Exploring Degradation of Intrinsically Disordered Protein Yes-Associated Protein Induced by Proteolysis TArgeting Chimeras.

Yes-associated protein (YAP) is a key oncogene in the Hippo tumor suppression pathway, historically challenging to target due to its intrinsically disordered nature. Leveraging recent advances in high-throughput screening that identified several YAP binders, we employed proteolysis-targeting chimera technology to develop a series of YAP degraders. Utilizing NSC682769, a known YAP binder, linked with VHL ligand 2 or pomalidomide via diverse linkers, we synthesized degraders including YZ-6. This degrader not only recruits the E3 ligase VHL for the rapid and sustained degradation of YAP but also effectively inhibits its nuclear localization, curtailing YAP/TEAD-mediated transcription in cancer cell lines such as NCI-H226 and Huh7. This dual action significantly diminishes YAP's oncogenic activity, contributing to the potent antiproliferative effects observed both in vitro and in a Huh7 xenograft mouse model. These results underscore the potential of PROTAC-mediated YAP degradation as a strategy for treating YAP-driven cancers.

Acetyl-CoA Carboxylase Proteolysis-Targeting Chimeras: Conceptual Design and Application as Insecticides.

Novel approaches for pest control are essential to ensure a sufficient food supply for the growing global population. The development of new insecticides must meet rigorous regulatory requirements for safety and address the resistance issues of existing insecticides. Proteolysis-targeting chimeras (PROTACs), originally developed for human diseases, show promise in agriculture. They offer innovative insecticides tailored to overcome resistance, opening avenues for agricultural applications. In this study, we developed small-molecule degraders by incorporating pomalidomide as an E3 ligand. These degraders were linked to a ligand (spirotetratmat enol) targeting the ACC protein through a flexible chain, aiming to achieve the efficient control of insects. Compounds 9a-9d were designed, synthesized, and evaluated for biological activities and mechanisms. Among them, 9b exhibited superior potency against Aphis craccivora (LC50 = 107.8 µg mL-1) compared to others and effectively degraded ACC proteins through the ubiquitin-proteasome system. These findings highlight the potential of utilizing PROTAC-based approaches in the development of insecticides for efficient pest control.

Revisiting the antiangiogenic mechanisms of fluorinated thalidomide derivatives.

Introduction of fluorine into bioactive molecules has attracted much attention in drug development. For example, tetrafluorination of the phthalimide moiety of immunomodulatory drugs (IMiDs) has a strong beneficial effect on the ability to inhibit angiogenesis. The neomorphic activity of E3 ligase complexes is induced by the binding of IMiDs to cereblon. We investigated that a set of eight thalidomide analogs, comprising non- and tetrafluorinated counterparts, did not induce the degradation of neomorphic substrates (IKZF3, GSPT1, CK1α, SALL4). Hence, the antiangiogenic activity of fluorinated IMiDs was not triggered by neosubstrate degradation features. A fluorine scanning of non-traditional IMiDs of the benzamido glutarimide chemotype was performed. By measuring the endothelial cell tube formation, no angiogenesis inhibitors were identified, confirming the narrow structure-activity window of IMiD-induced antiangiogenesis.

Preclinical Evaluation of a Novel Series of Polyfluorinated Thalidomide Analogs in Drug-Resistant Multiple Myeloma.

Immunomodulatory imide drugs (IMiDs) play a crucial role in the treatment landscape across various stages of multiple myeloma. Despite their evident efficacy, some patients may exhibit primary resistance to IMiD therapy, and acquired resistance commonly arises over time leading to inevitable relapse. It is critical to develop novel therapeutic options to add to the treatment arsenal to overcome IMiD resistance. We designed, synthesized, and screened a new class of polyfluorinated thalidomide analogs and investigated their anti-cancer, anti-angiogenic, and anti-inflammatory activity using in vitro and ex vivo biological assays. We identified four lead compounds that exhibit potent anti-myeloma, anti-angiogenic, anti-inflammatory properties using three-dimensional tumor spheroid models, in vitro tube formation, and ex vivo human saphenous vein angiogenesis assays, as well as the THP-1 inflammatory assay. Western blot analyses investigating the expression of proteins downstream of cereblon (CRBN) reveal that Gu1215, our primary lead candidate, exerts its activity through a CRBN-independent mechanism. Our findings demonstrate that the lead compound Gu1215 is a promising candidate for further preclinical development to overcome intrinsic and acquired IMiD resistance in multiple myeloma.

