Stable H-bond networks are crucial for selective CDK4 inhibition revealed from comprehensive in silico investigation

https://doi.org/10.1016/j.compbiolchem.2022.107699Get rights and content

Highlights

  • The interactions between CDK isoforms, and their selective inhibitors are investigated to elucidate the selectivity mechanism.

  • Crucial amino acids involved in molecular interaction are explored, to reveal the main contributor to molecular interactions.

  • A variety of methods, including MD simulation, ASM, QM/MM calculation, etc., were employed to verify the hypothesis.

Abstract

CDK1 and CDK4 are highly similar isoforms but with apparently diverse cellular functions, which makes it fundamental to discover selective CDK4 inhibitors that could accurately control the process of cell cycle of the specific organization so as to restore normal physiological state. In current research, interaction modes of CDK1 and CDK4 inhibitors were investigated through combined in silico strategies to elucidate the selectivity mechanism against CDK4 over CDK1, revealing that H-bond networks formed with key amino acids such as LYS33 and LEU83 of CDK1 and VAL93 of CDK4 are crucial for CDK4 selective inhibition, which would provide a theoretical basis for the design of selective CDK4 inhibitors.

Introduction

The cyclin-dependent kinase (CDK) are heterodimers that consist of a catalytic subunit and regulatory cyclin subunit which are required for CDK activation (Garcia-Gutierrez et al., 2019). CDK proteins play an integral role in the regulation of the eukaryotic cell cycle, including apoptosis, transcription, differentiation, and neuronal function (Malumbres, 2014, Lim and Kaldis, 2013, Coxon et al., 2017), and the expression out of control promotes aberrant cell proliferation widely recognized hallmark cancer (Vogelstein et al., 2013, Marra and Curigliano, 2019). CDK1 controls cell cycle G1/s and G2/m phase transition and regulates about 75 important proteins in different aspects of cell cycle by phosphorylation/dephosphorylation and cyclin binding (Nagato et al., 2019, Etzkorn and Zhao, 2015, Enserink and Kolodner, 2010). Moreover, the over-expression of CDK1 in tumor tissues makes it become a good anti-cancer target (Zhang et al., 2011, Schwermer et al., 2015, Marlier et al., 2018, Liu et al., 2008, Galindo-Moreno et al., 2017).

Another isoform CDK4 is positively regulated by D-type cyclins to drive cell cycle progression transiting from G1-to-S phase (Asghar et al., 2015, Knudsen et al., 2020), which is crucial for normal and cancer cell proliferation (O'Leary et al., 2016, Colleoni et al., 2017). The activity of these kinase complexes were kept in check by endogenous CDK4 inhibitors (CDKN2A) which limit inappropriate proliferation due to oncogenic signaling (Witkiewicz et al., 2011, Guerini et al., 2020). However, CDK4 mediated phosphorylation initiates the inactivation of the retinoblastoma tumor suppressor (RB) and enables the expression of downstream genes that drive progression through the cell cycle and cell division (Knudsen and Witkiewicz, 2016). RB-pathway impacts tumor metabolism, immunological features of the tumor microenvironment, and complex epigenetic states in a context-dependent fashion although much attention has been focused on cell cycle control mediated by RB, which the context dependent features across human tumors remains poorly understood (Pancholi et al., 2020, Knudsen and Witkiewicz, 2017, Knudsen et al., 2019, Dick et al., 2018, Chaikovsky and Sage, 2018).

CDK1 and CDK4 have similar structures with different cellular functions, and selective CDK inhibitors can precisely control cell cycle processes in specific tissues to restore normal physiological conditions while reducing side effects. Thus, multiple computational approaches, such as molecular docking, molecular dynamics simulation, MM-GBSA calculation and quantum mechanism (QM) technologies, to name a few, were applied to reveal the different interaction modes between highly selective CDK1 and CDK4 inhibitors (Fig. 1), which would shed a light on a better access for designing and developing novel selective CDK inhibitors.

Section snippets

Software

Schrödinger suite 2018–1, LigandScout 4.3, Discovery Studio 3.0, Multiwfn 3.8, VMD 1.0, and PyMOL were used in the study.

Protein and ligand preparation

Multiple CDK1 structures were downloaded from the RCSB Protein Databank (https://www.rcsb.org) with PDB codes: 4Y72, 5HQ0, 5LQF, 6GU2, 6GU3, 6GU4, 6GU6 (Wood et al., 2019, Brown et al., 2015), to summarize the key amino acids involved in CDK1 inhibitor binding. The crystal structure of CDK4 was released as apo protein without any bound ligand (PDB code: 6P8E), thus the most

Investigation of CDK protein structures

After careful investigation of multiple CDK1 crystal structures, amino acid LEU83 was disclosed as a conserved residue within the CDK1 pocket (Table 1). Since currently the available CDK4 crystal structures are apo proteins without co-crystal compounds to analyze the crucial amino acids involved within the active site, we searched the SWISS-MODEL website based on CDK4 sequence (Uniprot code: P24385), and found CDK6 (PDB Code: 2EUF) exhibited the highest sequence identity of 71.19% towards CDK4 (

Conclusion

CDK1 and CDK4 isoforms own highly similar structures, but exhibit obvious clinical distinctions due to their diverse pharmacological profiles. The key characteristic binding patterns towards their specific inhibitors were comprehensively explored through the combined application of molecular docking, molecular dynamics simulations, alanine scanning mutagenesis, and Hirshfeld surface technologies, revealing that LYS33 and LEU83 of CDK1 and VAL93 of CDK4 remarkably affect the selectivity by

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The work was financially supported by the Overseas Expertise Introduction Project for Discipline Innovation (Grant No. D20029), Program for Innovative Talents of Higher Education of Liaoning (2012520005), and Education Department of Liaoning (2020LJC05).

Author statement

I have made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work. And I have drafted the work or revised it critically for important intellectual content. And I

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