Research paper
Phthalimide conjugations for the degradation of oncogenic PI3K

https://doi.org/10.1016/j.ejmech.2018.03.066Get rights and content

Highlights

  • A series of pathalimide conjugations were developed based on PI3K inhibitor.

  • Several compounds inhibited the proliferation of the liver cancer cell lines HepG2.

  • Compounds D-F degraded PI3K protein in time-/concentration-dependent mode.

  • Compounds D-F regulated the expression of proteins downstream of PI3K signaling.

  • Compound D induced autophagy instead of apoptosis and cell cycle arrest.

Abstract

PI3K/Akt/mTOR pathway is crucial for carcinogenesis and its inhibitors have made a great progress in cancer treatment. However, there is still a great developing space for PI3K inhibitors as the acquired drug resistance hindered their application in clinical. Proteolysis-targeting chimeras (PROTACs) with the potential to handle the challenges faced in drug development could be an alternative therapeutic strategy. Moreover, the past two years have witnessed remarkable advances in the development of phthalimide conjugation as a strategy for the degradation instead of inhibition of the targets, including BET family proteins, Sirtuin 2, CDK 9, Smad 3, and BCR-ABL proteins. Here, we designed and synthesized a series of potential small molecular PROTACs for the degradation of PI3K. Four compounds induced remarkable PI3K degradation and down-regulated the phosphorylation of Akt, S6K and GSK-3β in liver cancer cells HepG2. Furthermore, the representative compound D proved to inhibit tumor cells proliferation by the induction of autophagy instead of apoptosis or cell cycle arrest.

Introduction

Phosphatidylinositol 3-kinases (PI3Ks) are a family of intracellular lipid kinases, which have crucial functions in the survival, proliferation, growth, differentiation and migration of cells [[1], [2], [3], [4], [5], [6]]. According to the primary structural characteristics and substrate specificity, PI3Ks are usually divided into three classes, among which Class I PI3Ks were the most commonly studied enzyme [2,7]. Class I PI3Ks can be activated by G protein-coupled receptor (GPCR) or receptor tyrosine kinase (RTK), thus transducing signals from growth factors and cytokines into intracellular receptors [8]. The activated Class I PI3Ks can phosphorylate inositol lipids to form the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), which further activate the serine-threonine protein kinase Akt and other downstream targets to regulate cellular processes. In mammals, based on the modes of regulation, Class I PI3Ks are divided into IA and IB subclasses [9]. Class IA PI3Ks are heterodimers that consist of a p110 catalytic subunit and a p85 regulatory subunit. There are three highly homologous isoforms of p110, including α, β, and δ, constituting the corresponding enzymes PI3Kα, PI3Kβ, and PI3Kδ, respectively. Class IB PI3Ks, expressed in white blood cells, are heterodimers that consist of a catalytic subunit p110γ and a regulatory subunit p101 or p84 [10].

As PI3K plays an important role in carcinogenesis, it has become a famous anticancer target and its inhibitors have achieved great success in chemotherapy [9,[11], [12], [13], [14], [15]]. ZSTK474 (Fig. 1) is a pan Class I PI3K inhibitor, which indicates high selectivity over other classes of PI3Ks and protein kinases [16]. It inhibited the growth of many human cancer cell lines and block cell cycle progression at G1 phase [17]. Moreover, it inhibited migration and invasion of prostate cancer cells PC-3, and also induced autophagy in breast cancer cells MCF-7. ZSTK474 is now in phase I/II clinical trails for the treatment of solid tumors. However, acquired resistance has been observed in tumor cells after long-term treatment with ZSTK474.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional compounds, which consist of two recruiting ligands connected by a linker [[18], [19], [20], [21], [22], [23], [24]]. One ligand binds specific to the protein of interest (POI), while the other one recruits an E3 ligase. By hijacking the E3 ligase around target protein, this strategy can selectively knock down the level of POI. Unlike small-molecule inhibitors, which only control a specific activity, PROTACs can lead to the loss function of many activities [25]. This approach has been successfully applied to the degradation of PI3K in a reported work [26,27]. The peptidic PROTACs showed dose-dependent toxicity in MCF-7 and induced reduction of tumor size in a mouse xenograft model. However, as peptide lacked traditional drug-like properties, the development of small-molecule candidates with increased stability, better biodistribution and improved potency are urgently needed. Pomalidomide was identified as the ligand of cereblon (CRBN), a component of the cullin-4-containig E3 ubiquitin ligase complex (CRL4), which is ubiquitously expressed in physiologic and pathophysiologic tissues [[28], [29], [30]]. Phthalimide conjugation as a strategy for target degradation instead of inhibition has been successful in targets, including bromodomain and extraterminal (BET) family proteins [[31], [32], [33]], Sirtuin 2 [34], CDK 9 [35], Smad 3 [36], and BCR-ABL proteins [37]. Our group has been doing some researches on protein degradation [38] and small molecular anticancer agents including PI3K inhibitors [39], which were proved to be efficient anticancer therapies. Based on pomalidomide and ZSTK474, we have designed a series of new small-molecule PROTACs for degradation of PI3K, which is pioneering.

Section snippets

Design strategy

The X-ray structure of ZSTK474 binding with PI3K revealed the important interactions of the three substituent groups on triazine with the protein's ATP-site. The lone pairs of the imidazole interacted with Lys779 residue, which contributes to the tight binding. One of the morpholine oxygen atoms forms a key hydrogen bond with Val828 of the PI3K hinge region, while the other one doesn't observed any obvious interaction with any amino acid. Inspired by the structure characteristics, we assumed

Conclusion

Based on the strategy of PROTAC, a series of phthalimide conjugations were synthesized and their biological mechanisms were evaluated. Most of the compounds exhibited potent enzyme binding ability and inhibited the proliferation of HepG2. Four compounds proved to time-/concentration-dependently degrade PI3K protein and mediated the expression of downstream proteins, including p-Akt, p-S6K, and p-GSK-3β. Further study on representative compound D indicated our compound inhibited cancer cell

4-Bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)butanamide (2a)

To a solution of 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (1, 1 mmol) in dry THF (4 mL) was added into 4-bromobutanoyl chloride (3 mmol). The reaction was heated to reflux for 4 h. The reaction mixture was concentrated and the residue was extracted with DCM. The organic layer was washed with saturated NaHCO3 and dried with anhydrous MgSO4. The residue was purified by chromatography (DCM/MeOH, 50:1) to give the light yellow solid 2a (0.24 g, 58% yield). MS (ESI) m/z [M+Na]+

Acknowledgements

The authors would like to thank the financial supports from Shenzhen Municipal Development and Reform Commission (Disciplinary Development Program for Chemical Biology), and Shenzhen Sci & Tech Bureau (CXZZ20150529165045064, JCYJ20160301153753269).

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