Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1 Inhibitors as an Example

Abstract

Phosphorylation events catalyzed by protein kinases represent one of the most prevalent as well as important regulatory posttranslational modifications, and dysregulation of protein kinases is associated with the pathogenesis of different diseases. Therefore, interest in developing potent small molecule kinase inhibitors has increased enormously within the last two decades. A critical step in the development of new inhibitors is cell-free in vitro testing with the intention to determine comparable parameters like the commonly used IC50 value. However, values described in the literature are often biased as experimental setups used for determination of kinase activity lack comparability due to different readout parameters, insufficient normalization or the sheer number of experimental approaches. Here, we would like to hold a brief for highly sensitive, radioactive-based in vitro kinase assays especially suitable for kinases exhibiting autophosphorylation activity. Therefore, we demonstrate a systematic workflow for complementing and validating results from high-throughput screening as well as increasing the comparability of enzyme-specific inhibitor parameters for radiometric as well as non-radiometric assays. Using members of the CK1 family of serine/threonine-specific protein kinases and established CK1-specific inhibitors as examples, we clearly demonstrate the power of our proposed workflow, which has the potential to support the generation of more comparable data for biological characterization of kinase inhibitors.

C5‐Iminosugar modification of casein kinase 1δ lead 3‐(4‐fluorophenyl)‐5‐isopropyl‐4‐(pyridin‐4‐yl)isoxazole promotes enhanced inhibitor affinity and selectivity

“…To examine the inhibitory properties of compounds 34 , 39 , and 48 toward the casein kinase 1 family members CK1δ, ε, and α, we employed suitable in vitro kinase assay conditions that have recently been optimized for the evaluation of CK1. [ 19 ] The assay enables the differentiation between kinase autophosphorylation and substrate phosphorylation, thus limiting the accumulation of false‐positive responses. In essence, the assay relies on the transfer of the radiolabeled phosphate group from [γ‐ 32 P]‐ATP to a substrate protein and careful measurement of each kinases’ initial velocity region as well as defining an ATP concentration equal to the corresponding K m(ATP) .…”

“…In vitro kinase assays were performed as described previously 19 . Briefly, a total volume of 15 µl containing 1 µg α‑casein, 20 nM CK1α or 70 nM CK1δ/ε, kinase buffer (composed of 25 mM Tris‐HCl (pH 7.0), 10 mM MgCl 2 , 0.1 mM EDTA), 2 µCi [γ‐ 32 P]‐ATP (Hartmann), and different potential inhibitors of CK1 in triplicates was used.…”

“…4 Hz,2H),2H and 7.22, AA'BB'X, J AB = J BF = 8.9 Hz, 2H), 5.09 (dd, J = 6.9, 3.4 Hz, 1H), 4.30 (dd, J = 9.0, 7.1 Hz, 1H), 4.03 (dd, J = 9.2, 3.3 Hz, 1H), 1.60 (s, 3H), 1.51 (s, 3H), and 1.29 (s br , 9H); 13 C NMR (500 MHz, DMSO-d 6 , 90°C) δ 168. 19,163.57/161.59 (d,J = 246.7 Hz),159.39,150.41,149.53 (2C),136.42,130.00/129.93 (d,J = 7.0 Hz,2C),124.09 (2C),123.94,115.46/115.29 (d,J = 21.9 Hz,2C),112.92,93.86,79.58,66.75,52.46,27.38 (3C) 7 Hz),160.02,150.38 (2C),138.69,138.47,130.42/130.36 (d,J = 8.4 Hz,2C),128.59 (2C),128.26 (2C),126.99,124.38 (3C),115.98/115.81 (d,J = 21.8 Hz,2C),113.87,76.75,62.24,60.17,57.27,46.59,28.98,25.73 (3C),17.90, 1 Hz),160.04,150.34 (2C),138.42,138.23,130.40/130.34 (d,…”

“…51-8.38 (m, 2H), 7.67-7.58 (m, 2H), 7.23 (AA'BB'X, J AB = 8.7, J AF = 5.3 Hz, 2H), 7.02 (AA'BB'X, J AB = J BF = 8. 7 Hz,2H),4.12 (dt,J = 4.7,3.9 Hz,1H),3.70 (dd,J = 11.8,7.4 Hz,1H),1H),3.09 (dd,J = 12.2,3.5 Hz,1H),3.04 (dd,J = 11.9,5.9 Hz,1H),2H),and 2.45 (dd,J = 16.4,8.1 Hz,1H); 19 F NMR (470 MHz, CD 3 OD) δ −76.87 and −112.03; 13 C NMR (125 MHz,CD 3 OD) δ 194.93,166.11,165.85/163.86 (d,J = 248.3 Hz),160.34,142.27 (2C),133.61,132.49/132.42 (d,J = 8.4 Hz,2C),132.08 (2C),116.81/116.63 (d,J = 22.1 Hz,2C),107.31,74.82,52.10,50.13,43.83,and 42.80 To a solution of 4-picolyl ketone (-)-25 (787 mg, 1.85 mmol) in methanol (30 ml) was added 10% palladium on charcoal (90 mg) and the atmosphere was exchanged with hydrogen. After being stirred at room temperature overnight and complete deprotection (TLC analysis), the reaction was filtered through Celite and the filtrate concentrated and dried under high vacuum.…”

“…Finally, all iminosugar-modified analogs of the lead compound 1 were tested for their activity against human CK1δ, -ε, and -α in established in vitro assays. [19] 2 | RESULTS AND DISCUSSION…”