2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases.

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).

Emerging Place of JAK Inhibitors in the Treatment of Inborn Errors of Immunity.

Among inborn errors of immunity (IEIs), some conditions are characterized by inflammation and autoimmunity at the front line and are particularly challenging to treat. Monogenic diseases associated with gain-of-function mutations in genes critical for cytokine signaling through the JAK-STAT pathway belong to this group. These conditions represent good candidates for treatment with JAK inhibitors. Type I interferonopathies, a group of recently identified monogenic auto-inflammatory diseases characterized by excessive secretion of type I IFN, are also good candidates with growing experiences reported in the literature. However, many questions remain regarding the choice of the drug, the dose (in particular in children), the efficacy on the various manifestations, the monitoring of the treatment, and the management of potent side effects in particular in patients with infectious susceptibility. This review will summarize the current experiences reported and will highlight the unmet needs.

Drug screening with zebrafish visual behavior identifies carvedilol as a potential treatment for an autosomal dominant form of retinitis pigmentosa.

Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.

In Silico Repurposing of J147 for Neonatal Encephalopathy Treatment: Exploring Molecular Mechanisms of Mutant Mitochondrial ATP Synthase.

BACKGROUND: Neonatal Encephalopathy (NE) is a mitochondrial ATP synthase (mATPase) disease, which results in the death of infants. The case presented here is reportedly caused by complex V deficiency as a result of mutation of Arginine to Cysteine at residue 329 in the mATPase. A recent breakthrough was the discovery of J147, which targets mATPase in the treatment of Alzheimer's disease. Based on the concepts of computational target-based drug design, this study investigated the possibility of employing J147 as a viable candidate in the treatment of NE. OBJECTIVE/METHODS: The structural dynamic implications of this drug on the mutated enzyme are yet to be elucidated. Hence, integrative molecular dynamics simulations and thermodynamic calculations were employed to investigate the activity of J147 on the mutated enzyme in comparison to its already established inhibitory activity on the wild-type enzyme. RESULTS: A correlated structural trend occurred between the wild-type and mutant systems whereby all the systems exhibited an overall conformational transition. Equal observations in favorable free binding energies further substantiated uniformity in the mobility, and residual fluctuation of the wild-type and mutant systems. The similarity in the binding landscape suggests that J147 could as well modulate mutant mATPase activity in addition to causing structural modifications in the wild-type enzyme. CONCLUSION: Findings suggest that J147 can stabilize the mutant protein and restore it to a similar structural state as the wild-type which depicts functionality. These details could be employed in drug design for potential drug resistance cases due to mATPase mutations that may present in the future.

Regulatory perspectives on next-generation sequencing and complementary diagnostics in Japan.

INTRODUCTION: The 'one biomarker/one drug' scenario is unsustainable because cancer is a complex disorder that involves a number of molecular defects. In the past decade, major technological advances have lowered the overall cost and increased the efficiency of next-generation sequencing (NGS). AREAS COVERED: We review recent regulations on NGS and complementary diagnostics in Japan, mainly focusing on high-quality studies that utilized these new diagnostic modalities and were published within the last 5 years. We highlight significant changes in regulation, and explain the direction of efforts to translate the results of NGS and complementary diagnostics into clinical practice. EXPERT OPINION: NGS holds a number of advantages over conventional companion and complementary diagnostics that enable simultaneous analyzes of multiple cancer genes to detect actionable mutations. Parallel technological developments and regulatory changes have led to the rapid adoption of NGS into clinical practice. NGS-based genomic data have been leveraged to better understand the characteristics of a disease that affects its patient's response to a given therapy. As NGS-based tests become more widespread, however, Japanese authorities will face significant challenges particularly with respect to the complexity of genomic data, which will have to be managed if NGS is to benefit patients.

[Update on the treatment of RASopathies]. / Actualizacion del tratamiento de las rasopatias.

INTRODUCTION: The term 'RASopathies' covers a series of diseases that present mutations in the genes that code for the proteins of the RAS/MAPK pathway. These diseases include neurofibromatosis type 1, Noonan syndrome, Legius syndrome, LEOPARD syndrome, Costello syndrome and cardiofaciocutaneous syndrome. Involvement of the RAS/MAPK pathway not only increases predisposition to develop tumours, but also determines the presence of phenotypic anomalies and alterations in learning processes. AIM: To review the use of therapeutic strategies with mechanisms that have a selective action on RASopathies. DEVELOPMENT: The fact that the RAS pathway is involved in a third of all neoplasms has led to the development and study of different drugs at this level. Some of these pharmaceutical agents have been tested in RASopathies, mainly in neurofibromatosis type 1. Here we analyse the use of different antitarget treatments: drugs that act on the membrane receptors, such as tyrosine kinase inhibitors, in the mTOR pathway or MEK inhibitors. These latter have shown potential benefits in recent studies conducted on different RASopathies. CONCLUSIONS: Today, thanks to the results from the first studies conducted with MEK inhibitor based mainly on animal models, a number of promising clinical trials are being carried out.

