A Modular Chemistry Platform for the Development of a Cereblon E3 Ligase-Based Partial PROTAC Library

Pr oteolysis ta rgeting c himeras (PROTACs) are a promising therapeutic strategy to selectively promote the degradation of protein targets by exploiting the ubiquitin-proteasome system. Among the limited number of E3 ligase ligands discovered for the PROTAC technology, ligands of cereblon (CRBN) E3 ligase, such as pomalidomide, thalidomide, or lenalidomide, are the most frequently used for the development of PROTACs. Our group previously reported that a phenyl group could be tolerated on the C4-position of lenalidomide as the ligand of CRBN to develop PROTACs. Herein, we report a modular chemistry platform for the efficient attachment of various ortho-, meta-, and para-substituted phenyls to the C4-position of the lenalidomide via Suzuki cross-coupling reaction, which allows the systematic investigation of the linker effect for the development of PROTACs against any target. We examined the substrate scope by preparing twelve lenalidomide-derived CRBN E3 ligase ligands with different linkers.     

Native Mass Spectrometry for the Study of PROTAC GNE-987-Containing Ternary Complexes

PRO teolysis TA rgeting C himeras (PROTACs) promote the degradation, rather than inhibition, of a drug target as a mechanism for therapeutic treatment. Bifunctional PROTAC molecules allow simultaneous binding of both the target protein and an E3-Ubiquitin ligase, bringing the two proteins into close spatial proximity to allow ubiquitinylation and degradation of the target protein via the cell's endogenous protein degradation pathway. We utilized native mass spectrometry (MS) to study the ternary complexes promoted by the previously reported PROTAC GNE-987 between Brd4 bromodomains 1 and 2, and Von Hippel Lindeau E3-Ubiquitin Ligase. Native MS at high resolution allowed us to measure ternary complex formation as a function of PROTAC concentration to provide a measure of complex affinity and stability, whilst simultaneously measuring other intermediate protein species. Native MS provides a high-throughput, low sample consumption, direct screening method to measure ternary complexes for PROTAC development.     

Jostling for Position: Optimizing Linker Location in the Design of Estrogen Receptor‐Targeting PROTACs

Estrogen receptor‐α (ER) antagonists have been widely used for breast cancer therapy. Despite initial responsiveness, hormone‐sensitive ER‐positive cancer cells eventually develop resistance to ER antagonists. It has been shown that in most of these resistant tumor cells, the ER is expressed and continues to regulate tumor growth. Recent studies indicate that tamoxifen initially acts as an antagonist, but later functions as an ER agonist, promoting tumor growth. This suggests that targeted ER degradation may provide an effective therapeutic approach for breast cancers, even those that are resistant to conventional therapies. With this in mind, we previously demonstrated that proteolysis targeting chimeras (PROTACs) effectively induce degradation of the ER as a proof‐of‐concept experiment. Herein we further refined the PROTAC approach to target the ER for degradation. The ER‐targeting PROTACs are composed of an estradiol on one end and a hypoxia‐inducing factor 1α (HIF‐1α)‐derived synthetic pentapeptide on the other. The pentapeptide is recognized by an E3 ubiquitin ligase called the von Hippel Lindau tumor suppressor protein (pVHL), thereby recruiting the ER to this E3 ligase for ubiquitination and degradation. Specifically, the pentapeptide is attached at three different locations on estradiol to generate three different PROTAC types. With the pentapeptide linked through the C7α position of estradiol, the resulting PROTAC shows the most effective ER degradation and highest affinity for the estrogen receptor. This result provides an opportunity to develop a novel type of ER antagonist that may overcome the resistance of breast tumors to conventional drugs such as tamoxifen and fulvestrant (Faslodex).     

HaloTag-Targeted Sirtuin-Rearranging Ligand (SirReal) for the Development of Proteolysis-Targeting Chimeras (PROTACs) against the Lysine Deacetylase Sirtuin 2 (Sirt2)**

We have discovered the sirtuin-rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD⁺-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis-targeting chimera (PROTAC) enabled small-molecule-induced degradation of Sirt2. Herein, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can also be harnessed for small-molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be used as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.     

