Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin-dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology
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eLife assessment
This important study reports selective CDKL5 inhibitors that may be of high interest to investigate the role of this kinase in disease (particularly, in CDKL5 deficiency disorder) and to address unsolved issues of inconsistency in the phenotypic characterization of CDKL5-deficient knockout mice. The evidence supporting the conclusions is convincing, with rigorous biochemical, in vitro and ex vivo assays. The work will be of interest to cell and medical biologists and epileptologists working in the fields of epilepsy and neural excitation.
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Abstract
Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 ( CDKL5 ) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual, and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD has indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3β, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3β activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3β activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3β. These compounds are very soluble in water but blood–brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces postsynaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity, and human neuropathology.
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Author Response
Reviewer #3 (Public Review):
In this manuscript, Castano et al generate and test a small molecule inhibitor of CDKL5, an Xlinked kinase whose loss-of-function is the cause of a severe neurodevelopmental disorder. Since the current knowledge of CDKL5 functions mainly rely on genetic models it is still unclear which effects are caused directly by CDKL5 loss and which can be ascribed to indirect effects. A specific inhibitor would therefore be an important tool for the field.
Castano and colleagues therefore tested a panel of twenty kinase inhibitors for their capacity to block phosphorylation of a EB2, a bona fide CDKL5 substrate, in rat neurons. Among the three that could inhibit EB2 phosphorylation at low concentrations, one was found to inhibit CDKL5 while not affecting GSK3 kinases, which share significant …
Author Response
Reviewer #3 (Public Review):
In this manuscript, Castano et al generate and test a small molecule inhibitor of CDKL5, an Xlinked kinase whose loss-of-function is the cause of a severe neurodevelopmental disorder. Since the current knowledge of CDKL5 functions mainly rely on genetic models it is still unclear which effects are caused directly by CDKL5 loss and which can be ascribed to indirect effects. A specific inhibitor would therefore be an important tool for the field.
Castano and colleagues therefore tested a panel of twenty kinase inhibitors for their capacity to block phosphorylation of a EB2, a bona fide CDKL5 substrate, in rat neurons. Among the three that could inhibit EB2 phosphorylation at low concentrations, one was found to inhibit CDKL5 while not affecting GSK3 kinases, which share significant homology to CDKL5. Considering that genetic studies have previously linked CDKL5 to excitatory synaptic transmission, acute hippocampal slices were exploited to test the consequences of CDKL5 inhibition. While CDKL5 loss in the past was found to affect both AMPA- and NMDA-Rs, the small molecule-based inhibition affected only AMPA-R responses at the post-synaptic level. Since pharmacokinetic analyses showed that the inhibitor has a low capacity for brain penetration the molecule remains limited for testing the acute inhibition of CDKL5 in vitro and ex vivo. Such a tool represents an important aspect in the CDKL5 field and the findings suggesting a direct role of CDKL5 in regulating AMPA-R functions are interesting. However, the manuscript could be improved to render it more readable.
Thank you for this positive feedback and we hope that our adjustments improve the readability.
The description of the binding and orthogonal assays, which are the basis for the selection of the small molecule inhibitor, is not straightforward to understand for non-expert readers and could be improved.
We have added additional text to the Methods and Results to better explain the assays.
While the in vitro and ex vivo assays are well presented, it is not clear why the myelin basic protein is used as a substrate for CDKL5 in the in vitro kinase assays. Does this protein contain a CDKL5 consensus site?
To execute the in vitro kinase assays, myelin basic protein (Active Motif, 31314) was employed as a substrate for recombinant CDKL5. Myelin basic protein is used as a substrate for multiple kinases, both serine/threonine and tyrosine kinases, to enable in vitro kinase assays due to the presence of multiple sites for phosphorylation. As such, we and others have used this protein as a kinase substrate for evaluating kinase activity[2, 4]. MBP does not contain a CDKL5 consensus site of RPXS/T*, and as such could be considered a less than ideal substrate to study CDKL5 activity, however for in vitro kinase assays MBP is still suitable as it can be phosphorylated by CDKL5. In addition, CDKL5 is known to phosphorylate substrates that do not contain a consensus motif[3].