TITLE: Actualizacion del tratamiento de las rasopatias.Introduccion. El termino 'rasopatias' agrupa una serie de enfermedades que presentan mutaciones en genes que codifican las proteinas de la via RAS/MAPK. Estas enfermedades incluyen la neurofibromatosis de tipo 1, el sindrome de Noonan, el sindrome de Legius, el sindrome LEOPARD, el sindrome de Costello y el sindrome cardiofaciocutaneo. La afectacion de la via RAS/MAPK no solo aumenta la predisposicion a desarrollar tumores, sino que tambien determina la presencia de anomalias fenotipicas y alteraciones en los procesos de aprendizaje. Objetivo. Revisar el papel del uso de estrategias terapeuticas con mecanismos de accion selectivo en las rasopatias. Desarrollo. El hecho de que la via RAS participe en un tercio de las neoplasias ha motivado el desarrollo y el estudio de distintos farmacos a este nivel. Algunos de estos farmacos han sido probados en las rasopatias, principalmente en la neurofibromatosis de tipo 1. Analizamos el uso de distintos tratamientos antidiana: farmacos que actuan en los receptores de membrana, como los inhibidores de la tirosincinasa, en la via mTOR o los inhibidores de MEK. Existe un potencial beneficio de estos ultimos en estudios recientes realizados en distintas rasopatias. Conclusiones. Actualmente, gracias a los resultados de los primeros trabajos desarrollados con inhibidor de MEK basados principalmente en modelos animales, se estan realizando multiples ensayos clinicos prometedores.

Unrelated donor hematopoietic stem cell transplantation for the treatment of non-malignant genetic diseases: An alemtuzumab based regimen is associated with cure of clinical disease; earlier clearance of alemtuzumab may be associated with graft rejection.

Hematopoietic stem cell transplantation (HSCT) with matched unrelated donors (MUD), offers potentially curative therapy for patients with non-malignant genetic diseases. In this pilot study conducted from 2006 to 2014, we report the outcomes of 15 patients with non-malignant genetic diseases who received a myeloablative regimen with a reduced cyclophosphamide dose, adjunctive serotherapy and MUD HSCT [intravenous alemtuzumab (52 mg/m(2) ), busulfan (16 mg/kg), fludarabine (140mg/m(2) ), and cyclophosphamide (105 mg/kg)]. Graft-versus-host-disease (GVHD) prophylaxis consisted of tacrolimus/cyclosporine and methylprednisolone. Median (range) time to neutrophil engraftment (>500 cells/µL) and platelet engraftment (>20,000/mm(3) ) were 15 (12-28) and 25 (17-30) days, respectively. At a median follow-up of 2 (0.2-5.4) years, the overall survival (OS) was 93.3% (95% CI: 0.61-0.99) and disease-free survival (DFS) was 73.3% (95% CI: 0.44-0.89). Among this small sample, earlier alemtuzumab clearance was significantly associated with graft rejection (P = 0.047), earlier PHA response (P = 0.009) and a trend toward earlier recovery of recent thymic emigrants (RTE) (P = 0.06). This regimen was associated with durable donor engraftment and relatively low rates of regimen related toxicity (RRT); future alemtuzumab pharmacokinetic studies may improve outcomes, by allowing targeted alemtuzumab clearance to reduce graft rejection and promote more rapid immune reconstitution.

Labeled stem cells as disease models and in drug discovery.

Human pluripotent stem cells provide unique possibilities for in vitro studies of human cells in basic research, disease modeling as well as in industrial applications. By introducing relevant genome engineering technology, and thereby creating, for example, reporter cell lines, one will facilitate and improve safety pharmacology, toxicity testing, and can help the scientists to better understand pathological processes in humans. This review discusses how the merger of these two fields, human pluripotent stem cells and genome engineering, form extremely powerful tools and how they have been implemented already within the scientific community. In sharp contrast to immortalized human cell lines, which are both easy to expand and very simple to transfect, the genetically modified pluripotent stem cell line can be directed to a specific cell lineage and provide the user with highly relevant information. We highlight some of the challenges the field had to solve and how new technology advancements has removed the early bottlenecks.

SLCO1B1 haplotypes are not associated with atorvastatin-induced myalgia in Brazilian patients with familial hypercholesterolemia.

