Elsevier

European Journal of Medicinal Chemistry

Volume 164, 15 February 2019, Pages 602-614
European Journal of Medicinal Chemistry

Research paper
Covalent docking modelling-based discovery of tripeptidyl epoxyketone proteasome inhibitors composed of aliphatic-heterocycles

https://doi.org/10.1016/j.ejmech.2018.12.064Get rights and content

Highlights

  • Covalent docking was applied for the guidance of proteasome inhibitors designing.

  • Compound 11h exhibited good proteasome inhibitory potency and biological properties.

  • Compound 11h may perform a new binding mode with proteasome.

Abstract

The potential of specific proteasome inhibitors to act as anti-cancer agents has attracted intensive investigations. The proteasome can be covalently inhibited by epoxyketone derivatives via a two-step reaction. Several computational approaches have been developed to mimic the covalent binding event. Compound 1 composed of a six-membered heterocyclic ring was designed by using covalent docking. With a possible different binding mode from the clinical compound Carfilzomib, it occupied the S5 pocket of 20S proteasome and showed favorable inhibitory activity. Subsequently optimization and evaluation were taken place. Among these compounds, 11h demonstrated extraordinary in vitro inhibitory activity and selectivity, and good in vivo proteasome inhibitory activity, a favorable pharmacokinetic profile and xenograft tumor inhibition. The possible binding pattern of compound 11h against proteasome was further fully explored via calculations, providing a theoretical basis for finding potent proteasome inhibitors.

Introduction

The ubiquitin-proteasome pathway is responsible for approximately 80–90% of eukaryotic intracellular protein degradation [[1], [2], [3], [4]]. In particular, accumulating evidences illustrate that various regulatory proteins associated with cell growth, differentiation, and apoptosis in cancer cells are controlled by the proteasome [3,4]. Accordingly, proteasome inhibitors have gained extensive interests as anti-cancer treatments [[5], [6], [7]]. To date, numerous peptidyl proteasome inhibitors, which can be divided into boric acid, epoxyketone, vinyl sulfone and aldehyde derivatives, have been identified and developed [5,8]. In general, the warhead of these compounds can be attacked by a threonine residual of the proteasome, forming reversible/irreversible covalent bond [8]. To date, several compounds (including MG-132, Bortezomib, Carfilzomib and Marizomib, Fig. 1) have been extensively evaluated as anti-cancer agents. However, adverse effects and drug resistance have been frequently observed in clinical practice, including peripheral neuropathy, cardiovascular effects [9,10] and p-glycoprotein mediated drug resistance in certain cell lines [11]. Thus, there remains a strong demand for the development of novel proteasome inhibitors with enhanced potency, efficacy, fewer side effects and reduced drug resistance.

In eukaryotic proteasomes, three β-type subunits (β1, β2 and β5) of the 20S proteasome contain proteolytically active centers [12,13], amongst which the β5 subunit has been shown to be the rate-limiting proteolytic step. Crystal structures of bortezomib- and carfilzomib-bound proteasomes revealed that both compounds bind to the β5 subunit, which is the primary target for proteasome inhibition [14]. Although the structure of the β5 subunit is known, structure-based drug design for the development of new covalent proteasome inhibitors has not been performed in detail, as calculating the binding energy for an irreversibly bound protein complex remains a challenging issue.

Recently, computational approaches have been developed to mimic the covalent binding event, resulting in several covalent molecular docking programs [15,16]. These can be implemented during computer-aided drug design to describe covalent interactions between inhibitors and biological targets. Using this method, we can successfully predict the binding energy between a nucleophilic receptor and electrophilic ligand. Blake et al. [17], Schroeder et al. [18] and Taunton et al. [19] performed pioneering studies in which a covalent docking method was applied during virtual drug screening to discover new irreversible inhibitors, revealing a new strategy for covalent inhibitors drug design. For the proteasome, Zhu et al. [20] and Zhang et al. [21] demonstrated the binding pockets and key residues of the 20S proteasome that can be targeted for, providing a potential way to design more potent inhibitors of the 20S proteasome. However, the binding mode between the 20S proteasome and peptide epoxyketone (EK) was not fully explored, despite disclosure of the crystal structure of carfilzomib-bound to the proteasome. It was reported that the EK forms two covalent bonds with the proteasome via a two-step reaction [22,23]. As shown in Fig. 2, the hydroxyl group of a Thr1 residue in the 20S proteasome attacks the ketone (Osingle bondC bond formation) of EK fragment, leading to the formation of a Hemiketal intermediate. The amino group then attacks the epoxy group from either side (Nsingle bondC bond formation), generating a stable six-membered 1,4-morpholine ring or seven-membered 1,4-oxazepane ring [22,23]. It would be of interest to perform covalent docking studies between the 20S proteasome and EK-containing proteasome inhibitors, this can provide guidance for altering the skeleton and/or sidechains of EK-containing proteasome inhibitors.

