Synthesis and Properties of a Selective Inhibitor of Homeodomain–Interacting Protein Kinase 2 (HIPK2)

<div><p>Homeodomain-interacting protein kinase 2 (HIPK2) is a Ser/Thr kinase controlling cell proliferation and survival, whose investigation has been hampered by the lack of specific inhibitors able to dissect its cellular functions. SB203580, a p38 MAP kinase inhibitor, has been used as a tool to inhibit HIPK2 in cells, but here we show that its efficacy as HIPK2 inhibitor is negligible (IC₅₀>40 µM). In contrast by altering the scaffold of the promiscuous CK2 inhibitor TBI a new class of HIPK2 inhibitors has been generated. One of these, TBID, displays toward HIPK2 unprecedented efficacy (IC₅₀ = 0.33 µM) and selectivity (Gini coefficient 0.592 out of a panel of 76 kinases). The two other members of the HIPK family, HIPK1 and HIPK3, are also inhibited by TBID albeit less efficiently than HIPK2. The mode of action of TBID is competitive with respect to ATP, consistent with modelling. We also provide evidence that TBID is cell permeable by showing that HIPK2 activity is reduced in cells treated with TBID, although with an IC₅₀ two orders of magnitude higher (about 50 µM) than in vitro.</p></div

Cell treatment with TBID inhibits endogenous HIPK.

<p>A. HIPK2 was immunoprecipitated from lysates of HepG2 cells treated with different concentrations of TBID or the inactive analog <b>5e</b>, as indicated; HIPK2 activity was measure towards the specific peptide substrate, as detailed in Materials and Methods. The amount of immunoprecipitated HIPK2 is shown by WB. B. CEM cells were treated as indicated, then 10 µg of cell lysate proteins were analysed by WB with an antibody against pS46 of p53, anti-total p53, or anti-actin, as loading control. A representative experiment is shown on the left, while a histogram is presented on the right where Sp46 p53 of three separate experiments has been quantified, normalized to the p53 total level, and reported as means±SEM. C. Cell viability was assessed by the method of MTT, treating HepG2 or CEM cells as indicated (left panel) or by evaluating the amount of the caspase substrate PARP (HepG2 cells, right panel), whose reduction would denote apoptosis. The recognized band corresponds to the full length PARP protein of 116 kDa.</p

Lorenz curves, Gini coefficients and hit rates for TBID and TBI.

<p>For details see experimental section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089176#pone.0089176-Graczyk1" target="_blank">[25]</a>. Lorenz curves were drawn from the selectivity profile of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089176#pone-0089176-g004" target="_blank">Figure 4</a> and from analogous data published in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089176#pone.0089176-Pagano1" target="_blank">[19]</a> for TBID and TBI, respectively.</p

Phosphorylation of Akt1 and Akt2 peptide variants by CK2.

<p>(A) and (B): Peptides at the indicated concentrations were incubated with CK2 for 10 min, under conditions described in Materials and Methods. Results are reported as % of the cpm value obtained with Akt1 wt peptide (means of 4 experiments ± SEM). (C): CK2 was incubated with 2 mM Akt2 peptide for 20 or 120 min in a radioactive mixture; incubations were also performed in the absence of peptide, to evaluate possible contribution of CK2 autophosphorylation. Samples were hydrolyzed to obtain free phospho-aminoacids, that were separated by high voltage electrophoresis and detected by digital autoradiography. Migrations of standard phospho-aminoacids, as well as the starting point of the electrophoresis, are indicated.</p

IC₅₀ (µM) of 2-aryl-4,5,6,7-tetrabromoisoindoline-1,3-dione derivatives (Figure 1) for HIPK2, CK2, PIM1, CK1; TBB and TBI values are drawn from [19].

<p>n.d. = not determined.</p

Comparison of Akt2 sequences in different species.

<p>Analysis was performed by means of Basic Local Alignment Search Tool (BLAST) software of Uniprot. A 29-aminoacid fragment around S131 Akt2 of human sequences was used as reference. Invariable residues at positions between n-3 and n+3 are in bold, while variations are underlined. The common name of species with T at n+1 position, as in human, are underlined.</p

Kinetic analysis of HIPK2 inhibition by TBID.

<p>Inhibition of HIPK2 by TBID is competitive with respect to the phosphodonor substrate ATP. Kinetics were performed as described in Materials and Method either in the absence or in the presence of the indicated TBID concentrations. The data represent means of triplicate experiments with SEM never exceeding 15%.</p

List of the peptides used in this work, with sequence (one-letter code) and nomenclature adopted.

<p>The parental peptides are indicated as wild type (wt). The CK2 target site Akt1 S129 and its homologous residue Akt2 S131 are underlined. Positions not conserved in the two Akt isoforms (human sequence), and substitutions that swap Akt1/Akt2 positions in the variant peptides, are in bold. The same colors denote substitutions that swap Akt1/Akt2 positions. Green indicates other substitutions. Three N-terminal Arginine residues (R3) were introduced for technical reasons (see Methods).</p

Effect of n+1 T on Akt2 secondary structure.

<p>(A) Predicted secondary structure for Akt1 / Akt2 peptide variants: α-helix is indicated by red underlining. The results from different algorithms (see Methods) were evaluated and considered acceptable only when consensus was at least 90%, with the exception of Akt2TS peptide, where (*) indicates that a consensus of 70% was considered. (B) Docking sampling of the most energetically favorable complexes clusterized by an RMSD < 3Å. (C) Protein-protein docking analysis and Molecular Dynamics simulation. Comparison between the Akt1 peptide (Violet) and Akt2 peptide (orange) docked against CK2α (green, PDB code: 3Q04); in the inset, the detailed interaction of Akt1 and CK2α substrate binding site is magnified.</p

Comparison of Akt1 sequences in different species.

<p>Analysis was performed by means of Basic Local Alignment Search Tool (BLAST) software of Uniprot. A 29-aminoacid fragment around S129 Akt1 of human sequences was used as reference. Invariable residues at positions between n-3 and n+3 are in bold, while variations are underlined.</p