SBLs); REL and AVI showed moderate activity against the Class C AmpC from P. aeruginosa and the Class D OXA-10/-48 SBLs, but outperformed tazobactam and AAI. All tested inhibitors lacked activity against Class B MBLs. In the presence of REL, IPM, but not AAI susceptibility increased against KPC-positive and OXA-48-positive isolates. Both aztreonam-AVI and ceftolozane-tazobactam were more efficacious than IPM-REL. In all the tested combinations, AAI was a more effective inhibitor of class A β-lactamases (ESBLs) than the established inhibitors. Conclusions: The results lead to the proposal of alternative combination therapies involving REL and AAI to potentiate the use of β-lactams against clinical Gram-negative isolates expressing a variety of β-lactamases. These results highlight the potential of novel combinations for combating strains not covered by existing therapies.</p

UK mosquitoes are competent to transmit Usutu virus at native temperatures

Usutu virus (USUV) is an emerging zoonotic virus transmitted primarily by Culex mosquitoes. Since its introduction into Europe from Africa during the late 20th century, it has caused mortality within populations of passerine birds and captive owls, and can on occasion lead to disease in humans. USUV was first detected in the UK in 2020 and has become endemic, having been detected in either birds and/or mosquitoes every subsequent year. Importantly, the vector competence of indigenous mosquitoes for the circulating UK (London) USUV strain at representative regional temperatures is still to be elucidated. This study assessed the vector competence of five field-collected mosquito species/biotypes, Culex pipiens biotype molestus, Culex pipiens biotype pipiens, Culex torrentium, Culiseta annulata and Aedes detritus for the London USUV strain, with infection rates (IR) and transmission rates (TR) evaluated between 7 and 28 days post-infection. Infection and transmission were observed in all species/biotypes aside from Ae. detritus and Cx. torrentium. For Cx. pipiens biotype molestus, transmission potential suggests these populations should be monitored further for their role in transmission to humans. Furthermore, both Cx. pipiens biotype pipiens and Cs. annulata were shown to be competent vectors at 19 °C indicating the potential for geographical spread of the virus to other UK regions

In vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii

Objectives: Widespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants. Methods: A thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity. Results: MIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin. Conclusions: Its superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics

Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential beta-lactamase stable beta-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.Peer reviewe