Research paperExploring the PROTAC degron candidates: OBHSA with different side chains as novel selective estrogen receptor degraders (SERDs)
Graphical abstract
A series of novel SERDs with excellent ER degradation efficacy have been discovered. These findings simplified the structure of currently available degrons and provide new possibility for discovering novel PROTACs.
Introduction
It is well-known that the overexpression of estrogen receptor α (ERα) may lead to ER positive breast cancer, which accounts for 70% of breast cancer [1,2]. Contraposing this target, clinical pharmacists generally use endocrine therapy, in which the drugs can be classified into two types: aromatase inhibitors (AIs) [[3], [4], [5]] and selective estrogen receptor modulators (SERMs) [2,[6], [7], [8]]. However, the recurrence of breast cancer is difficult to treat, and the long-term use of SERMs, such as tamoxifen, is prone to lead to the development ovarian cancer and drug resistance [[9], [10], [11]], which is usually fatal. For patients who are on long-term medication, the limited number of effective drugs encourages the development of new compounds that reduce the likelihood of disease recurrence and drug resistance through various means.
Inducing protein degradation by small molecules has recently become a hot spot for drug discovery [12,13]. To date, as the emerging and efficient treatments for breast cancer, there have been two approaches targeting ERα protein degradation: selective estrogen receptor downregulators (SERDs) and proteolysis targeting chimeras (PROTACs). SERDs are recognized as pure ER antagonists that can cause spatial structural instability and activate the ubiquitination pathway to degrade ERα protein [[14], [15], [16], [17], [18]]. In 2007, the FDA approved the first SERD fulvestrant for the treatment of advanced breast cancer [19]. Nevertheless, this SERD's low oral bioavailability limited its application [20]. Therefore, medicinal chemists hope to obtain SERDs with better oral bioavailability through structural modification and optimization. To date, a variety of SERDs have been identified [[21], [22], [23], [24], [25], [26]], most of which have different side chains, e.g., long alkyl side chains, acrylic side chains, and basic side chains (Fig. 1). Fulvestrant (Fig. 1, compound 1) is one typical drug that introduced a sulfinyl pentafluoro alkyl chain onto a E2 skeleton; the hydrophobic chain was exposed to the surface of the protein when binding to ER and destabilized the active ligand binding domain (LBD) conformer, thereby leading to protein degradation. Therefore, Kurihara et al. reported that a derivative of tamoxifen with a long alkyl side chain (Fig. 1, compound 2) could effectively reduce ER protein levels in breast cancer cells and have an antagonistic effect [26]. Later, these researchers expanded the range of currently available SERDs by attaching a decyl group to the amine moiety of raloxifene and revealed that the ER degradation efficacy of this compound is 1 μM [25]. GDC0810 (Fig. 1, compound 3), a second-generation SERD, contains an acrylic side chain, which directly interacted with the peptide backbone of ERα and induced a conformational change that exposed a hydrophobic surface on the receptor [27]. GDC0810 has undergone a clinical phase II trial and was effective in endocrine therapy to treat advanced metastatic ER+ breast cancer; unfortunately, its clinical trial was recently discontinued. In 2018, Smith's group reported that derivatives of EM-652 that contained a basic side chain were highly potent and efficacious SERDs [18]. The best compound (Fig. 1, compound 4) of this series demonstrated robust activity with a 91% ERα degradation efficacy in a xenograft model of tamoxifen-resistant breast cancer. Moreover, the crystal structure study revealed that the side chains of these compounds may play an essential role in the degradation of ER, e.g., the acrylic acid side chain of 3-type compounds formed a hydrogen bond with D351 in the ligand binding domain of ER, which blocked the proper positioning of the critical helix 12. Thus, hydrophobic patches were exposed in solution, the receptor was recognized by the ubiquitin-proteasome system and degradation proceeded [28]. For fulvestrant-type SERDs, when binding to ER, helix 12 appeared to be completely distorted, and the hydrophobic regions were exposed to solution for ubiquitination targeting. As a result, the side chain is a major driving factor of SERDs and plays a critical role in the degradation of ER.
Another method for compounds capable of inducing protein degradation is proteolysis targeting chimeras (PROTACs). PROTAC is a bifunctional compound possesses a ligand for a targeted protein and a recognition motif for E3 ubiquitin ligase recruitment that ultimately promotes ubiquitination-dependent proteasomal degradation of the target protein. In 2010, an ER-targeting PROTAC that consisted of an estradiol and a hypoxia-inducing factor 1α (HIF-1α)-derived synthetic pentapeptide was reported [29], which showed good ER binding affinity (10% binding affinity relative to E2) and effective ER degradation efficacy (60% ER degradation). Kurihara et al. disclosed a complex comprised of 4-hydroxytamoxifen and bestatin (an inhibitor of the cIAP1) via an alkyl linker [30]. This complex induced cIAP1-mediated ubiquitination of ERα with proteasomal degradation at 10 μM. Recently, Li et al. reported a novel ERα-targeting PROTAC that was composed of an N-terminal aspartic acid cross-linked stabilized peptide ERα modulator (TD-PERM) and the Von Hippel-Lindau (VHL) E3 ubiquitin ligase through a pentapeptide [31]. The TD-PROTAC approach utilized a peptide stabilization strategy, which could provide peptide conjugates with satisfactory stability and cellular uptake. However, the effective dose for degradation is 20 μM, which still needs improvement. Generally, PROTACs are under rapid development as a novel and promising technology for drug discovery [[32], [33], [34]]. Nonetheless, there is a very narrow selection of the structural species for PROTACs targeting ER degradation, and their low potency limits their clinical trials.
Despite the different mechanisms for degrading ER by SERDs and PROTACs, these compounds have a molecular skeleton that induces ER degradation and could be called a degron. A degron is reported to be a part of the ligand-induced degradation (LID) domain in known works, and it functions as a cryptic degradation sequence [35,36]. Recently, Sharma's group reported a series of new SERDs that extended the currently available library of PROTAC-type scaffolds, which may be useful for the degradation of a variety of other therapeutically important proteins [16]. The researchers defined the special motif that can induce ER degradation as a degron in both SERDs and PROTAC molecules. As the skeletons of the PROTAC degrons are limited and always complicated, there is an urge to explore novel degrons with diverse and simple structures that allow the ER degrader to display drug-like properties and more potent efficacy. In 2012, our group reported oxabicycloheptene sulfonamide (OBHSA, Fig. 2) compounds as full ER antagonists [37], and further study on the crystal structures of complexes of these ligands with ERα revealed a new mechanism of action of these SERD compounds [38]. The large R2 groups (N-trifluoroethyl group and N-ethyl group) clashed strongly with Leu525, inducing a 2.5 Å shift in h11 and leading to the complete disorder of the C terminus of h11. Thus, h12 Leu544 moved out of the hydrophobic groove. In addition, the 4-methoxyphenyl substitution flipped the ethyl and aryl groups around the sulfonamide linker and interacted with the loop between h7 and h8 by attacking the backbone carbonyl of Glu419. Therefore, under the influence of the disordered h11 and h12 exposed in the hydrophilic environment, the ER protein would become a target for ubiquitination. Considering the important role played by the sulfonamide moiety in this new mechanism, we turn our attention to the phenol group on the other side, hoping to develop new degrons on the phenolic group that would contribute to the degradation activity. As part of our long-term interests in the development of ER ligands [[39], [40], [41]], we utilized the OBHSA as a core structure and explored alternatives of degrons to, for example, the basic side chains, long alkyl acid side chains, and glycerol ether side chains, and then, we investigated their inhibition efficacy of MCF-7 cell proliferation and ERα degradation activity. In the process, we uncovered some remarkable structure-activity relationships (SARs) in which compounds 17d, 17e and 17g containing basic side chains with N-trifluoroethyl and para methoxy substituents displayed outstanding ERα degradation efficacy and anti-proliferation activity. Further docking analysis illustrates that the basic side chain could distort helix 3 by forming a hydrogen bond with Thr347 and a hydrophobic repulsion with the backbone of Met343. The N-trifluoroethyl group and the para methoxyl group may also be beneficial to the ERα degradation efficacy by influencing the loop of helix 7 and helix 8. The results indicate that the basic side chain proved to be the best degron and provides new possibilities in the development of more effective PROTACs.
