High-Throughput and High-Content Screening for Huntington’s Disease Therapeutics
ABSTRACT Since the isolation of the gene and mutation that cause Huntington’s disease (HD) more than a decade ago, there has been optimism that this knowledge would lead to the rapid discovery of therapeutic agents for this fatal and incurable disease . In fact, considerable effort has been invested in HD drug discovery, predominantly in academic environments but also in the biopharmaceutical industry to some certain extent. Historically, however, interest in HD research and development in large pharmaceutical firms has been limited by the relatively small size of the HD patient population (~30,000 affected persons in United States), which has led to the perception that the market size for HD might be too small to justify the investment of substantial resources necessary to bring a new drug to clinical trials. However, there is a growing appreciation for the actual market size of “first in class” drugs for otherwise unaddressable diseases, as well as for the idea that HD may prove to be a paradigmatic disease for other, much more prevalent neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease . One hurdle in drug discovery for most neurodegenerative disorders is their incompletely understood, multigenic, and multifactorial etiology. Thus, animal models of these diseases may often recapitulate only some aspects of such diseases and rarely reproduce the full pathophysiology of the human diseases , . In contrast, HD is caused by a well-defined mutation in a single autosomal gene that has a dominant and fully penetrant phenotype. This simplicity in the genetics of HD has allowed the creation of a variety of cells cultures and transgenic animal models for HD in which there can be greater confidence that these models do capture important aspects of human disease initiation and progression . This chapter will focus on the development and implementation of high-throughput and high-content in vitro assays for the discovery of HD therapeutics. High-throughput screening (HTS) of small molecules allows the rapid interrogation of the effects of thousands to hundreds of thousands of small molecules in a variety of in vitro and cell-based assays, whereas high-content screening (HCS) approaches may sacrifice some of these high-throughput capabilities in return for great biological and phenotypic complexity in the assay endpoints used. Why do we need such high-throughput and high-content methods? Simply because we do not currently have sufficient knowledge of the molecular targets and pathways that may be therapeutic for HD nor do we know how to design a priori custom small molecule compounds that will be guaranteed to have the desired effect on such biological targets. Hence rapid testing of many tens of thousands or more drug molecule candidates in HD models offers the potential for systematized serendipity, that we will encounter effective compounds in such discovery campaigns that will prove to be clinically relevant, using controllable and predictable in vitro screening processes. Active compounds emerging from HTS and HCS screens—termed “hits”—are the templates on which eventually drug “leads” are developed through further combinatorial and medicinal chemistry efforts Figure 5.1. Such efforts in the field have identified a number of hits that are being pursued as drug leads (see Chapters 8 and 12, this volume). In the following sections we will describe different strategies and approaches in the design and implementation of HTS and HCS screens for HD, and will also discuss the development of prioritization schemes for potential drug leads identified, future screening approaches, and the use of these compounds for gaining new insights into mechanisms underlying HD.
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ABSTRACT: The engineered antibody approach to Huntington's disease (HD) therapeutics is based on the premise that significantly lowering the levels of the primary misfolded mutant protein will reduce abnormal protein interactions and direct toxic effects of the misfolded huntingtin (HTT). This will in turn reduce the pathologic stress on cells, and normalize intrinsic proteostasis. Intracellular antibodies (intrabodies) are single-chain (scFv) and single-domain (dAb; nanobody) variable fragments that can retain the affinity and specificity of full-length antibodies, but can be selected and engineered as genes. Functionally, they represent a protein-based approach to the problem of aberrant mutant protein folding, post-translational modifications, protein-protein interactions, and aggregation. Several intrabodies that bind on either side of the expanded polyglutamine tract of mutant HTT have been reported to improve the mutant phenotype in cell and organotypic cultures, fruit flies, and mice. Further refinements to the difficult challenges of intraneuronal delivery, cytoplasmic folding, and long-term efficacy are in progress. This review covers published studies and emerging approaches on the choice of targets, selection and engineering methods, gene and protein delivery options, and testing of candidates in cell and animal models. The resultant antibody fragments can be used as direct therapeutics and as target validation/drug discovery tools for HD, while the technology is also applicable to a wide range of neurodegenerative and other diseases that are triggered by toxic proteins.Progress in Neurobiology 11/2011; 97(2):190-204. DOI:10.1016/j.pneurobio.2011.11.004 · 10.30 Impact Factor
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ABSTRACT: Pluripotent stem cell (PSC) technologies are becoming a key asset for deciphering pathological cascades and for developing new treatments against many neurodegenerative disorders, including Huntington's disease (HD). This perspective discusses the challenges and opportunities facing the use of PSCs for treating HD, focusing on four major applications: namely, the use of PSCs as a substitute source of human striatal cells for current HD cell therapy, as a cellular model of HD for the validation of human-specific gene therapies, for deciphering molecular mechanisms underlying HD, and in drug discovery.Cell stem cell 08/2012; 11(2):153-61. DOI:10.1016/j.stem.2012.07.015 · 22.15 Impact Factor
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ABSTRACT: Huntington’s disease (HD) is the most frequent autosomal dominant neurodegenerative disease with a known gene locus and product. HD likely affects a few hundred thousand subjects worldwide – either in its diagnosed or prodromal stage or by the burden to live with the knowledge of carrying the gene or being at risk. So far no disease modifying (neuroprotective) therapy is available. However, the availability of several transgenic or knock-in animal models, the rising clinical knowledge about the biology and phenotype of the disease in both manifest and pre-diagnosed stages through large observational and biomarker studies, and the progress in drug development and validation processes contributed to a considerable progress in the field. Several possible targets for a therapy based on identified mechanisms of action aiming at either delaying or preventing the progression of the disease are in preclinical or early clinical development. Selected approaches are discussed and the clinical infrastructure available to conduct clinical trials across the globe is outlined. Novel clinical trial endpoints such as quantitative motor (Q-Motor) assessments and further evidence for imaging measures as reliable endpoints for proof of concept studies are reviewed. Gaps and possible bridges for preclinical and clinical development are discussed and facts and opinions on areas needing further attention are presented. HD harbors unique opportunities: it is a promising model to establish a proof of concept for a disease modifying therapy in a neurodegenerative disease and the feasibility to treat premanifest gene carriers may even allow to delay or prevent the manifestation of the disease.12/2012; 2(4):241–248. DOI:10.1016/j.baga.2012.09.004