Cephalosporin-NO-donor prodrug PYRRO-C3D shows β-lactam-mediated activity against Streptococcus pneumoniae biofilms

Bacterial biofilms show high tolerance towards antibiotics and are a significant problem in clinical settings where they are a primary cause of chronic infections. Novel therapeutic strategies are needed to improve anti-biofilm efficacy and support reduction in antibiotic use. Treatment with exogenous nitric oxide (NO) has been shown to modulate bacterial signaling and metabolic processes that render biofilms more susceptible to antibiotics. We previously reported on cephalosporin-3\u27-diazeniumdiolates (C3Ds) as NO-donor prodrugs designed to selectively deliver NO to bacterial infection sites following reaction with β-lactamases. With structures based on cephalosporins, C3Ds could, in principal, also be triggered to release NO following β-lactam cleavage mediated by transpeptidases/penicillin-binding proteins (PBPs), the antibacterial target of cephalosporin antibiotics. Transpeptidase-reactive C3Ds could potentially show both NO-mediated anti-biofilm properties and intrinsic (β-lactam-mediated) antibacterial effects. This dual-activity concept was explored using Streptococcus pneumoniae, a species that lacks β-lactamases but relies on transpeptidases for cell-wall synthesis. Treatment with PYRRO-C3D (a representative C3D containing the diazeniumdiolate NO donor PYRRO-NO) was found to significantly reduce viability of planktonic and biofilm pneumococci, demonstrating that C3Ds can elicit direct, cephalosporin-like antibacterial activity in the absence of β-lactamases. While NO release from PYRRO-C3D in the presence of pneumococci was confirmed, the anti-pneumococcal action of the compound was shown to arise exclusively from the β-lactam component and not through NO-mediated effects. The compound showed similar potency to amoxicillin against S. pneumoniae biofilms and greater efficacy than azithromycin, highlighting the potential of C3Ds as new agents for treating pneumococcal infections

pH-dependent interactions of coacervate-forming histidine-rich peptide with model lipid membranes

Peptide-based liquid droplets (coacervates) produced by spontaneous liquid-liquid phase separation (LLPS), have emerged as a promising class of drug delivery systems due to their high entrapping efficiency and the simplicity of their formulation. However, the detailed mechanisms governing their interaction with cell membranes and cellular uptake remain poorly understood. In this study, we investigated the interactions of peptide coacervates composed of HBpep—peptide derived from the histidine-rich beak proteins (HBPs) of the Humboldt squid—with model cellular membranes in the form of supported lipid bilayers (SLBs). We employed quartz crystal microbalance with dissipation monitoring (QCM-D), neutron reflectometry (NR) and atomistic molecular dynamics (MD) simulations to reveal the nature of these interactions in the absence of fluorescent labels or tags. HBpep forms small oligomers at pH 6 whereas it forms µm-sized coacervates at physiological pH. Our findings reveal that both HBpep oligomers and HBpep-coacervates adsorb onto SLBs at pH 6 and 7.4, respectively. At pH 6, when the peptide carries a net positive charge, HBpep oligomers insert into the SLB, facilitated by the peptide’s interactions with the charged lipids and cholesterol. Importantly, however, HBpep coacervate adsorption at physiological pH, when it is largely uncharged, is fully reversible, suggesting no significant lipid bilayer rearrangement. HBpep coacervates, previously identified as efficient drug delivery vehicles, do not interact with the lipid membrane in the same manner as traditional cationic drug delivery systems or cell-penetrating peptides. Based on our findings, HBpep coacervates at physiological pH cannot cross the cell membrane by a simple passive mechanism and are thus likely to adopt a non-canonical cell entry pathway

The design and synthesis of novel anti-malarial agents

A new convergent methodology has been developed for the synthesis of the anti-malarial lead compound ADAM (24). A series of 41 derivatives were successfully synthesized, 35 of which were new compounds; all of which related to ADAM (24). All the synthesized derivatives were screened against in vitro Plasmodium falciparum K1, of which 11 showed significant activity in the low micromolar range, including compound (46) (IC50 0.3 ?g/ml), (80) (0.7 ?g/ml). and (88) (1.4 ?g/ml). A preliminary structure activity relationship analysis study was performed, which suggested that while 4,6-diaminopyrimidine analogs are inactive, if one of the two amines are alkylated then anti-malarial activity is returned. In order to generate a more thorough analysis, more compounds need to be synthesized by modifying the side chain. The SAR study also suggests that the heteroatom nitrogens in the pyrimidine ring are required for anti-malarial activity. The entire range of derivatives based on 5-benzoyl-4,6-dichloropyrimidine are inactive. The replacement of the phenyl substituent with an isopropyl group resulted in good anti-malarial activity but less than the parent compound. Novel synthetic dimerising methodology mediated by thallium(III) trifluoroacetate has been developed for the synthesis of biindoles. The methodology included the development of 2,2- and 2,7-biindoles, and 2,7,7,7-triindole. A series of 41 new indole derivatives were synthesized and screened for anti-malarial activity, of which three showed significant anti-malarial activity. During this study, the dimerisation of indole mediated by triflic anhydride was demonstrated for the first time. There are two other interesting synthetic results were observed, the triflic anhydride-mediated bi-pyrroloindole formation and the formation of the 1,2-dihydropyridineindole. Most compounds synthesized here were further tested in a series of additional biological assays, and a number of them showed activity against HIV reverse transcriptase, integrase, tuberculosis, anti-fungal and anti-cancer. In particular, the biaryl derivatives showed moderate activity against tuberculosis and fungal, which may be able to investigate further. A couple of biaryl compounds also showed significant anti-cancer activity. [Note: this abstract contained scientific formulae that would not come across on this form. Please see the 01Front files abstract for the full details.

