High-throughput drug screens for amyotrophic lateral sclerosis drug discovery

ABSTRACT

Introduction: Amyotrophic lateral sclerosis (ALS) is a rapid adult-onset neurodegenerative disorder characterised by the progressive loss of upper and lower motor neurons. Current treatment options are limited for ALS, with very modest effects on survival. Therefore, there is a unmet need for novel therapeutics to treat ALS.

Areas covered: This review highlights the many diverse high-throughput screening platforms that have been implemented in ALS drug discovery. The authors discuss cell free assays including in silico and protein interaction models. The review also covers classical in vitro cell studies and new cell technologies, such as patient derived cell lines. Finally, the review looks at novel in vivo models and their use in high-throughput ALS drug discovery

Expert opinion: Greater use of patient-derived in vitro cell models and development of better animal models of ALS will improve translation of lead compounds into clinic. Furthermore, AI technology is being developed to digest and interpret obtained data and to make ‘hidden knowledge’ usable to researchers. As a result, AI will improve target selection for high-throughput drug screening (HTDS) and aid lead compound optimisation. Furthermore, with greater genetic characterisation of ALS patients recruited to clinical trials, AI may help identify responsive genetic subtypes of patients from clinical trials.     

肌萎缩侧索硬化的药物治疗进展

肌萎缩侧索硬化(ALS)是一种运动神经元的进行性变性疾病。其发病机制未明,目前缺乏特效治疗。我们对近年来治疗ALS药物:抗兴奋性氨基酸毒性、神经营养因子、抗氧化、抗炎性类药物等的研究进展进行了综述。     

The cyanobacteria derived toxin Beta-N-methylamino-L-alanine and amyotrophic lateral sclerosis

There is mounting evidence to suggest that environmental factors play a major role in the development of neurodegenerative diseases like ALS (Amyotrophic Lateral Sclerosis). The non-protein amino acid beta-N-methylamino-L-alanine (BMAA) was first associated with the high incidence of Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) in Guam, and has been implicated as a potential environmental factor in ALS, Alzheimer's disease, and other neurodegenerative diseases. BMAA has a number of toxic effects on motor neurons including direct agonist action on NMDA and AMPA receptors, induction of oxidative stress, and depletion of glutathione. As a non-protein amino acid, there is also the strong possibility that BMAA could cause intraneuronal protein misfolding, the hallmark of neurodegeneration. While an animal model for BMAA-induced ALS is lacking, there is substantial evidence to support a link between this toxin and ALS. The ramifications of discovering an environmental trigger for ALS are enormous. In this article, we discuss the history, ecology, pharmacology and clinical ramifications of this ubiquitous, cyanobacteria-derived toxin.     

Non-mammalian animal models of Parkinson's disease for drug discovery

Importance of the field: Parkinson's disease (PD) is a prevalent neurodegenerative disease affecting millions of predominantly elderly individuals worldwide. Despite intensive efforts devoted to drug discovery, the disease remains incurable. Compounding this problem is the current lack of a truly representative mammalian model of PD. However, a number of non-mammalian models of PD have been created in recent years that hold tremendous promise to accelerate our understanding of the disease as well as to transform the drug discovery process.

Areas covered in this review: This review provides an overview of the various Caenorhabditis elegans and Drosophila genetic models of PD that have been generated to date and discusses the utility of these model systems in the identification of molecules of potential therapeutic value for the PD patient.

What the reader will gain: Readers will appreciate the strengths (and limitations) of C. elegans and Drosophila in modeling salient features of the disease as well as their usefulness in uncovering novel gene–gene interaction and pathways relevant to PD pathogenesis. Readers will also appreciate how technological advancements have allowed the direct evaluation of novel compounds in these living models of PD in a virtually high-throughput manner.

Take home message: Non-mammalian models of PD provide a valuable in vivo platform for drug screening. Unlike cell-based systems, these living models with an intact nervous system allow for a more meaningful evaluation of the neuroprotective properties of genetic and chemical modifiers to be conducted.     

