Structural Basis for Variations in Polo-like Kinase 1 Conformation and Intracellular Stability Induced by ATP-Competitive and Novel Noncompetitive Abbapolin Inhibitors

Abstract

Polo-like kinase 1 (PLK1) is an essential protein kinase with multiple roles in mitotic progression. PLK1 consists of a kinase domain (KD) and a phosphopeptide-binding polobox domain (PBD), which is responsible for substrate recognition and subcellular localization. The regulation of PLK1 involves an autoinhibitory conformation in which KD and PBD interact. Our previous work identified PBD-binding molecules termed abbapolins that inhibit the cellular phosphorylation of a PLK1 substrate and induce the loss of intracellular PLK1.Here, we describe a comparison of the abbapolin activity with that of KD inhibitors to gain insight into conformational features of PLK1. As measured by a cellular thermal shift assay, abbapolins produce ligand-induced thermal stabilization of PLK1. In contrast, KD inhibitors decreased the soluble PLK1, suggesting that catalytic-site binding causes a less thermally stable PLK1 conformation. Binding measurements with full-length PLK1 and a KD inhibitor also demonstrated a conformational change. Interestingly, the cellular consequences of KD versus PBD engagement contrast as KD binding causes the accumulation of intracellular PLK1, whereas PBD binding produces a striking loss of nuclear PLK1. These data are consistent with the relief of autoinhibited PLK1 by KD binders; an explanation for these observations is presented using structures for the catalytic domain and full-length PLK1 predicted by AlphaFold. Collectively, the results highlight an underappreciated aspect of targeting PLK1, namely, conformational perturbations induced by KD versus PBD binding. In addition to their significance for PBD-binding ligands, these observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 noncatalytic functions, which may explain their lack of clinical efficacy to date.

Affinity enhancement of polo-like kinase 1 polo box domain-binding ligands by a bivalent approach using a covalent kinase-binding component

“…This is consistent with previous reports suggesting that ATP competitive compounds increase the affinity of the PBD for PBD-binding phosphopeptides. 46,47 The bivalent compounds 3 , 5 , 7 , and 8 showed attenuated PBD-binding affinities by treatment with Wortmannin (IC 50 values for Wortmannin (+) are as follows; 3 : 2.8 nM (Exp. 1)/3.8 nM (Exp.…”

Identification of PLK1-PBD Inhibitors from the Library of Marine Natural Products: 3D QSAR Pharmacophore, ADMET, Scaffold Hopping, Molecular Docking, and Molecular Dynamics Study

“…The process of molecular docking involves computer simulation the recognition of molecules in order to identify the optimal binding conformation of proteins and their ligands and to ensure the lowest binding free energy of the complex. Abbapolin [16], a new inhibitor of modified PLHSpTA, was used as the positive compound, and -CDOCKER_ENERGY was used as the scoring criterion. The docking score was higher than or equal to that of the molecule with higher activity than abbapolin, and three molecules with higher scores than the positive compound were identified, namely molecules 90, 95, and 97.…”

“…The docking priority was configured to high quality, the spatial hot-spot number was set to 100, and the ligand conformation generation method was specified as BEST for the operation. To differentiate and ascertain the molecules with superior target binding activity, we opted for the novel inhibitor abbapolin [16], which exhibits inhibitory activity, for our molecular docking studies. Compounds with docking scores exceeding a certain threshold were deemed to warrant further research.…”