Fabrication and characterization of apremilast-loaded zinc oxide-mesoporous silica nanoparticles for psoriasis treatment.

Aim: The study was aimed to formulate and evaluate apremilast-loaded zinc oxide-mesoporous silica nanoparticles for treatment of psoriasis. Materials & methods: Mesoporous silica nanoparticles were prepared by using sol-gel method and evaluated for particle size, in vitro drug release, in vitro cytotoxicity study and in vivo pharmacodynamic study. Results: The synthesized mesoporous silica nanoparticles showed particle size of 319.9 ± 3.9 nm, with 24 ± 0.217% of loading capacity. In vitro cytotoxicity study on A-431 cell line showed increased anti-psoriatic activity of apremilast-loaded zinc oxide-mesoporous silica nanoparticles. In vivo pharmacodynamic study and histological studies showed improved efficacy of drug in imiquimod-induced psoriasis mice model. Conclusion: The apremilast-loaded zinc oxide-mesoporous silica nanoparticles showed improved therapeutic efficacy, suggesting that they are promising approach for topical treatment of psoriasis.

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Strengthening Molecular Glues: Design Strategies for Improving Thalidomide Analogs as Cereblon Effectors and Anticancer Agents.

In the two decades since a novel thalidomide analog was last approved, many promising drug candidates have emerged with remarkable potency as targeted protein degraders. Likewise, the advent of PROTACs for suppressing 'undruggable' protein targets reinforces the need for new analogs with improved cereblon affinity, target selectivity and drug-like properties. However, thalidomide and its approved derivatives remain plagued by several shortcomings, such as structural instability and poor solubility. Herein, we present a review of strategies for mitigating these shortcomings and highlight contemporary drug discovery approaches that have generated novel thalidomide analogs with enhanced efficacy as cereblon effectors and/or anticancer agents.

Old drug, new use: The thalidomide-based fluorescent probe for cysteine detection and imaging in living cells.

Thalidomide, as a high-profile cereblon (CRBN) ligand, has attracted much attention because of its ability to target protein degradation. In this study, we are committed to developing a new fluorescent probe THD-1 based on thalidomide, aiming at improving the performance of cysteine fluorescent probe in optical properties and biocompatibility. The experimental results showed that THD-1, as a cysteine fluorescent probe, owned the characteristics of obvious colorimetric change, fast response time, good selectivity and high sensitivity. The mechanism of THD-1 sensing cysteine was further verified to ensure its reliability and effectiveness. It was also worth mentioning that THD-1 was successfully applied to the biological imaging of cysteine in living A549 cells, which highlighted its value in practical application. Overall, thalidomide, as a clinically approved drug, not only enriches the fluorescent skeleton library, but also paves a new way for the further development of fluorescent probes.

A Validated Chiral Chromatography Method for Enantiomeric Separation of Pomalidomide in Human Plasma.

In the present work, new chiral stationary phase high-performance liquid chromatography (CSP-HPLC) method was established and validated for the quantification of pomalidomide (PMD) enantiomers in human plasma. The chromatographic enantiomeric separation was achieved on a Daicel-CSP, Chiralpack IA 4.6 × 250 mm, 5 µm; because of its advantages of high degree of retention, high resolution capacity, better reproducibility, ability to produce lower back pressure and low degree of tailing. The mobile phase was maintained as methanol: glacial acetic acid (499.50 ml:50 µL). Ultraviolet wavelength for detection was 220 nm. PMD enantiomer-I and enantiomer-II were separated at 8.83 and 15.34 min, respectively. Limit of detection and limit of quantification for each enantiomer and the calibration curve of standard PMD was linear in range between 10-5,000 ng mL-1. The method was validated according to The International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH(Q2R1)) specific guidelines. We found no interference peak with PMD chromatogram obtained. This is a simple, reliable and specific method for detection and quantification of enantiomer of PMD in human plasma sample.

