Pain, although unpleasant, is an essential warning mechanism against injury and damage of the organism. An intricate network of specialised sensors and transmission systems contributes to reception, ...transmission and central sensitization of pain. Here, we briefly introduce some of the main aspects of pain signal transmission, including nociceptors and nociceptive signals, mechanisms of inflammatory and neuropathic pain, and the situation of diabetes-associated neuropathic pain. The role of glia-astrocytes, microglia, satellite glia cells-and their specific channels, transporters and signaling pathways is described. A focus is on the contribution of inhibitory synaptic signaling to nociception and a possible role of glycine receptors in glucose-mediated analgesia and treatment-induced diabetic neuropathy. Inhibitory receptors such as GABA
- and glycine receptors are important contributors to nociceptive signaling; their contribution to altered pain sensation in diabetes may be of clinical relevance, and they could be promising therapeutic targets towards the development of novel analgesics.
Viroporins are indispensable for viral replication. As intracellular ion channels they disturb pH gradients of organelles and allow Ca2+ flux across ER membranes. Viroporins interact with numerous ...intracellular proteins and pathways and can trigger inflammatory responses. Thus, they are relevant targets in the search for antiviral drugs. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underlies the world-wide pandemic of COVID-19, where an effective therapy is still lacking despite impressive progress in the development of vaccines and vaccination campaigns. Among the 29 proteins of SARS-CoV-2, the E- and ORF3a proteins have been identified as viroporins that contribute to the massive release of inflammatory cytokines observed in COVID-19. Here, we describe structure and function of viroporins and their role in inflammasome activation and cellular processes during the virus replication cycle. Techniques to study viroporin function are presented, with a focus on cellular and electrophysiological assays. Contributions of SARS-CoV-2 viroporins to the viral life cycle are discussed with respect to their structure, channel function, binding partners, and their role in viral infection and virus replication. Viroporin sequences of new variants of concern (α–ο) of SARS-CoV-2 are briefly reviewed as they harbour changes in E and 3a proteins that may affect their function.
SARS-CoV-2 has been responsible for the major worldwide pandemic of COVID-19. Despite the enormous success of vaccination campaigns, virus infections are still prevalent and effective antiviral ...therapies are urgently needed. Viroporins are essential for virus replication and release, and are thus promising therapeutic targets. Here, we studied the expression and function of recombinant ORF3a viroporin of SARS-CoV-2 using a combination of cell viability assays and patch-clamp electrophysiology. ORF3a was expressed in HEK293 cells and transport to the plasma membrane verified by a dot blot assay. Incorporation of a membrane-directing signal peptide increased plasma membrane expression. Cell viability tests were carried out to measure cell damage associated with ORF3a activity, and voltage-clamp recordings verified its channel activity. The classical viroporin inhibitors amantadine and rimantadine inhibited ORF3a channels. A series of ten flavonoids and polyphenolics were studied. Kaempferol, quercetin, epigallocatechin gallate, nobiletin, resveratrol and curcumin were ORF3a inhibitors, with IC
values ranging between 1 and 6 µM, while 6-gingerol, apigenin, naringenin and genistein were inactive. For flavonoids, inhibitory activity could be related to the pattern of OH groups on the chromone ring system. Thus, the ORF3a viroporin of SARS-CoV-2 may indeed be a promising target for antiviral drugs.
Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics and chemotherapy in the treatment of multidrug‐resistant pathogens and drug‐resistant cancers. Clinical ...application of AMPs is limited due to low stability and inefficient transport. Encapsulation in nanocarriers may improve their therapeutic potential. Chitosan nanoparticles (CS‐NPs) are efficient carriers for proteins and peptides, improving the treatment of microbial infections and targeted drug delivery. We examined toxicity against cancer cell lines and antibacterial activities of the pleurocidin‐like AMP NRC‐07 upon encapsulation in CS‐NPs by ionotropic gelation. The biological activities of various formulations of free and encapsulated NRC‐07 and free nanoparticles were evaluated against Pseudomonas aeruginosa and breast cancer cells, using assays for cell viability and lactate dehydrogenase cytolysis with non‐cancer cell lines as controls. NRC‐07‐containing nanoparticles decreased the bacterial and cancer cell viability in a concentration‐dependent manner. Activities of encapsulated peptide were >2‐fold higher than those of free NRC‐07 peptide. Unloaded CS‐NPs and free peptide were not cytotoxic against control cells. Encapsulation of NRC‐07 into CS‐NPs enhanced the antibacterial and selective cytotoxicity of the peptide, possibly enhancing anticancer activities. Encapsulation presents a promising tool for the development of efficient drug delivery systems.
