Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models.

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

Harnessing the Physiological Functions of Cellular Prion Protein in the Kidneys: Applications for Treating Renal Diseases.

A cellular prion protein (PrPC) is a ubiquitous cell surface glycoprotein, and its physiological functions have been receiving increased attention. Endogenous PrPC is present in various kidney tissues and undergoes glomerular filtration. In prion diseases, abnormal prion proteins are found to accumulate in renal tissues and filtered into urine. Urinary prion protein could serve as a diagnostic biomarker. PrPC plays a role in cellular signaling pathways, reno-protective effects, and kidney iron uptake. PrPC signaling affects mitochondrial function via the ERK pathway and is affected by the regulatory influence of microRNAs, small molecules, and signaling proteins. Targeting PrPC in acute and chronic kidney disease could help improve iron homeostasis, ameliorate damage from ischemia/reperfusion injury, and enhance the efficacy of mesenchymal stem/stromal cell or extracellular vesicle-based therapeutic strategies. PrPC may also be under the influence of BMP/Smad signaling and affect the progression of TGF-ß-related renal fibrosis. PrPC conveys TNF-α resistance in some renal cancers, and therefore, the coadministration of anti-PrPC antibodies improves chemotherapy. PrPC can be used to design antibody-drug conjugates, aptamer-drug conjugates, and customized tissue inhibitors of metalloproteinases to suppress cancer. With preclinical studies demonstrating promising results, further research on PrPC in the kidney may lead to innovative PrPC-based therapeutic strategies for renal disease.

Tackling prion diseases: a review of the patent landscape.

Introduction: Prion diseases are a class of rare and fatal neurodegenerative diseases for which no cure is currently available. They are characterized by conformational conversion of cellular prion protein (PrPC) into the disease-associated 'scrapie' isoform (PrPSc). Under an etiological point of view, prion diseases can be divided into acquired, genetic, and idiopathic form, the latter of which are the most frequent.Areas covered: Therapeutic approaches targeting prion diseases are based on the use of chemical and nature-based compounds, targeting either PrPC or PrPSc or other putative player in pathogenic mechanism. Other proposed anti-prion treatments include passive and active immunization strategies, peptides, aptamers, and PrPC-directed RNA interference techniques. The treatment efficacy has been mainly assessed in cell lines or animal models of the disease testing their ability to reduce prion accumulation.Expert opinion: The assessed strategies focussing on the identification of an efficient anti-prion therapy faced various issues, which go from permeation of the blood brain barrier to immunological tolerance of the host. Indeed, the use of combinatory approaches, which could boost a synergistic anti-prion effect and lower the potential side effects of single treatments and may represent an extreme powerful and feasible way to tackle prion disease.

Vaporized Hydrogen Peroxide and Ozone Gas Synergistically Reduce Prion Infectivity on Stainless Steel Wire.

Prions are infectious agents causing prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans. Several cases have been reported to be transmitted through medical instruments that were used for preclinical CJD patients, raising public health concerns on iatrogenic transmissions of the disease. Since preclinical CJD patients are currently difficult to identify, medical instruments need to be adequately sterilized so as not to transmit the disease. In this study, we investigated the sterilizing activity of two oxidizing agents, ozone gas and vaporized hydrogen peroxide, against prions fixed on stainless steel wires using a mouse bioassay. Mice intracerebrally implanted with prion-contaminated stainless steel wires treated with ozone gas or vaporized hydrogen peroxide developed prion disease later than those implanted with control prion-contaminated stainless steel wires, indicating that ozone gas and vaporized hydrogen peroxide could reduce prion infectivity on wires. Incubation times were further elongated in mice implanted with prion-contaminated stainless steel wires treated with ozone gas-mixed vaporized hydrogen peroxide, indicating that ozone gas mixed with vaporized hydrogen peroxide reduces prions on these wires more potently than ozone gas or vaporized hydrogen peroxide. These results suggest that ozone gas mixed with vaporized hydrogen peroxide might be more useful for prion sterilization than ozone gas or vaporized hydrogen peroxide alone.

Ellagic acid and pentagalloylglucose are potential inhibitors of prion protein fibrillization.

