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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. Protein degraders are an emerging drug modality; however, their properties lie beyond typical drug-like space. Here the authors report optimisation via structure-based exit vector and linker design towards the VHL-recruiting PROTAC ACBI2, an orally bioavailable and selective degrader of SMARCA2.

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.

Bifunctional targeted protein degraders, also known as Proteolysis Targeting Chimeras (PROTACs), are an emerging drug modality that may offer a new approach for treating neurodegenerative diseases. Identifying chemical starting points for PROTACs remains a largely empirical process and the design rules for identifying Central Nervous System (CNS) active PROTACs have yet to be established. Here we demonstrate a concept of using orthogonally reactive linker reagents, that allow the construction of screening libraries whereby the E3 ligase binder, the target protein binder and the linker can be simultaneously varied and tested directly in cellular assays. This approach enabled the discovery of Glycogen Synthase Kinase 3 (GSK3) PROTACs which are CNS in vivo active in female mice. Our findings provide opportunities to investigate the role of GSK3 paralogs in cellular and in vivo disease models and for the rapid discovery of in vivo quality bifunctional chemical probes for CNS disease concepts. The authors in this work identify an in vivo CNS active bifunctional degrader of GSK3. This was discovered via development of orthogonally reactive linker chemistry and a direct-to-biology screen that was able to provide hits of in vivo chemical probe quality.

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBN midi , a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBN midi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBN midi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics. Structure-based drug discovery of cereblon (CRBN)-recruiting protein degraders has been to date challenging due to limitations with current constructs for recombinant protein expression. In this work, the authors design and validate a truncated CRBN construct, CRBNmidi, that enables crystallization and biophysical characterization of CRBN-binding ligands and degraders.

Targeted protein degradation using proteolysis-targeting chimeras (PROTACs) offers a promising strategy to eliminate previously undruggable proteins. PROTACs are bifunctional molecules that link a target protein with an E3 ubiquitin ligase, enabling the formation of a ternary complex that promotes ubiquitination and subsequent proteasomal degradation. Although many ternary complex structures are available, understanding how structural features relate to PROTAC function remains challenging due to the dynamic nature of these complexes. Here we show that the interface between the target protein SMARCA2 and the E3 ligase VHL is conformationally flexible and stabilized by interactions involving disordered loops. Using molecular dynamics simulations and X-ray crystallography of SMARCA2–VHL complexes bound to five different PROTACs, we find that interfacial residues often adopt energetically suboptimal, or ‘frustrated,’ configurations. We further show that the degree of frustration correlates with experimentally measured cooperativity for a set of 11 PROTACs. These findings suggest that quantifying interface frustration provides a rational, structure-based approach to guiding PROTAC design. PROTACs induce degradation by bridging a target protein and E3 ubiquitin ligase. Here, authors show that protein interface frustration correlates with cooperativity, offering a structural metric to prioritize PROTAC candidates prior to synthesis.

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. A structure-based design allows the development of a potent PROTAC to degrade BAF ATPase subunits SMARCA2 and SMARCA4 via recruitment of E3 ubiquitin ligase VHL and induce cancer cell death.

Diseases of the central nervous system represent an increasing area of unmet medical need. Despite high levels of investment from the pharmaceutical industry few disease modifying drugs have been discovered with the most common cause of late stage clinical failure being a lack of efficacy. In order to enable greater understanding of disease mechanisms and the ability to validate specific biological targets for disease intervention, there is a need for medicinal chemists to identify high quality chemical compounds which can reliably probe new therapeutic concepts. Herein, I report three approaches towards this goal. In the first case, a structure based approach allowed the de novo design of a novel class of D-amino acid oxidase (DAAO) inhibitors. This work gave rise to three novel series of DAAO inhibitors, all of which were capable of addressing some or all of the issues that had limited previous attempts to inhibit this high profile CNS target. Furthermore, subsequent optimization by colleagues of chemical leads I discovered and detailed within this thesis, led to the identification of clinical candidate TAK-831. In addition, novel inhibitors for Cholesterol-24-hydroxylase (CH24H) are shown, developed via a property guided approach. The advantages of balancing physicochemical properties throughout lead molecules are demonstrated, giving rise to compounds with in vivo activity in rodent models of spatial learning and memory deficit for this novel drug target. Finally, a strategy for the efficient synthesis of diverse and highly lead-like screening libraries is presented specifically with the objective of providing improved molecular starting points for CNS drug discovery.