New therapy options for amyotrophic lateral sclerosis

Introduction: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease leading almost irrevocably to paralysis and death within 5 years after the first symptoms. Since the approval of riluzole, all other therapeutic trials have been negative, including many that followed hopeful preclinical and early clinical data. New approaches are needed to uncover effective treatments for this still-devastating disease.

Areas covered: The review summarizes the current approaches to clinical drug development in ALS. It focuses on several new trials listed on PubMed Central or the National Institutes of Health online trial registry. New targets for therapeutic intervention in ALS include skeletal muscle, energetic metabolism and cell replacement. Two different approaches are directed at muscle: interventions that influence proteins near the neuromuscular junction such as Nogo-A; in contrast to drugs pointed toward disease physiology, therapies that directly increase strength. Other trials are evaluating nutritional interventions. Current cell therapy strategies utilize various types of stem cells to study disease pathophysiology, support neurons or surrounding cells through gene therapy or release of neurotrophic factors, or directly replace cells. The review includes a section on known genetic influences in ALS and future directions for the field.

Expert opinion: These new interventions have important implications for the direction of ALS research. Investigators are focusing less on physiological mechanisms inside the neuron, a process that has proved unfruitful for nearly two decades, and more on concepts that have not been examined previously. These studies will surely add to the overall understanding of ALS. Future research will test ways to reduce gene expression in those with known mutations, as well as means to reduce the spread of aggregated protein.     

Stem cell treatments for amyotrophic lateral sclerosis: a critical overview of early phase trials

Introduction: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of cortical, brainstem, and spinal motor neurons; it causes progressive muscle weakness and atrophy, respiratory failure, and death. No currently available treatment either stops or reverses this disease. Therapeutics to slow, stop, and reverse ALS are needed. Stem cells may be a viable solution to sustain and nurture diseased motor neurons. Several early-stage clinical trials have been launched to assess the potential of stem cells for ALS treatment.

Areas covered: This review covers the key advances from early phase clinical trials of stem cell therapy for ALS and identifies promising avenues and key challenges.

Expert opinion: Clinical trials in humans are still in the nascent stages of development. It will be critical to ensure that powered, well-controlled trials are conducted, that optimal treatment windows are identified, and that the ideal cell type, cell dose, and delivery site and method are determined. Several trials have used more invasive procedures, and ethical concerns of sham procedures on patients in the control arm and on their safety should be considered.     

New therapeutic targets for amyotrophic lateral sclerosis

Introduction: Amyotrophic lateral sclerosis (ALS) is one of the most devastating neurological disorders, affecting approximately half a million people worldwide. Currently there is no cure or prevention for ALS. Although ALS is a rare condition, it places a tremendous socioeconomic burden on patients, family members, caregivers and health systems.

Areas covered: The review examines the mechanisms that may contribute to motor neuron degeneration in ALS, among which oxidative damage, glutatamate excitoxicity, mitochondrial dysfunction, impaired axonal transport, apoptotic cell death, growth factor deficiency, glial cell pathology and abnormal RNA metabolism are potential targets for ALS treatment. The article provides an overview of clinical trials performed to date in attempts to treat ALS with regard to molecular mechanisms and pathways they act on. It also discusses new trials based on recently developed molecular biology techniques.

Expert opinion: Despite significant effectiveness of several potential therapeutics observed in preclinical trials, the results were not translatable to patients with ALS. The development of effective treatments of ALS strictly depends on understanding the primary cause of the disease. This goal will only be achieved when we identify the trigger point for motor neuron death in ALS.     

