Intrinsically cell-penetrating multivalent and multitargeting ligands for myotonic dystrophy type 1.

Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. Here, we report intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of our multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1 Drosophila was also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics.

RNA Toxicity and Perturbation of rRNA Processing in Spinocerebellar Ataxia Type 2.

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of a CAG repeat in Ataxin-2 (ATXN2) gene. The mutant ATXN2 protein with a polyglutamine tract is known to be toxic and contributes to the SCA2 pathogenesis. OBJECTIVE: Here, we tested the hypothesis that the mutant ATXN2 transcript with an expanded CAG repeat (expATXN2) is also toxic and contributes to SCA2 pathogenesis. METHODS: The toxic effect of expATXN2 transcripts on SK-N-MC neuroblastoma cells and primary mouse cortical neurons was evaluated by caspase 3/7 activity and nuclear condensation assay, respectively. RNA immunoprecipitation assay was performed to identify RNA binding proteins (RBPs) that bind to expATXN2 RNA. Quantitative PCR was used to examine if ribosomal RNA (rRNA) processing is disrupted in SCA2 and Huntington's disease (HD) human brain tissue. RESULTS: expATXN2 RNA induces neuronal cell death, and aberrantly interacts with RBPs involved in RNA metabolism. One of the RBPs, transducin ß-like protein 3 (TBL3), involved in rRNA processing, binds to both expATXN2 and expanded huntingtin (expHTT) RNA in vitro. rRNA processing is disrupted in both SCA2 and HD human brain tissue. CONCLUSION: These findings provide the first evidence of a contributory role of expATXN2 transcripts in SCA2 pathogenesis, and further support the role of expHTT transcripts in HD pathogenesis. The disruption of rRNA processing, mediated by aberrant interaction of RBPs with expATXN2 and expHTT transcripts, suggest a point of convergence in the pathogeneses of repeat expansion diseases with potential therapeutic implications. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Integrating Display and Delivery Functionality with a Cell Penetrating Peptide Mimic as a Scaffold for Intracellular Multivalent Multitargeting.

The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to develop a series of multivalent ligand constructs that bind to the expanded dCTG (CTG(exp)) and rCUG nucleotide repeats (CUG(exp)) known to cause myotonic dystrophy type I (DM1), an incurable neuromuscular disease. By assembling this polyvalent construct, the hydrophobic ligands are solubilized and delivered into cell nuclei, and their enhanced binding affinity leads to the inhibition of ribonuclear foci formation and a reversal of splicing defects, all at low concentrations. Some of the multivalent ligands are shown to inhibit selectively the in vitro transcription of (CTG·CAG)74, to reduce the concentration of the toxic CUG RNA in DM1 model cells, and to show phenotypic improvement in vivo in a Drosophila model of DM1. This strategy may be useful in drug design for other trinucleotide repeat disorders and more broadly for intracellular multivalent targeting.

Rationally designed small molecules that target both the DNA and RNA causing myotonic dystrophy type 1.

Single-agent, single-target therapeutic approaches are often limited by a complex disease pathobiology. We report rationally designed, multi-target agents for myotonic dystrophy type 1 (DM1). DM1 originates in an abnormal expansion of CTG repeats (CTG(exp)) in the DMPK gene. The resultant expanded CUG transcript (CUG(exp)) identified as a toxic agent sequesters important proteins, such as muscleblind-like proteins (MBNL), undergoes repeat-associated non-ATG (RAN) translation, and potentially causes microRNA dysregulation. We report rationally designed small molecules that target the DM1 pathobiology in vitro in three distinct ways by acting simultaneously as transcription inhibitors, by inhibiting aberrant protein binding to the toxic RNA, and by acting as RNase mimics to degrade the toxic RNA. In vitro, the agents are shown to (1) bind CTG(exp) and inhibit formation of the CUG(exp) transcript, (2) bind CUG(exp) and inhibit sequestration of MBNL1, and (3) cleave CUG(exp) in an RNase-like manner. The most potent compounds are capable of reducing the levels of CUG(exp) in DM1 model cells, and one reverses two separate CUG(exp)-induced phenotypes in a DM1 Drosophila model.

