PROTAC-Mediated Degradation of Estrogen Receptor in the Treatment of Cancer

Important Compound Classes

Title

Compounds and Their Use in Treating Cancer

Patent Application Number

WO 2019/123367 A1

Publication Date

June 27, 2019

Priority Application

US 62/608,847

Priority Date

December 21, 2017

Inventors

Yang, B.; Kettle, J. G.; Hayhow, T. G. C.; Rasmusson, T. G.; Nissink, J. W. M.; Fallan, C.; Lamont, G. M.

Assignee Company

AstraZeneca AB; 151-85 Södertälje (SE).

Disease Area

Cancer

Biological Target

Estrogen receptor alpha (ERα)

Summary

Breast cancer is the second most common and the most occurring type of cancer among women. In 2019, an estimated 268,600 invasive and 62,930 noninvasive (in situ) new cases of breast cancer are expected to be diagnosed in women. For men, it is estimated that 2,670 new cases of invasive breast cancer will be diagnosed in the U.S. A man’s lifetime risk of breast cancer is about 1 in 883 (https://www.breastcancer.org/symptoms/understand_bc/statistics, accessed 08/25/2019). About 5–10% of breast cancers can be hereditary and may also be linked to gene mutations. The BRCA1 and BRCA2 genes are the most common mutations, and women with these mutations have up to 72% and 69% lifetimes risk of developing breast cancer, respectively. Thus, active research is devoted in understanding the physiological and biochemical processes, including reproduction, proliferation, and development, among others.

Nuclear receptors (NRs) are receptor proteins involved in regulating a wide range of physiological functions, including the endocrine-disrupting chemicals (EDCs). These NRs can bind to human NRs and cause inappropriate activation or inhibition of gene transcription, which can result in adverse biological and physiological effects involving the development, cell differentiation, reproduction, nervous system function, and immune responses in mammals. The two nuclear estrogen receptors (ERα and ERβ) mediate the biological effects of the estrogen hormones, and ERα is a key member of the nuclear receptor ER protein family that controls a variety of physiological and developmental processes, which has made it an attractive therapeutic target for diseases, including breast cancer and osteoporosis. ERα is often overexpressed in breast cancer cells and promotes their estrogen-dependent proliferation. Patients with ERα-positive breast cancer may benefit from the mainstay treatment involving endocrine therapies such as the tamoxifen inhibitors. However, patients ultimately develop relapse due to acquired or de novo resistance, and advanced breast cancer remains incurable. Numerous resistance mechanisms to antiestrogens have been suggested, including mutations in ERα, changes in the expression of ERα-cofactors, and activation of signaling pathways. Nonetheless, ERα activity is still required, and this highlights the continual importance of ERα in tumorigenesis.

There is considerable interest in the therapeutic use of selective estrogen receptor modulators (SERMs) such as estradiol, and there are a number of SERMs that are currently in clinical use, including tamoxifen for the treatment of hormone-dependent breast cancer, and lasofoxidine, raloxifene, and basidoxifene for the prevention of osteoporosis. Estrogen receptor alpha (ERα, ESRl, NR3A) and estrogen receptor beta (ERβ, ESR2, NR3b) are steroid hormone receptors that are members of the large nuclear receptor family. The role of ERβ in cancer is unclear, but it is generally thought that the expression of ERβ has antiproliferative effects in breast cancer cells. In prostate cancer there is some evidence that certain isoforms of ERβ are oncogenic, and its expression has been reported to have potentially protective effect in normal cells. Conversely, the ERα is composed of six functional domains (named A–F) and is classified as a ligand-dependent transcription factor because after its association with the specific ligand, the complex binds to genomic sequences, named estrogen receptor elements (ERE), and interacts with coregulators to modulate the transcription of target genes.

The C and E domains of ERα and ERβ are quite conserved (96% and 55% amino acid identity, respectively); however, the conservation of the A/B, D, and F domains is poor (below 30% amino acid identity). Both receptors are involved in the regulation and in the development of the female reproductive tract. In addition, they play important roles in the central nervous system, cardiovascular system, and bone metabolism. The genomic action of ERs occurs in the nucleus of the cell when the receptor binds to EREs directly or indirectly. In the absence of ligand, ERs are associated with heat shock proteins, and the associated chaperone machinery stabilizes the ligand binding domain (LBD), which makes it accessible to ligands. Liganded ER dissociates from the heat shock proteins leading to a conformational change in the receptor and allows dimerization, DNA binding, interaction with coactivators or corepressors, and modulation of target gene expression.