肌萎缩侧索硬化患者低密度脂蛋白的氧化敏感性

目的测定肌萎缩侧索硬化（ALS）患者血中低密度脂蛋白（LDL）体外氧化延迟时间的变化。方法根据EL Escorial诊断标准选取ALS 19例,其中男15例,女4例,并与19例年龄、影响因素等相当的健康成人相对照。以铜氧化共轭双烯法测定血中LDL体外氧化延迟时间。结果 ALS组血中LDL体外氧化延迟时间较对照组明显减低,分别为（59.32±8.65）min与（83.16±8.37）min,差异有统计学意义（P〈0.05）。结论增高的氧化压力与降低的抗氧化能力在ALS的发病机制中具有一定的作用,提示有效的抗氧化治疗可能对控制疾病的发展及改善预后具有一定的临床意义。     

Excessive and precocious glutamate release in a mouse model of amyotrophic lateral sclerosis

The release of [3H]D-aspartate ([3H]D-ASP) or [3H]GABA evoked by glycine and that of [3H]D-ASP or [3H]glycine evoked by GABA from spinal cord synaptosomes were studied in SOD1-G93A(+) mice, a transgenic model of amyotrophic lateral sclerosis, SOD1(+) mice and SOD1(-)/G93A(-) animals. Mutant mice were killed at advanced phase of pathology or during the presymptomatic period. In SOD1(-)/G93A(-) or SOD1(+) mice glycine evoked [(3)H]d-ASP and [(3)H]GABA release, while GABA caused [3H]D-ASP, but not [3H]glycine, release. The glycine-evoked release of [3H]D-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]D-ASP release, but not that of [3H]glycine, were more pronounced in SOD1-G93A(+) than in SOD1(+) mice. Furthermore, these potentiations were already present in asymptomatic 30- to 40-day-old mice. Basal [3H]D-ASP release was also higher in SOD1-G93A(+) than SOD1(+) or SOD1(-)/G93A(-) mice. The release of endogenous glutamate and GABA was also enhanced in asymptomatic animals; the glycine-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in SOD1(+) animals. The effects of glycine and GABA were insensitive to receptor blockers, but sensitive to transporter inhibitors, indicating coexistence of glutamate and glycine transporters and of glutamate and GABA transporters on glutamate-releasing terminals. The glutamate release machinery seems excessively functional in SOD1-G93A(+) animals.     

Using Caenorhabditis elegans for antimicrobial drug discovery

Introduction: The number of microorganism strains with resistance to known antimicrobials is increasing. Therefore, there is a high demand for new, non-toxic and efficient antimicrobial agents. Research with the microscopic nematode Caenorhabditis elegans can address this high demand for the discovery of new antimicrobial compounds. In particular, C. elegans can be used as a model host for in vivo drug discovery through high-throughput screens of chemical libraries.

Areas covered: This review introduces the use of substitute model hosts and especially C. elegans in the study of microbial pathogenesis. The authors also highlight recently published literature on the role of C. elegans in drug discovery and outline its use as a promising host with unique advantages in the discovery of new antimicrobial drugs.

Expert opinion: Caenorhabditis elegans can be used, as a model host, to research many diseases, including fungal infections and Alzheimer's disease. In addition, high-throughput techniques for screening chemical libraries can also be facilitated. Nevertheless, C. elegans and mammals have significant differences that both limit the use of the nematode in research and the degree by which results can be interpreted. That being said, the use of C. elegans in drug discovery still holds promise and the field continues to grow, with attempts to improve the methodology already underway.     

Developing animal models of Zika virus infection for novel drug discovery

Introduction: Just before the Brazilian outbreak, Zika virus was related to a mild infection, causing fever and skin rash. Congenital Zika Syndrome was first described in Brazil, causing microcephaly and malformations in newborns. Three years after the outbreak, the mechanisms of Zika pathogenesis are still not completely elucidated. Moreover, as of today, there is still no approved vaccine that can be administered to the susceptible population. Considering the unmet clinical need, animal models represent an unprecedented opportunity to study Zika pathophysiology and test drugs for the treatment and prevention of vertical transmission.

Areas covered: The authors explore the current knowledge about Zika through animal models and advancements in drug discovery by highlighting drugs with the greatest potential to treat ZIKV infection and block vertical transmission.