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eLife assessment
This important study reports selective CDKL5 inhibitors that may be of high interest to investigate the role of this kinase in disease (particularly, in CDKL5 deficiency disorder) and to address unsolved issues of inconsistency in the phenotypic characterization of CDKL5-deficient knockout mice. The evidence supporting the conclusions is convincing, with rigorous biochemical, in vitro and ex vivo assays. The work will be of interest to cell and medical biologists and epileptologists working in the fields of epilepsy and neural excitation.
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Reviewer #1 (Public Review):
Castano et al. report a screening to search for selective CDKL5 inhibitors. After profiling an extensive library of selective cyclin-dependen kinase inhibitors, the authors synthesized and characterized high-affinity selective inhibitors of CDKL5. Kinome-wide studies were performed to verify selectivity. Preliminary PK studies were realized in rodents, including the determination of total brain/plasma ratio associated with two dose levels and microsomal stability. When applied directly to rat hippocampal brain slices, one of the inhibitors (CAF-382) reduced post-synaptic function of AMPA-type glutamate receptors dose-dependently, and also reduced hippocampal long-term potentiation. CAF-382 could be a valuable tool to further investigate the role of CDKL5 in disease, although some potential applications may …
Reviewer #1 (Public Review):
Castano et al. report a screening to search for selective CDKL5 inhibitors. After profiling an extensive library of selective cyclin-dependen kinase inhibitors, the authors synthesized and characterized high-affinity selective inhibitors of CDKL5. Kinome-wide studies were performed to verify selectivity. Preliminary PK studies were realized in rodents, including the determination of total brain/plasma ratio associated with two dose levels and microsomal stability. When applied directly to rat hippocampal brain slices, one of the inhibitors (CAF-382) reduced post-synaptic function of AMPA-type glutamate receptors dose-dependently, and also reduced hippocampal long-term potentiation. CAF-382 could be a valuable tool to further investigate the role of CDKL5 in disease, although some potential applications may be limited by the seemingly low brain bioavailability of this compound.
The conclusions of this paper are in general terms well supported by data, but some aspects of the discussion of the results could be extended.
-
Reviewer #2 (Public Review):
In the present study, Castano et al. discovered a chemical inhibitor that is specifically effective against the kinase activity of CDKL5 and applied it in the in vitro and the brain slice culture to reveal the acute effects of the loss of function (LOF) of CDKL5. LOF has been modeled in gene knockout mice, but these are loss-of-function models with the added developmental time effects of the absence of CDKL5 from developmental stages. The present authors' approach is the fastest timescale study to date, examining CDKL5 LOF effects in seconds to minutes.
The authors showed that chemical inhibition of CDKL5 kinase activity suppresses postsynaptically derived LTP in rat brain slice experiments, indicating that the previously controversial results of CDKL5 LOF on LTP in knockout mice and rats are possibly due to …
Reviewer #2 (Public Review):
In the present study, Castano et al. discovered a chemical inhibitor that is specifically effective against the kinase activity of CDKL5 and applied it in the in vitro and the brain slice culture to reveal the acute effects of the loss of function (LOF) of CDKL5. LOF has been modeled in gene knockout mice, but these are loss-of-function models with the added developmental time effects of the absence of CDKL5 from developmental stages. The present authors' approach is the fastest timescale study to date, examining CDKL5 LOF effects in seconds to minutes.
The authors showed that chemical inhibition of CDKL5 kinase activity suppresses postsynaptically derived LTP in rat brain slice experiments, indicating that the previously controversial results of CDKL5 LOF on LTP in knockout mice and rats are possibly due to combined effects of the loss of the kinase and compensation by other factors.
The authors employed state-of-the-art methodologies and presented their data clearly and convincingly.
-
Reviewer #3 (Public Review):
In this manuscript, Castano et al generate and test a small molecule inhibitor of CDKL5, an X-linked kinase whose loss-of-function is the cause of a severe neurodevelopmental disorder. Since the current knowledge of CDKL5 functions mainly rely on genetic models it is still unclear which effects are caused directly by CDKL5 loss and which can be ascribed to indirect effects. A specific inhibitor would therefore be an important tool for the field.