Native mass spectrometry of complexes formed by molecular glues reveals stoichiometric rearrangement of E3 ligases.

In this application of native mass spectrometry (nMS) to investigate complexes formed by molecular glues (MGs), we have demonstrated its efficiency in delineating stoichiometric rearrangements of E3 ligases that occur during targeted protein degradation (TPD). MGs stabilise interactions between an E3 ligase and a protein of interest (POI) targeted for degradation, and these ternary interactions are challenging to characterise. We have shown that nMS can unambiguously identify complexes formed between the CRBN : DDB1 E3 ligase and the POI GSPT1 upon the addition of lenalidomide, pomalidomide or thalidomide. Ternary complex formation was also identified involving the DCAF15 : DDA1 : DDB1 E3 ligase in the presence of MG (E7820 or indisulam) and POI RBM39. Moreover, we uncovered that the DCAF15 : DDA1 : DDB1 E3 ligase self-associates into dimers and trimers when analysed alone at low salt concentrations (100 mM ammonium acetate) which dissociate into single copies of the complex at higher salt concentrations (500 mM ammonium acetate), or upon the addition of MG and POI, forming a 1 : 1 : 1 ternary complex. This work demonstrates the strength of nMS in TPD research, reveals novel binding mechanisms of the DCAF15 E3 ligase, and its self-association into dimers and trimers at reduced salt concentration during structural analysis.

Formulation characterization of lecithin organogel as topical drug delivery system for psoriasis: In-vitro permeation and preclinical evaluation.

Psoriasis is a chronic inflammatory and proliferative skin disease that causes pathological skin changes and has a substantial impact on the quality of patient life. Apremilast was approved by the US Food and Drug Administration as an oral medication for psoriasis and is beneficial in mild to moderate conditions for chronic usage. However, 5%-7% of withdrawals were reported due to severe side effects. To address the issue, a localized drug delivery strategy via the topical route may be a viable approach. However, poor physicochemical properties make it vulnerable to passing through the skin, requiring a specialized drug delivery system to demonstrate its full potential via a topical route like lecithin organogel. The formulation was optimized by screening the suitable lecithin type and non-polar solvents based on the gel formation ability of lecithin and the solubility of apremilast in the solvent. The pseudo-ternary diagram was used to optimize the water content required to form the gel. The optimized gel was found to be shear thinning characterized for rheological parameters, in-vitro diffusion studies, and in-vitro skin distribution studies. Preclinical studies in Imiquimod-induced mice showed a better reduction in severity index, cytokine levels, and epidermal hyperplasia from the lecithin organogel group compared to the apremilast oral administration and marketed standard topical gel group. Based on these results, lecithin organogel can be considered a promising approach to deliver molecules like apremilast by topical route in psoriatic-like conditions.

Continuous evolution of compact protein degradation tags regulated by selective molecular glues.

Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo-electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40's activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons.

Protein degraders - from thalidomide to new PROTACs.

Recently, the development of protein degraders (protein-degrading compounds) has prominently progressed. There are two remarkable classes of protein degraders: proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs). Almost 70 years have passed since thalidomide was initially developed as a sedative-hypnotic drug, which is currently recognized as one of the most well-known MGDs. During the last two decades, a myriad of PROTACs and MGDs have been developed, and the molecular mechanism of action (MOA) of thalidomide was basically elucidated, including identifying its molecular target cereblon (CRBN). CRBN forms a Cullin Ring Ligase 4 with Cul4 and DDB1, whose substrate specificity is controlled by its binding ligands. Thalidomide, lenalidomide and pomalidomide, three CRBN-binding MGDs, were clinically approved to treat several intractable diseases (including multiple myeloma). Several other MGDs and CRBN-based PROTACs (ARV-110 and AVR-471) are undergoing clinical trials. In addition, several new related technologies regarding PROTACs and MGDs have also been developed, and achievements of protein degraders impact not only therapeutic fields but also basic biological science. In this article, I introduce the history of protein degraders, from the development of thalidomide to the latest PROTACs and related technologies.

