William Farnaby

Bifunctional degrader molecules, known as proteolysis-targeting chimeras (PROTACs), function by recruiting a target to an E3 ligase, forming a target/PROTAC/ligase ternary complex. Despite the importance of this key intermediate species, no detailed validation of a method to directly determine binding parameters for ternary complex kinetics has been reported, and it remains to be addressed whether tuning the kinetics of PROTAC ternary complexes may be an effective strategy to improve the efficiency of targeted protein degradation. Here, we develop an SPR-based assay to quantify the stability of PROTAC-induced ternary complexes by measuring for the first time the kinetics of their formation and dissociation in vitro using purified proteins. We benchmark our assay using four PROTACs that target the bromodomains (BDs) of bromodomain and extraterminal domain proteins Brd2, Brd3, and Brd4 to the von Hippel–Lindau E3 ligase (VHL). We reveal marked differences in ternary complex off-rates for different PROTACs that exhibit either positive or negative cooperativity for ternary complex formation relative to binary binding. The positively cooperative degrader MZ1 forms comparatively stable and long-lived ternary complexes with either Brd4BD2 or Brd2BD2 and VHL. Equivalent complexes with Brd3BD2 are destabilized due to a single amino acid difference (Glu/Gly swap) present in the bromodomain. We observe that this difference in ternary complex dissociative half-life correlates to a greater initial rate of intracellular degradation of Brd2 and Brd4 relative to Brd3. These findings establish a novel assay to measure the kinetics of PROTAC ternary complexes and elucidate the important kinetic parameters that drive effective target degradation.

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.

Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders.

Abstract Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that converts cholesterol into 24 S -hydroxycholesterol, the primary mechanism of cholesterol catabolism in the brain. The therapeutic potential of CH24H activation has been extensively investigated, whereas the effects of CH24H inhibition remain poorly characterized. In this study, the therapeutic potential of CH24H inhibition was investigated using a newly identified small molecule, soticlestat (TAK-935/OV935). The biodistribution and target engagement of soticlestat was assessed in mice. CH24H-knockout mice showed a substantially lower level of soticlestat distribution in the brain than wild-type controls. Furthermore, brain-slice autoradiography studies demonstrated the absence of [ 3 H]soticlestat staining in CH24H-knockout mice compared with wild-type mice, indicating a specificity of soticlestat binding to CH24H. The pharmacodynamic effects of soticlestat were characterized in a transgenic mouse model carrying mutated human amyloid precursor protein and presenilin 1 (APP/PS1-Tg). These mice, with excitatory/inhibitory imbalance and short life-span, yielded a remarkable survival benefit when bred with CH24H-knockout animals. Soticlestat lowered brain 24 S -hydroxycholesterol in a dose-dependent manner and substantially reduced premature deaths of APP/PS1-Tg mice at a dose lowering brain 24 S -hydroxycholesterol by approximately 50%. Furthermore, microdialysis experiments showed that soticlestat can suppress potassium-evoked extracellular glutamate elevations in the hippocampus. Taken together, these data suggest that soticlestat-mediated inhibition of CH24H may have therapeutic potential for diseases associated with neural hyperexcitation.