Prion diseases are fatal neurodegenerative diseases caused by the conformational transition of the cellular prion protein (PrPC) to the abnormal pathological prion protein (PrPSc). In this work, the effects of ellagic acid (EA) and pentagalloylglucose (PGG) on prion protein (PrP) fibrillization were investigated. Fluorescence quenching experiments indicated that both EA and PGG could specifically interact with native human PrP with binding affinities of 1.92 × 105 and 2.36 × 105 L·mol-1, respectively. Thioflavin-T (ThT) fluorescence assays showed that the binding of EA or PPG could effectively inhibit the nucleation and elongation of PrP fibrilization and reduce the amount of PrP fibrils generated. EA and PGG could also lead to a significant disaggregation of PrP fibrils. Circular dichroism (CD) measurements suggested that EA- or PPG-bound PrP could preserve a higher content of α-helical structures than ß-sheet-rich PrP fibrils. The PrP aggregates formed in the presence of EA or PGG showed lower resistance to proteinase K (PK) digestion. Overall, the present work reported the inhibitory effect of EA and PGG on PrP fibrillization. These two natural polyphenols could be potential prodrug molecules for the prevention and treatment of prion diseases.

Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice.

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.

Anti-PrPC antibody rescues cognition and synapses in transgenic alzheimer mice.

Objective: Amyloid-beta oligomers (Aßo) trigger the development of Alzheimer's disease (AD) pathophysiology. Cellular prion protein (PrPC) initiates synaptic damage as a high affinity receptor for Aßo. Here, we evaluated the preclinical therapeutic efficacy of a fully human monoclonal antibody against PrPC. This AZ59 antibody selectively targets the Aßo binding site in the amino-terminal unstructured domain of PrPC to avoid any potential risk of direct toxicity. Methods: Potency of AZ59 was evaluated by binding to PrPC, blockade of Aßo interaction and interruption of Aßo signaling. AZ59 was administered to mice by weekly intraperitoneal dosing and brain antibody measured. APP/PS1 transgenic mice were treated with AZ59 and assessed by memory tests, by brain biochemistry and by histochemistry for Aß, gliosis and synaptic density. Results: AZ59 binds PrPC with 100 pmol/L affinity and blocks human brain Aßo binding to PrPC, as well as prevents synaptotoxic signaling. Weekly i.p. dosing of 20 mg/kg AZ59 in a murine form achieves trough brain antibody levels greater than 10 nmol/L. Aged symptomatic APP/PS1 transgenic mice treated with AZ59 for 5-7 weeks show a full rescue of behavioral and synaptic loss phenotypes. This recovery occurs without clearance of plaque pathology or elimination of gliosis. AZ59 treatment also normalizes synaptic signaling abnormalities in transgenic brain. These benefits are dose-dependent and persist for at least 1 month after the last dose. Interpretation: Preclinical data demonstrate that systemic AZ59 therapy rescues central synapses and memory function from transgenic Alzheimer's disease pathology, supporting a disease-modifying therapeutic potential.

Melatonin Promotes Apoptosis of Oxaliplatin-resistant Colorectal Cancer Cells Through Inhibition of Cellular Prion Protein.

BACKGROUND/AIM: Drug resistance restricts the efficacy of chemotherapy in colorectal cancer. However, the detailed molecular mechanism of drug resistance in colorectal cancer cells remains unclear. MATERIALS AND METHODS: The level of cellular prion protein (PrPC) in oxaliplatin-resistant colorectal cancer (SNU-C5/Oxal-R) cells was assessed. RESULTS: PrPC level in SNU-C5/Oxal-R cells was significantly increased compared to that in wild-type (SNU-C5) cells. Superoxide dismutase and catalase activities were higher in SNU-C5/Oxal-R cells than in SNU-C5 cells. Treatment of SNU-C5/Oxal-R cells with oxaliplatin and melatonin reduced PrPC expression, while suppressing antioxidant enzyme activity and increasing superoxide anion generation. In SNU-C5/Oxal-R cells, endoplasmic reticulum stress and apoptosis were significantly increased following co-treatment with oxaliplatin and melatonin compared to treatment with oxaliplatin alone. CONCLUSION: Co-treatment with oxaliplatin and melatonin increased endoplasmic reticulum stress in and apoptosis of SNU-C5/Oxal-R cells through inhibition of PrPC, suggesting that PrPC could be a key molecule in oxaliplatin resistance of colorectal cancer cells.

Molecular Dynamics Simulation Study on the Binding and Stabilization Mechanism of Antiprion Compounds to the "Hot Spot" Region of PrPC.