Controlling PROTACs with Light

Proteolysis targeting chimeras, PROTACs, are emerging as a powerful strategy for exerting exogenous control over protein levels, allowing small molecules to exploit the ubiquitin–proteasome pathway for targeted protein degradation. This highlight focuses on the fusion of photochemistry with these bifunctional compounds, which has provided a novel pathway for spatiotemporally tuning the activation of PROTACs in the form of their photocaged and photoswitchable versions. Photocaged PROTACs consist of a hindered optolabile group that detaches only upon irradiation at a specific wavelength, releasing the active PROTAC. These modified PROTACs are inactive in the dark. Photoswitchable PROTACs are photoisomerizable molecules with azobenzene linkages that are active in either the cis or trans form and inactive in the other. The isomers interconvert upon irradiation with an appropriate wavelength of light and relax to the thermodynamically stable isomer in the dark or with another wavelength of light. Although photocaged PROTACs only permit activation control for protein degradation, photoswitching PROTACs offer reversible activation and deactivation by using suitable wavelengths of light.     

A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity**

Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4^CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4^CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4^CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4^CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.     

The tale of proteolysis targeting chimeras (PROTACs) for Leucine-Rich Repeat Kinase 2 (LRRK2)

Here we present the rational design and synthetic methodologies towards proteolysis-targeting chimeras (PROTACs) for the recently-emerged target leucine-rich repeat kinase 2 (LRRK2). Two highly potent, selective, brain-penetrating kinase inhibitors were selected, and their structure was appropriately modified to assemble a cereblon-targeting PROTAC. Biological data show strong kinase inhibition and the ability of the synthesized compounds to enter the cells. However, data regarding the degradation of the target protein are inconclusive. The reasons for the inefficient degradation of the target are further discussed.     

Targeted Protein Degradation as a Promising Tool for Epigenetic Upregulation of Fetal Hemoglobin

The level of fetal hemoglobin (HbF) is an important disease modifier for β-thalassemia and sickle cell disease patients. Indeed, genetic tinkering with the HbF repression machinery has demonstrated great potential for disease mitigation. Such genetic treatments are costly and the high incidence of β-hemoglobinopathies in low-income countries, therefore, calls for the development of affordable, off-the-shelf, oral treatments. The use of PROTAC ( PR oteolysis TA rgeting C himeras) technology to influence the epigenetic mechanisms involved in HbF suppression may provide a solution. In this minireview, we briefly explain the HbF repression network highlighting the epigenetic factors that could be targeted for degradation by PROTACs. We hope that this review will inspire clinicians, molecular and chemical biologists to collaborate and contribute to this fascinating field, which should ultimately deliver drugs that reactivate HbF expression with high specificity and low toxicity.     

Delivering on Cell-Selective Protein Degradation Using Chemically Tailored PROTACs

PROTACs (Proteolysis-Targeting Chimeras) have emerged as a groundbreaking class of chemical tools that facilitate the degradation of target proteins by leveraging the ubiquitin-proteasome system (UPS). However, the effective utilization of PROTACs in chemical biology studies and therapeutics encounters significant challenges when it comes to achieving cell-selective protein degradation and in vivo applications. This review article aims to shed light on recent advancements in the development of Pro-PROTACs, which exhibit controlled protein degradation capabilities in response to external stimuli or disease-related endogenous biochemical signals. The article delves into the specific chemical strategies employed to regulate the interaction between PROTACs and E3 ubiquitin ligases or target proteins. These strategies enable spatial and temporal control over the protein degradation potential of Pro-PROTACs. Furthermore, the review summarizes recent investigations regarding the delivery of PROTACs using biodegradable nanoparticles for in vivo applications and targeted protein degradation. Such delivery systems hold great promise for enabling efficient and selective protein degradation in vivo. Lastly, the article provides a perspective on the future design of multifunctional PROTACs and their intracellular delivery mechanisms, with a particular focus on achieving cell-selective protein degradation.     