A novel missense mutation in CCDC88C activates the JNK pathway and causes a dominant form of spinocerebellar ataxia.

BACKGROUND: Spinocerebellar ataxias (SCAs) are a group of clinically and genetically diverse and autosomal-dominant disorders characterised by neurological deficits in the cerebellum. At present, there is no cure for SCAs. Of the different distinct subtypes of autosomal-dominant SCAs identified to date, causative genes for only a fraction of them are currently known. In this study, we investigated the cause of an autosomal-dominant SCA phenotype in a family that exhibits cerebellar ataxia and pontocerebellar atrophy along with a global reduction in brain volume. METHODS AND RESULTS: Whole-exome analysis revealed a missense mutation c.G1391A (p.R464H) in the coding region of the coiled-coil domain containing 88C (CCDC88C) gene in all affected individuals. Functional studies showed that the mutant form of CCDC88C activates the c-Jun N-terminal kinase (JNK) pathway, induces caspase 3 cleavage and triggers apoptosis. CONCLUSIONS: This study expands our understanding of the cause of autosomal-dominant SCAs, a group of heterogeneous congenital neurological conditions in humans, and unveils a link between the JNK stress pathway and cerebellar atrophy.

Targeting toxic RNAs that cause myotonic dystrophy type 1 (DM1) with a bisamidinium inhibitor.

A working hypothesis for the pathogenesis of myotonic dystrophy type 1 (DM1) involves the aberrant sequestration of an alternative splicing regulator, MBNL1, by expanded CUG repeats, r(CUG)(exp). It has been suggested that a reversal of the myotonia and potentially other symptoms of the DM1 disease can be achieved by inhibiting the toxic MBNL1-r(CUG)(exp) interaction. Using rational design, we discovered an RNA-groove binding inhibitor (ligand 3) that contains two triaminotriazine units connected by a bisamidinium linker. Ligand 3 binds r(CUG)12 with a low micromolar affinity (K(d) = 8 ± 2 µM) and disrupts the MBNL1-r(CUG)12 interaction in vitro (K(i) = 8 ± 2 µM). In addition, ligand 3 is cell and nucleus permeable, exhibits negligible toxicity to mammalian cells, dissolves MBNL1-r(CUG)(exp) ribonuclear foci, and restores misregulated splicing of IR and cTNT in a DM1 cell culture model. Importantly, suppression of r(CUG)(exp) RNA-induced toxicity in a DM1 Drosophila model was observed after treatment with ligand 3. These results suggest ligand 3 as a lead for the treatment of DM1.

Crystallization and preliminary crystallographic analysis of the central domain of Drosophila Dribble, a protein that is essential for ribosome biogenesis.

Dribble (DBE) is a Drosophila protein that is essential for ribosome biogenesis. Bioinformatics analysis revealed a folded central domain of DBE which is flanked by structural disorder in the N- and C-terminal regions. The protein fragment spanning amino-acid residues 16-197 (DBE(16-197)) was produced for structural determination. In this report, the crystallization and preliminary X-ray diffraction data analysis of the DBE(16-197) protein domain are described. Crystals of DBE(16-197) were grown by the sitting-drop vapour-diffusion method at 289 K using ammonium phosphate as a precipitant. The crystals belonged to space group P2(1)2(1)2(1). Data were collected that extended to beyond 2 A resolution.

Exclusion of unsuitable CNS drug candidates based on their physicochemical properties and unbound fractions in biomatrices for brain microdialysis investigations.

Microdialysis has been the only direct method of continuously measuring the unbound drug concentrations in extracellular fluid at a specific brain region with respect to time in the same animal. However, not every compound is suitable for microdialysis system as demonstrated by their inconsistent "by gain" and "by loss" in-vitro microdialysis probe recoveries leading to over- or under- estimated in-vivo concentrations. Therefore, our current study was proposed aiming to develop simple exclusion criteria for drug candidates that are not suitable for microdialysis system investigation. Through literature research, the properties ((LogP, pKa, water solubility and unbound fraction in plasma and brain) of drugs that have been reported for microdialysis studies were summarized. The exclusion criteria were developed by evaluating the impact of such properties on the consistency of in-vitro "by gain" and "by loss" recoveries of microdialysis probe. As a result, forty-five compounds were identified from literatures, among which doxorubicin, docetaxel, omeprazole, donepezil and phenytoin were found to have inconsistent in-vitro "by gain" and "by loss" microdialysis probe recoveries and subsequently selected for the exclusion criteria analysis. It was found that compounds with limited water solubility (less than 1 g/L) and unbound fraction in plasma (fu,plasma less than 30%) and brain homogenate (fu,brain less than 10%) were more likely to have inconsistent "by gain" and "by loss" microdialysis probe recoveries. Our proposed exclusion criteria were further validated using carbamazepine (limited water solubility only), DB213 (limited fu,brain only) and piperine (both limited water solubility and limited fu,plasma, fu,brain). Our current proposed exclusion criteria will help excluding the CNS drug candidates that are highly unlikely suitable for brain microdialysis approach leading to a better success rate in brain microdialysis approach development.

A Three-Way Combinatorial CRISPR Screen for Analyzing Interactions among Druggable Targets.

We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson's disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.

A mannose/glucose-specific lectin from Chinese evergreen chinkapin (Castanopsis chinensis).

A mannose/glucose-specific lectin has been purified from Chinese evergreen chinkapin (Castanopsis chinensis) seeds, one of the most popular foods in East Asia. This lectin, designated as CCL, exhibited hemagglutinating activity in mouse and rabbit erythrocytes. It displayed a single band with a molecular mass of 29 kDa in SDS-PAGE and a 120-kDa peak in gel-filtration on Superdex-200. Its hemagglutinating activity was stable in the pH range 6-12 and at temperatures below 60 degrees C. The N-terminal amino acid sequence of CCL differed from those of other lectins in the same family. CCL inhibited the proliferation of HepG2 cells and adult emergence in fruitflies. CCL exhibited mitogenic activity toward mouse splenocytes, and induced nitric oxide production from mouse peritoneal macrophages but was devoid of inhibitory activity toward mycelial growth and HIV-1 reverse transcriptase.

The role of ubiquitin linkages on alpha-synuclein induced-toxicity in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is a common movement disorder marked by the loss of dopaminergic (DA) neurons in the brain stem and the presence of intraneuronal inclusions designated as Lewy bodies (LB). The cause of neurodegeneration in PD is not clear, but it has been suggested that protein misfolding and aggregation contribute significantly to the development of the disease. Misfolded and aggregated proteins are cleared by ubiquitin proteasomal system (UPS) and autophagy lysosomal pathway (ALP). Recent studies suggested that different types of ubiquitin linkages can modulate these two pathways in the process of protein degradation. In this study, we found that co-expression of ubiquitin can rescue neurons from alpha-syn-induced neurotoxicity in a Drosophila model of PD. This neuroprotection is dependent on the formation of lysine 48 polyubiquitin linkage which is known to target protein degradation via the proteasome. Consistent with our results that we observed in vivo, we found that ubiquitin co-expression in the cell can facilitate cellular protein degradation by the proteasome in a lysine 48 polyubiquitin-dependent manner. Taken together, these results suggest that facilitation of proteasomal protein degradation can be a potential therapeutic approach for PD.

Sodium dodecyl sulfate-insoluble oligomers are involved in polyglutamine degeneration.

In polyglutamine (polyQ) degeneration, disease protein that carries an expanded polyQ tract is neurotoxic. Expanded polyQ protein exists in different conformations that display distinct solubility properties. In this study, an inducible transgenic Drosophila model is established to define the pathogenic form of polyQ protein at an early stage of degeneration in vivo. We show that microscopic polyQ aggregates are neither pathogenic nor protective. Further, no toxic effect of sodium dodecyl sulfate (SDS) -soluble polyQ protein is observed in our model. By means of filtration, 2 forms of SDS-insoluble protein species are identified according to their size. Coexpression of an ATPase-defective form of the molecular chaperone Hsc70 (Hsc70-K71S) selectively reduces the abundance of the large SDS-insoluble polyQ species, but such modulation has no modifying effects on degeneration. Notably, we detect a distinct Hsc70-K71S-resistant, small, SDS-insoluble polyQ oligomeric species that is closely correlated with degeneration. Our data highlight the toxic role of SDS-insoluble oligomers in polyQ degeneration in vivo.