Synthesis of Perdeuterated Linoleic Acid-d31 and Chain Deuterated 1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine-d62

Herein, we report a gram-scale synthesis of perdeuterated linoleic acid-d31. The starting materials for the synthesis are two saturated fatty acids, azelaic acid-d14 and pentanoic acid-d9, which can be obtained by metal catalysed hydrothermal hydrogen-deuterium exchange. The synthesis utilises the fatty acids directly via decarboxylative coupling. Copper catalysed coupling of a terminal alkyne intermediate with a propargyl bromide derivative affords a skipped diyne, which can be reduced using P-2 nickel to obtain the desired cis,cis-diene geometry. The subsequent synthesis of the tail-deuterated phospholipid, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine-d62 (PLPC-d62) is also described. Optimised reaction conditions were developed to access this phospholipid and its regioisomeric purity was characterised by two complementary mass spectrometry techniques.</p

Measuring the Interactions and Influence of Amphipathic Copolymers with Lipid Monolayers and Bilayers as Models of Biological Membranes

Amphipathic copolymers are useful materials for nanomedicine, owing to their ability to self-assemble into nanoparticles, act as surfactants for inorganic materials, or for their favorable interactions with lipid membranes. Despite their widespread use, there is still a range of questions about the physicochemical properties that are necessary to drive their interactions at biological interfaces. To fully understand these interactions requires a diverse range of complementary analytical techniques. In this work, a library of neutral amphipathic methacrylate copolymers is synthesized by reversible addition-fragmentation chain-transfer polymerization (RAFT) polymerization, to investigate the effect of polymer composition and nature of the hydrophobic comonomer on interactions with model lipid membranes. These materials are shown to interact with Langmuir lipid monolayers, and neutron reflectometry demonstrates that hydrophobic interactions lead to the polymers intercalating with the monolayers. More complex models of lipid bilayers are studied using an in situ quartz crystal microbalance (QCM) model and shows while the composition and hydrophobic comonomer affect the stability of these interactions, there is no effect on the viscoelasticity of the lipid membranes. The in-depth understanding of these interfacial interactions afforded by this suite of analytical tools will allow for more complex copolymers to be studied, providing a greater understanding of key processes in nanomedicine, such as cellular entry and endosomal escape.</p

Dependence of organic interlayer diffusion on glass-transition temperature in OLEDs

Organic light-emitting diodes (OLEDs) are subject to thermal stress from Joule heating and the external environment. In this work, neutron reflectometry (NR) was used to probe the effect of heat on the morphology of thin three-layer organic films comprising materials typically found in OLEDs. It was found that layers within the films began to mix when heated to approximately 20 °C above the glass-transition temperature (T) of the material with the lowest T. Diffusion occurred when the material with the lowest T formed a supercooled liquid, with the rates of interdiffusion of the materials depending on the relative T's. If the supercooled liquid formed at a temperature significantly lower than the T of the higher-T material in the adjacent layer, then pseudo-Fickian diffusion occurred. If the two T's were similar, then the two materials can interdiffuse at similar rates. The type and extent of diffusion observed can provide insight into and a partial explanation for the "burn in" often observed for OLEDs. Photoluminescence measurements performed simultaneously with the NR measurements showed that interdiffusion of the materials from the different layers had a strong effect on the emission of the film, with quenching generally observed. These results emphasize the importance of using thermally stable materials in OLED devices to avoid film morphology changes

Effects of MnDPDP andICRF-187 on Doxorubicin-Induced Cardiotoxicityand Anticancer Activity1

Oxidative stress participates in doxorubicin (Dx)–induced cardiotoxicity. The metal complex MnDPDP and its metaboliteMnPLED possess SOD-mimetic activity, DPDP and PLED have, in addition, high affinity for iron. Mice wereinjected intravenously with MnDPDP, DPDP, or dexrazoxane (ICRF-187). Thirty minutes later, mice were killed, theleft atria were hung in organ baths and electrically stimulated, saline or Dx was added, and the contractility wasmeasured for 60 minutes. In parallel experiments, 10 μM MnDPDP or MnPLED was added directly into the organbath. The effect of MnDPDP on antitumor activity of Dx against two human tumor xenografts (MX-1 and A2780)was investigated. The in vitro cytotoxic activity was studied by co-incubating A2780 cells with MnDPDP, DPDP,and/or Dx. Dx caused a marked reduction in contractile force. In vivo treatment with MnDPDP and ICRF-187 attenuatedthe negative effect of Dx. When added directly into the bath, MnDPDP did not protect, whereas MnPLEDattenuated the Dx effect by approximately 50%. MnDPDP or ICRF-187 did not interfere negatively with the antitumoractivity of Dx, either in vivo or in vitro. Micromolar concentrations of DPDP but not MnDPDP displayed anin vitro cytotoxic activity against A2780 cells. The present results show that MnDPDP, after being metabolized toMnPLED, protects against acute Dx cardiotoxicity. Both in vivo and in vitro experiments show that cardioprotectiontakes place without interfering negatively with the anticancer activity of Dx. Furthermore, the results suggest thatthe previously described cytotoxic in vivo activity of MnDPDP is an inherent property of DPDP. Translational Oncology (2012) 5, 252–259funding agencies|Medical Research Council of Southeast Sweden|FORSS-85191|PledPharma AB||</p