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

The acetylation patterns of histones H3 and H4 along Vicia faba chromosomes are different

The original publication can be found at www.springerlink.comThe acetylation pattern of H3 was studied on field bean chromosomes by means of indirect immunofluorescence using polyclonal antibodies recognizing H3 isoforms acetylated at lysine positions 9/18, 14 and 23. H3 was found to be hypoacetylated at lysine residues 9/18 and 14 within the heterochromatic regions composed of tandem repetitive Fok-I elements. Hyperacetylation of these residues was observed at the nucleolar organizing region (NOR) and in heterochromatic regions composed of repeats other than Fok-I elements. In contrast, H4 was underacetylated (H4.Ac5, 8, 12) or uniformly acetylated (H4.Ac16) at all heterochromatic regions, and acetylated above the average at all four lysines only within the NOR. Acetylation of lysine-23 of H3 was uniform, except for the NOR that showed no fluorescence. Inhibition of deacetylase during and after replication of heterochromatin by trichostatin A had no influence on the acetylation status of H3 but mediated an increase in acetylation of lysines 5, 12 and 16 of H4 above the average in the field bean heterochromatin. Thus, the chromosomal acetylation patterns of H4 and H3 of this species revealed common and divergent features. Whereas the acetylation level of H4 correlates well with the potential transcriptional activity and inversely with the time of replication of defined chromatin domains of Vicia faba, this is not generally true for H3.N. D. Belyaev, A. Houben, P. Baranczewski and I. Schuber

Histone H4 acetylation in plant heterochromatin is altered during the cell cycle

The original publication can be found at www.springerlink.comUsing polyclonal antibodies directed against acetylated isoforms of histone H4 (H4 acetylated at lysine positions 5, 8, 12, 16 and H4 tetraacetylated), indirect immunofluorescence revealed hyperacetylation for all H4 variants at the nucleolus organizer region (NOR) of metaphase chromosomes of the field bean Vicia faba. The transcriptionally inactive and late-replicating heterochromatin regions proved to be hypoacetylated at lysine positions 5, 8 and 12. The remaining chromatin showed average fluorescence. These patterns were altered when deacetylase was blocked by exposure of root tip meristems to trichostatin A for more than 2 h prior to fixation. Under these conditions, all lysine positions, except lysine 8, appeared to be hyperacetylated at the NOR and in addition at the prominent heterochromatin domains. This observation represents a hitherto unique switch of histone acetylation pattern during the cell cycle. This is apparently caused by deposition of acetylated H4.Ac5, 12 and 16 or by acetylation directly after replication, which later on becomes reduced (H4.Ac16) or even reversed (H4.Ac5 and 12) by deacetylase before cells enter mitosis.Nikolai D. Belyaev, Andreas Houben, Pawel Baranczewski and Ingo Schuber

Improved predictions of time-dependent drug-drug interactions by determination of cytosolic drug concentrations

The clinical impact of drug-drug interactions based on time-dependent inhibition of cytochrome P450 (CYP) 3A4 has often been overpredicted, likely due to use of improper inhibitor concentration estimates at the enzyme. Here, we investigated if use of cytosolic unbound inhibitor concentrations could improve predictions of time-dependent drug-drug interactions. First, we assessed the inhibitory effects of ten time-dependent CYP3A inhibitors on midazolam 1′-hydroxylation in human liver microsomes. Then, using a novel method, we determined the cytosolic bioavailability of the inhibitors in human hepatocytes, and used the obtained values to calculate their concentrations at the active site of the enzyme, i.e. the cytosolic unbound concentrations. Finally, we combined the data in mechanistic static predictions, by considering different combinations of inhibitor concentrations in intestine and liver, including hepatic concentrations corrected for cytosolic bioavailability. The results were then compared to clinical data. Compared to no correction, correction for cytosolic bioavailability resulted in higher accuracy and precision, generally in line with those obtained by more demanding modelling. The best predictions were obtained when the inhibition of hepatic CYP3A was based on unbound maximal inhibitor concentrations corrected for cytosolic bioavailability. Our findings suggest that cytosolic unbound inhibitor concentrations improves predictions of time-dependent drug-drug interactions for CYP3A

Transfer of cetirizine/levocetirizine into human breast milk and estimation of drug exposure to infants through breastfeeding - a human lactation study from the ConcePTION project.

Data on drug transfer into human breast milk is sparse. This study aimed to quantify concentrations of cetirizine and levocetirizine in breast milk and to estimate drug exposure to infants. Breastfeeding women at least eight weeks postpartum and using cetirizine or its pure (R)-enantiomer levocetirizine were eligible to participate. Breast milk samples were collected at six predefined times during a dose interval (0, 2, 4, 8, 12 and 24 hours after drug intake) at steady state. Infant drug exposure was estimated by calculating the absolute infant dose (AID) and the weight-adjusted relative infant dose (RID). In total, 32 women were eligible for final inclusion, 31 women using cetirizine and one woman using levocetirizine. Means of the individual maximum and average cetirizine milk concentrations were 41.0 μg/L and 16.8 μg/L, respectively. Maximum concentrations occurred on average 2.4 hours after intake, and the mean half-life in milk was 7.0 h. Estimated AID and RID for cetirizine in a day were 2.5 μg/kg and 1.9%, respectively. The corresponding values for levocetirizine were 1.1 μg/kg and 1.9%. No severe adverse events were reported. Our findings demonstrate that the transfer of cetirizine and levocetirizine into breast milk is low and compatible with breastfeeding

Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound

Rift Valley fever virus (RVFV) is a mosquito-borne hemorrhagic fever virus affecting both humans and animals with severe morbidity and mortality and is classified as a potential bioterror agent due to the possible aerosol transmission. At present there is no human vaccine or antiviral therapy available. Thus, there is a great need to develop new antivirals for treatment of RVFV infections. Benzavir-2 was previously identified as potent inhibitor of human adenovirus, herpes simplex virus type 1, and type 2. Here we assess the anti-RVFV activity of benzavir-2 together with four structural analogs and determine pre-clinical pharmacokinetic parameters of benzavir-2. In vitro, benzavir-2 efficiently inhibited RVFV infection, viral RNA production and production of progeny viruses. In vitro, benzavir-2 displayed satisfactory solubility, good permeability and metabolic stability. In mice, benzavir-2 displayed oral bioavailability with adequate maximum serum concentration. Oral administration of benzavir-2 formulated in peanut butter pellets gave high systemic exposure without any observed toxicity in mice. To summarize, our data demonstrated potent anti-RVFV activity of benzavir-2 in vitro together with a promising pre-clinical pharmacokinetic profile. This data support further exploration of the antiviral activity of benzavir-2 in in vivo efficacy models that may lead to further drug development for human use