Comparing Selection on S. aureus between Antimicrobial Peptides and Common Antibiotics

With a diminishing number of effective antibiotics, there has been interest in developing antimicrobial peptides (AMPs) as drugs. However, any new drug faces potential bacterial resistance evolution. Here, we experimentally compare resistance evolution in Staphylococcus aureus selected by three AMPs (from mammals, amphibians and insects), a combination of two AMPs, and two antibiotics: the powerful last-resort vancomycin and the classic streptomycin. We find that resistance evolves readily against single AMPs and against streptomycin, with no detectable fitness cost. However the response to selection from our combination of AMPs led to extinction, in a fashion qualitatively similar to vancomycin. This is consistent with the hypothesis that simultaneous release of multiple AMPs during immune responses is a factor which constrains evolution of AMP resistant pathogens.     

Discovery of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as potent agonists of TGR5 via sequential combinatorial libraries

Optimization of a high-throughput screening hit led to the discovery of a new series of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as highly potent agonists of TGR5. This novel chemotype was rapidly developed through iterative combinatorial library synthesis. It was determined that in vitro agonist potency correlated with functional activity data from human peripheral blood monocytes.     

Peripherally acting novel lipo-endomorphin-1 peptides in neuropathic pain without producing constipation

We previously described two novel analogues of endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂, 1), modified with an 8-carbon lipoamino acid (C8LAA) with or without replacement of Tyr¹ with 2,6-dimethyltyrosine (Dmt) at the N-terminus of the peptide (compounds 3 and 4, respectively). They were shown to be more stable and permeable, and acted as potent μ-opioid receptor agonists. In this study we report that the C8LAA modification resulted in successful systemic delivery of both analogues. They produced potent dose-dependent pain relief in a chronic constriction injury-rat model of neuropathic pain after intravenous administration with ED₅₀ values obtained at 6.58 (±1.22) μmol/kg for 3 and 6.18 (±1.17) μmol/kg for 4. Using two different rat models of constipation that assess the effects of μ-opioid receptor agonists on stool hydration and gastro-intestinal motility, compound 3 produced insignificant constipation at 16 μmol/kg, whereas morphine elicited significant constipation at 2 μmol/kg. Compound 3 in contrast to morphine, did not attenuate the hypercapnic ventilatory response at 5 μmol/kg, a dose that fully alleviated hindpaw sensitivity at the time of peak effect in CCI-rats. This finding revealed the lack of respiratory depression effect at antinociceptive dose.     

Amphitropic proteins: regulation by reversible membrane interactions (Review)

The ability of apocytochrome c and the heme containing respiratory chain component, cytochrome c, to induce fusion of phosphatidylcholine (PC) small unilamellar vesicles containing 0–50 mol% negatively charged lipids was examined. Both molecules mediated fusion of phosphatidylserine (PS):PC 1:1 vesicles as measured by energy transfer changes between fluorescent lipid probes in a concentration- and pH-dependent manner, although cytochrome c was less potent and interacted over a more limited pH range than the apocytochrome c. Maximal fusion occurred at pH 3, far below the pK_a of the 19 lysine groups contained in the protein (pl = 10.5). A similar pH dependence was observed for vesicles containing 50 mol% cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylinositol (PI) in PC but the apparent pK_a values varied somewhat. In the absence of vesicles, the secondary structure of apocytochrome c was unchanged over this pH range, but in the presence of negatively charged vesicles, the polypeptide underwent a marked conformational change from random coil to α-helix. By comparing the pH dependencies of fusion induced by poly-L-lysine and apocytochrome c, we concluded that the pH dependence derived from changes in the net charge on both the vesicles and apocytochrome c. Aggregation could occur under conditions where fusion was imperceptible. Fusion increased with increasing mole ratio of PS. Apocytochrome c did induce some fusion of vesicles composed only of PC with a maximum effect at pH 4. Biosynthesis of cytochrome c involves translocation of apocytochrome c from the cytosol across the outer mitochondrial membrane to the outer mitochondrial space where the heme group is attached. The ability of apocytochrome c to induce fusion of both PS-containing and PC-only vesicles may reflect characteristics of protein/membrane interaction that pertain to its biological translocation.     

