Cephalosporin-3’-diazeniumdiolate NO-donor prodrug PYRRO-C3D enhances azithromycin susceptibility of non-typeable Haemophilus influenzae biofilms

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Objectives: PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO)-donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against non-typeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. Methods: The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free LC/MS proteomic analyses were performed to identify differentially expressed proteins following NO treatment. Results: PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log-fold reduction in viability (p<0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log reduction was observed (p<0.01). Label-free proteomics showed that NO increased expression of sixteen proteins involved in metabolic and transcriptional/translational functions. Conclusions: NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm associated antibiotic tolerance

ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition.

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body

The Synthesis and Testing of Arenearylpyrimidylmethanes (AAPM) as Anti-Malarial Agents

The anti-malarial activity of the arenearylpyrimidylmethane (AAPM) class of compounds emerged from database searching of a pharmacophore. A new 2 step synthesis of the AAPM scaffold is reported and subsequent substitution yielded a short synthesis of the lead anti-malarial compound. The presence of atropisomerism in this class of compounds is also reported for the first time

Oxidative coupling of indoles using thallium(III) trifluoroacetate

The oxidative coupling of polysubstituted electron-rich indoles mediated by thallium trifluoroacetate was found to be a facile, clean, and high yielding reaction. Indolic coupling sites were determined by the nature of the substituents present, with dimerisation at the indole 2-position being the dominant outcome. Indoles bearing two potential reaction sites with similar reactivity were additionally found to undergo heterocoupling

Cephalosporin-3\u27-diazeniumdiolates:Targeted NO-donor prodrugs for dispersing bacterial biofilms

Biofilms are sessile communities of microbial cells contained within a self-produced exopolymeric matrix. Bacteria encased in biofilms exhibit upwards of 10–1000-fold higher resistance to biocides and traditional antibiotics than their planktonic counterparts (i.e. floating, unattached), and they are less susceptible to host immune defenses.[1, 2] Accordingly, chronic antimicrobial-tolerant bacterial infections are often biofilmbased (e.g. infections on indwelling medical devices and incurable Pseudomonas aeruginosa respiratory infections in cystic fibrosis (CF) sufferers).[3] Currently there are few effective therapeutics for clearing biofilm-based infections, and a critical need exists for new agents and treatment strategie

ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body