The precise mechanism in which ER affects gene transcription is poorly understood; however, it appears to be mediated by numerous nuclear factors that are recruited by the DNA bound receptor. The recruitment of coregulators is primarily mediated by the AF1 and AF2 protein surfaces. A large number of compounds that bind to ER, including the endogenous ligand estradiol, act as receptor agonists, while others competitively inhibit estradiol binding and act as receptor antagonists. These compounds can be divided into two classes based on their functional effects. The selective estrogen receptor modulators (SERMS) such as tamoxifen have the ability to act as both receptor agonists and antagonists depending on the cellular and promoter context as well as the ER isoform targeted. For instance, tamoxifen acts as an antagonist in the breast but acts as a partial agonist in the bone, cardiovascular system, and uterus. SERMS appear to act as AF2 antagonists and derive their partial agonist characteristics through AF1. The second group are considered full antagonists such as fulvestrant, which blocks the estrogen activity via the complete inhibition of AF1 and AF2 domains.

Fulvestrant is a selective ER downregulator (SERD) ERα antagonist with no known confounding agonist properties and has the unique ability to increase the rate of turnover of the receptor, which limits the amount available for further activation and has demonstrated superiority over the aromatase inhibitor, anastrozole. This has led to a focus on SERDs as a strategy for inhibition and removal of the ERα. However, fulvestrant lacks oral bioavailability and is administered intramuscularly in doses of 500 mg once a month, which has intensified the need for oral bioavailable drug that could lead to a more complete removal of ERα and a more durable response in patients.

Intracellular levels of ERα are down-regulated in the presence of estradiol through the ubiquitin/proteasome (Ub/26 S) pathway. The polyubiquitinylation of liganded ERα is catalyzed by at least three enzymes; the ubiquitin-activating enzyme E1 activated ubiquitin, which is conjugated by E2 conjugating enzyme with lysine residues through an isopeptide bond by the E3 ubiquitin ligase, and the polyubiquitinated ERα is then directed to the proteasome for degradation. The traditional approach to inhibiting transcriptional activity of ERα is generally based on modulating the conformational states of ERα with various unnatural ligands, and the main drawbacks of this classical strategy as described above is drug resistance. Consequently, PROTAC technology, as a strategy to directly degrade the ERα target protein, could be beneficial. PROTACs are heterobifunctional molecules containing two small molecule binding moieties, which are joined by a linker, and the PROTAC in the cell seeks out and selectively binds to the target protein of interest. The PROTAC then recruits a specific E3 ligase to the target protein to form a ternary complex with both the target protein and the E3 ligase in close proximity. Furthermore, the E3 ligase then recruits an E2 conjugating enzyme to the ternary complex, which allow ubiquitination of the target protein by the E2 ligase and then dissociates from the ternary complex to eventually initiate another catalytic cycle. In this Patent Highlight, the designated PROTACs targeting ER for degradation contain an ER ligand at one of the linkers and an E3 ligase such as the von Hippel–Lindau tumor suppressor (VHL) ligand at the other. ER PROTAC selectively recruits VHL E3 ligase to ER in the cells, which leads to the degradation of the ER by the Ub/26S system. The PROTACs compounds exhibit antitumor activity with the ability to degrade the estrogen receptor in a number of different breast cancer cell-lines such as MCF-7, CAMA-1, and BT474 cell-lines.

Definitions

R¹ = H, Me;

A and G are independently CR² or N;

R² = H, F, Cl, Me, or MeO;

D and E = CR³ or N;

R³ = H, F, Cl, CN;

R⁴ = R⁵ = H, Me, or F;

R⁶ = H, Me, F, CH₂F, CHF₂, CF₃, CN, CH₂CN, CH₂OH, CH₂OMe, and so forth;

R⁷ = H, Me, −CH₂NHMe, −CH₂NMe₂, or CH₂NH₂;

X = −O–, −CH=CH–C(O)NH–, −NHC(O)–, −C(O)NH–, or −pyrrolidinyl-NHMeC(O)–.

Key Structures

Biological Assay

Biochemical assay for estrogen receptor alpha ligand binding was assessed in competitive assay using a LantahScreen time-resolved fluorescence resonance energy transfer (TR-FRET). MCF-7 ER degradation was assessed in cell-based immune-fluorescence assay using the MCF-7 human ductal carcinoma breast cell line.

Biological Data

The compounds in this Patent Highlight show biological activity greater or equal to 90% ERα degradation at 0.3 μM in the MCF-7 degradation cellular assay. ER degradation by fulvestrant at 0.3 μM is defined as 100%, compounds with 90–99% ERα degradation are marked “•”, and compounds with greater or equal to 100% ERα degradation are marked “••”.

Recent Review Articles

• 1.Flanagan J. J.; Neklesa T. K.. Mol. Cell Endocrinol. 2019, 493, 110452.
• 2.Saha T.; Makar S.; Swetha R.; Gutti G.; Singh S. K.. Eur. J. Med. Chem. 2019, 177, 116.
• 3.Waks A. G.; Winer E. P.. JAMA 2019, 321, 288.
• 4.Verma A.; Schwartz Z.; Boyan B. D.. Steroids 2019, 150, 108447.

The author declares no competing financial interest.