Sigmoid Emax Modeling To Define the Fixed Concentration of Enmetazobactam for MIC Testing in Combination with Cefepime.

The use of carbapenem antibiotics to treat infections caused by Enterobacterales expressing increasingly aggressive extended-spectrum ß-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 checkerboard MIC profiles were obtained for a challenge panel of cefepime-resistant ESBL-producing clinical isolates of Klebsiella pneumoniae. Sigmoid maximum effect (Emax) modeling described cefepime MICs as a function of enmetazobactam concentration with no bias. A concentration of 8 µg/ml enmetazobactam proved sufficient to restore >95% of cefepime antibacterial activity in vitro against >95% of isolates tested. These results support a fixed concentration of 8 µg/ml of enmetazobactam for MIC testing.

Novel Specific Metallo-ß-Lactamase Inhibitor ANT2681 Restores Meropenem Activity to Clinically Effective Levels against NDM-Positive Enterobacterales.

The global dissemination of metallo-ß-lactamase (MBL)-producing carbapenem-resistant Enterobacterales (CRE) is a serious public health concern. Specifically, NDM (New Delhi MBL) has been a major cause of carbapenem therapy failures in recent years, particularly as effective treatments for serine-ß-lactamase (SBL)-producing Enterobacterales are now commercially available. Since the NDM gene is carried on promiscuous plasmids encoding multiple additional resistance determinants, a large proportion of NDM-CREs are also resistant to many commonly used antibiotics, resulting in limited and suboptimal treatment options. ANT2681 is a specific, competitive inhibitor of MBLs with potent activity against NDM enzymes, progressing to clinical development in combination with meropenem (MEM). Susceptibility studies have been performed with MEM-ANT2681 against 1,687 MBL-positive Enterobacterales, including 1,108 NDM-CRE. The addition of ANT2681 at 8 µg/ml reduced the MEM MIC50/MIC90 from >32/>32 µg/ml to 0.25/8 µg/ml. Moreover, the combination of 8 µg/ml of both MEM and ANT2681 inhibited 74.9% of the Verona integron-encoded MBL (VIM)-positive and 85.7% of the imipenem hydrolyzing ß-lactamase (IMP)-positive Enterobacterales tested. The antibacterial activity of MEM-ANT2681 against NDM-CRE compared very favorably to that of cefiderocol (FDC) and cefepime (FEP)-taniborbactam, which displayed MIC90 values of 8 µg/ml and 32 µg/ml, respectively, whereas aztreonam-avibactam (ATM-AVI) had a MIC90 of 0.5 µg/ml. Particularly striking was the activity of MEM-ANT2681 against NDM-positive Escherichia coli (MIC90 1 µg/ml), in contrast to ATM-AVI (MIC90 4 µg/ml), FDC (MIC90 >32 µg/ml), and FEP-taniborbactam (MIC90 >32 µg/ml), which were less effective due to the high incidence of resistant PBP3-insertion mutants. MEM-ANT2681 offers a potential new therapeutic option to treat serious infections caused by NDM-CRE.

Discovery of ANT3310, a Novel Broad-Spectrum Serine ß-Lactamase Inhibitor of the Diazabicyclooctane Class, Which Strongly Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacterales and Acinetobacter baumannii.

The diazabicyclooctanes (DBOs) are a class of serine ß-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of ß-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of ß-lactam antibiotics against carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO "critical priority pathogen" producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure-activity relationship, leading to the discovery of a novel DBO, ANT3310, which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.

Evaluation of in vitro activity of iclaprim in combination with other antimicrobials against pulmonary pathogens: a pilot study.

In this pilot study, the in vitro antimicrobial activity of iclaprim, a diaminopyrimidine, tested in combination with other antimicrobials against recent and common Gram-positive and Gram-negative respiratory pathogens, was examined by the checkerboard method. The range of minimal inhibitory concentrations (MICs) for iclaprim against all bacteria tested in the study was 0.03 to >128 µg ml-1. Iclaprim exhibited synergy with sulfamethoxazole against 11 of the 16 bacterial strains tested, with mean fractional inhibitory concentration index (FICI) values of 0.2-0.5. Synergy with sulfamethoxazole was demonstrated against all Gram-positive bacteria and selected Gram-negative bacteria. Neither synergy nor antagonism was observed for combinations of iclaprim with ampicillin, meropenem, tetracycline, levofloxacin, aztreonam, piperacillin/tazobactam, colistin, cefepime or gentamicin against any of the bacterial strains tested. The significant reduction in the MIC values observed with the combination of iclaprim and sulfamethoxazole demonstrates that this regimen could be effective against common Gram-positive and selected Gram-negative respiratory bacteria.

Elucidating the role of (p)ppGpp in mycobacterial persistence against antibiotics.

