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. 2023 Oct 27;24(21):15639.
doi: 10.3390/ijms242115639.

Leveraging the Fragment Molecular Orbital Method to Explore the PLK1 Kinase Binding Site and Polo-Box Domain for Potent Small-Molecule Drug Design

Haiyan Jin  1 , Jongwan Kim  2   3 , Onju Lee  1 , Hyein Kim  3 , Kyoung Tai No  1   2   3   4
Affiliations

Leveraging the Fragment Molecular Orbital Method to Explore the PLK1 Kinase Binding Site and Polo-Box Domain for Potent Small-Molecule Drug Design

Haiyan Jin et al. Int J Mol Sci. .

Abstract

Polo-like kinase 1 (PLK1) plays a pivotal role in cell division regulation and emerges as a promising therapeutic target for cancer treatment. Consequently, the development of small-molecule inhibitors targeting PLK1 has become a focal point in contemporary research. The adenosine triphosphate (ATP)-binding site and the polo-box domain in PLK1 present crucial interaction sites for these inhibitors, aiming to disrupt the protein's function. However, designing potent and selective small-molecule inhibitors can be challenging, requiring a deep understanding of protein-ligand interaction mechanisms at these binding sites. In this context, our study leverages the fragment molecular orbital (FMO) method to explore these site-specific interactions in depth. Using the FMO approach, we used the FMO method to elucidate the molecular mechanisms of small-molecule drugs binding to these sites to design PLK1 inhibitors that are both potent and selective. Our investigation further entailed a comparative analysis of various PLK1 inhibitors, each characterized by distinct structural attributes, helping us gain a better understanding of the relationship between molecular structure and biological activity. The FMO method was particularly effective in identifying key binding features and predicting binding modes for small-molecule ligands. Our research also highlighted specific "hot spot" residues that played a critical role in the selective and robust binding of PLK1. These findings provide valuable insights that can be used to design new and effective PLK1 inhibitors, which can have significant implications for developing anticancer therapeutics.