Expert opinion: Some drugs used to treat other infections have been repurposed to treat Zika infection, reducing the cost and time for clinical application. One promising example is Sofosbuvir, which protected mice models against Zika pathogenesis by preventing vertical transmission. Importantly, there is a lack on exploration on the long-term effects of Zika Congenital Syndrome, as well as the possible ways to treat its sequelae.     

Therapeutic developments in amyotrophic lateral sclerosis

There is currently no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating disorder of the human nervous system that, due to motoneurone degeneration, causes progressive loss of muscle function and death. The relentless progression of ALS and the uniformly poor prognosis have been unhindered by a variety of therapeutic agents tested in previous clinical studies. Recently, two drugs, namely riluzole and recombinant human insulin-like growth factor-I (IGF-1), have been reported to benefit patients with ALS by improving survival or slowing disease progression. Several other drugs, such as gabapentin and various neurotrophic factors, are being investigated in on-going clinical trials. Therapeutic developments in ALS have been hampered by the fact that the precise cause of the disease remains unknown. In addition, there are considerable variations in disease related characteristics among patients, rendering accurate measurements of disease progression difficult. Advances in theories of pathogenesis, such as genetic factors, glutamate excitotoxicity, oxidative stress, autoimmune mechanism and cytoskeletal abnormality will help guide the development of future therapies. Newer approaches to therapy may include suitable glutamate antagonists, small molecules that augment neurotrophic factor function, and anti-oxidants. Combination therapy of effective agents should be considered.     

Honokiol alleviated neurodegeneration by reducing oxidative stress and improving mitochondrial function in mutant SOD1 cellular and mouse models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis(ALS) is a progressive neurodegenerative disease affecting both upper and lower motor neurons(MNs) with large unmet medical needs. Multiple pathological mechanisms are considered to contribute to the progression of ALS, including neuronal oxidative stress and mitochondrial dysfunction. Honokiol(HNK) has been reported to exert therapeutic effects in several neurologic disease models including ischemia stroke, Alzheimer’s disease and Parkinson’s disease. Here we found ...     

散发性肌萎缩侧索硬化的临床特征

目的探讨散发性肌萎缩侧索硬化（SALS）的临床特征,为早期准确诊断SALS提供依据。方法回顾分析近年来我院收治的40例SALS患者的临床资料,对其发病特点、症状、体征及实验室检查进行统计分析。结果SALS平均发病年龄52．4岁,30岁以前和75岁以后发病少见。男女发病比例5：3。SALS在临床上隐袭起病居多．但也可呈亚急性起病。上肢远端乏力或肌肉萎缩为最常见的起病症状,其发展一般从起病部位水平或垂直的累及邻近的运动神经元．并发呼吸肌麻痹是SALS最常见的死因。SALS的诊断仍然主要依靠临床表现。EMG检查对确诊SALS的病变范围和诊断无临床表现的隐匿损伤部位有重要意义。MRI检查对鉴别颈椎病及脊髓压迫症、脊髓肿瘤和脊髓空洞症的诊断具有重要的辅助价值。结论SALS以中年发病为主．男性多见．其发展遵循一定的规律。EMG和MRI检查对SALS的诊断和鉴别诊断有重要意义。     

化学蛋白质组学与药物靶点的发现

小分子药物靶点的发现对于生物和医学的研究者而言,是一项既重要又艰巨的任务,医学和药学界研究工作者急切需要发现和确认新的靶点。为了克服药物靶点确认的瓶颈,已经发展了许多新技术用以研究小分子化合物与蛋白质分子间的相互作用,其中包括化学蛋白质组学方法。化学蛋白质组是全蛋白质组学研究的一个亚类,化学蛋白质组学是利用能够与靶蛋白质发生特异性相互作用的化学小分子来干扰和探测蛋白质组,在分子水平上系统揭示特定蛋白质的功能以及蛋白质与化学小分子的相互作用,从而准确找到药物作用靶点的组学研究方法。化学蛋白质组学技术和方法不断成熟,在药物作用靶点的发现、确认和药物多靶点研究等方面都将起到重要的作用,并将大大提高药物发现的效率。     

