SMARCA2 x SMARCA4 x Ubiquitin ligase

Reversible protein phosphorylation plays a critical role in the regulation of various cellular processes, including intracellular signaling and protein–protein interaction. Major causes of tumorigenesis and cancer malignancy are consequences of aberrant phosphorylation and dephosphorylation of proteins in humans. Eukaryotic cells compact their DNA, the full genetic code for life in very small volume in the cell nucleus. Furthermore, complexing chromosomal DNA connects with core histone proteins to form chromatin, which allows the double-stranded DNA to be wrapped around an octameric core of histone proteins. Chromosomal DNA can pack tightly against one another and may impose a steric barrier that prevents transcription, replication, recombination, and DNA repair machineries from binding to the DNA. Accessibility to the DNA is facilitated by two classes of enzymes, ATP-dependent nucleosome remodelers and histone-modifying enzymes: the mammalian switch/sucrose nonfermentable (SWI/SNF) complexes and polycomb repressor complex 2 (PRC2). The mammalian SWI/SNF complexes (also called BAF complex for Brg-/Brama-associated factor complex) regulate chromatin remodeling and are powered by two closely related ATPase subunits, SMARCA4 and SMARCA2 (BRAHMA or BRM). SMARCA4 and SMARCA2 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, members 4 and 2, respectively) are coexpressed in most tissue types with the exception of embryonic stem cells in which SMARCA4 is the only ATPase expressed unit. SWI/SNF complexes utilize the energy derived from ATP hydrolysis to achieve the coordinate mobilization of nucleosomes and the modulation of chromatin accessibility. Chromatin remodeling complexes (CRCs) utilize the energy of ATP hydrolysis to disrupt DNA–nucleosome contacts, also move nucleosomes along the DNA, and eject or exchange nucleosomes. SWI/SNF complexes are required for several aspects of development and more recently have been strongly implicated in human disease. Mutations in the genes encoding the canonical SWI/SNF subunits are observed in nearly 20% of all cancers, including lung, ovarian, uterine, gastric, cervical, and esophageal. SMARCA4 is frequently mutated in primary tumors, while SMARCA2 inactivation is infrequent in tumor development. SMARCA2 has been shown to be an essential gene in SMARCA4-related cancer cell lines. Thus, SMARCA2 may be targeted in SMARCA4-related or -deficient cancers. However, lack of specificity and the inability to target and modulate SMARCA2 remains an obstacle to the development of effective treatments. Consequently, small-molecules that target SMARCA2 and potentiate the VHL’s substrate specifically may help provide promising therapeutic agents. The present Patent Highlight describes bifunctional (proteolysis targeting chimeric, PROTAC) compounds which function to recruit endogenous proteins to an E3 ubiquitin ligase for degradation. The PROTAC compounds have a broad range of pharmacological activities, and an effective amount is used for the treatment or amelioration of disease conditions such as cancer, including SMARCA4-related or -deficient cancer. Furthermore, the PROTAC compounds comprise of an E3 ubiquitin ligase binding moiety (ligand for an E3 ubiquitin ligase or “ULM” group) and a moiety that binds a target protein (protein/polypeptide targeting ligand or “PTM” group) such as the target protein/polypeptide that is placed in close proximity to the ubiquitin ligase to effect degradation of that protein. The ubiquitination ligase modulator (ULM) can be Von Hippel–Lindau E3 ubiquitin ligase (VHL) binding moiety (VLM). Furthermore, a chemical linker “L” is a bond that couples PTM to ULM. The linker may contain ether, amide, alkane, aromatic, heteroaromatic, cyclic, thioether, and so forth.

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.