Bacterial persistence, the ability of bacteria to survive high concentrations of antibiotics for extended periods of time, is an important contributing factor to therapy failure and development of chronic and recurrent infections. Several recent studies have suggested that this persistence is mediated primarily by (p)ppGpp, through its interactions with toxin-antitoxin modules and polyphosphates. In this study, we address whether these key players play a role in mycobacterial persistence against antibiotics. We targeted these specific pathways in Mycobacterium smegmatis by constructing deletion strains of (p)ppGpp synthetase/hydrolase (relA), polyphosphate kinases (ppk1 and ppk2), exopolyphosphatases (ppx1 and ppx2), and the lon protease. None of these mutant strains exhibited altered levels of persisters against isoniazid and ciprofloxacin, when compared with wild-type strain. Even under conditions in which the stringent response usually gets activated, these strains displayed wild-type persister levels. Interestingly, we also found that unlike Escherichia coli, maintaining M. smegmatis in exponential phase by repeated passaging does not eliminate persisters suggesting that at least against the antibiotics tested, stationary-phase dependent persisters (type I) are not the major contributors. Thus, our data demonstrate that multiple mechanisms of antibiotic persistence exist and that these vary widely among different bacterial species. © 2018 IUBMB Life, 70(9):836-844, 2018.

Amyloid-beta oligomerization is associated with the generation of a typical peptide fragment fingerprint.

Amyloid-beta (Aß) peptide oligomerization plays a central role in the pathogenesis of Alzheimer's disease (AD), and Aß oligomers are collectively considered an appealing therapeutic target for the treatment of AD. However, the molecular mechanisms leading to the pathologic accumulation of oligomers are unclear, and the exact structural composition of oligomers is being debated. Using targeted and quantitative mass spectrometry, we reveal site-specific Aß autocleavage during the early phase of aggregation, producing a typical Aß fragment signature and that truncated Aß peptides can form stable oligomeric complexes with full-length Aß peptide. We show that the use of novel anti-Aß antibodies raised against these truncated Aß isoforms allows for monitoring and targeting the accumulation of truncated Aß fragments. Antibody-enabled screening of transgenic models of AD as well as human postmortem brain tissue and cerebrospinal fluid revealed that aggregation-associated Aß cleavage is a highly relevant clinical feature of AD.

Nanoparticle conjugation and pulmonary delivery enhance the protective efficacy of Ag85B and CpG against tuberculosis.

Vaccines that drive robust T-cell immunity against Mycobacterium tuberculosis (Mtb) are needed both for prophylactic and therapeutic purposes. We have recently developed a synthetic vaccine delivery platform with Pluronic-stabilized polypropylene sulfide nanoparticles (NPs), which target lymphoid tissues by their small size (∼ 30 nm) and which activate the complement cascade by their surface chemistry. Here we conjugated the tuberculosis antigen Ag85B to the NPs (NP-Ag85B) and compared their efficacy in eliciting relevant immune responses in mice after intradermal or pulmonary administration. Pulmonary administration of NP-Ag85B with the adjuvant CpG led to enhanced induction of antigen-specific polyfunctional Th1 responses in the spleen, the lung and lung-draining lymph nodes as compared to soluble Ag85B with CpG and to the intradermally-delivered formulations. Mucosal and systemic Th17 responses were also observed with this adjuvanted NP formulation and vaccination route, especially in the lung. We then evaluated protection induced by the adjuvanted NP formulation following a Mtb aerosol challenge and found that vaccination with NP-Ag85B and CpG via the pulmonary route displayed a substantial reduction of the lung bacterial burden, both compared to soluble Ag85B with CpG and to the corresponding intradermally delivered formulations. These findings highlight the potential of administrating NP-based formulations by the pulmonary route for TB vaccination.

Luminescent lanthanide helicates self-assembled from ditopic ligands bearing phosphonic acid or phosphoester units.

A series of hexadentate ditopic receptors incorporating benzimidazole moieties have been designed, which are fitted with phosphonic acid or phosphoethylester coordinating units. In addition, poly(oxyethylene) pendants have been introduced on the benzimidazole backbone of two ligands to increase water solubility. The ligands self-assemble with lanthanide ions under stoichiometric conditions, yielding triple-stranded homobimetallic helicates, as ascertained by mass spectrometry and UV-visible titrations. The helicates display large thermodynamic stability, for example, log beta(23) approximately 21-24 for all the Eu(III) complexes. Photophysical measurements reveal sensitization of the metal-centered luminescence in the europium and terbium complexes, which is modulated by the nature of the ligand. Hydration numbers determined by the lifetime method are essentially zero. The Eu((5)D(0)) lifetimes are long and reach values up to 3.2 ms, while quantum yields as high as 25% are obtained in water at pH 7.4. Back transfer limits the sensitization efficiency for Tb(III) luminescence, and both lifetimes and quantum yields are much smaller. The properties of the helicates are discussed with respect to those self-assembled from ligands bearing carboxylate coordinating units.