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26e engages the WDR domain of DCAF1 in cells. In a cellular thermal shift assay (CETSA) as described in the materials and methods, the amount of unaggregated flag tagged WDR domain of DCAF1 was quantified. (A) 26e significantly stabilized the Flag-Hb-DCAF1-WD at 40 μM. (B) Titration of 26e showed a dose-dependent pattern of DCAF1-WD stability. Different compound concentrations (40 to 0.2 μM) of 26e were heated at 61 °C for 3.5 min. (C) When compared to the DMSO treatment, 26e thermally stabilized the WDR domain of DCAF1 to a greater extent than 3d, causing a 3.6 and 1.7 °C shift in the aggregation temperature of Flag-Hb-DCAF1_WD at 40 μM. Results shown as average ± SD (n = 3) (D) 26e thermally stabilized Flag-Hb-DCAF1_WD in a dose-dependent manner with an EC 50 value of 10.5 μM. Results shown as average ± SD (n = 3). CDC40 was used as a negative control. Note that in C, cells are subjected to a gradient of temperatures (46 to 66 °C). Across this range of temperatures, 3d thermally stabilizes the WDR domain of DCAF1, but the stabilization does not extend beyond 61 °C. In D, the protein is kept at 61 °C, and consistent with our observation in C, 3d was not expected to stabilize the WDR domain at any concentration.
Source publication
DCAF1 is a substrate receptor of two distinct E3 ligases (CRL4DCAF1 and EDVP), plays a critical physiological role in protein degradation, and is considered a drug target for various cancers. Antagonists of DCAF1 could be used toward the development of therapeutics for cancers and viral treatments. We used the WDR domain of DCAF1 to screen a 114-bi...
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Context 1
... This approach allows the assessment of DCAF1 stability upon compound treatment by monitoring the change in denaturation temperature. Using this approach, 26e significantly (>2 °C) stabilized HiBiT-DCAF1-WDR at 40 μM ( Figure 7A, C) and was also dosedependent ( Figure 7B, D). Furthermore, 26e thermally stabilized the WDR domain of DCAF1 to a greater extent than 3d, with an EC 50 value of 10.5 μM (Figure 7C,D). ...
Context 2
... This approach allows the assessment of DCAF1 stability upon compound treatment by monitoring the change in denaturation temperature. Using this approach, 26e significantly (>2 °C) stabilized HiBiT-DCAF1-WDR at 40 μM ( Figure 7A, C) and was also dosedependent ( Figure 7B, D). Furthermore, 26e thermally stabilized the WDR domain of DCAF1 to a greater extent than 3d, with an EC 50 value of 10.5 μM (Figure 7C,D). ...
Context 3
... this approach, 26e significantly (>2 °C) stabilized HiBiT-DCAF1-WDR at 40 μM ( Figure 7A, C) and was also dosedependent ( Figure 7B, D). Furthermore, 26e thermally stabilized the WDR domain of DCAF1 to a greater extent than 3d, with an EC 50 value of 10.5 μM (Figure 7C,D). However, both compounds failed to thermally stabilize the fulllength DCAF1 (HiBiT-DCAF1_FL) at 40 μM ( Figure S6). ...
Citations
... There is growing interest in ligand discovery for WDRs from the context of proximity pharmacology. For example, the WDRs DCAF1 [14][15][16][17] , DCAF11 18 , and DCAF16 19 are substrate recognition subunits of Cullin E3 ubiquitin ligases 20 , and several interact with DDB1 21 . ...
... Overall, 14 apo WDR structures were generated to support structure-guided drug discovery efforts (Fig. 2a, Tables S5, S6). The protocols for expressing, purifying and crystallizing the apo WDR domains greatly facilitated the rapid characterization and advancement of ligands identified through DEL-ML as described below 14,27 . ...
... ; https://doi.org/10.1101/2024.03.03.583197 doi: bioRxiv preprint referred to as Positive Training Examples (PTEs) (Fig. 2d, Table S9). Models that performed well on the test set were then used to predict the binding of commercially available compounds from large virtual libraries such as Enamine REAL and Mcule databases 14,25,27 . Depending on the number of predictions and in-stock availability, between 33 and 200 predicted compounds were purchased for 18 targets (16 WDRs) totaling ~3000 compounds in all (Figs 2e, S1, Table S10). ...
Protein class-focused drug discovery has a long and successful history in pharmaceutical research, yet most members of druggable protein families remain unliganded, often for practical reasons. Here we combined experiment and computation to enable discovery of ligands for WD40 repeat (WDR) proteins, one of the largest human protein families. This resource includes expression clones, purification protocols, and a comprehensive assessment of the druggability for hundreds of WDR proteins. We solved 21 high resolution crystal structures, and have made available a suite of biophysical, biochemical, and cellular assays to facilitate the discovery and characterization of small molecule ligands. To this end, we use the resource in a hit-finding pilot involving DNA-encoded library (DEL) selection followed by machine learning (ML). This led to the discovery of first-in-class, drug-like ligands for 9 of 20 targets. This result demonstrates the broad ligandability of WDRs. This extensive resource of reagents and knowledge will enable further discovery of chemical tools and potential therapeutics for this important class of proteins.
