Modulating Androgen Receptor in the Therapeutic Intervention for Prostate Cancer and Kennedy’s Disease

Important Compound Classes

Title

Bifunctional Compounds and Methods for Targeted Ubiquitination of Androgen Receptor

Patent Publication Number

US 2020/0282068 A1 and WO 2020160295 A1

Publication Date

September 10, 2020 and August 06, 2020

Priority Application

US 16/876,949 and 62/798,554

Priority Date

May 18, 2020 and January 30, 2019

Inventors

Desantis, J.; Vaz, R. J. and Kim, I.

Assignee Company

Montelino Therapeutics, LLC, Southborough, MA, United States 01772 (US).

Disease Area

Androgen Receptor

Biological Target

Prostate Cancer

Summary

There are over 3.1 million American men living with prostate cancer in the United States. The American Cancer Society estimates that 248,530 men will have prostate cancer in 2021. Androgen ablation therapy (ADT) has been one of the central therapeutic strategies against advanced prostate cancer for nearly 70 years. However, patients receiving ADT have good initial response, but most will inevitably develop into more aggressive castration-resistant prostate cancer (CRPC) after 2–3 years. In addition, mechanistic studies have shown that androgen receptor (AR) signaling remains active even in these hormone-refractory cancers, which suggests that AR remains an important target for prostate cancer (PCa) and CRPC treatment.

The androgen receptor (AR) or nuclear receptor subfamily 3, group C gene (4NR3C4) and its endogenous ligands play a pivotal role in the growth and development of the normal prostate gland and also the androgen-dependent (AD) or androgen-independent (AI) progression of the PCa. AR is a member of the steroid receptor superfamily, which is expressed in many human tissues, among which breast cancer (BC) tissue has the third-highest expression of AR. Between 70% and 90% of BC patients overexpress AR, which suggests that AR might also be a predictive or prognostic factor and a drug target in BC. Furthermore, androgens are also known to play a role in female cancers. Ovarian cancer has elevated levels of androgens, which is associated with an increased risk of the development of ovarian cancer.

AR is activated by androgens such as testosterone and dihydrotestosterone. The androgens, as well as antagonists such as enzalutamide, compete with the androgens that bind to the ligand binding domain (LBD). The nuclear receptors are composed of an N-terminal domain (NTD), a central DNA binding domain (DBD), a short hinge region, and C-terminal domain that contains a hormone ligand binding pocket and the activation function-2 (AF2) site.

The activation of AR in the cytoplasm is associated with chaperone proteins that maintain agonist binding conformation. Upon binding of an androgen, AR undergoes a series of conformational changes, disassociation from chaperones, dimerization, and translocation into the nucleus, where it further interacts with coactivator proteins at the AF2 site. Consequently, it triggers the recruitment of RNA polymerase II and other factors to form a functional transcriptional complex with the AR. In CRPC, drug resistance can manifest through AR-LBD mutations which convert AR antagonists into agonists or by the expression of AR-variants lacking the LBD.

Recent studies have shown that patients on enzalutamide and abiraterone with a splice variant of AR, labeled as AR-V7, had lower PSA response rates, shorter PSA progression-free survival, and shorter overall survival. AR-V7 lacks the LBD, which is the target of enzalutamide and testosterone, but AR-V7 remains constitutively active as a transcription factor. As a result, other approaches to antagonize the AR receptor as well as AR-V7 is desirable.

Although several methods exist for the manipulation of protein levels, the bifunctional proteolysis targeting chimeric molecules (PROTACs) have generated high interest in drug discovery. PROTACs consist of a ligand which recognizes the target protein that is linked to a ligand and binds to a specific E3 ubiquitin ligase. The bifunctional molecule binds to the target protein and the E3 ligase, enabling the transfer of ubiquitin to the target protein from the ligase, which may have a suitable acceptor on the target protein.

The “molecular glue” concept consists of a molecule that is attached to the E3 ligase, which recruit the target protein to the E3 ligase followed by the ubiquitin transfer and degradation of the target. After binding to the E3 ligase, the ensuing moiety then recruits the protein to be degraded. Protein ubiquitination is a multistep process whereby a ubiquitin protein is successively relayed between different classes of enzymes (El, E2, E3) in order to tag a cellular substrate. An E3 ligase recruits a specific downstream target protein and mediates the transfer of the ubiquitin from the E2 enzyme to the terminal substrate through either a covalent or noncovalent mechanism. On the other hand, each ubiquitin is ligated to a protein through either a peptide bond with the N-terminal amino group or an isopeptide bond formed between a side chain, an amino group of a select lysine residue on the target protein and the ubiquitin. The combination of the target ligand and the ubiquitin recruiting ligand, the linker length and conformational flexibility also contributes to the effectiveness of the degradation molecule. Thus, the mechanism depends on the availability of a lysine residue on the surface of the protein close to the targeted protein ligand binding pocket. Proposed treatment for CRPC is based on bifunctional molecules that couples various E3 ligases to the AR antagonists binding at the AR LBD site.

The deubiquitinating enzymes (DUBs) play a key role in ubiquitin homeostasis by regulating the nature and extent of substrate ubiquitination as well as the availability of free ubiquitin. The human genome encodes over 90 DUBs that fall into 5 structural classes. In this Patent Highlight, PROTAC molecules do not target the LBD site but the central DNA binding domain (DBD). The molecules simultaneously bind AR and an E3 ubiquitin ligase and also promote ubiquitination of AR-V7, possibly AR, and lead to degradation of AR-V7 and AR by the proteasome. In addition, an effective amount of these compounds is used for the treatment or amelioration of a disease condition including cancer, e.g., prostate cancer, and Kennedy’s disease.

Key Structures

Biological Assay

Electrophoresis, visualized by using an enhanced chemiluminescence (ECL) kit. Cell proliferation assay, apoptosis assay, and PCR based detection system. In vivo mouse tumor model using compound 26 showed substantial reduced tumor mass beginning around 3 weeks of treatment through 7 weeks at 8.3 and 0.83 mg/kg.

Biological Data

The table below demonstrate that exemplary compounds decreased cell count in a concentration dependent manner where +++ = cell count decreased by more than 50%; ++ = cell count decreased less than 50%; + = cell count decrease between 0 and 20%.

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The author declares no competing financial interest.