Efficient brain uptake and distribution of an expanded CAG RNA inhibitor DB213 via intranasal administration.

DB213 is an expanded CAG RNA inhibitor targeting polyglutamine diseases. This current study aims to investigate biopharmaceutic characteristics of DB213 as well as its brain uptake and distribution in C57 wild type mice, R6/2 Huntington's disease mice and Sprague-Dawley (SD) rats via intranasal administration. The biopharmaceutic characteristics of DB213 were investigated in vitro using Calu-3/MDCK/HEK293 cell lines and brain slices for its membrane transport, equilibrium dialysis for its plasma protein/brain tissue bindings and liver/brain microsomes incubation for its enzyme kinetics profiles. In vivo study of DB213 brain distribution was conducted in rats via intravenous and intranasal routes at 50 mg/kg followed by its brain uptake evaluation in mice at 25 mg/kg via intranasal route. In vitro membrane transport studies found that DB213 not only had a limited passive diffusion with a Papp (a→b) value of 1.75 × 10-6 cm/s in Calu-3 cell monolayer model but also was substrate of MRP2, MRP3, and amino acid transporter. Furthermore, DB213 demonstrated higher binding towards brain homogenate (80%) than plasma (10%) with limited metabolism in liver and brain. After intranasal administration of DB213, both olfactory bulb and trigeminal nerve served as its entry points to reach brain as demonstrated in rats while efficient brain uptake was observed in mice. In summary, limited nasal epithelium permeability and MRP2/MRP3 mediated efflux transport of DB213 could be overcome by its influx transport via amino acid transporter and minimal liver and brain metabolism, which further contribute to its rapid brain uptake and distribution in mice and rats.

Maternal oxytocin responsiveness improves specificity of positive social memory recall.

BACKGROUND: Oxytocin is related to positive parenting behaviours and social cognition. Long-term relationships are partly influenced by the social memory of a person. Positive social memory with the attachment figure may play a mediating role between oxytocin responsiveness and positive parenting behaviours. METHODOLOGY: The study recruited 61 pairs of married mothers and preschool children from a community in Hong Kong. Sociodemographic background and neuroticism data of the respondents were collected in a laboratory. Salivary oxytocin and current mood rating were obtained 10 and 15 min before and after free play, respectively. After collecting the second salivary oxytocin samples, the mothers engaged in a parenting recall task. RESULTS: The mothers with high oxytocin responsiveness recalled previous positive social events with great detail and used uncontrollability attribution to explain such positive events. DISCUSSION: Oxytocin responsiveness influenced the recall of positive social events and attribution. This influence may enhance the sensitivity and positive behaviours of parenting.

Statistical Design of Experiment (DoE) based development and optimization of DB213 in situ thermosensitive gel for intranasal delivery.

DB213 is an HIV-1 replication inhibitor targeting the Central Nervous System for the treatment of HIV-associated neurocognitive disorders. Current study aims to develop an in situ thermosensitive gelling system for intranasal delivery of DB213 facilitated by Statistical Design of Experiment (DoE) to conduct a more efficient experimentation by extracting the maximum amount of information from limited experiments. In our current study, information was extracted from twenty-five experimental designs from MODDE® Software and a mathematical model was successfully developed to predict formulations to achieve desired performance as well as to analyze relationships between the amount of Pluronic F-127, Pluronic F-68, Chitosan, DB213 and the performances of in situ thermosensitive gels. Based on DoE, in situ thermosensitive gels of 1% DB213 (F1) and 5% DB213 (F2) were developed for further in vivo bioavailability and brain uptake evaluations in Sprague-Dawley rats and C57BL/6 mice, respectively. In comparison to DB213 water solution, intranasal administrations of F1 at 1 mg/kg in rats and F2 at 25 mg/kg in mice demonstrated relative bioavailabilities of 145% and 165% with significant increase in brain uptake.

Pharmacokinetics and brain uptake of HIV-1 replication inhibitor DB213 in Sprague-Dawley rats.

