Philipp Knechtle

The use of carbapenem antibiotics to treat infections caused by Enterobacterales expressing increasingly aggressive extended-spectrum β-lactamases (ESBLs) has contributed to the emergence of carbapenem resistance. Enmetazobactam is a novel ESBL inhibitor being developed in combination with cefepime as a carbapenem-sparing option for infections caused by ESBL-producing Enterobacterales .

Cefepime-enmetazobactam is a novel β-lactam–β-lactamase inhibitor combination with broad-spectrum antimicrobial activity against a range of multidrug-resistant Enterobacteriaceae . This agent is being developed for a range of serious hospital infections. An understanding of the extent of partitioning of β-lactam–β-lactamase inhibitor combinations into the human lung is required to better understand the potential role of cefepime-enmetazobactam for the treatment of nosocomial pneumonia.

Klebsiella pneumoniae that produce extended spectrum beta lactamases (ESBLs) are a persistent public health threat. There are relatively few therapeutic options and there is undue reliance on carbapenems. Alternative therapeutic options are urgently required. A combination of cefepime and the novel beta lactamase inhibitor enmetazobactam is being developed for treatment of serious infections caused by ESBL-producing organisms. The pharmacokinetics-pharmacodynamics (PK-PD) of cefepime-enmetazobactam against ESBL-producing K. pneumoniae was studied in a neutropenic murine pneumonia model. Dose ranging studies were performed. Dose fractionation studies were performed to define the relevant PD index for the inhibitor. The partitioning of cefepime and enmetazobactam into the lung was determined by comparing area under the concentration time curve (AUC) in plasma and epithelial lining fluid. The magnitude of drug exposure for cefepime-enmetazobactam required for logarithmic killing in the lung was defined using 3 ESBL-producing strains. Cefepime 100 mg/kg q8h i.v. had minimal antimicrobial effect. When this background regimen of cefepime was combined with enmetazobactam half-maximal effect was induced with enmetazobactam 4.71 mg/kg q8h i.v. The dose fractionation study suggest both fT>threshold and fAUC:MIC are potentially relevant PD indices. The AUC_ELF:AUC_plasma for cefepime and enmetazobactam was 73.4% and 61.5%, respectively. A ≥2-log kill in the lung was achieved with a plasma and ELF cefepime fT>MIC of ≥20% and enmetazobactam fT>2 mg/L of ≥20% of the dosing interval. These data and analyses provide the underpinning evidence for the combined use of cefepime and enmetazobactam for nosocomial pneumonia.

Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae are classified as critical priority pathogens, with extended-spectrum β-lactamases (ESBLs) as principal resistance determinants. Enmetazobactam (formerly AAI101) is a novel ESBL inhibitor developed in combination with cefepime for empirical treatment of serious Gram-negative infections in settings where ESBLs are prevalent. Cefepime-enmetazobactam has been investigated in a phase 3 trial in patients with complicated urinary tract infections or acute pyelonephritis.

Third-generation cephalosporin resistance among Enterobacteriaceae , mediated by the spread of extended-spectrum β-lactamases (ESBLs), is a very serious medical concern with limited therapeutic options. Enmetazobactam (formerly AAI101) is a novel penicillanic sulfone β-lactamase inhibitor active against a wide range of ESBLs. The combination of enmetazobactam and cefepime has entered phase 3 development in patients with complicated urinary tract infections.

