Table 3.
Summary of compounds with thermal shifts of at least 3 °C (ΔTm ≥ 3 °C), total predicted ligand-protein binding energies in ATP-binding site and percent phosphorylation activity up to 75% for recombinant hNeks 1, 6, and 7. The percent phosphorylation is according to a compound final concentration of 2×, 1×, and 0.5× the ATP Km[apparent] concentration of each kinase. The ATP Km[apparent] concentrations found for Neks are as follows: 0.625 µM for hNek6(S206A) and hNek7wt, and 25 µM for hNek1(Δ262-1258)-(T162A). Only percentage of phosphorylation values with standard errors < 20 are depicted in the table. Names of compounds corresponding to ΔTm ≥ 4.0 °C and phosphorylation ≤ 75% are depicted in bold and were selected as the best candidates for hNeks.
| Nek | CAS Number | Compound Description | ΔTm (°C) | Docked/Predicted Binding Energy (kcal/mol) | Phosphorylation (%) 2× b | Phosphorylation (%) 1× b | Phosphorylation (%) 0.5× b |
|---|---|---|---|---|---|---|---|
| Nek1(Δ262-1258)-(T162A) | 4129-56-6 | JNK Inhibitor II | 4.0 | −7.345 | 71.5 ± 0.1 | 87.3 ± 18.3 | - |
| Nek6(S206A) | 244148-46-7 | Isogranulatimide a | 6.5 | −8.917 | - | 74.9 ± 10.4 | 100.2 ± 1.3 |
| Nek7wt | 220792-57-4 | Aminopurvalanol A | 3.0 | −8.626 | 41.0 ± 13.5 | 35.9 ± 3.3 | 64.4 ± 3.4 |
| Nek7wt | 487021-52-3 | GSK-3b Inhibitor VIII | 3.3 | −7.572 | 58.1 ± 3.2 | 47.5 ± 4.4 | 44.3 ± 3.3 |
| Nek7wt | 404828-08-6 | GSK-3 Inhibitor XIII | 4.3 | −8.629 | 46.3 ± 2.9 | - | 43.3 ± 10.1 |
a: Isogranulatimide was selected for hNek6(S206A), although corresponding to a minimum percentage of phosphorylation of 74.9 ± 10.4; b: S.E. of two measurements.