The pleurocidin‐like antimicrobial peptide NRC‐07 was encapsulated in chitosan nanoparticles (CS‐NP) by ionotropic gelation and examined for its toxicity against cancer cell lines and antibacterial activities. Encapsulation of NRC‐07 into CS‐NPs enhanced the antibacterial and selective cytotoxicity of the peptide, possibly enhancing anticancer activities.
SARS-CoV-2 has caused a worldwide pandemic since December 2019 and the search for pharmaceutical targets against COVID-19 remains an important challenge. Here, we studied the envelope protein E of ...SARS-CoV and SARS-CoV-2, a highly conserved 75-76 amino acid viroporin that is crucial for virus assembly and release. E protein channels were recombinantly expressed in HEK293 cells, a membrane-directing signal peptide ensured transfer to the plasma membrane.
Viroporin channel activity of both E proteins was investigated using patch-clamp electrophysiology in combination with a cell viability assay. We verified inhibition by classical viroporin inhibitors amantadine, rimantadine and 5-(N,N-hexamethylene)-amiloride, and tested four ivermectin derivatives.
Classical inhibitors showed potent activity in patch-clamp recordings and viability assays. In contrast, ivermectin and milbemycin inhibited the E channel in patch-clamp recordings but displayed only moderate activity on the E protein in the cell viability assay, which is also sensitive to general cytotoxic activity of the tested compounds. Nemadectin and ivermectin aglycon were inactive. All ivermectin derivatives were cytotoxic at concentrations > 5 µM, i.e. below the level required for E protein inhibition.
This study demonstrates direct inhibition of the SARS-CoV-2 E protein by classical viroporin inhibitors. Ivermectin and milbemycin inhibit the E protein channel but their cytotoxicity argues against clinical application.
Severe acute respiratory syndrome coronavirus (SARS-CoV), an enveloped single-stranded positive-sense RNA virus, is a member of the genus
Betacoronavirus
, family Coronaviridae. The SARS-CoV envelope ...protein E is a small (∼8.4 kDa) channel-forming membrane protein whose sequence is highly conserved between SARS-CoV and SARS-CoV-2. As a viroporin, it is involved in various aspects of the virus life cycle including assembly, budding, envelope formation, virus release, and inflammasome activation. Here, SARS-CoV E protein was recombinantly expressed in HEK293 cells and channel activity and the effects of viroporin inhibitors studied using patch-clamp electrophysiology and a cell viability assay. We introduced a membrane-directing signal peptide to ensure transfer of recombinant E protein to the plasma membrane. E protein expression induced transmembrane currents that were blocked by various inhibitors. In an ion-reduced buffer system, currents were proton-dependent and blocked by viroporin inhibitors rimantadine and amantadine. I-V relationships of recombinant E protein were not pH-dependent in a classical buffer system with high extracellular Na
+
and high intracellular K
+
. E-protein mediated currents were inhibited by amantadine and rimantadine, as well as 5-(N,N-hexamethylene)amiloride (HMA). We tested a total of 10 flavonoids, finding inhibitory activity of varying potency. Epigallocatechin and quercetin were most effective, with IC
50
values of 1.5 ± 0.1 and 3.7 ± 0.2 nM, respectively, similar to the potency of rimantadine (IC
50
= 1.7 ± 0.6 nM). Patch-clamp results were independently verified using a modified cell viability assay for viroporin inhibitors. These results contribute to the development of novel antiviral drugs that suppress virus activity and proliferation.