Structural transitions in the prion protein from the cellular form, PrPC, into the pathological isoform, PrPSc, are regarded as the main cause of the transmissible spongiform encephalopathies, also known as prion diseases. Hence, discovering and designing effective antiprion drugs that can inhibit PrPC to PrPSc conversion is regarded as a promising way to cure prion disease. Among several strategies to inhibit PrPC to PrPSc conversion, stabilizing the native PrPC via specific binding is believed to be one of the valuable approaches and many antiprion compounds have been reported based on this strategy. However, the detailed mechanism to stabilize the native PrPC is still unknown. As such, to unravel the stabilizing mechanism of these compounds to PrPC is valuable for the further design and discovery of antiprion compounds. In this study, by molecular dynamics simulation method, we investigated the stabilizing mechanism of several antiprion compounds on PrPC that were previously reported to have specific binding to the "hot spot" region of PrPC. Our simulation results reveal that the stabilization mechanism of specific binding compounds can be summarized as (I) to stabilize both the flexible C-terminal of α2 and the hydrophobic core, such as BMD42-29 and GN8; (II) to stabilize the hydrophobic core, such as J1 and GJP49; (III) to stabilize the overall structure of PrPC by high binding affinity, as NPR-056. In addition, as indicated by the H-bond analysis and decomposition analysis of binding free energy, the residues N159 and Q160 play an important role in the specific binding of the studied compounds and all these compounds interact with PrPC in a similar way with the key interacting residues L130 in the ß1 strand, P158, N159, Q160, etc. in the α1-ß2 loop, and H187, T190, T191, etc. in the α2 C-terminus although the compounds have large structural difference. As a whole, our obtained results can provide some insights into the specific binding mechanism of main antiprion compounds to the "hot spot" region of PrPC at the molecular level and also provide guidance for effective antiprion drug design in the future.

Amyloid-ß Receptors: The Good, the Bad, and the Prion Protein.

Several different receptor proteins have been identified that bind monomeric, oligomeric, or fibrillar forms of amyloid-ß (Aß). "Good" receptors internalize Aß or promote its transcytosis out of the brain, whereas "bad" receptors bind oligomeric forms of Aß that are largely responsible for the synapticloss, memory impairments, and neurotoxicity that underlie Alzheimer disease. The prion protein both removes Aß from the brain and transduces the toxic actions of Aß. The clustering of distinct receptors in cell surface signaling platforms likely underlies the actions of distinct oligomeric species of Aß. These Aß receptor-signaling platforms provide opportunities for therapeutic intervention in Alzheimer disease.

Discovery of Novel Anti-prion Compounds Using In Silico and In Vitro Approaches.

Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrP(C)) to the pathogenic form, PrP(Sc). Compounds that inhibit this process by blocking conversion to the PrP(Sc) could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrP(C) interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrP(Sc) levels and one of these compounds also showed a high binding affinity for PrP(C). These results provide a promising starting point for the development of anti-prion compounds.

Dextran sulfate sodium inhibits amyloid-ß oligomer binding to cellular prion protein.

Amyloid-ß peptide (Aß), especially its oligomeric form, is believed to play an important role in the pathogenesis of Alzheimer's disease (AD). To this end, the binding of Aß oligomer to cellular prion protein (PrP(C)) plays an important role in synaptic dysfunction in a mouse model of AD. Here, we have screened for compounds that inhibit Aß oligomer binding to PrP(C) from medicines already used clinically (Mizushima Approved Medicine Library 1), and identified dextran sulfate sodium (DSS) as a candidate. In a cell-free assay, DSS inhibited Aß oligomer binding to PrP(C) but not to ephrin receptor B2, another endogenous receptor for Aß oligomers, suggesting that the drug's action is specific to the binding of Aß oligomer to PrP(C) . Dextran on the other hand did not affect this binding. DSS also suppressed Aß oligomer binding to cells expressing PrP(C) but not to control cells. Furthermore, while incubation of mouse hippocampal slices with Aß oligomers inhibited the induction of long-term potentiation, simultaneous treatment with DSS restored the long-term potentiation. As DSS has already been approved for use in patients with hypertriglyceridemia, and its safety in humans has been confirmed, we propose further analysis of this drug as a candidate for AD treatment. Amyloid-ß peptide (Aß) oligomer-binding to cellular prion protein (PrP(C) ) is important in synaptic dysfunction in Alzheimer's disease (AD). We found here that dextran sulfate sodium (DSS) inhibits Aß oligomer binding to PrP(C) . Simultaneous treatment of hippocampal slices with DSS restored long-term potentiation (LTP) in the presence of Aß oligomers. Since DSS has already been approved for clinical use, we propose this drug is a candidate drug for AD treatment.