Optochemical Control of Protein Degradation

Optochemical approach has been successfully utilized to regulate cellular protein degradation with a high resolution of spatiotemporal control. In this highlight, we discuss two recent developments of combining reversible optochemical functionalities with the bifunctional proteolysis targeting chimeras, or PROTACs to achieve light-controlled degradation of protein targets of interest. PHOTACs are azobeneze-containing molecules that are inactive as trans forms and active as cis forms, switchable upon pulse-irradiation with either an activating 390 nm light or a deactivating 525 nm light. In contrast, photoPROTACs are o-F₄-azobenzene-containing molecules that can be switched between active trans isomers and inactive cis isomers by a single irradiation event using 415 or 530 nm light. Combining these two optochemically controlled PROTAC systems has the potential to achieve orthogonal control on protein degradation.     

dSTORM-Based Single-Cell Protein Quantitative Analysis Can Effectively Evaluate the Degradation Ability of PROTACs

As an emerging therapeutic strategy, proteolysis-targeting chimeras (PROTACs) have been proven to be superior to traditional drugs in many aspects. However, due to their unique mechanism of action, existing methods for evaluating the degradation still have many limitations, which seriously restricts the development of PROTACs. In this methodological study, using direct stochastic optical reconstruction microscopy (dSTORM)-based single-cell protein quantitative analysis, we systematically investigated the dynamic degradation characteristics of FLT3 protein during PROTACs treatment. We found that the distribution of FLT3 varies between FLT3-ITD mutation and FLT3-WT cells. PROTACs had an obvious time-course effect on protein degradation and present two distinct phases; this provided a basis for deciding when to evaluate protein degradation. High concentrations of PROTACs were more effective than long-time administration because a higher D_max was achieved. Two-color dSTORM-based colocalization analysis efficiently detected the proportion of ternary complexes, making it very useful in screening PROTACs. Taken together, our findings show that the dSTORM method is an ideal tool for evaluating PROTACs and will accelerate the development of new PROTACs.     

Hydrogen Peroxide-Inducible PROTACs for Targeted Protein Degradation in Cancer Cells

Proteolysis-targeting chimeras (PROTACs) provide a powerful technique to degrade targeted proteins utilizing the cellular ubiquitin-proteasome system. The major concern is the host toxicity resulting from their poor selectivity. Inducible PROTACs responding to exogenous stimulus, such as light, improve their specificity, but it is difficult for photo-activation in deep tissues. Herein, we develop H₂O₂-inducible PROTAC precursors 2 / 5 , which can be activated by endogenous H₂O₂ in cancer cells to release the active PROTACs 1 / 4 to effectively degrade targeted proteins. This results in the intended cytotoxicity towards cancer cells while targeted protein in normal cells remains almost unaffected. The higher Bromodomain-containing protein 4 (BRD4) degradation activity and cytotoxicity of 2 towards cancer cells is mainly due to the higher endogenous concentration of H₂O₂ in cancer cells (A549 and H1299), characterized by H₂O₂-responsive fluorescence probe 3 . Western blot assays and cytotoxicity experiments demonstrate that 2 degrades BRD4 more effectively and is more cytotoxic in H₂O₂-rich cancer cells than in H₂O₂-deficient normal cells. This method is also extended to estrogen receptor (ER)-PROTAC precursor 5 , showing H₂O₂-dependent ER degradation ability. Thus, we establish a novel strategy to induce targeted protein degradation in a H₂O₂-dependent way, which has the potential to improve the selectivity of PROTACs.     

Synthesis and Evaluation of Cereblon-Recruiting HaloPROTACs

Target validation is key to the development of protein degrading molecules such as proteolysis-targeting chimeras (PROTACs) to identify cellular proteins amenable for induced degradation by the ubiquitin-proteasome system (UPS). Previously the HaloPROTAC system was developed to screen targets of PROTACs by linking the chlorohexyl group with the ligands of E3 ubiquitin ligases VHL and cIAP1 to recruit target proteins fused to the HaloTag for E3-catalyzed ubiquitination. Reported here are HaloPROTACs that engage the cereblon (CRBN) E3 to ubiquitinate and degrade HaloTagged proteins. A focused library of CRBN-pairing HaloPROTACs was synthesized and screened to identify efficient degraders of EGFP-HaloTag fusion with higher activities than VHL-engaging HaloPROTACs at sub-micromolar concentrations of the compound. The CRBN-engaging HaloPROTACs broadens the scope of the E3 ubiquitin ligases that can be utilized to screen suitable targets for induced protein degradation in the cell.     

PROTAC Compatibilities,Degrading Cell-Surface Receptors,and the Sticky Problem of Finding a Molecular Glue

PROteolysis TArgeting Chimeras (PROTACs) are emerging as critical tools in biomedicinal chemistry and drug design, but they have limitations. These limitations include a lack of guiding principles when selecting suitable E3 ligases to induce ubiquitinylation, and problems degrading cell-surface receptors. This Highlight outlines some recent advances that circumvent some of these limitations, and new alternative methods to induce selective protein degradation.     

Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC₅₀ of 0.030 µM and CC₅₀ of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.     

Effect of the polyester chemical structure on the stability of polyester–melamine coatings when exposed to accelerated weathering

The effect of the polyester chemical structure on the degradation of polyester/melamine coatings was studied. Four different polyesters were synthesized using different diacid monomers: (i) isophthalic acid (IPA) and mixtures of hexahydrophthalic anhydride (HHPA) with (ii) terephthalic acid (TA), (iii) phthalic anhydride (PAN) and (iv) 1,4 cyclohexanedicarboxylic acid (1,4-CHDA). Varnishes were prepared using melamine resin and submitted to accelerated degradation cycles. The process was monitored in terms of photooxidation index (POI), based on FTIR analysis, and in terms of changes in films’ hardness, level of gloss and surface morphology. The monomers which contributed the most to polymer degradation were the couple HHPA and 1,4-CHDA. The most important factor to degradation was the high number of hydrogen atoms attached to tertiary and secondary carbons in the polymer structure, due to their high sensitivity to abstraction, which favors the photooxidation reactions. The monomer IPA presented the lowest POI and the highest gloss retention.     

Strategies to Reduce the On-Target Platelet Toxicity of Bcl-xL Inhibitors: PROTACs,SNIPERs and Prodrug-Based Approaches

Apoptosis is a highly regulated cellular process. Aberration in apoptosis is a common characteristic of various disorders. Therefore, proteins involved in apoptosis are prime targets in multiple therapies. Bcl-x_L is an antiapoptotic protein. Compared to other antiapoptotic proteins, the expression of Bcl-x_L is common in solid tumors and, to an extent, in some leukemias and lymphomas. The overexpression of Bcl-x_L is also linked to survival and chemoresistance in cancer and senescent cells. Therefore, Bcl-x_L is a promising anticancer and senolytic target. Various nanomolar range Bcl-x_L inhibitors have been developed. ABT-263 was successfully identified as a Bcl-x_L/Bcl-2 dual inhibitor. But it failed in the clinical trial (phase-II) because of its on-target platelet toxicity, which also implies an essential role of Bcl-x_L protein in the survival of human platelets. Classical Bcl-x_L inhibitor designs utilize occupancy-driven pharmacology with typical shortcomings (such as dose-dependent off-target and on-target platelet toxicities). Hence, event-driven pharmacology-based approaches, such as proteolysis targeting chimeras (PROTACs) and SNIPERs (specific non-genetic IAP-based protein erasers) have been developed. The development of Bcl-x_L based PROTACs was expected, as 600 E3-ligases are available in humans, while some (such as cereblon (CRBN), von Hippel-Lindau (VHL)) are relatively less expressed in platelets. Therefore, E3 ligase ligand-based Bcl-x_L PROTACs (CRBN: XZ424, XZ739; VHL: DT2216, PZ703b, 753b) showed a significant improvement in platelet therapeutic index than their parent molecules ( ABT-263 : DT2216, PZ703b, 753b, XZ739, PZ15227; A1155463 : XZ424). Other than their distinctive pharmacology, PROTACs are molecularly large, which limits their cell permeability and plays a role in improving their cell selectivity. We also discuss prodrug-based approaches, such as antibody-drug conjugates ( ABBV-155 ), phosphate prodrugs ( APG-1252 ), dendrimer conjugate ( AZD0466 ), and glycosylated conjugates ( Nav-Gal ). Studies of in-vitro, in-vivo, structure-activity relationships, biophysical characterization, and status of preclinical/clinical inhibitors derived from these strategies are also discussed in the review.     

Getting a Grip on the Undrugged: Targeting β-Catenin with Fragment-Based Methods

Aberrant WNT pathway activation, leading to nuclear accumulation of β-catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of β-catenin and subsequent nuclear translocation. Restoring cellular degradation of β-catenin represents a potential therapeutic strategy. Here, we report the fragment-based discovery of a small molecule binder to β-catenin, including the structural elucidation of the binding mode by X-ray crystallography. The difficulty in drugging β-catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X-ray co-crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein–protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards β-catenin proteolysis targeting chimeras (PROTACs) as alternative modality.