Reduced cell surface expression or the malfunctioning of ion channels gives rise to a group of disorders known as channelopathies. To treat the underlying cause, the delivery and/or expression of a ...functional ion channel into the cell membrane of the cell of interest is required. Unfortunately, for most channelopathies, current treatment options are only symptomatic and treatments that rectify the underlying damage are still lacking. Within this context, approaches that rely on gene and protein therapy are required. Gene therapy would allow the expression of a functional protein, provided that the cellular machinery in the diseased cell could correctly fold and traffic the protein to the cell membrane. Whereas protein therapy would allow the direct delivery of a functional protein, provided that the purification process does not affect protein function and a suitable delivery vehicle for targeted delivery is used. In this review, we provide an overview of channelopathies and available symptomatic treatments. The current state of gene therapy approaches mainly using viral vectors is discussed, which is followed by the role of nanomedicine in protein therapy and how nanomedicine could be exploited for the delivery of functional ion channels to diseased cells.
The inhibitory glycine receptor is a member of the Cys-loop superfamily of ligand-gated ion channels. It is the principal mediators of rapid synaptic inhibition in spinal cord and brainstem and plays ...an important role in the modulation of higher brain functions including vision, hearing and pain signaling. Glycine receptor function is controlled by only few agonists, while the number of antagonists, and positive or biphasic modulators is steadily increasing. These modulators are important for the study of receptor activation and regulation, and have found clinical interest as potential analgesics and anticonvulsants. High-resolution structures of the receptor have become available recently, adding to our understanding of structure-function relationships and revealing agonistic, inhibitory and modulatory sites on the receptor protein. This review presents an overview of compounds that activate, inhibit, or modulate glycine receptor function in vitro and in vivo.
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Channelopathies are disorders caused by reduced expression or impaired function of ion channels. Most current therapies rely on symptomatic treatment without addressing the underlying ...cause. We have recently established proof of principle for delivery of functional ion channel protein into the membrane of target cells using fusogenic liposomes incorporating glycine receptor (GlyR)-containing cell membrane fragments (CMF) that were formulated by thin film hydration. Here, the effect of liposome size and the formulation technique on the performance of the delivery vehicle was assessed. Three types of liposomes were prepared using lecithin and cholesterol, (i) small (SL), and (ii) large (LL) liposomes made by thin film hydration, and (iii) small liposomes prepared by vortex agitation (V-SL). All liposomes were evaluated for their ability to (i) incorporate GlyR-rich CMF, (ii) fuse with the cell membrane of target cells and (iii) deliver functional GlyR, as assessed by patch-clamp electrophysiology. SL prepared by thin film hydration offered the most effective delivery of glycine receptors that gave clear glycine-mediated currents in target cells. LL showed higher incorporation of CMF, but did not effectively fuse with the target cell membrane, while V-SL did not incorporate sufficient amounts of CMF. Additionally, SL showed minimalin vivotoxicity upon intrathecal injection in mice. Thus, liposome-mediated delivery of membrane proteins may be a promising therapeutic approach for the treatment of channelopathies.
Mg–Ca alloys have recently attracted great attention towards the research in the field of orthopedic biodegradable implants. This study presents an in vitro degradation assessment of Mg–0.8Ca ...(0.8 wt. % of Ca) alloy in Hank's balanced salt solution (HBSS). Immersion, hydrogen evolution and electrochemical behavior was studied as well as the cytotoxicity of the degradation products. Morphology and phase composition of the corrosion products were studied using SEM, EDX and XRD techniques. Degradation in HBSS resulted in the formation of the needle-shaped carbonated hydroxyapatite which was similar to the biological apatite in the human bone. Degradation kinetics showed that Mg–0.8Ca alloy had approximately 3-fold faster degradation rate than the pure Mg (1.08 ± 0.38 mm/year for Mg–0.8Ca and 0.35 ± 0.17 mm/year for pure Mg), as observed in two independent experiments. Both, pure Mg and Mg–0.8Ca alloy were biocompatible, generating no cytotoxic degradation products against human-derived HEK 293 cells. Thus, the Mg–0.8Ca alloy was found to be a promising biodegradable implant in terms of bioactivity and compatibility with human cell lines. Depending on the application of the implant and the estimated healing time of the bone, the desired degradation rate of an implant can be controlled by the Mg–Ca composition of such alloys.
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