Affinity capillary electrophoresis for identification of active drug candidates in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal dominantly inherited degenerative disease with a slow progression. At the present, there is no commercially available treatment, but sustained effort is currently undertaken for the development of a promising lead compound. In the present paper we report the development of a fast, versatile, and cost-effective affinity capillary electrophoresis (ACE) method for the screening and identification of potential drug candidates targeting pathological ARN probes relevant for DM1. The affinity studies were conducted in physiologically relevant conditions using 50 mM HEPES buffer (pH 7.4) in a fused silica capillary dynamically coated with poly(ethylene oxide), by testing a library of potential ligands against (CUG)50 RNA as target probe with a total run time of 4-5 h/ligand. For the most promising ligands, their affinity parameters were assessed and some results formerly reported on the affinity of pentamidine (PTMD) and neomycin against CUG repeats were confirmed. To the best of the authors' knowledge, the estimated binding stoichiometry for some of the tested compounds (i.e., ~ 121:1 for PTMD against the tested RNA probe) is reported for the first time. Additionally, the potential of a novel pentamidine like compound, namely 1,2-ethane bis-1-amino-4-benzamidine (EBAB) with much lower in vivo toxicity than its parent compound has also been confirmed studying its effect on a live cell model by fluorescence microscopy. Further tests, such as the evaluation of the rescue in the mis-splicing of the involved genes, can be performed to corroborate the potential therapeutic value of EBAB in DM1 treatment. Graphical abstract ᅟ.

Subtly Modulating Glycogen Synthase Kinase 3 ß: Allosteric Inhibitor Development and Their Potential for the Treatment of Chronic Diseases.

Glycogen synthase kinase 3 ß (GSK-3ß) is a central target in several unmet diseases. To increase the specificity of GSK-3ß inhibitors in chronic treatments, we developed small molecules allowing subtle modulation of GSK-3ß activity. Design synthesis, structure-activity relationships, and binding mode of quinoline-3-carbohydrazide derivatives as allosteric modulators of GSK-3ß are presented here. Furthermore, we show how allosteric binders may overcome the ß-catenin side effects associated with strong GSK-3ß inhibition. The therapeutic potential of some of these modulators has been tested in human samples from patients with congenital myotonic dystrophy type 1 (CDM1) and spinal muscular atrophy (SMA) patients. We found that compound 53 improves delayed myogenesis in CDM1 myoblasts, while compounds 1 and 53 have neuroprotective properties in SMA-derived cells. These findings suggest that the allosteric modulators of GSK-3ß may be used for future development of drugs for DM1, SMA, and other chronic diseases where GSK-3ß inhibition exhibits therapeutic effects.

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations.

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I.

Myotonic dystrophy type I (DM1) is a disabling neuromuscular disease with no causal treatment available. This disease is caused by expanded CTG trinucleotide repeats in the 3' UTR of the dystrophia myotonica protein kinase gene. On the RNA level, expanded (CUG)n repeats form hairpin structures that sequester splicing factors such as muscleblind-like 1 (MBNL1). Lack of available MBNL1 leads to misregulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1. Many studies aiming to identify small molecules that target the (CUG)n-MBNL1 complex focused on synthetic molecules. In an effort to identify new small molecules that liberate sequestered MBNL1 from (CUG)n RNA, we focused specifically on small molecules of natural origin. Natural products remain an important source for drugs and play a significant role in providing novel leads and pharmacophores for medicinal chemistry. In a new DM1 mechanism-based biochemical assay, we screened a collection of isolated natural compounds and a library of over 2100 extracts from plants and fungal strains. HPLC-based activity profiling in combination with spectroscopic methods were used to identify the active principles in the extracts. The bioactivity of the identified compounds was investigated in a human cell model and in a mouse model of DM1. We identified several alkaloids, including the ß-carboline harmine and the isoquinoline berberine, that ameliorated certain aspects of the DM1 pathology in these models. Alkaloids as a compound class may have potential for drug discovery in other RNA-mediated diseases.

Studying a Drug-like, RNA-Focused Small Molecule Library Identifies Compounds That Inhibit RNA Toxicity in Myotonic Dystrophy.

There are many RNA targets in the transcriptome to which small molecule chemical probes and lead therapeutics are desired. However, identifying compounds that bind and modulate RNA function in cellulo is difficult. Although rational design approaches have been developed, they are still in their infancies and leave many RNAs "undruggable". In an effort to develop a small molecule library that is biased for binding RNA, we computationally identified "drug-like" compounds from screening collections that have favorable properties for binding RNA and for suitability as lead drugs. As proof-of-concept, this collection was screened for binding to and modulating the cellular dysfunction of the expanded repeating RNA (r(CUG)(exp)) that causes myotonic dystrophy type 1. Hit compounds bind the target in cellulo, as determined by the target identification approach Competitive Chemical Cross-Linking and Isolation by Pull-down (C-ChemCLIP), and selectively improve several disease-associated defects. The best compounds identified from our 320-member library are more potent in cellulo than compounds identified by high-throughput screening (HTS) campaigns against this RNA. Furthermore, the compound collection has a higher hit rate (9% compared to 0.01-3%), and the bioactive compounds identified are not charged; thus, RNA can be "drugged" with compounds that have favorable pharmacological properties. Finally, this RNA-focused small molecule library may serve as a useful starting point to identify lead "drug-like" chemical probes that affect the biological (dys)function of other RNA targets by direct target engagement.

