Identification of Aminothienopyridazine Inhibitors of Tau Assembly by Quantitative High-Throughput Screening

Center for Neurodegenerative Disease Research, Institute on Aging, and Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Biochemistry (Impact Factor: 3.02). 08/2009; 48(32):7732-45. DOI: 10.1021/bi9006435
Source: PubMed


Inclusions comprised of fibrils of the microtubule- (MT-) associated protein tau are found in the brains of those with Alzheimer's disease (AD) and other neurodegenerative tauopathies. The pathology that is observed in these diseases is believed to result from the formation of toxic tau oligomers or fibrils and/or from the loss of normal tau function due to its sequestration into insoluble deposits. Hence, small molecules that prevent tau oligomerization and/or fibrillization might have therapeutic value. Indeed, examples of such compounds have been published, but nearly all have properties that render them unsuitable as drug candidates. For these reasons, we conducted quantitative high-throughput screening (qHTS) of approximately 292000 compounds to identify drug-like inhibitors of tau assembly. The fibrillization of a truncated tau fragment that contains four MT-binding domains was monitored in an assay that employed complementary thioflavin T fluorescence and fluorescence polarization methods. Previously described classes of inhibitors as well as new scaffolds were identified, including novel aminothienopyridazines (ATPZs). A number of ATPZ analogues were synthesized, and structure-activity relationships were defined. Further characterization of representative ATPZ compounds showed they do not interfere with tau-mediated MT assembly, and they are significantly more effective at preventing the fibrillization of tau than the Abeta(1-42) peptide which forms AD senile plaques. Thus, the ATPZ molecules described here represent a novel class of tau assembly inhibitors that merit further development for testing in animal models of AD-like tau pathology.

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    • "Taken together, this suggests that the in vivo drug discovery approach demonstrated here could significantly reduce post-screening costs for identifying quality leads from the initial candidate pool. Success in the identification of novel valid compounds for the potential development of a DM1 treatment suggests that the method developed could be adapted to any particular type of alternative splicing deregulation (exon skipping, intron retention and exon extension, among others) linked to human disease, such as in myotonic dystrophy type 2 (DM2) (Udd et al., 2011), progeria (Beard et al., 2008), Alzheimer's disease (Crowe et al., 2009) or cancer (Clower et al., 2010), where missplicing events are already well-described and for which key disease aspects are conserved in D. melanogaster (Pandey and Nichols, 2011). We foresee its extended use in genetic screens focused on better understanding the mechanisms of splicing misregulation in human disease. "
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    ABSTRACT: Alternative splicing of pre-messenger RNAs is an important mechanism to achieve correct cellular function in higher eukaryotes. It is known that a growing number of human genetic diseases involve important splicing defects directly connected to pathologic signs. In myotonic dystrophy type 1 (DM1) it is proposed that several clinical manifestations are consequence of tissue-specific missplicing of numerous genes, triggered by a RNA gain-of-function and resultant deregulation of specific RNA-binding factors, being remarkable the nuclear sequestration of muscleblind-like family factors (MBNL1-3). Thus, identification of chemical modulators of splicing events is thought to have beneficial impacts towards the development of first valid therapy for DM1 patients. For this purpose, we generated and validated transgenic flies, which contained a luciferase reporter-based system coupled to the expression of MBNL1-reliant splicing events deregulated in DM1 patients on a relevant disease tissue (spliceosensor flies). We then developed an innovative 96-well plate screening platform to carry out in vivo high throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities) several reliable splicing modulators (hits) were identified. Hit validation steps recognized separate DM1-linked therapeutic traits for some of them, which corroborated the feasibility of the approach described herein for revealing promising drug candidates by indirect improving of DM1 missplicing. The powerful Drosophila-based tool for innovative chemical screening is suitable for its use with other disease models displaying abnormal alternative splicing coupled to a luminescent reporter-based system as final read-out, thus offering widespread uses in drug discovery.
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    • "identifier: NCT01689246). Also, diverse possible candidates were suggested as tau aggregation inhibitor, including anthraquinones, aminothienopyridazines, polyphenols and phenothiazines (68, 90, 91, 92). These compounds, however, need more verification because they failed to show consistent efficacies in in vivo studies. "
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    • "Therefore, counterscreens are needed to ensure that compounds block early steps in the aggregation process. One example is to tag tau with a fluorescent moiety and use fluorescence polarization as a readout, as increased size of the assemblies (e.g., monomer to dimer to trimer, etc.) results in an increased signal [168]. "
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    ABSTRACT: The abnormal deposition of proteins in and around neurons is a common pathological feature of many neurodegenerative diseases. Among these pathological proteins, the microtubule-associated protein tau forms intraneuronal filaments in a spectrum of neurological disorders. The discovery that dominant mutations in the MAPT gene encoding tau are associated with familial frontotemporal dementia strongly supports abnormal tau protein as directly involved in disease pathogenesis. This and other evidence suggest that tau is a worthwhile target for the prevention or treatment of tau-associated neurodegenerative diseases, collectively called tauopathies. However, it is critical to understand the normal biological roles of tau, the specific molecular events that induce tau to become neurotoxic, the biochemical nature of pathogenic tau, the means by which pathogenic tau exerts neurotoxicity, and how tau pathology propagates. Based on known differences between normal and abnormal tau, a number of approaches have been taken toward the discovery of potential therapeutics. Key questions still remain open, such as the nature of the connection between the amyloid- β protein of Alzheimer's disease and tau pathology. Answers to these questions should help better understand the nature of tauopathies and may also reveal new therapeutic targets and strategies.
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