... Li et al. 27 19 13 ChEMBL (version 33) 28 -989 ...
Among the plethora of E3 Ligases, only a few have been utilized for the novel PROTAC technology. However, extensive knowledge of the preparation of E3 ligands and their utilization for PROTACs is already present in several databases. Here we provide, together with an analysis of functionalized E3 ligands, a comprehensive list of trained ML models to predict the probability to be an E3 ligase binder. We compared the different algorithms based on the different description schemes used and identified that the pharmacophoric-based ML approach was the best. Due to the peculiar pharmacophores present in E3 ligase binders and the presence of an explainable model, we were able to show the capability of our ErG model to filter compound libraries for fast virtual screening or focused library design. A particular focus was also given to target E3 ligase prediction and to find a subset of candidate E3 ligase binders within known public and commercial compound collections.
Advances in genome sequencing and editing technologies have enriched our understanding of the biochemical pathways that drive tumorigenesis. Translating this knowledge into new medicines for cancer treatment, however, remains challenging, and many oncogenic proteins have proven recalcitrant to conventional approaches for chemical probe and drug discovery. Here, we discuss how innovations in chemical proteomics and covalent chemistry are being integrated to identify and advance first-in-class small molecules that target cancer-relevant proteins. Mechanistic studies have revealed that covalent compounds perturb protein functions in cancer cells in diverse ways that include the remodeling of protein–protein and protein–RNA complexes, as well as through alterations in posttranslational modification. We speculate on the attributes of chemical proteomics and covalent chemistry that have enabled targeting of previously inaccessible cancer-relevant pathways and consider technical challenges that remain to be addressed in order to fully realize the druggability of the cancer proteome.
Dynamic combinatorial chemistry (DCC) leverages a reversible reaction to generate compound libraries from constituting building blocks under thermodynamic control. The position of this equilibrium can be biased by addition of a target macromolecule towards enrichment of bound ligands. While DCC has been applied to select ligands for a single target protein, its application to identifying chimeric molecules inducing proximity between two proteins is unprecedented. In this proof‐of‐concept study, we develop a DCC approach to select bifunctional proteolysis targeting chimeras (PROTACs) based on their ability to stabilize the ternary complex. We focus on VHL‐targeting Homo‐PROTACs as model system, and show that the formation of a VHL2:Homo‐PROTAC ternary complex reversibly assembled using thiol‐disulfide exchange chemistry leads to amplification of potent VHL Homo‐PROTACs with degradation activities which correlated well with their biophysical ability to dimerize VHL. Ternary complex templated dynamic combinatorial libraries allowed identification of novel Homo‐PROTAC degraders. We anticipate future applications of ternary‐complex directed DCC to early PROTAC screenings and expansion to other proximity‐inducing modalities beyond PROTACs.
Dynamic combinatorial chemistry (DCC) leverages a reversible reaction to generate compound libraries from constituting building blocks under thermodynamic control. The position of this equilibrium can be biased by addition of a target macromolecule towards enrichment of bound ligands. While DCC has been applied to select ligands for a single target protein, its application to identifying chimeric molecules inducing proximity between two proteins is unprecedented. In this proof‐of‐concept study, we develop a DCC approach to select bifunctional proteolysis targeting chimeras (PROTACs) based on their ability to stabilize the ternary complex. We focus on VHL‐targeting Homo‐PROTACs as model system, and show that the formation of a VHL2 : Homo‐PROTAC ternary complex reversibly assembled using thiol‐disulfide exchange chemistry leads to amplification of potent VHL Homo‐PROTACs with degradation activities which correlated well with their biophysical ability to dimerize VHL. Ternary complex templated dynamic combinatorial libraries allowed identification of novel Homo‐PROTAC degraders. We anticipate future applications of ternary‐complex directed DCC to early PROTAC screenings and expansion to other proximity‐inducing modalities beyond PROTACs.
Ubiquitination, a highly adaptable post-translational modification, plays a pivotal role in maintaining cellular protein homeostasis, encompassing cancer chemoresistance-associated proteins. Recent findings have indicated a potential correlation between perturbations in the ubiquitination process and the emergence of drug resistance in CRC cancer. Consequently, numerous studies have spurred the advancement of compounds specifically designed to target ubiquitinates, offering promising prospects for cancer therapy. In this review, we highlight the role of ubiquitination enzymes associated with chemoresistance to chemotherapy via the Wnt/β-catenin signaling pathway, epithelial–mesenchymal transition (EMT), and cell cycle perturbation. In addition, we summarize the application and role of small compounds that target ubiquitination enzymes for CRC treatment, along with the significance of targeting ubiquitination enzymes as potential cancer therapies.
Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.