Trastornos de sueño en la distrofia miotónica tipo 1 / Sleep disorders in myotonic dystrophy type I

Evaluamos los trastornos de sueño en ocho pacientes con distrofia miotónica tipo 1 confirmada genéticamente, con edades comprendidas entre 38 y 58 años. Los pacientes fueron estudiados mediante polisomnografía y test de latencias múltiples de sueño. Encontramos excesiva somnolencia diurna en el 75% de los pacientes, no justificable por los eventos respiratorios durante el sueño. El modafinil mejoró este síntoma en todos ellos (AU)

We investigated the sleep disorders in eight patients with genetically confirmed myotonic dystrophy type 1, aged from 38 to 58 years. Patients were studied with nocturnal polysomnogram and multiple sleep latency tests. We found excessive daytime sleepiness in 75% of them, not accountable for respiratory events during sleep. Modafinil improved this symptom in all of them (AU)

Functional deterioration and selenium-vitamin E treatment in myotonic dystrophy. A placebo-controlled study.

OBJECTIVES: The aim of the investigations was to study the effect of a combined selenium and vitamin E treatment in patients with myotonic dystrophy. DESIGN: A double-blind placebo-controlled trial. SUBJECTS: Twenty-seven patients with myotonic dystrophy divided into an experimental (n = 13) and a control (n = 14) group. INTERVENTIONS: The experimental group was given increasing doses for 4 months up to a maximum of 1.6 mg selenium and 800 mg vitamin E daily and the control group a corresponding number of placebo tablets. The total treatment period was 2 years. MAIN OUTCOME MEASURES: Muscle strength (knee extension, knee flexion, hand-grip), maximal walking speed for 30 m, function in daily activities (disability), well-being and cognitive functioning. RESULTS: Before treatment, muscle strength showed moderately or markedly reduced values as did maximal walking speed. Disability was most markedly demonstrated in instrumental ADL-activities. Spatial ability was reduced in the majority of patients. During the 2-year follow-up period, the hand-grip strength and maximal walking speed decreased in both groups (P < 0.01). The reduction was not significantly different according to an 'intention-to-treat-analysis'. However, there was a significantly larger decline (P < 0.01) in the left-hand grip strength in the control group when all the patients with complete treatment compliance were included. CONCLUSIONS: A useful set of measurements has been developed for following functional deterioration in myotonic dystrophy. No conclusive evidence of beneficial effects of selenium and vitamin E treatment has been produced.

Effect of sodium selenite and vitamin E treatment in myotonic dystrophy.

Fifteen patients with myotonic dystrophy, seven men and eight women, of age range 15-55 years, were treated with sodium selenite and vitamin E for 2 years. The patients were examined using a number of tests to assess muscle force and function at the start of the treatment period and 6, 12 and 24 months thereafter. Myotonia gives an increased relaxation time. The latter was measured in the adductor pollicis. There was a significant decrease in half relaxation time before treatment compared with the values during treatment. In summary, the results indicate that selenium and vitamin E therapy may have an effect on myotonia. The effects of treatment on motor functions were minimal, and overall motor performance was not improved.

Nifedipine in the treatment of myotonia in myotonic dystrophy.

Abnormal calcium transport may be implicated in the membrane defect in myotonic dystrophy. A single blind crossover trial of placebo (t.i.d.), nifedipine 10 mg (t.i.d.) and nifedipine 20 mg (t.i.d.), was performed in 10 patients with myotonic dystrophy. The severity of myotonia was assessed by measuring finger extension time after maximum voluntary finger flexion. A significant improvement in myotonia, after nifedipine, was recorded by this technique and supported by a subjective improvement in 50% of patients and clinical improvement of greater than 20% in five patients. Initial grip strength and muscle fatiguability measured by grip strength ergometry were not significantly altered.

Myotonic dystrophy treated with selenium and vitamin E.

We have previously reported the successful treatment of a patient with myotonic dystrophy with selenium and vitamin E. This paper deals with the treatment of a further five patients with myotonic dystrophy in different stages. All five patients improved subjectively and objectively in two or more respects. All improved their grip strength according to vigorimeter measurements (Martin), two normalized their gait, another two can now sit down on their heels and stand up, one patient can now walk on his toes, one can now get up from lying on the floor without using a chair and two patients have improved their physical capacity. Patients in early stages of the disease improved faster and more markedly than those in late stages. Electromyographical measurements also showed improvements, in that the myotonic discharges had diminished. The daily dose was 4 mg of Na2SeO3 and 600 mg of vitamin E. Serum concentration of selenium increased in all patients at the beginning of the treatment, but stabilized at a level slightly above the normal. No side-effects were observed.

Selenium therapy of myotonic dystrophy.

A patient with myotonic dystrophy (MD) has been treated for two years with selenium and vitamin E. The serum selenium concentration and other relevant variables were continuously studied. The patient showed a marked subjective and objective improvement with increased muscular strength and regression of pathological electromyographic findings. To our knowledge, this is the first report of successful treatment of MD.