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John J. Flanagan

Bio: John J. Flanagan is an academic researcher from Amicus Therapeutics. The author has contributed to research in topics: Pharmacological chaperone & Enzyme replacement therapy. The author has an hindex of 25, co-authored 51 publications receiving 3274 citations. Previous affiliations of John J. Flanagan include University of California, Los Angeles & Dartmouth College.


Papers
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Journal ArticleDOI
TL;DR: Major improvements to the proteolysis targeting chimeras (PROTACs) method are described, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation.
Abstract: The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR

799 citations

Journal ArticleDOI
04 May 2007-Cell
TL;DR: It is demonstrated that dendrite self-avoidance in Drosophila da sensory neurons requires cell-recognition molecules encoded by the Dscam locus, which encodes a vast number of cell-surface proteins of the immunoglobulin superfamily.

346 citations

Journal ArticleDOI
03 Sep 2004-Cell
TL;DR: It is demonstrated that different isoforms exhibit different binding specificity, and it is proposed that this preferential homophilic binding specificity regulates interactions between cells and contributes to the formation of complex patterns of neuronal connections.

307 citations

Journal ArticleDOI
02 Sep 2004-Neuron
TL;DR: It is proposed that different extracellular domains of Dscam share a common function and that differences in isoforms expressed on the surface of neighboring axons influence interactions between them.

212 citations

Journal ArticleDOI
TL;DR: The data indicate that DGJ merits further evaluation as a treatment for patients with Fabry disease with various missense mutations, and elevated GL-3 levels in responsive Fabry fibroblasts were reduced after DGJ incubation, indicating that increased mutant α-Gal A levels can reduce accumulated substrate.
Abstract: Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene encoding α-galactosidase A (α-Gal A), with consequent accumulation of its major glycosphingolipid substrate, globotriaosylceramide (GL-3). Over 500 Fabry mutations have been reported; approximately 60% are missense. The iminosugar 1-deoxygalactonojirimycin (DGJ, migalastat hydrochloride, AT1001) is a pharmacological chaperone that selectively binds α-Gal A, increasing physical stability, lysosomal trafficking, and cellular activity. To identify DGJ-responsive mutant forms of α-Gal A, the effect of DGJ incubation on α-Gal A levels was assessed in cultured lymphoblasts from males with Fabry disease representing 75 different missense mutations, one insertion, and one splice-site mutation. Baseline α-Gal A levels ranged from 0 to 52% of normal. Increases in α-Gal A levels (1.5- to 28-fold) after continuous DGJ incubation for 5 days were seen for 49 different missense mutant forms with varying EC50 values (820 nmol/L to >1 mmol/L). Amino acid substitutions in responsive forms were located throughout both structural domains of the enzyme. Half of the missense mutant forms associated with classic (early-onset) Fabry disease and a majority (90%) associated with later-onset Fabry disease were responsive. In cultured fibroblasts from males with Fabry disease, the responses to DGJ were comparable to those of lymphoblasts with the same mutation. Importantly, elevated GL-3 levels in responsive Fabry fibroblasts were reduced after DGJ incubation, indicating that increased mutant α-Gal A levels can reduce accumulated substrate. These data indicate that DGJ merits further evaluation as a treatment for patients with Fabry disease with various missense mutations.

162 citations


Cited by
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Journal ArticleDOI
TL;DR: RNAscope is described, a novel RNA ISH technology with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology and may enable rapid development of RNAISH-based molecular diagnostic assays.

1,929 citations

Journal ArticleDOI
16 May 2013-Nature
TL;DR: It is shown that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.
Abstract: Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.

1,776 citations

Journal ArticleDOI
TL;DR: An overview of the role of autophagy in neurodegenerative disease is provided, focusing particularly on less frequently considered lysosomal clearance mechanisms and their considerable impact on disease.
Abstract: This Review provides an overview of the role of autophagy, a key lysosomal degradative process, in neurodegenerative diseases. The study of various neurodegenerative diseases has shown that defects in autophagy can arise at different points in the pathway, and this has implications for the successful modulation of autophagy for therapeutic purposes. The Review also discusses the latest developments in targeting alterations in autophagy as a therapeutic strategy for neurodegenerative diseases.

1,643 citations

Journal ArticleDOI
TL;DR: Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global approaches that promise to reveal details of the nature and operation of cellular codes that are constituted by combinations of regulatory elements in pre-mRNA substrates and by cellular complements of splicing regulators, which together determine regulated splicing pathways.
Abstract: In violation of the 'one gene, one polypeptide' rule, alternative splicing allows individual genes to produce multiple protein isoforms - thereby playing a central part in generating complex proteomes. Alternative splicing also has a largely hidden function in quantitative gene control, by targeting RNAs for nonsense-mediated decay. Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global approaches. These promise to reveal details of the nature and operation of cellular codes that are constituted by combinations of regulatory elements in pre-mRNA substrates and by cellular complements of splicing regulators, which together determine regulated splicing pathways.

1,334 citations

Journal ArticleDOI
TL;DR: It is becoming apparent that molecules previously known for their role in patterning can also direct axonal outgrowth and a sophisticated and dynamic set of cues that enable a growth cone to successfully navigate to its destination, modulating its response to changing environmental cues along its pathway.

1,009 citations