PURPOSE: Recent studies reported the association of SLCO1B1 haplotypes with the development of musculoskeletal side effects during simvastatin use. The aim was to evaluate the pharmacogenetic association of SLCO1B1 haplotypes with atorvastatin-induced myalgia in a sample of individuals on high-dose atorvastatin regimens. METHODS: One hundred and forty-three patients with familial hypercholesterolemia were followed for at least 12 months while receiving atorvastatin. Genotypes for the rs2306283 (c.A388G) and rs4149056 (c.T521C) polymorphisms were detected by high-resolution melting analysis. These markers form four distinct haplotypes (*1A, *1B, *5 and *15). RESULTS: During the follow-up period, 14 (9.8%) patients developed myalgia and 16 (11.2%) presented CK levels more than 3 times the upper limit of the normal range. No association of the SLCO1B1 rs2306283 and rs4149056 genotypes or haplotypes with the presence of myalgia or creatine kinase (CK) values was found. Presence of rs2306283 AG + GG genotypes was not associated with increased risks of myalgia or abnormal CK values (OR 2.08, 95% CI 0.62-7.00, p = 0.24 and OR 0.51, 95% CI 0.21-1.26, p = 0.15 respectively). The presence of rs4149056 TC + CC genotypes was also not associated with increased risk of myalgia or abnormal CK values (OR 2.24, 95% CI 0.47-10.72, p = 0.31 and OR 1.51, 95% CI 0.57-3.96, p = 0.41 respectively). CONCLUSIONS: Our findings reaffirm that the SLCO1B1 genetic risk appears to be greater in those patients receiving simvastatin compared with those receiving atorvastatin. This suggests that the importance of SLCO1B1 haplotypes depends on the specific statin that has been used.

Human pluripotent stem cells for genetic disease modeling and drug screening.

Considerable hope surrounds the use of disease-specific pluripotent stem cells, which can differentiate into any cell type, as starting materials to generate models of human disease that will allow exploration of pathological mechanisms and the search for new treatments. Disease-specific human embryonic stem cells have provided a useful source for studying certain disease states. However, reprogramming of human somatic cells that use readily accessible tissue, such as skin or blood, to generate embryonic-like induced pluripotent stem cells has opened new perspectives for modeling and understanding a larger number of human pathologies. Here, we examine the challenges in creating a disease model from human pluripotent stem cells, and describe their use to model both cell-autonomous and non-cell-autonomous mechanisms, the need for adequate control experiments and the genetic limitations of human induced pluripotent stem cells. Progress in these areas will substantially accelerate effective application of disease-specific human pluripotent stem cells for drug screening.

Toward a therapeutic strategy for polyalanine expansions disorders: in vivo and in vitro models for drugs analysis.

Molecular pathogenesis of congenital disorders associated with polyalanine expansions has been investigated for several years. Despite different pathological hallmarks characterize each polyalanine disease, they share common features, mainly represented by aggregates containing the mutant proteins, usually mislocated inside the cellular compartments, along with ubiquitin and proteasome components. Recently, particular interest has been raised by investigations on molecules able to restore both correct localization and function of the expanded proteins. Here we report a list of drugs whose effects have been assayed both in in vitro and in vivo models of polyalanine disorders, such as the oculopharyingeal muscular dystrophy, congenital central hypoventilation syndrome, synpolydactyly and in cell and animal models carrying specific artificial mutations. In particular, we have reviewed, for each polyalanine mutant protein, the molecules tested, cellular models under investigation, drugs effects on aggregation and underlying mechanisms.

[Plants, medicine and genetics, which applications for tomorrow?]. / Plantes, médicaments et génétique: Quelles applications pour demain?

From 60,000 B.C. to the 19th century, plants were the main source for human drugs. Plant-derived pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis at the beginning of the 19th century. However, 50% of small molecules used for cancer therapy or the most efficient drugs for flu or paludism treatment are still extracted or derived from plants. Furthermore, strategies which aim at improving plant expression systems for biopharmaceutical production are currently making them safe and extremely cost-effective alternatives to microbial and mammalian cell cultures for the production of either simple or highly complex recombinant therapeutic proteins.

High-dose vitamin therapy stimulates variant enzymes with decreased coenzyme binding affinity (increased K(m)): relevance to genetic disease and polymorphisms.

As many as one-third of mutations in a gene result in the corresponding enzyme having an increased Michaelis constant, or K(m), (decreased binding affinity) for a coenzyme, resulting in a lower rate of reaction. About 50 human genetic dis-eases due to defective enzymes can be remedied or ameliorated by the administration of high doses of the vitamin component of the corresponding coenzyme, which at least partially restores enzymatic activity. Several single-nucleotide polymorphisms, in which the variant amino acid reduces coenzyme binding and thus enzymatic activity, are likely to be remediable by raising cellular concentrations of the cofactor through high-dose vitamin therapy. Some examples include the alanine-to-valine substitution at codon 222 (Ala222-->Val) [DNA: C-to-T substitution at nucleo-tide 677 (677C-->T)] in methylenetetrahydrofolate reductase (NADPH) and the cofactor FAD (in relation to cardiovascular disease, migraines, and rages), the Pro187-->Ser (DNA: 609C-->T) mutation in NAD(P):quinone oxidoreductase 1 [NAD(P)H dehy-drogenase (quinone)] and FAD (in relation to cancer), the Ala44-->Gly (DNA: 131C-->G) mutation in glucose-6-phosphate 1-dehydrogenase and NADP (in relation to favism and hemolytic anemia), and the Glu487-->Lys mutation (present in one-half of Asians) in aldehyde dehydrogenase (NAD + ) and NAD (in relation to alcohol intolerance, Alzheimer disease, and cancer).