Based on the crystal structure of the carfilzomib-bound proteasome complex (PDB: 4R67), and as part of our continuous studies on molecular docking and the discovery of proteasome inhibitors [5,[24], [25], [26], [27], [28], [29], [30], [31]]. In this study, we designed EK derivatives as proteasome inhibitors based on the usage of covalent docking approaches. This led to the identification of a shortened tripeptide skeleton and more flexible terminal moiety that may occupy a new pocket that differs from the binding mode of Carfilzomib (Fig. 3(A)). We fully explored the structure-activity relationships of these new tripeptide derivatives, in vitro and in vivo proteasome inhibitory activities. Furthermore, the drug profiles and in vivo anticancer activity of a promising candidate (compound 11h) was extensively evaluated, which was found to be more potent than carfilzomib. Finally, we fully explored the covalent bond forming process between 11h and the proteasome, resulting in a stable 11h-bound proteasome complex linked by a seven-membered 1,4-oxazepane ring.

Section snippets

Covalent docking-based rational design of tripeptidyl epoxyketone derivatives as proteasome inhibitors

According to the crystal structure of the human 20S proteasome in complex with carfilzomib (PDB: 4R67) and through the analysis of different binding pockets [22], the interaction mode indicated that the hydrophobic P4 phenyl group occupied the S4 pocket, the L-leucine residue P3 pointed into the S3 pocket, the S2 pocket was able to accommodate the P2 phenylalanine moiety and the P1 leucyl group of carfilzomib was oriented towards the S1 pocket, which maintained a high binding affinity of

Conclusion

Through the investigation of the interaction mode of carfilzomib with proteasome, compound 1 composed of a six-membered heterocyclic ring was designed on the basis of covalent docking, further biological evaluation demonstrated a promising proteasome inhibitory potency. After initial exploration, a series of tripeptidyl EK derivatives were synthesized and tested. Compound 11h was selected for further evaluation on selectivity, anti-tumor proliferation, in vivo proteasome inhibition,

General experimental information

All reagents and solvents were used as purchased from commercial sources. Chromatography was performed using silica gel (200–300 mesh). All reactions were monitored by TLC, using silica gel plates with fluorescence F254 and UV light visualization. 1H NMR and 13C NMR spectra were obtained on a Bruker Advance III 500 with use of CDCl3, CD3OD, (CD3)2CO or DMSO‑d6 as solvent. Chemical shifts are referenced to the residual solvent peak and reported in ppm (d scale) and all coupling constant (J)

Author contributions

X.D. and J.Z. designed and synthesized the compounds, analyzed the data. L.X. performed the biological experiments, J.C. analyzed the data and drafted the manuscript, G.C. performed the docking calculations. X.H., L.S. and A.G. helped to analyze the data. Y.Z., T.L., Y.H. and J.L. conceived the study.

Funding sources

National Natural Science Foundation of China; the Key Project of Zhejiang Provincial Natural Science Foundation of China; the Science and Technology Commission of Shanghai Municipality; Strategic Priority Research Program of the Chinese Academy of Sciences; National Major Scientific and Technological Special Project for “Significant New Drugs Development”.

Declaration of interests

The authors declare no competing financial interest.

Acknowledgements

We thank Jianyang Pan (Research and Service Center, College of Pharmaceutical Sciences, Zhejiang University) for performing NMR spectrometry for structure elucidation. This work was supported by grant from the National Natural Science Foundation of China (81673294), the Key Project of Zhejiang Provincial Natural Science Foundation of China (LZ15H300001), the Science and Technology Commission of Shanghai Municipality (17431903000), the "Personalized Medicines---Molecular Signature-based Drug

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    X.D., J.Z. and L.X. contributed equally to this work.

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