Enmetazobactam, formerly AAI101, is a novel penicillanic acid sulfone extended-spectrum β-lactamase (ESBL) inhibitor. The combination of enmetazobactam with cefepime has entered clinical trials to assess safety and efficacy in patients with complicated urinary tract infections. Here, the in vitro activity of cefepime-enmetazobactam was determined for 1,993 clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa collected in the United States and Europe during 2014 and 2015. Enmetazobactam at a fixed concentration of 8 μg/ml lowered the cefepime MIC₉₀ from 16 to 0.12 μg/ml for Escherichia coli, from >64 to 0.5 μg/ml for Klebsiella pneumoniae, from 16 to 1 μg/ml for Enterobacter cloacae, and from 0.5 to 0.25 μg/ml for Enterobacter aerogenes. Enmetazobactam did not enhance the potency of cefepime against P. aeruginosa. Applying the Clinical and Laboratory Standards Institute susceptible-dose-dependent (SDD) breakpoint of 8 μg/ml to cefepime-enmetazobactam for comparative purposes resulted in cumulative inhibitions of 99.9% for E. coli, 96.4% for K. pneumoniae, 97.0% for E. cloacae, 100% for E. aerogenes, 98.1% for all Enterobacteriaceae assessed, and 82.8% for P. aeruginosa. Comparator susceptibilities for all Enterobacteriaceae were 99.7% for ceftazidime-avibactam, 96.2% for meropenem, 90.7% for ceftolozane-tazobactam, 87% for cefepime (SDD breakpoint), 85.7% for piperacillin-tazobactam, and 81.2% for ceftazidime. For the subset of ESBL-producing K. pneumoniae isolates, the addition of 8 μg/ml enmetazobactam to cefepime lowered the MIC₉₀ from >64 to 1 μg/ml, whereas the shift for 8 μg/ml tazobactam was from >64 to 8 μg/ml. Cefepime-enmetazobactam may represent a novel carbapenem-sparing option for empirical treatment of serious Gram-negative infections in settings where ESBL-producing Enterobacteriaceae are expected.

Human fungal infections represent a therapeutic challenge. Although effective strategies for treatment are available, resistance is spreading, and many therapies have unacceptable side effects. A clear need for novel antifungal targets and molecules is thus emerging. Here, we present the identification and characterization of the plant-derived diyne-furan fatty acid EV-086 as a novel antifungal compound. EV-086 has potent and broad-spectrum activity in vitro against Candida , Aspergillus , and Trichophyton spp., whereas activities against bacteria and human cell lines are very low. Chemical-genetic profiling of Saccharomyces cerevisiae deletion mutants identified lipid metabolic processes and organelle organization and biogenesis as targets of EV-086. Pathway modeling suggested that EV-086 inhibits delta-9 fatty acid desaturation, an essential process in S. cerevisiae , depending on the delta-9 fatty acid desaturase OLE1 . Delta-9 unsaturated fatty acids—but not saturated fatty acids—antagonized the EV-086-mediated growth inhibition, and transcription of the OLE1 gene was strongly upregulated in the presence of EV-086. EV-086 increased the ratio of saturated to unsaturated free fatty acids and phosphatidylethanolamine fatty acyl chains, respectively. Furthermore, EV-086 was rapidly taken up into the lipid fraction of the cell and incorporated into phospholipids. Together, these findings demonstrate that EV-086 is an inhibitor of delta-9 fatty acid desaturation and that the mechanism of inhibition might involve an EV-086–phospholipid. Finally, EV-086 showed efficacy in a guinea pig skin dermatophytosis model of topical Trichophyton infection, which demonstrates that delta-9 fatty acid desaturation is a valid antifungal target, at least for dermatophytoses.

Background: Natural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host.Results: As an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5–7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology.Conclusion: The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds

Members of the dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) family perform a variety of functions in eukaryotes. We used gene disruption, targeted pharmacologic inhibition, and genome-wide transcriptional profiling to dissect the function of the Yak1 DYRK in the human fungal pathogen Candida albicans. C. albicans strains with mutant yak1 alleles showed defects in the yeast-to-hypha transition and in maintaining hyphal growth. They also could not form biofilms. Despite their in vitro filamentation defect, C. albicans yak1Δ/yak1Δ mutants remained virulent in animal models of systemic and oropharyngeal candidiasis. Transcriptional profiling showed that Yak1 was necessary for the up-regulation of only a subset of hypha-induced genes. Although downstream targets of the Tec1 and Bcr1 transcription factors were down-regulated in the yak1Δ/yak1Δ mutant, TEC1 and BCR1 were not. Furthermore, 63% of Yak1-dependent, hypha-specific genes have been reported to be negatively regulated by the transcriptional repressor Tup1 and inactivation of TUP1 in the yak1Δ/yak1Δ mutant restored filamentation, suggesting that Yak1 may function upstream of Tup1 in governing hyphal emergence and maintenance.