Please cite this article in press as: Ferguson et al., Discovery of Covalent CDK14 Inhibitors with Pan-TAIRE Family Specificity, Cell Chemical Biology
2019), https://doi.org/10.1016/j.chembiol.2019.02.015
(
progression, in agreement with cell-cycle analysis data. These
phenotypes warrant further investigation.
B In Vitro Phosphorylation of Synthetic Peptides followed
by MS/MS
Finally, the availability of a narrow-spectrum TAIRE family
kinase tool compound enables study of this class of kinases
upon acute inhibition, complementing previous genetic studies.
As there is evidence that the TAIRE kinases have some redun-
dancies, the ability to rapidly inhibit these kinases in concert
may also aid in initial substrate and functional annotation.
B Chemistry Synthetic Schemes
B General Chemistry Methods
B Experimental Details for Individual Compound Syn-
thesis
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Antiproliferation Assay
B In Vitro Kinase Assays
B NanoBRET Assay
SIGNIFICANCE
B Intact Mass and Compound Labeling
B FACS
CDK14 and other TAIRE cyclin-dependent kinase family
members (CDKs 15–18) are frequent drug off-targets, despite
their biological functions remaining poorly understood. Here
we describe the development of FMF-04-159-2, a tool com-
pound that specifically targets CDK14 covalently. FMF-04-
B Immunohistochemistry
B Proteomics and Phospho-proteomics
d DATA AND SOFTWARE AVAILABILITY
SUPPLEMENTAL INFORMATION
1
59-2hasa TAIRE kinase-biasedreversible bindingselectivity
profile, with substantial off-target activity only detected
against CDK2. In addition to validating LRP6 as a substrate
of CDK14 in mitosis, unbiased investigation into the conse-
quences of pan-TAIRE/CDK2 and covalent CDK14 inhibition
through phospho-proteomics suggested that CDK14 plays a
supporting role in cell-cycle regulation. We characterize the
cellular consequences of covalent CDK14 kinase inhibition,
and describe characteristics of putative substrates of these
kinases. When used in combination with FMF-04-159-R, or a
compound washout control, FMF-04-159-2 is a valuable tool
for further study of CDK14 kinase function.
ACKNOWLEDGMENTS
The authors would like to acknowledge Eric Wang and Milka Kostic for helpful
comments on the manuscript. The authors would also like to acknowledge
Matthew Robers and the scientists at Promega, for helpful discussion and pro-
tocol sharing related to the NanoBRET cellular target engagement assays. This
work was supported by R35CA197588, P50-GM107618, U24-DK116204
R21CA188881, R01CA219850, the DFCI Strategic Research Initiative, and
the Chleck Family Foundation, United States.
AUTHOR CONTRIBUTIONS
STAR+METHODS
F.M.F. designed and synthesized all compounds. Z.M.D. designed and con-
ducted the experiments, and performed data analysis. S.B.F. and C.M.B. con-
ducted MS experiments. M.J.B. and M.K. conducted the proteomics and
phospho-proteomics experiments. J.L.J. and T.M.Y. conducted phospho-
array experiments and peptide motif analysis. N.D.K. and T.S. performed all
compound docking and modeling studies. F.M.F. and Z.M.D. wrote the
manuscript, with guidance from N.S.G., J.A.M., L.C.C. and P.K.S. supervised
the research. All authors gave feedback on the manuscript.
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
B Pull Down / Cellular Target Engagement Protocol
B Antiproliferation Assay Protocol
DECLARATION OF INTERESTS
L.C.C. is a founder and member of the Scientific Advisory Board (SAB) and
holds equity in Agios Pharmaceuticals and Petra Pharmaceuticals. L.C.C. is
also a member of the Board of Directors (BOD) of Agios and an observer on
the BOD of Petra. Petra provides partial support for his laboratory. Both Agios
and Petra are developing drugs for cancer therapies.
B In Vitro Kinase Assays
B Intact Mass Spectrometry Analysis
B Nano-LC/MS Analysis of Labeled CDK14
B Cell Line Generation
B KiNativ Live Cell Profiling Protocol
B Fluorescence-Activated Cell Sorting Analysis (FACS)
for Cell Cycle Analysis
J.M. is a member of the SAB of Devices.
N.S.G. is a founder, SAB member, and equity holder in Gatekeeper, Syros,
Petra, C4, B2S, and Soltego. The Gray lab receives or has received research
funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Her2llc,
Deerfield, and Sanofi.
B Inhibitor Treatment and Western Blotting Experiments
B NanoBRET Live Cell Target Engagement
B Immunohistochemistry for Phalloidin Staining
B Double Thymidine Synchronization with Compound
Treatment
N.S.G., F.M.F., and Z.M.D. are inventors on a patent application covering
chemical matter in this publication owned by Dana-Farber Cancer Institute.
P.K.S. is a founder, SAB member, and equity holder in Merrimack Pharma-
ceutical and Glencoe Software; he is on the BOD of Applied Biomath and the
SAB of RareCyte. In the last 5 years the Sorger lab has received research fund-
ing from Novartis and Merck. P.K.S. declares that none of these relationships
are directly or indirectly related to the content of this manuscript.
B Peptide Library Assay for Phospho-Motif Identification
B Proteomics and Phospho-proteomics Cell Lysis, Pro-
tein Digest and TMT Labeling
B Proteomics and Phospho-proteomics Phosphopep-
tide Enrichment
Received: September 19, 2018
Revised: January 23, 2019
Accepted: February 24, 2019
Published: March 28, 2019
B Proteomics and Phospho-proteomics Mass Spec-
trometry Analysis
12 Cell Chemical Biology 26, 1–14, June 20, 2019