Luminescent reporter cells enable the identification of broad-spectrum antivirals against emerging viruses.

The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

A proopiomelanocortin-derived peptide sequence enhances plasma stability of peptide drugs.

Bioactive peptide drugs hold promise for therapeutic application due to their high potency and selectivity but display short plasma half-life. Examination of selected naturally occurring peptide hormones derived from proteolytic cleavage of the proopiomelanocortin (POMC) precursor lead to the identification of significant plasma-stabilizing properties of a 12-amino acid serine-rich orphan sequence NSSSSGSSGAGQ in human γ3-melanocyte-stimulating hormone (MSH) that is homologous to previously discovered NSn GGH (n = 4-24) sequences in owls. Notably, transfer of this sequence to des-acetyl-α-MSH and the therapeutically relevant peptide hormones neurotensin and glucagon-like peptide-1 likewise enhance their plasma stability without affecting receptor signaling. The stabilizing effect of the sequence module is independent of plasma components, suggesting a direct effect in cis. This natural sequence module may provide a possible strategy to enhance plasma stability, complementing existing methods of chemical modification.

Direct and retrograde transduction of nigral neurons with AAV6, 8, and 9 and intraneuronal persistence of viral particles.

Recombinant adeno-associated viral (AAV) vectors of serotypes 6, 8, and 9 were characterized as tools for gene delivery to dopaminergic neurons in the substantia nigra for future gene therapeutic applications in Parkinson's disease. While vectors of all three serotypes transduced nigral dopaminergic neurons with equal efficiency when directly injected to the substantia nigra, AAV6 was clearly superior to AAV8 and AAV9 for retrograde transduction of nigral neurons after striatal delivery. For sequential transduction of nigral dopaminergic neurons, the combination of AAV9 with AAV6 proved to be more powerful than AAV8 with AAV6 or repeated AAV6 administration. Surprisingly, single-stranded viral genomes persisted in nigral dopaminergic neurons within cell bodies and axon terminals in the striatum, and intact assembled AAV capsid was enriched in nuclei of nigral neurons, 4 weeks after virus injections to the substantia nigra. 6-Hydroxydopamine (6-OHDA)-induced degeneration of dopaminergic neurons in the substantia nigra reduced the number of viral genomes in the striatum, in line with viral genome persistence in axon terminals. However, 6-OHDA-induced axonal degeneration did not induce any transsynaptic spread of AAV infection in the striatum. Therefore, the potential presence of viral particles in axons may not represent an important safety issue for AAV gene therapy applications in neurodegenerative diseases.

Cytotoxicity of Triptolide and Triptolide loaded polymeric micelles in vitro.

Triptolide (TP), a diterpenoid triepoxide purified from the Chinese herb Tripterygium wilfordii Hook F is characterized by strong anti-tumor effects on various cancer cells. Except its anti-tumor effects, TP also shows multiple pharmacological side activities, such as anti-inflammatory, immune-suppressive and male anti-fertility. In order to reduce these side effects, especially the immuno-suppressive activity when used to cure cancer, a novel polymeric micelle system containing TP (TP-PM) was constructed. The immune-modulation effects of TP-PM have been evaluated by previous studies. In this study, we compared the cytotoxicity of TP and TP-PM on Jurkat and HT29 cells. Therefore, we determined the cell viability, membrane integrity, cell proliferation, apoptosis, and caspase 3/7 activity after exposure to TP and TP-PM. The results demonstrated that actually low concentrated TP and TP-PM could induce an inhibition of cell growth and proliferation as well as membrane damage in both tumor cell lines. TP and TP-PM induced apoptosis and caused activation of caspase 3/7 even at low concentrations. Both formulations destroyed the membrane of Jurkat cells, nevertheless, TP-PM showed stronger pernicious effects. In general, TP-PM induced in both tested cell lines stronger effects than TP. Therefore, polymeric micelles can be considered as promising carriers for TP in cancer therapy.

Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells.

The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

Targeted human serum albumin nanoparticles for specific uptake in EGFR-Expressing colon carcinoma cells.

The specific application and transport of drugs into malignant tissue is a critical point during diagnosis and therapy. Nanoparticles are known as excellent drug carrier systems and offer the possibility of surface modification with targeting ligands, leading to a specific accumulation in the targeted tissue. First, the specificity of such a carrier system has to be proven. In this study, cetuximab-modified nanoparticles based on biodegradable human serum albumin (HSA) are investigated regarding their cellular binding and intracellular accumulation. Different EGFR-expressing colon carcinoma cells were used to test possible cytotoxic potential, specific binding and intracellular accumulation. A specific accumulation targeting the EGFR could be shown. These results emphasize that cetuximab-modified HSA-nanoparticles are a promising carrier system for later drug transport. To our knowledge, this is the first study investigating the specific accumulation of HSA nanoparticles into different EGFR-expressing colon carcinoma cells. FROM THE CLINICAL EDITOR: In this study, cetuximab-modified nanoparticles based on human serum albumin (HSA) are investigated regarding their cellular binding and intracellular accumulation. The results suggest that these nanoparticles are a promising carrier system for EGFR overexpressing colon carcinoma cells.

Induction of corticospinal regeneration by lentiviral trkB-induced Erk activation.

Several experimental manipulations of the CNS environment successfully elicit regeneration of sensory and bulbospinal motor axons but fail to elicit regeneration of corticospinal axons, suggesting that cell-intrinsic mechanisms limit the regeneration of this critical class of motor neurons. We hypothesized that enhancement of intrinsic neuronal growth mechanisms would enable adult corticospinal motor axon regeneration. Lentiviral vectors were used to overexpress the BDNF receptor trkB in layer V corticospinal motor neurons. After subcortical axotomy, trkB transduction induced corticospinal axon regeneration into subcortical lesion sites expressing BDNF. In the absence of trkB overexpression, no regeneration occurred. Selective deletion of canonical, trkB-mediated neurite outgrowth signaling by mutation of the Shc/FRS-2 activation domain prohibited Erk activation and eliminated regeneration. These findings support the hypothesis that the refractory regenerative state of adult corticospinal axons can be attributed at least in part to neuron-intrinsic mechanisms, and that activation of ERK signaling can elicit corticospinal tract regeneration.

Netrin-1 is a novel myelin-associated inhibitor to axon growth.

We investigated the influence of the bifunctional guidance molecule netrin-1 on axonal growth in the injured adult spinal cord. In the adult, netrin-1 is expressed on mature oligodendrocytes, cells of the central canal, and the meninges. Netrin-1 protein in white matter is selectively enriched adjacent to paranodal loops of myelin in nodes of Ranvier. The repulsion-mediating netrin-1 uncoordinated-5 (UNC5) receptors are expressed by neurons of the corticospinal and rubrospinal projections, and by intrinsic neurons of the spinal cord, both before and after spinal cord injury. Neutralization of netrin-1 in myelin prepared from adult rat spinal cord using UNC5 receptor bodies increases neurite outgrowth from UNC5-expressing spinal motor neurons in vitro. Furthermore, axon regeneration is inhibited in a netrin-1-enriched zone, devoid of other myelin-associated inhibitors, within spinal cord lesion sites in vivo. We conclude that netrin-1 is a novel oligodendrocyte-associated inhibitor that can contribute to axonal growth failure after adult spinal cord injury.