The current study aims to investigate the pharmacokinetics and brain uptake of HIV-1 replication inhibitor DB213 via a developed LC/MS/MS analytical method. A sensitive, selective, accurate and reliable LC/MS/MS method for determination and quantification of DB213 in rat plasma and brain was developed and validated. A triple quadrupole mass spectrometer equipped with electrospray ionization (ESI) source was applied for the detection of DB213 and benzamidine (Internal Standard). The analytes were quantified by using multiple reaction monitoring (MRM) mode with m/z 333.4→86.1 and m/z 121.2→104 for DB213 and benzamidine respectively. Chromatographic separation of DB213 and benzamidine was achieved on a SunFire C8 (4.6×250mm, i.d. 5µm) analytical column with gradient elution of a mobile phase consisted of acetonitrile and 20mM ammonium formate buffer (containing 0.5% formic acid). The method achieved good linearity from 1.95∼1000ng/ml (r(2)=0.999) in plasma and 0.98∼125ng/ml (r(2)=0.999) in brain. The validated method was successfully applied to plasma pharmacokinetics (PK) and brain uptake of intravenous administration of DB213 water solution (1mg/kg) to Sprague-Dawley rats. It was found that the area under the plasma concentration-time curve from 0 to 360min (AUC0→360min) was 184422.1±42450.8ngmin/ml and the elimination half-life of DB213 after intravenous administration was 70.9±16.1min. In addition, DB213 has demonstrated a potential to cross the blood-brain barrier via intravenous administration with a brain tissue concentration of 11.3±3.6ng/g peaked at 30min post-dosing.

A Potent Inhibitor of Protein Sequestration by Expanded Triplet (CUG) Repeats that Shows Phenotypic Improvements in a Drosophila Model of Myotonic Dystrophy.

Myotonic dystrophy is the most common form of adult-onset muscular dystrophy, originating in a CTG repeat expansion in the DMPK gene. The expanded CUG transcript sequesters MBNL1, a key regulator of alternative splicing, leading to the misregulation of numerous pre-mRNAs. We report an RNA-targeted agent as a possible lead compound for the treatment of myotonic dystrophy type 1 (DM1) that reveals both the promise and challenges for this type of small-molecule approach. The agent is a potent inhibitor of the MBNL1-rCUG complex with an inhibition constant (Ki ) of 25±8 nm, and is also relatively nontoxic to HeLa cells, able to dissolve nuclear foci, and correct the insulin receptor splicing defect in DM1 model cells. Moreover, treatment with this compound improves two separate disease phenotypes in a Drosophila model of DM1: adult external eye degeneration and larval crawling defect. However, the compound has a relatively low maximum tolerated dose in mice, and its cell uptake may be limited, providing insight into directions for future development.

Isorhynchophylline, a natural alkaloid, promotes the degradation of alpha-synuclein in neuronal cells via inducing autophagy.

Accumulation of α-synuclein (α-syn) in the brain is a pathogenic feature and also a causative factor of Parkinson disease. Isorhynchophylline (IsoRhy) is a major tetracyclic oxindole alkaloid isolated from the Chinese herbal medicine Uncaria rhynchophylla (Miq.)Jacks (Gouteng in Chinese), which has been used for the treatment of neurological diseases in East Asia for centuries. Here we report a novel function of IsoRhy as a neuronal autophagy inducer. IsoRhy induced autophagy in different neuronal cell lines, including N2a, SH-SY5Y and PC12 cells, and also in primary cortical neurons. Furthermore, IsoRhy induced autophagy in the fat bodies of Drosophila. IsoRhy promoted clearance of wild-type, A53T and A30P α-syn monomers, α-syn oligomers and α-syn/synphilin-1 aggresomes in neuronal cells via the autophagy-lysosome pathway. More importantly, IsoRhy was able to decrease the expression levels of wild-type and A53T α-syn protein in differentiated human dopaminergic neurons. Notably, IsoRhy-induced autophagy was independent of the mTOR pathway but dependent on the function of Beclin 1. Taken together, data from this study raise the possibility that oxindole alkaloid derivatives may serve as a means to stimulate autophagy in neuronal cells, thereby exerting preventive and therapeutic values against neurodegenerative diseases such as Parkinson disease by reducing pathogenic protein aggregates in neurons.