Keywords: fragment molecular orbital method; molecular dynamics simulation; polo-like kinase 1; protein-protein interaction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Domain information of the five human polo-like kinase (PLK) families and full-length structure of the PLK1 predicted by Alphafold2. (A) Numbers represent the amino acid sequence numbers. (B) Illustration of the kinase (blue) and polo-box (orange) domains.
Figure 2
Figure 2
Structure-based selectivity pattern considerations. (A) Superposition of 11 crystal structures in the holo-form of human PLK1. Based on para and meta, PLK1 kinase domain inhibitors can be divided into two groups. The molecules of A group are represented in blue, and B in light pink. The inhibitors from the B group are PLK1 selective inhibitors toward PLK2 and 3. They all interact with Glu140 in PLK1. (B) Two-dimensional chemical structure of inhibitors from two groups. Hinge-binding regions are highlighted in light yellow, solvent exposure regions in light blue, and phosphate regions in light pink.
Figure 3
Figure 3
Characteristics of the ATP-binding pocket of the kinase domain in polo-like kinase 1 (PLK1). (A) Crystal structure of the human PLK1 kinase domain with ATP (PDB ID: 2OU7). The ATP-binding site is divided into adenine, ribose, and phosphate pockets and solvent Channels 1 and 2. (B) Comparison of the conformation change of the two crystal structures of the PLK1 complex with two inhibitors, BI2536 and Onvansertib. Nonselective inhibitor BI2536 only occupies solvent Channel 1, but potent PLK1 selective inhibitor Onvansertib binds solvent Channels 1 and 2.
Figure 4
Figure 4
Fragment Molecular Orbital (FMO) analysis of ATP. (A) FMO results of the crystal structure of the PLK1 complex with the ATP analog. The ligand is blue; key residues are green. (B) PIE values of the significant residues in the ATP-binding site. (C) PIE decomposition analysis of these critical interactions.
Figure 5
Figure 5
FMO analysis of BI2536 and Onvansertib. (A) FMO results of the crystal structure of the PLK1 complex with BI2536. The ligand is blue; the key residues are green. (B) FMO results of the crystal structure of the PLK1 complex with Onvansertib. The ligand is light pink, key residues are green, and nitrogen and oxygen atoms are blue and red. (A,B) Middle bar plots describe the PIE values of the significant residues in the ATP-binding site, whereas bottom bar plots describe the PIE decomposition analysis of these critical interactions.
Figure 6
Figure 6
Docking structure and FMO analysis of GSK461364. (A) Docking structure of GSK461364. The ligand is purple; key residues are green. (B) Docking structure of GSK461364 overlayed with the crystal structure of Onvansertib (light pink). (C) Bar plot describing the PIE values of the significant residues in the ATP-binding site and GSK461364. (D) The bar plot describes the PIE decomposition analysis of these critical interactions.
Figure 7
Figure 7
Superposition of the kinase domain of PLK1–3. Superposition of the crystal structures of the PLK1-3 and sequence alignment. Four residues occur around the ATP-binding site. Glu140 of PLK1 has a negative charge, except His169 and His149 of PLK2, and PLK3 have a positive charge.
Figure 8
Figure 8
Heatmap of the protein–ligand contact from the molecular dynamic simulation of 13 ligands. Percentages represent interactions occurring during the simulation. Direct interaction <30% is yellow, 30% to 60% is green, and >60% is red. The water bridge is light blue.
Figure 9
Figure 9
WaterMap analysis of the ATP-binding pocket of the kinase domain. Hydration sites are represented as spheres, with colors reflecting the predicted associated free energies. Green spheres signify favorable free energies, whereas red spheres indicate unfavorable free energies. (A,B) Onvansertib (light pink) is overlayed in the WaterMap of the crystal structure of BI2536, focusing on the hydration site of the phosphate-binding site and solvent Channel 2. (C,D) WaterMap of the crystal structure of Onvansertib.
Figure 10
Figure 10
Characteristics of the PBD. Pockets of the PBD are divided into phosphate (blue), pyrrolidine (orange), and Tyr-rich (magenta) pockets.
Figure 11
Figure 11
Hot-spot analysis of substrate peptide complex with the PBD of PLK1. (A) The substrate peptide was divided into nine fragments: Ala1, p-Thr2, Ser3, His4, Leu5, Pro6, Pro7, Asp8, and Phe9 (green and yellow sticks). (B) PLK1 PBD hot-spot residues are blue, orange, and light pink sticks. PIE values are described in the right table.
Figure 12
Figure 12
Hot-spot analysis of the 4j complex with the PBD of PLK1. (A) Fragments of 4j: p-Thr, Ser, F-Akyl, Leu, and Pro (green and yellow sticks). (B) Significant interaction residues in the PBD pocket are blue, orange, and light pink sticks. PIE values are described in the right table.
Figure 13
Figure 13
Hot-spot analysis of the 4a complex with the PBD of PLK1. (A) Fragments of 4a: p-Thr, Ser, F-CH3, Leu, and Pro (green and yellow sticks). (B) Significant interaction residues in the PBD pocket are blue, orange, and light pink sticks. PIE values are described in the right table.
Figure 14
Figure 14
FMO analysis of KBJK557 and KBJK-4a. (A) FMO results of the structure from Frame 489 of the molecular dynamic simulation. The ligand is blue; key protein residues are blue, orange, and light pink sticks. (B) Bar plot describing the PIE values of the significant residues in the PBD. (C) Bar plot describing the PIE decomposition analysis of these critical interactions. (D) FMO results of the docking structure of the KBJK-4a. The ligand is blue; key protein residues are blue, orange, and light pink sticks. (E) Bar plot describing the PIE values of the significant residues in the PBD. (F) Bar plot describing the PIEDA of these critical interactions.
Figure 15
Figure 15
Heatmap of the FMO results of five structures. The FMO results of the complex structure of the PBD of PLK1 complex with substrate peptide, 4j and 4a, KBJK557, and KBJK-4a. The critical residues are highlighted blue for the phosphate pocket, orange for the pyrrolidine pocket, and pink for the Tyr-rich pocket. The pair interaction energy was summed for each residue. Darker red indicates a lower energy value.
Figure 16
Figure 16
Heatmap of the protein–ligand contact from the molecular dynamic simulation of 5 ligands. Percentages represent interactions occurring during the simulation. The critical residues are highlighted blue for the phosphate pocket, orange for the pyrrolidine pocket, and pink for the Tyr-rich pocket. Direct interaction <30% is yellow, 30% to 60% is green, and >60% is red. The water bridge is light blue.
Figure 17
Figure 17
WaterMap analysis of the binding pocket of the PBD. Hydration sites are represented as spheres, with colors reflecting their predicted free energies. Green spheres signify favorable free energies; red spheres indicate unfavorable free energies. (A) WaterMap analysis of the 4a (lime) binding structure in the PBD. (B) Focusing on the Tyr-rich pocket in the 4a (cyan surface) water map analysis with 4j superposition (orange stick). (C) WaterMap analysis of the crystal structure of the 4j binding in the PBD. (D) WaterMap focusing on KBJK557 (surfaced with a partial charge) and hydration sites in the Tyr-rich pocket.
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This research was supported by the Yonsei University graduate school “IntegrativeBiotechnology & TranslationalMedicine” and the Establishment and demonstration of a biomaterial data platform, P0014714.

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