Section snippets
Chemistry
The synthesis of the final OBHSA derivatives 17a-t involved a Diels-Alder reaction between a series of furan analogs and ethylene sulfonamide derivatives (Scheme 4), and the furan intermediate 7 was synthesized according to our previous work [42]. To introduce different degrons onto the OBHSA pharmacophore core, we synthesized the key intermediates 8a-j, and the synthetic route is shown in Scheme 1. The furan analogs 8a-g were obtained by Williamson etherification reactions with different
Conclusions
As drug resistance is continuously being identified in the treatment of breast cancer, new strategies based on small molecule-induced protein degradation have developed rapidly. However, most of the candidates involve very limited scaffolds and are still in clinical trials, and none of them has been approved for marketing. Therefore, there is still an urgent need to develop novel compounds with good ER degradation efficacy. In this work, we directed our attention toward discovering new degrons
General chemical methods
Starting materials, reagents and solvents are purchased from commercial sources and used directly unless otherwise noted. THF, DCM and acetonitrile are redistilled and dried to avoid water. Glassware was oven-dried, assembled while hot, and cooled under an inert atmosphere. Reaction progress was monitored using analytical thin-layer chromatography (TLC). Visualization was achieved by UV light (254 nm and 365 nm). Silica gel (230–400 mesh) was used for column chromatography purifications. A
Acknowledgements
We are grateful to the NSFC (81773557 and 81573279), the Major Project of Technology Innovation Program of Hubei Province (2016ACA126, 2016ACA148, 2018ACA123), the NSFHP (2017CFA024), the Fundamental Research Funds for the Central Universities of China (2042017kf0288), and the Open Research Fund Program of the Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals for supporting this research.
References (45)
- et al.
Endocrine treatment in breast cancer: cure, resistance and beyond
Cancer Treat Rev.
(2016) - et al.
Identification of novel indole based heterocycles as selective estrogen receptor modulator
Bioorg. Chem.
(2018) - et al.
Mechanisms of resistance in estrogen receptor positive breast cancer: overcoming resistance to tamoxifen/aromatase inhibitors
Curr. Opin. Pharmacol.
(2018) - et al.
The changing role of ER in endocrine resistance
Breast
(2015) - et al.
Treating gynecologic malignancies with selective estrogen receptor downregulators (SERDs): promise and challenges
Mol. Cell. Endocrinol.
(2015) - et al.
Synthesis and evaluation of raloxifene derivatives as a selective estrogen receptor down-regulator
Bioorg. Med. Chem.
(2016) - et al.
Design and synthesis of tamoxifen derivatives as a selective estrogen receptor down-regulator
Bioorg. Med. Chem. Lett
(2014) - et al.
Structural basis for an unexpected mode of SERM-mediated ER antagonism
Mol. Cell.
(2005) - et al.
Design and synthesis of estrogen receptor degradation inducer based on a protein knockdown strategy
Bioorg. Med. Chem. Lett
(2012) - et al.
Estrogen receptor sensing in living cells by a high affinity turn-on fluorescent probe
Sensor. Actuator. B Chem.
(2018)
Structural underpinnings of oestrogen receptor mutations in endocrine therapy resistance
Nat. Rev. Canc.
Synthesis of triphenylethylene bisphenols as aromatase inhibitors that also modulate estrogen receptors
J. Med. Chem.
Novel aromatase inhibitors by structure-guided design
J. Med. Chem.
Design, synthesis, and structure-activity relationships of azolylmethylpyrroloquinolines as nonsteroidal aromatase inhibitors
J. Med. Chem.
Estrogen and its receptors in cancer
Med. Res. Rev.
Endocrine disrupting chemicals targeting estrogen receptor signaling: identification and mechanisms of action
Chem. Res. Toxicol.
D538G mutation in estrogen receptor-alpha: a novel mechanism for acquired endocrine resistance in breast cancer
Cancer Res.
Induced protein degradation: an emerging drug discovery paradigm
Nat. Rev. Drug Discov.
Small-molecule modulation of protein homeostasis
Chem. Rev.
Oral selective estrogen receptor downregulators (SERDs), a breakthrough endocrine therapy for breast cancer
J. Med. Chem.
New class of selective estrogen receptor degraders (SERDs): expanding the toolbox of PROTAC degrons
ACS Med. Chem. Lett.
Discovery of LSZ102, a potent, orally bioavailable selective estrogen receptor degrader (SERD) for the treatment of estrogen receptor positive breast cancer
J. Med. Chem.
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2021, European Journal of Medicinal ChemistryCitation Excerpt :Similar principles were applied to PROTAC-9 and PROTAC-10, although synthetic feasibility and commercial material availability were also in consideration for this case. For linker length, we used a mix of long and short linkers (9–17 atoms) according to multiple publications on the optimization of PROTAC linkers [60–67]. All reactions were carried out in flame-dried flasks with magnetic stirring.
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The authors contributed equally to this work.