Research paper
Design and synthesis of tripeptidyl furylketones as selective inhibitors against the β5 subunit of human 20S proteasome

https://doi.org/10.1016/j.ejmech.2020.112160Get rights and content

Highlights

  • A series of tripeptidic proteasome inhibitors with furylketone as C-terminus were found acting selectively and reversibly against the β5 subunit.

  • The optimized Compd. 11m was as potent as MG132 at both enzymatic and cellular levels.

  • The pharmacokinetic profiles of 11m in rats suggested a broader tissue distribution than carfilzomib.

  • 11m inhibits tumor growth in xenograft model with potency not surpassing carfilzomib.

Abstract

A series of tripeptidic proteasome inhibitors with furylketone as C-terminus were designed and synthesized. Biochemical evaluations against β1, β2 and β5 subunits revealed that they acted selectively on β5 subunit with IC50s against chymotrypsin-like (CT-L) activity in micromolar range. LC-MS/MS analysis of the ligand-20S proteasome mixture showed that the most potent compound 11m (IC50 = 0.18 μM) made no covalent modification on 20S proteasome. However, it was identified acting in a slowly reversible manner in wash-out assay and the reversibility was much lower than that of MG132, suggesting the possibility of these tripeptidic furylketones forming reversible covalent bonds with 20S proteasome. Several compounds were selected for anti-proliferative assay towards multiple cancer cell lines, and compound 11m displayed comparable potency to positive control (MG132) in all cell lines tested. Furthermore, the pharmacokinetic (PK) data in rats indicated 11m behaved similarly (Cmax, 2007 μg/L; AUC0−t, 680 μg/L·h; Vss, 0.66 L/kg) to the clinical used agent carfilzomib. All these data suggest 11m is a good lead compound to be developed to novel anti-tumor agent.

Introduction

In eukaryotic cells, the amount of each intracellular protein depends on the balance of its rates of synthesis and degradation. This homeostasis is maintained by two proteolysis systems, namely the ubiquitin-proteasome system (UPS) and the lysosomal degradation pathway [1]. The UPS is responsible for the specific degradation of short-lived regulatory proteins and the removal of damaged soluble proteins, and thus tightly controls a broad array of basic cellular processes including gene transcription, signal transduction, DNA repair, cell cycle regulation, apoptosis, and immune response [2]. The catalytic degradation of polyubiquitinated substrates in the ubiquitin-proteasome pathway (UPP) is performed by 26S proteasome which is a barrel-shaped, multi-catalytic protease complex consisting of a 20S core particle (CP) and two 19S regulatory caps [3]. The 20S CP of the proteasome comprises 28 protein subunits assembled into four heptameric rings: two outer α-rings (α1-α7) and two inner β-rings (β1-β7) [4,5]. The outer α subunits are proteolytically inactive and serve a regulatory function, while the two inner rings each possess three catalytic active β subunits [[6], [7], [8]]. Each of the active β subunits is responsible for a single type of proteolytic activity: β1 for caspase-like (C-L), β2 for trypsin-like (T-L), and β5 for chymotrypsin-like (CT-L) activity. Site-directed mutagenesis analysis of the yeast proteasome has revealed that CT-L activity of proteasome had the greatest impact on proteolysis [9], and inhibition of tumor cellular CT-L activity is a strong stimulus that induces apoptosis [[10], [11], [12]]. These observations have promoted development of numerous synthetic inhibitors of the β5 subunit [13], among which bortezomib, carfilzomib and ixazomib (Fig. 1A) have been successfully approved by the US Food and Drug Administration (FDA) in the year of 2003, 2012, and 2015, respectively, for the treatment of multiple myeloma [14].

Recently, we have reported a series of C-terminal furylketone-based peptidic inhibitors against β5 subunit proposing that the C-terminal furylketone could serve as a warhead that form covalent bond with the catalytically active residue, namely Thr1, of the β5 subunit and therefore, could produce high affinity and inhibitory potency. However, only moderate potency was observed on the most potent compound 10b’ (Fig. 1B) with IC50 of 1.64 ± 0.44 μM in enzymatic assay [15]. Docking and dynamic simulations suggested that the distance between the C-terminal furylketone of 10b′ and the –OH of residue Thr1 was too large (4.9 Å) to form covalent bond due to the torsional strain brought about from the P2 and P3 substitutions. To further explore our hypothesis regarding binding mode, more active compounds are required. In the present study, we extended substitutions on the peptidic backbone at R2 to R4 (Fig. 1C) aiming at optimized fitting into the S2 to S4 sub-pockets of β5 subunit and therefore, enhanced potency could be achieved as was expected for covalent inhibitors [16]. The binding mode was also discussed based on computational simulations together with wash-out assay and LC-MS/MS analysis.

Selected compounds were evaluated for anti-proliferating effect against a panel of cancer cell lines using MG132 (Fig. 1D) as positive control, meanwhile, their cytotoxicities were also measured in nonmalignant cells (HEK293).

Section snippets

Chemistry

The design of substitutions at R2 to R4 (Fig. 1C) on the tripeptidic scaffold was performed according to the structure-activity relationship (SAR) studies on our previously described tripeptidyl furylketones [15,17]. Overall, the best fit into the S2-S4 sub-pockets were explored.

The designed furylketone-based tripeptides (7a-7j, 11a-11o) were acquired through two different synthetic routes. Initially, the furan-based leucine 3 was synthesized starting from commercially available Boc-protected l

Conclusion

We designed and synthesized a series of tripeptidic furylketones as 20S proteasome inhibitors that selectively inhibited the CT-L activity. The results of LC-MS/MS analyses and wash-out assay indicated they inhibit the β5 subunit in reversible manner. Docking study suggested the distance between the C-terminal furylketone moiety and the catalytic Thr1 allowed the formation of hemiketal which endow these furylketones “rapid loading and slow dissociation” kinetics. Although not as potent as

Chemistry

Commercially available solvents and reagents were used directly without further purification. Reaction progress was monitored by thin-layer chromatography (TLC) performed on silica gel GF254 purchased from Qingdao Haiyang Chemical Co. (Qingdao, China). Melting points (mp) were obtained on an XT4A apparatus and were uncorrected.1H NMR and 13C NMR spectra were recorded at 400/100 MHz on a Bruker Avance III 400 spectrometer with TMS as internal standard. Low-resolution mass spectra were obtained

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.

Acknowledgment

This work was generously supported by the National Natural Science Foundation of China (81872731, 21977006, 21807006). The authors also appreciate Dr. Hao Liang from the College of Chemistry and Molecular Engineering, Peking University for his kind technical help on computational simulations.

References (31)

  • D. Voges et al.

    The 26S proteasome: a molecular machine designed for controlled proteolysis

    Annu. Rev. Biochem.

    (1999)
  • M. Groll et al.

    Structure of 20S proteasome from yeast at 2.4 angstrom resolution

    Nature

    (1997)
  • F. Kopp et al.

    Subunit arrangement in the human 20S proteasome

    Proc. Natl. Acad. Sci. U.S.A.

    (1997)
  • P. Zwickl et al.

    Critical elements in proteasome assembly

    Nat. Struct. Biol.

    (1994)
  • E. Seemuller et al.

    Proteasome from thermoplasm-acidophilum - a threonine protease

    Science

    (1995)
  • Cited by (0)

    1

    These authors contributed equally to this work.

    2

    Dr. Qi Sun is now working at BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China.

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