Castano and colleagues therefore tested a panel of twenty kinase inhibitors for their capacity to block phosphorylation of a EB2, a bona fide CDKL5 substrate, in rat neurons. Among the three that could inhibit EB2 phosphorylation at low concentrations, one was found to inhibit CDKL5 while not affecting GSK3 kinases, which share significant homology to CDKL5. …
Reviewer #3 (Public Review):
In this manuscript, Castano et al generate and test a small molecule inhibitor of CDKL5, an X-linked kinase whose loss-of-function is the cause of a severe neurodevelopmental disorder. Since the current knowledge of CDKL5 functions mainly rely on genetic models it is still unclear which effects are caused directly by CDKL5 loss and which can be ascribed to indirect effects. A specific inhibitor would therefore be an important tool for the field.
Castano and colleagues therefore tested a panel of twenty kinase inhibitors for their capacity to block phosphorylation of a EB2, a bona fide CDKL5 substrate, in rat neurons. Among the three that could inhibit EB2 phosphorylation at low concentrations, one was found to inhibit CDKL5 while not affecting GSK3 kinases, which share significant homology to CDKL5. Considering that genetic studies have previously linked CDKL5 to excitatory synaptic transmission, acute hippocampal slices were exploited to test the consequences of CDKL5 inhibition. While CDKL5 loss in the past was found to affect both AMPA- and NMDA-Rs, the small molecule-based inhibition affected only AMPA-R responses at the post-synaptic level. Since pharmacokinetic analyses showed that the inhibitor has a low capacity for brain penetration the molecule remains limited for testing the acute inhibition of CDKL5 in vitro and ex vivo. Such a tool represents an important aspect in the CDKL5 field and the findings suggesting a direct role of CDKL5 in regulating AMPA-R functions are interesting. However, the manuscript could be improved to render it more readable.
The description of the binding and orthogonal assays, which are the basis for the selection of the small molecule inhibitor, is not straightforward to understand for non-expert readers and could be improved.
While the in vitro and ex vivo assays are well presented, it is not clear why the myelin basic protein is used as a substrate for CDKL5 in the in vitro kinase assays. Does this protein contain a CDKL5 consensus site?
-
eLife assessment
This important study reports selective CDKL5 inhibitors that may be of high interest to investigate the role of this kinase in disease (particularly, in CDKL5 deficiency disorder) and to address unsolved issues of inconsistency in the phenotypic characterization of CDKL5-deficient knockout mice. The evidence supporting the conclusions is convincing, with rigorous biochemical, in vitro and ex vivo assays. The work will be of interest to cell and medical biologists and epileptologists working in the fields of epilepsy and neural excitation.
-
Reviewer #1 (Public Review):
Castano et al. report a screening to search for selective CDKL5 inhibitors. After profiling an extensive library of selective cyclin-dependen kinase inhibitors, the authors synthesized and characterized high-affinity selective inhibitors of CDKL5. Kinome-wide studies were performed to verify selectivity. Preliminary PK studies were realized in rodents, including the determination of total brain/plasma ratio associated with two dose levels and microsomal stability. When applied directly to rat hippocampal brain slices, one of the inhibitors (CAF-382) reduced post-synaptic function of AMPA-type glutamate receptors dose-dependently, and also reduced hippocampal long-term potentiation. CAF-382 could be a valuable tool to further investigate the role of CDKL5 in disease, although some potential applications may …
Reviewer #1 (Public Review):
Castano et al. report a screening to search for selective CDKL5 inhibitors. After profiling an extensive library of selective cyclin-dependen kinase inhibitors, the authors synthesized and characterized high-affinity selective inhibitors of CDKL5. Kinome-wide studies were performed to verify selectivity. Preliminary PK studies were realized in rodents, including the determination of total brain/plasma ratio associated with two dose levels and microsomal stability. When applied directly to rat hippocampal brain slices, one of the inhibitors (CAF-382) reduced post-synaptic function of AMPA-type glutamate receptors dose-dependently, and also reduced hippocampal long-term potentiation. CAF-382 could be a valuable tool to further investigate the role of CDKL5 in disease, although some potential applications may be limited by the seemingly low brain bioavailability of this compound.
The conclusions of this paper are in general terms well supported by data, but some aspects of the discussion of the results could be extended.