Use of monocyte/endothelial cell co-cultures (in vitro) and a subcutaneous implant mouse model (in vivo) to evaluate a degradable polar hydrophobic ionic polyurethane

Potential benefits of co-culturing monocytes (MC) with vascular smooth muscle cells have been reported on for tissue engineering applications with a degradable, polar, hydrophobic, and ionic polyurethane (D-PHI). Since the interaction of MC and endothelial cells (EC) within the blood vessel endothelium is also a process of wound repair it was of interest to investigate their function when cultured on the synthetic D-PHI materials, prior to considering the materials' use in vascular engineering. The co-culture (MC/EC) in vitro studies were carried out on films in 96 well plates and porous scaffold disks were prepared for implant studies in an in vivo subcutaneous mouse model. After 7 days in culture, the MC/EC condition was equal to EC growth but had lower esterase activity (a measure of degradative potential), no pro-inflammatory TNF-α and a relatively high anti-inflammatory IL-10 release while the ECs maintained their functional marker CD31. After explantation of the porous scaffolds, a live/dead stain showed that the cells infiltrating the scaffolds were viable and histological stains (May-Grunwald, Trichrome) demonstrated tissue in growth and extracellular matrix synthesis. Lysates from the implant scaffolds analyzed with a cytokine antibody array showed decreased pro-inflammatory cytokines (IL-6, TNF-α, GM-CSF), increased anti-inflammatory cytokines (IL-10, IL-13, TNF-RI), and increased chemotactic cytokines (MCP-1, MCP-5, RANTES). The low foreign body response elicited by D-PHI when implanted in vivo supported the in vitro studies (EC and MC co-culture), demonstrating that D-PHI promoted EC growth along with an anti-inflammatory MC, further demonstrating its potential as a tissue engineering scaffold for vascular applications.     

Microfluidic chip-based nanoelectrode array as miniaturized biochemical sensing platform for prostate-specific antigen detection

A microfluidic biosensor chip with an embedded three-electrode configuration is developed for the study of the voltammetric response of a nanoelectrode array with controlled inter-electrode distance in a nanoliter-scale sample volume. The on-chip three-electrode cell consists of a 5 × 5 array of Au working nanoelectrodes with radii between 60 and 120 nm, a Cl(2)-plasma-treated Ag/AgCl reference electrode, and a Au counter electrode. The nanoelectrode array is fabricated by creating high-aspect-ratio pores through an alumina insulating layer using an I(2) gas-assisted focused-ion-beam (FIB) milling, ion beam sculpting, and electrodeposition of Au. The glass substrate with the electrode pattern is assembled with a polydimethylsiloxane (PDMS) microchannel slab giving a volume of 180 nL for each channel. Cyclic voltammetry calibration with a standard redox species exhibits a significant increase of current density by two orders of magnitude compared to that obtained from a microelectrode. On-chip functionalization of the nanoelectrodes with a prostate-specific antigen (PSA) biosensor complex and detection of PSA based on a competitive immunoassay method are performed. The detection limit is approximately 10 pg/mL (～270 fM), which corresponds to roughly 30,000 copies of PSA in the microchannel test volume.     

Discovery, optimisation and in vivo evaluation of novel GPR119 agonists

GPR119 is increasingly seen as an attractive target for the treatment of type II diabetes and other elements of the metabolic syndrome. During a programme aimed at developing agonists of the GPR119 receptor, we identified compounds that were potent with reduced hERG liabilities, that had good pharmacokinetic properties and that displayed excellent glucose-lowering effects in vivo. However, further profiling in a GPR119 knock-out (KO) mouse model revealed that the biological effects were not exclusively due to GPR119 agonism, highlighting the value of transgenic animals in drug discovery programs.     

Discovery of non-glucoside SGLT2 inhibitors

A series of benzothiazinone and benzooxazinone derivatives were discovered as SGLT2 inhibitors. The optimization led to the discovery of compounds 31 and 32, which exhibited similar potency and better SGLT1 selectivity compared to dapagliflozin. These compounds may provide novel promising scaffolds, which are different from phlorizin-based SGLT2 inhibitors.