In the version of this article originally published, several lines of text in the last paragraph of the right column on page 1 of the PDF were transposed into the bottom paragraph of the left column. The affected text of the left column should read “The ATP-dependent activities of the BAF (SWI/SNF) chromatin remodeling complexes affect the positioning of nucleosomes on DNA and thereby many cellular processes related to chromatin structure, including transcription, DNA repair and decatenation of chromosomes during mitosis 12,13 .” The affected text of the right column should read “SMARCA2/4 BD inhibitors are thus precluded from use for the treatment of SMARCA4 mutant cancers but could provide attractive ligands for PROTAC conjugation. Small molecules binding to other bromodomains have been successfully converted into PROTACs by conjugating them with structures capable of binding to the E3 ligases von Hippel−Lindau (VHL) or cereblon 5,6,10,11,25,26,27 .” The errors have been corrected in the PDF version of the paper.

Targeted protein degradation of a protein of interest (POI) by Proteolysis Targeting Chimeras (PROTACs) is an attractive approach for dealing with formerly undruggable protein targets. PROTACs are heterobifunctional molecules that connect a POI-binding and an E3-ligase (E3) binding motif with a linker. The simultaneous binding and formation of a ternary POI::PROTAC::E3 complex (TC) induces proximity between the POI and the E3, allowing for POI-ubiquitination and subsequent induction of proteasomal degradation. Despite the availability of many three-dimensional structures of TCs, unveiling the structure-function relationships for the design of PROTACs remains a challenge. This is because the TCs can be dynamic with a complex conformational landscape that individual crystal structures may not capture. In this work, we used SMARCA2 as the POI and VHL as the E3-ligase and solved the X-ray crystal structures of the respective ternary complexes with four different PROTACs. Molecular dynamics (MD) simulations were used to show that the SMARCA2-VHL interface is flexible with multiple energy minima. The protein-protein (POI-E3) interactions are largely formed by residues located in structurally disordered loops in both VHL and SMARCA2. The residue pairs in the SMARCA2-VHL interface are 'frustrated', i.e., adopt a suboptimal energetic state. The number of frustrated residue pairs averaged over the MD ensemble shows a positive correlation with the experimentally determined cooperativity of the PROTACs. This indicates that protein-protein interface frustration can play an important role in PROTAC function. The TC ensembles of VHL, SMARCA2 and 11 different PROTACs were modeled by comparative modeling followed by MD. The frustration was subsequently calculated from the MD trajectories and correlated with the cooperativity. We found that identification of the dynamic protein-protein contacts and frustrated residue pairs in the interface can provide a rational framework for the structure-based design of PROTACs.Competing Interest StatementA.C. is a scientific founder and shareholder of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms. The Ciulli laboratory receives or has received sponsored research support from Almirall, Amgen, Amphista Therapeutics, Boehringer Ingelheim, Eisai, Merck KaaG, Nurix Therapeutics, Ono Pharmaceutical, and Tocris-Biotechne. Z.J., P.S., C.K.,P.G.,H.W.,G.B. and A.B. are current employees of Boehringer Ingelheim. E.D. is now an employee of Astra Zeneca. All other authors declare that they have no competing interests.

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using mathematical modelling of protein degradation, we demonstrate that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.Competing Interest StatementJE, AS, AK, TG, NC, TG, TC, GD, PG, NB, PE, CK and MK are direct employees of Boehringer Ingelheim RCV GmbH & Co KG. The Ciulli laboratory receives or has received sponsored research support from Almirall, Amgen, Amphista Therapeutics, Boehringer Ingelheim, Eisai, Merck KGaA, Nurix Therapeutics, Ono Pharmaceutical and Tocris-Biotechne. A.C. is a scientific founder, shareholder, and advisor of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms.