Journal: Nature Communications

Article Title: Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation

doi: 10.1038/s41467-021-26935-z

Figure Lengend Snippet: a Phylogenetic tree of human HIPK1–4 and DYRK1A kinases illustrating the homology of the catalytic domains. The average distance tree was calculated by percentage identity using Jalview . b Domain architecture of human HIPK1–4 and DYRK1A. HID homeoprotein-interacting domain, PEST proline, glutamate, serine, threonine-rich region, SQA serine, glutamine, alanine-rich region, DH DYRK homology box, HRD histidine-rich domain, ST serine/threonine-rich region. c Crystal structure of the human HIPK3 kinase domain (PDB: 7O7I). Key elements as the αC helix, the DFG motif, and the phosphorylated tyrosine within the activation loop are indicated. The CMGC insert region (residues 416–493) embedded between canonical helices αG and αH is colored from green to orange. d Coordination of the active center of the HIPK3 kinase. Electrostatic and hydrogen-bond interactions between the phosphorylated tyrosine of the activation loop, pY359, the RYYR element, the HAD motif with the general base, the DFG motif, the catalytic lysine K226 and the coordinating glutamate E241 of the αC helix are shown.

Article Snippet: The coding sequence of the human wild-type HIPK3 kinase domain (residues 159–562, UniProt accession number Q9H422) was PCR-amplified from Addgene plasmid #23467 and inserted into a pGEX-4T1 vector and a pACEBac1 vector, both containing an N-terminal GST-affinity tag followed by a TEV protease cleavage site.

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation

doi: 10.1038/s41467-021-26935-z

Figure Lengend Snippet: Sequence alignment of the kinase domain of human HIPKs and DYRK1A over the entire length of the crystallized HIPK3 construct (159–562). Secondary structure elements are indicated for HIPK3 as determined for the apo-HIPK3 structure. Characteristic sequence motifs, including the phosphorylated T-loop tyrosine, and functional regions are indicated. Serine/threonine or tyrosine residues found to be phosphorylated are circled magenta or green, respectively. Residues conserved in all kinases are boxed red, and those that are similar have red characters. The sequence alignment was prepared with MultAlin. The secondary structure alignment was prepared with ESPript. UniProt accession numbers are: Q86Z02 (HIPK1), Q9H2X6 (HIPK2), Q9H422 (HIPK3), Q8NE63 (HIPK4), and Q13627 (DYRK1A).

Article Snippet: The coding sequence of the human wild-type HIPK3 kinase domain (residues 159–562, UniProt accession number Q9H422) was PCR-amplified from Addgene plasmid #23467 and inserted into a pGEX-4T1 vector and a pACEBac1 vector, both containing an N-terminal GST-affinity tag followed by a TEV protease cleavage site.

Techniques: Sequencing, Construct, Functional Assay

Journal: Nature Communications

Article Title: Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation

doi: 10.1038/s41467-021-26935-z

Figure Lengend Snippet: a In HIPK2, DYRK1A, DYRK2, and DYRK3 the phosphorylated tyrosine residue of the activation loop forms salt bridges with the arginines of the R(Y/F)YR motif. In HIPK3 instead, pY359 interacts with R431 of the CMGC insert region. b Variations of the β-hairpin loop conformation in the CMGC insert region. Protein structures shown in this figure are HIPK2 (6P5S, palegreen), HIPK3 (7O7I, blue/green), DYRK1A (2VX3, magenta), DYRK2 (3K2L, yellow), and DYRK3 (5Y86, salmon).

Article Snippet: The coding sequence of the human wild-type HIPK3 kinase domain (residues 159–562, UniProt accession number Q9H422) was PCR-amplified from Addgene plasmid #23467 and inserted into a pGEX-4T1 vector and a pACEBac1 vector, both containing an N-terminal GST-affinity tag followed by a TEV protease cleavage site.

Techniques: Residue, Activation Assay

Journal: Nature Communications

Article Title: Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation

doi: 10.1038/s41467-021-26935-z

Figure Lengend Snippet: a Purification and activity measurements for all four HIPKs and DYRK1A. Recombinant protein kinases were either purified from Sf9 insect cells or from E. coli bacterial cells and analyzed by SDS-PAGE. Kinase activity was assessed using in vitro kinase assays with 0.2 mM [ 32 P]- γ -ATP, which was incubated for 30 min either without kinase, without substrate, as a kinase-dead mutation (HIPK1 D315N , HIPK2 D324N , HIPK3 D322N , and HIPK4 D136N ) or as wild-type kinase in the presence of 0.2 μM kinase and 10 μM His-c-Myc as a substrate. Measurements were performed as duplicates ( n = 2 biologically independent samples) and are depicted as mean. b Molecular masses of intact kinases determined by ESI–(LC)–MS indicate the total number of phosphorylations. Source data are provided as a .

Article Snippet: The coding sequence of the human wild-type HIPK3 kinase domain (residues 159–562, UniProt accession number Q9H422) was PCR-amplified from Addgene plasmid #23467 and inserted into a pGEX-4T1 vector and a pACEBac1 vector, both containing an N-terminal GST-affinity tag followed by a TEV protease cleavage site.

Techniques: Purification, Activity Assay, Recombinant, SDS Page, In Vitro, Incubation, Mutagenesis, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation

doi: 10.1038/s41467-021-26935-z

Figure Lengend Snippet: a A panel of 15 small-molecule inhibitors was tested at 1, 10, and 100 μM concentration for the inhibition of HIPK3 using radioactive kinase activity assays. Compounds were preincubated for 5 min with 0.2 μM kinase and 0.2 mM [ 32 P]-γ-ATP, followed by the addition of 10 μM His-c-Myc as a substrate and another incubation for 15 min. Measurements were performed as duplicates ( n = 2 biologically independent samples) and are depicted as mean. b In vitro kinase assays were performed as in ( a ) testing concentration series of the best five small-molecule inhibitors for HIPK3. All data are depicted as mean ± SD from duplicates ( n = 2 biologically independent samples). A sigmoidal fit was used to calculate IC 50 values. c In vitro kinase assays were performed as in ( a ) using concentration series of abemaciclib for the inhibition of all four HIPKs, DYRK1A, Cdk4/CycD3, Cdk6/CycD3, and Cdk9/CycT1. All data are depicted as mean ± SD from duplicates ( n = 2 biologically independent samples). Source data are provided as a .

Article Snippet: The coding sequence of the human wild-type HIPK3 kinase domain (residues 159–562, UniProt accession number Q9H422) was PCR-amplified from Addgene plasmid #23467 and inserted into a pGEX-4T1 vector and a pACEBac1 vector, both containing an N-terminal GST-affinity tag followed by a TEV protease cleavage site.

Techniques: Concentration Assay, Inhibition, Activity Assay, Incubation, In Vitro

Journal: Nature Communications

Article Title: Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation

doi: 10.1038/s41467-021-26935-z

Figure Lengend Snippet: a Overlay of the kinase domains of DYRK1A–abemaciclib (rose/green; PDB code 7O7K) and HIPK3–abemaciclib (light blue/cyan; PDB code 7O7J). b Binding modes of abemaciclib to HIPK3, DYRK1A, and Cdk6. Overlay of HIPK3–abemaciclib (PDB 7O7J), DYRK1A–abemaciclib (PDB 7O7K) and Cdk6–abemaciclib (PDB 5L2S) . Key active site residues are labeled, and hydrogen bonds are rendered as dotted lines. c Surface display of DYRK1A with space providing key residues N244 and D247 indicated. Isoleucine 165 is contributing with the largest buried surface area of all residues to the interaction with abemaciclib. d In CDK2 (PDB code 1QMZ) , K89 occupies the site where otherwise the piperazine ring of abemaciclib interacts with I165 of DYRK1A. This sequence variability might contribute to the interaction specificity of abemaciclib to CMGC kinases. e Sequence alignment of human DYRK1A, HIPK3, Cdk4, and Cdk6 in the interacting regions with abemaciclib. Residues in DYRK1A mediating direct interactions with abemaciclib are boxed light and dark rose according to the buried surface area (8–20 and >20 Å 2 , respectively).

Article Snippet: The coding sequence of the human wild-type HIPK3 kinase domain (residues 159–562, UniProt accession number Q9H422) was PCR-amplified from Addgene plasmid #23467 and inserted into a pGEX-4T1 vector and a pACEBac1 vector, both containing an N-terminal GST-affinity tag followed by a TEV protease cleavage site.

Techniques: Binding Assay, Labeling, Sequencing