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A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective

Han Wee Ong, Yi Liang, William Richardson, Emily R. Lowry, Carrow I. Wells, Xiangrong Chen, Margaux Silvestre, Kelvin Dempster, Josie A. Silvaroli, Jeffery L. Smith, Hynek Wichterle, Navjot S. Pabla, Sila K. Ultanir, Alex N. Bullock, David H. Drewry, View ORCID ProfileAlison D. Axtman
doi: https://doi.org/10.1101/2023.02.09.527935
Han Wee Ong
1Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America
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Yi Liang
1Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America
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William Richardson
2Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7DQ, United Kingdom
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Emily R. Lowry
3Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, New York, 10032, United States of America
4The Project ALS Therapeutics Core, Columbia University Irving Medical Center, New York, New York, 10032, United States of America
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Carrow I. Wells
1Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America
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Xiangrong Chen
2Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7DQ, United Kingdom
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Margaux Silvestre
5Kinases and Brain Development Laboratory, The Francis Crick Institute, London, NW1 1AT, United Kingdom
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Kelvin Dempster
5Kinases and Brain Development Laboratory, The Francis Crick Institute, London, NW1 1AT, United Kingdom
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Josie A. Silvaroli
6Division of Pharmaceutics and Pharmacology, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210, United States of America
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Jeffery L. Smith
1Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America
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Hynek Wichterle
7Departments of Pathology and Cell Biology, Neurology, Neuroscience, Rehabilitation and Regenerative Medicine, Columbia University Irving Medical Center, New York, New York, 10032, United States of America
8Center for Motor Neuron Biology and Disease, Columbia University Irving Medical Center, New York, New York, 10032, United States of America
9Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, New York, 10032, United States of America
4The Project ALS Therapeutics Core, Columbia University Irving Medical Center, New York, New York, 10032, United States of America
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Navjot S. Pabla
6Division of Pharmaceutics and Pharmacology, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210, United States of America
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Sila K. Ultanir
5Kinases and Brain Development Laboratory, The Francis Crick Institute, London, NW1 1AT, United Kingdom
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Alex N. Bullock
2Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7DQ, United Kingdom
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David H. Drewry
1Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America
10UNC Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America
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  • For correspondence: David.Drewry@unc.edu Alison.Axtman@unc.edu
Alison D. Axtman
1Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America
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  • ORCID record for Alison D. Axtman
  • For correspondence: David.Drewry@unc.edu Alison.Axtman@unc.edu
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ABSTRACT

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/β affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3α/β signaling and solved a co-crystal structure of analog 2 bound to CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/β. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3α/β promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Corrected Figure 6 legend

  • ABBREVIATIONS

    AKT
    protein kinase B
    Amph1
    amphiphysin
    ANOVA
    analysis of variance
    ARHGEF2
    Rho/Rac guanine nucleotide exchange factor 2
    CDK
    cyclin dependent kinase
    CDKL
    cyclin-dependent kinase-like
    CEP131
    centrosomal protein 131
    DIPEA
    N,N-diisopropylethyl amine
    DLG5
    Discs large MAGUK scaffold protein 5
    Dnmt1
    DNA methyltransferase
    DYRK2
    dual specificity tyrosine phosphorylation regulated kinase 2
    EB2
    microtubule end-binding protein 2
    ELOA
    elongin A
    Glu
    glutamic acid
    H2B
    histone H2B
    HCl
    hydrochloric acid
    HDAC4
    histone deacetylase 4
    HIPK2
    homeodomain interacting protein kinase 2
    iPSC
    induced pluripotent stem cell
    Lys
    lysine
    MAP1S
    microtubule associated protein 1S
    MeCP2
    Methyl-CpG-binding-protein
    NGL-1
    netrin-G ligand-1
    PDK1
    3-phosphoinositide-dependent kinase 1
    Rac1
    Ras-related C3 botulinum toxin substrate 1
    RT
    room temperature
    Sox9
    SRY-box transcription factor 9
    THF
    tetrahydrofuran
    Thr
    threonine
    Tyr
    tyrosine
    Val
    valine
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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    Posted February 11, 2023.
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    A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective
    Han Wee Ong, Yi Liang, William Richardson, Emily R. Lowry, Carrow I. Wells, Xiangrong Chen, Margaux Silvestre, Kelvin Dempster, Josie A. Silvaroli, Jeffery L. Smith, Hynek Wichterle, Navjot S. Pabla, Sila K. Ultanir, Alex N. Bullock, David H. Drewry, Alison D. Axtman
    bioRxiv 2023.02.09.527935; doi: https://doi.org/10.1101/2023.02.09.527935
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    A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective
    Han Wee Ong, Yi Liang, William Richardson, Emily R. Lowry, Carrow I. Wells, Xiangrong Chen, Margaux Silvestre, Kelvin Dempster, Josie A. Silvaroli, Jeffery L. Smith, Hynek Wichterle, Navjot S. Pabla, Sila K. Ultanir, Alex N. Bullock, David H. Drewry, Alison D. Axtman
    bioRxiv 2023.02.09.527935; doi: https://doi.org/10.1101/2023.02.09.527935

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