Third-generation sequencing techniques and applications to drug discovery

Introduction: There is an immediate need for functional and molecular studies to decipher differences between disease and ‘normal’ settings to identify large quantities of validated targets with the highest therapeutic utilities. Furthermore, drug mechanism of action and biomarkers to predict drug efficacy and safety need to be identified for effective design of clinical trials, decreasing attrition rates, regulatory agency approval process and drug repositioning. By expanding the power of genetics and pharmacogenetics studies, next-generation nucleic acid sequencing technologies have started to play an important role in all stages of drug discovery.

Areas covered: This article reviews the first- and second-generation sequencing technologies (SGSTs) and challenges they pose to biomedicine. The article then focuses on the emerging third-generation sequencing technologies (TGSTs), their technological foundations and potential contributions to drug discovery.

Expert opinion: Despite the scientific and commercial success of SGSTs, the goal of rapid, comprehensive and unbiased sequencing of nucleic acids has not been achieved. TGSTs promise to increase sequencing throughput and read lengths, decrease costs, run times and error rates, eliminate biases inherent in SGSTs and offer capabilities beyond nucleic acid sequencing. Such changes will have positive impact on all sequencing applications to drug discovery.     

An update on the use of C. elegans for preclinical drug discovery: screening and identifying anti-infective drugs

Introduction: The emergence of antibiotic-resistant and -tolerant bacteria is a major threat to human health. Although efforts for drug discovery are ongoing, conventional bacteria-centered screening strategies have thus far failed to yield new classes of effective antibiotics. Therefore, new paradigms for discovering novel antibiotics are of critical importance. Caenorhabditis elegans, a model organism used for in vivo, offers a promising solution for identification of anti-infective compounds.

Areas covered: This review examines the advantages of C. elegans-based high-throughput screening over conventional, bacteria-centered in vitro screens. It discusses major anti-infective compounds identified from large-scale C. elegans-based screens and presents the first clinically-approved drugs, then known bioactive compounds, and finally novel small molecules.

Expert opinion: There are clear advantages of using a C. elegans-infection based screening method. A C. elegans-based screen produces an enriched pool of non-toxic, efficacious, potential anti-infectives, covering: conventional antimicrobial agents, immunomodulators, and anti-virulence agents. Although C. elegans-based screens do not denote the mode of action of hit compounds, this can be elucidated in secondary studies by comparing the results to target-based screens, or conducting subsequent target-based screens, including the genetic knock-down of host or bacterial genes.     

Therapeutic developments in the treatment of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease characterised by the selective death of motor neurons. The mechanisms and processes responsible for the selective loss of motor neurons are still unknown, however several hypotheses have been put forward, including oxidative damage and/or toxicity from intracellular aggregates due to mutant superoxide dismutase-1 activity, axonal strangulation from cytoskeletal abnormalities, loss of trophic factor support and glutamate-mediated excitotoxicity. These theories are based on a better understanding of the genetics of amyotrophic lateral sclerosis and on biochemical and pathological analysis of post-mortem tissue. They have led to the development of appropriate animal and cell culture models, allowing the sequence of events in motor neuronal degeneration to be unravelled and potential therapeutic agents to be screened. Unfortunately, the majority of therapeutics found to be efficacious in the animal and cell culture models have failed in human trials. Riluzole is still the only proven therapy in humans, shown to extend survival of amyotrophic lateral sclerosis patients by ～ 3 months, but it has no effect on muscle strength. Other potential therapeutic approaches are being identified, including inhibition of caspase-mediated cell death, maintenance of mitochondrial integrity and energy production, regulation of glutamate homeostasis, reduction of inflammation and control of neurofilament synthesis. Hopefully, in the near future some new agents will be found that can alter the course of this devastating and fatal disease.