-
Reviewer #2 (Public Review):
In the present study, Castano et al. discovered a chemical inhibitor that is specifically effective against the kinase activity of CDKL5 and applied it in the in vitro and the brain slice culture to reveal the acute effects of the loss of function (LOF) of CDKL5. LOF has been modeled in gene knockout mice, but these are loss-of-function models with the added developmental time effects of the absence of CDKL5 from developmental stages. The present authors' approach is the fastest timescale study to date, examining CDKL5 LOF effects in seconds to minutes.
The authors showed that chemical inhibition of CDKL5 kinase activity suppresses postsynaptically derived LTP in rat brain slice experiments, indicating that the previously controversial results of CDKL5 LOF on LTP in knockout mice and rats are possibly due to …
Reviewer #2 (Public Review):
In the present study, Castano et al. discovered a chemical inhibitor that is specifically effective against the kinase activity of CDKL5 and applied it in the in vitro and the brain slice culture to reveal the acute effects of the loss of function (LOF) of CDKL5. LOF has been modeled in gene knockout mice, but these are loss-of-function models with the added developmental time effects of the absence of CDKL5 from developmental stages. The present authors' approach is the fastest timescale study to date, examining CDKL5 LOF effects in seconds to minutes.
The authors showed that chemical inhibition of CDKL5 kinase activity suppresses postsynaptically derived LTP in rat brain slice experiments, indicating that the previously controversial results of CDKL5 LOF on LTP in knockout mice and rats are possibly due to combined effects of the loss of the kinase and compensation by other factors.
The authors employed state-of-the-art methodologies and presented their data clearly and convincingly.
-
Reviewer #3 (Public Review):
In this manuscript, Castano et al generate and test a small molecule inhibitor of CDKL5, an X-linked kinase whose loss-of-function is the cause of a severe neurodevelopmental disorder. Since the current knowledge of CDKL5 functions mainly rely on genetic models it is still unclear which effects are caused directly by CDKL5 loss and which can be ascribed to indirect effects. A specific inhibitor would therefore be an important tool for the field.
Castano and colleagues therefore tested a panel of twenty kinase inhibitors for their capacity to block phosphorylation of a EB2, a bona fide CDKL5 substrate, in rat neurons. Among the three that could inhibit EB2 phosphorylation at low concentrations, one was found to inhibit CDKL5 while not affecting GSK3 kinases, which share significant homology to CDKL5. …
Reviewer #3 (Public Review):
In this manuscript, Castano et al generate and test a small molecule inhibitor of CDKL5, an X-linked kinase whose loss-of-function is the cause of a severe neurodevelopmental disorder. Since the current knowledge of CDKL5 functions mainly rely on genetic models it is still unclear which effects are caused directly by CDKL5 loss and which can be ascribed to indirect effects. A specific inhibitor would therefore be an important tool for the field.
Castano and colleagues therefore tested a panel of twenty kinase inhibitors for their capacity to block phosphorylation of a EB2, a bona fide CDKL5 substrate, in rat neurons. Among the three that could inhibit EB2 phosphorylation at low concentrations, one was found to inhibit CDKL5 while not affecting GSK3 kinases, which share significant homology to CDKL5. Considering that genetic studies have previously linked CDKL5 to excitatory synaptic transmission, acute hippocampal slices were exploited to test the consequences of CDKL5 inhibition. While CDKL5 loss in the past was found to affect both AMPA- and NMDA-Rs, the small molecule-based inhibition affected only AMPA-R responses at the post-synaptic level. Since pharmacokinetic analyses showed that the inhibitor has a low capacity for brain penetration the molecule remains limited for testing the acute inhibition of CDKL5 in vitro and ex vivo. Such a tool represents an important aspect in the CDKL5 field and the findings suggesting a direct role of CDKL5 in regulating AMPA-R functions are interesting. However, the manuscript could be improved to render it more readable.
The description of the binding and orthogonal assays, which are the basis for the selection of the small molecule inhibitor, is not straightforward to understand for non-expert readers and could be improved.
While the in vitro and ex vivo assays are well presented, it is not clear why the myelin basic protein is used as a substrate for CDKL5 in the in vitro kinase assays. Does this protein contain a CDKL5 consensus site?
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