In silico studies and fluorescence binding assays of potential anti-prion compounds reveal an important binding site for prion inhibition from PrP(C) to PrP(Sc).

To understand the pharmacophore properties of 2-aminothiazoles and design novel inhibitors against the prion protein, a highly predictive 3D quantitative structure-activity relationship (QSAR) has been developed by performing comparative molecular field analysis (CoMFA) and comparative similarity analysis (CoMSIA). Both CoMFA and CoMSIA maps reveal the presence of the oxymethyl groups in meta and para positions on the phenyl ring of compound 17 (N-[4-(3,4-dimethoxyphenyl)-1,3-thiazol-2-yl]quinolin-2-amine), is necessary for activity while electro-negative nitrogen of quinoline is highly favorable to enhance activity. The blind docking results for these compounds show that the compound with quinoline binds with higher affinity than isoquinoline and naphthalene groups. Out of 150 novel compounds retrieved using finger print analysis by pharmacophoric model predicted based on five test sets of compounds, five compounds with diverse scaffolds were selected for biological evaluation as possible PrP inhibitors. Molecular docking combined with fluorescence quenching studies show that these compounds bind to pocket-D of SHaPrP near Trp145. The new antiprion compounds 3 and 6, which bind with the interaction energies of -12.1 and -13.2 kcal/mol, respectively, show fluorescence quenching with binding constant (Kd) values of 15.5 and 44.14 µM, respectively. Further fluorescence binding assays with compound 5, which is similar to 2-aminothiazole as a positive control, also show that the molecule binds to the pocket-D with the binding constant (Kd) value of 84.7 µM. Finally, both molecular docking and a fluorescence binding assay of noscapine as a negative control reveals the same binding site on the surface of pocket-A near a rigid loop between ß2 and α2 interacting with Arg164. This high level of correlation between molecular docking and fluorescence quenching studies confirm that these five compounds are likely to act as inhibitors for prion propagation while noscapine might act as a prion accelerator from PrP(C) to PrP(Sc).

Comparison of the anti-prion mechanism of four different anti-prion compounds, anti-PrP monoclonal antibody 44B1, pentosan polysulfate, chlorpromazine, and U18666A, in prion-infected mouse neuroblastoma cells.

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP(Sc)) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP(Sc) formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP(C)) and PrP(Sc) in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP(Sc) levels without altering intracellular distribution of PrP(Sc). PPS did not change the distribution and levels of PrP(C), whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP(C) to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP(Sc) formation. In contrast, CPZ and U18666A initiated the redistribution of PrP(Sc) from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP(C). The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP(Sc) redistribution by CPZ or U18666A partly antagonized PrP(Sc) degradation, suggesting that the transfer of PrP(Sc) to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP(Sc) degradation. This study revealed that precise analysis of the intracellular dynamics of PrP(C) and PrP(Sc) provides important information for understanding the mechanism of anti-prion agents.

Therapeutic molecules and endogenous ligands regulate the interaction between brain cellular prion protein (PrPC) and metabotropic glutamate receptor 5 (mGluR5).

Soluble Amyloid-ß oligomers (Aßo) can trigger Alzheimer disease (AD) pathophysiology by binding to cell surface cellular prion protein (PrP(C)). PrP(C) interacts physically with metabotropic glutamate receptor 5 (mGluR5), and this interaction controls the transmission of neurotoxic signals to intracellular substrates. Because the interruption of the signal transduction from PrP(C) to mGluR5 has therapeutic potential for AD, we developed assays to explore the effect of endogenous ligands, agonists/antagonists, and antibodies on the interaction between PrP(C) and mGluR5 in cell lines and mouse brain. We show that the PrP(C) segment of amino acids 91-153 mediates the interaction with mGluR5. Agonists of mGluR5 increase the mGluR5-PrP(C) interaction, whereas mGluR5 antagonists suppress protein association. Synthetic Aßo promotes the protein interaction in mouse brain and transfected HEK-293 cell membrane preparations. The interaction of PrP(C) and mGluR5 is enhanced dramatically in the brains of familial AD transgenic model mice. In brain homogenates with Aßo, the interaction of PrP(C) and mGluR5 is reversed by mGluR5-directed antagonists or antibodies directed against the PrP(C) segment of amino acids 91-153. Silent allosteric modulators of mGluR5 do not alter Glu or basal mGluR5 activity, but they disrupt the Aßo-induced interaction of mGluR5 with PrP(C). The assays described here have the potential to identify and develop new compounds that inhibit the interaction of PrP(C) and mGluR5, which plays a pivotal role in the pathogenesis of Alzheimer disease by transmitting the signal from extracellular Aßo into the cytosol.

Computational studies on the prion protein.

Prion diseases are rare neurodegenerative diseases characterized by the conversion of the prion protein from its native state (PrP(C)) towards the so-called 'scrapie form', rich in ß-strands. Computational approaches, here briefly reviewed, are instrumental to understand the intrinsic instability of PrP(C) fold and how the latter is affected by mutations, binding of metals as well as by different environmental conditions, such as pH and temperature. These studies also provide a structural basis for the binding of anti-prion compounds, which may block the conversion to the scrapie form and, consequently, may inhibit fibril formation.

Strain specificity and drug resistance in anti-prion therapy.

Prion diseases are a group of fatal neurodegenerative diseases caused by the misfolding of cellular prion protein (PrP(C)) into pathogenic conformers (PrP(Sc)). Although no effective therapies for prion diseases are currently available, a number of small molecule inhibitors have been identified that are capable of reducing or eliminating PrP(Sc) in prion infected cells. However, recent experiments have shown that upon sustained treatment, prions have the capacity to evolve into drug resistant conformations. These studies suggest that the mechanism of prion strain adaptation involves rare conformational conversions followed by competitive selection among the heterogeneous pool of PrP(Sc) conformers. The plasticity of prion conformers makes PrP(Sc) a particularly challenging drug target and suggests that combination drug therapies or targeting of PrP(C) may be required for effective therapy. In this review, we highlight recent literature that demonstrate the phenomenon of prion drug resistance and strain specificity, and discuss potential ramifications for therapeutic efforts against prion diseases.

PrP(C) regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells.

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrP(SC)) has been studied in depth, the physiological role of PrP(C) remains elusive and controversial. PrP(C) is a cell-surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrP(C) in animals and in cellular models. In this article, we present PrP(C)-dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrP(C) over-expression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrP(C) silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrP(C) in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrP(C) in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrP(C) over-expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway.

Anti-prion drug mPPIg5 inhibits PrP(C) conversion to PrP(Sc).

Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrP(Sc), an abnormal isoform of the cellular protein PrP(C), is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrP(Sc) in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrP(C) to PrP(Sc) conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future.

Comprehensive study of the interaction between a potential antiprion cationic porphyrin and human prion protein at different pH by using multiple spectroscopic methods.

Cationic porphyrins are potential antiprion drugs; however, the action mechanisms remain poorly understood. Herein, the interaction between a cationic porphyrin and recombinant human prion protein (rPrP(C)) was comprehensively studied by using surface plasmon resonance (SPR), fluorescence, resonance light scattering (RLS), and circular dichroism (CD) spectroscopy. The experimental results showed that the interaction between the cationic porphyrin and rPrP(C) was pH dependent. The equilibrium association constants obtained from SPR spectroscopy were 4.12 × 10(3) M(-1) at pH 4.0, 1.74 × 10(5) M(-1) at pH 6.0, and 5.98 × 10(5) M(-1) at pH 7.0. The binding constants at 298 K obtained from the fluorescence quenching method were 7.286 × 10(4) M(-1) at pH 4.0 and 1.457 × 10(5) M(-1) at pH 6.0. The thermodynamic parameters such as enthalpy change, entropy change, and free energy change were calculated, and the results indicated hydrogen bonds and van der Waals interactions played a major role in the binding reaction. The RLS experiment was performed to study the influence of porphyrin on the rPrP(C) aggregation at different pH values. The CD experiments were conducted to investigate the effects of porphyrin on the secondary structure and thermal stability of rPrP(C). Finally, the comparison of SPR measurement and fluorescence quenching measurement was discussed.