Structural and biophysical comparisons of the pomalidomide- and CC-220-induced interactions of SALL4 with cereblon.

The design of cereblon-binding molecular glues (MGs) that selectively recruit a desired protein while excluding teratogenic SALL4 is an area of significant interest when designing therapeutic agents. Previous studies show that SALL4 is degraded in the presence of IKZF1 degraders pomalidomide, and to a lesser extent by CC-220. To expand our understanding of the molecular basis for the interaction of SALL4 with cereblon, we performed biophysical and structural studies demonstrating that SALL4 zinc finger domains one and two (ZF1-2) interact with cereblon (CRBN) in a unique manner. ZF1 interacts with the N-terminal domain of cereblon and ZF2 binds as expected in the C-terminal IMiD-binding domain. Both ZF1 and ZF2 contribute to the potency of the interaction of ZF1-2 with CRBN:MG complexes and the affinities of SALL4 ZF1-2 for the cereblon:CC-220 complex are less potent than for the corresponding pomalidomide complex. Structural analysis provides a rationale for understanding the reduced affinity of SALL4 for cereblon in the presence of CC-220, which engages both ZF1 and ZF2. These studies further our understanding of the molecular glue-mediated interactions of zinc finger-based proteins with cereblon and may provide structural tools for the prospective design of compounds with reduced binding and degradation of SALL4.

Simultaneous quantification of thalidomide, lenalidomide and pomadomide in plasma by LC-MS/MS.

OBJECTIVE: To develop a new method for quantitatively analyzing three immunomodulators (thalidomide, lenalidomide and pomadomide) by liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: Using thalidomide-d4 as internal standard, the three analytes were separated on Agilent Zorbax SB-C18(2.1 mm × 100 mm, 3.5 µm, Agilent, USA) column and monitored in multiple reactions monitoring mode in Agilent G6460A triple quadrupole mass spectrometer operating in positive ionization mode. The sample was pretreated by protein precipitation using methanol at 3-fold volume to sample. The mobile phase was comprised of 0.1% formic acid in water (phase A) and acetonitrile (phase B) and was delivered in gradient elution program. The flow rate was 0.3 mL/min, and the injection volume was 5 µL. RESULTS: The accuracy and stability of the method are within ±15.0%, and the precision is not >15.0%. The recoveries were 85.04% âˆ¼ 119.07%, and the matrix effect was 73.68% âˆ¼ 116.75%. Specificity, linearity, LLOQ, carry-over and dilution were all in line with the requirements of pharmacopeia and guidelines. The peak concentrations of thalidomide, lenalidomide shows huge inter-individual differences. CONCLUSIONS: This newly developed method was sensitive, simple, and robust and can be used in therapeutic drug monitoring of three immunomodulators in multiple myeloma patients.

Molecular glue CELMoD compounds are regulators of cereblon conformation.

Cereblon (CRBN) is a ubiquitin ligase (E3) substrate receptor protein co-opted by CRBN E3 ligase modulatory drug (CELMoD) agents that target therapeutically relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBN's thalidomide-binding domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We performed cryo-electron microscopy analyses of the DNA damage-binding protein 1 (DDB1)-CRBN apo complex and compared these structures with DDB1-CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an open conformation to the canonical closed conformation. The neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy and informing structure-guided design strategies to improve therapeutic efficacy.

Computational investigations of indanedione and indanone derivatives in drug discovery: Indanone derivatives inhibits cereblon, an E3 ubiquitin ligase component.

BACKGROUND: Cereblon, an extensively studied multifunctional protein, is a Cullin 4-RING E3 ubiquitin ligase complex component. Cereblon is a well-known target of thalidomide and its derivatives. Cereblon is involved in multiple myeloma cell apoptosis. When ligands such as thalidomide and lenalidomide bind to cereblon, it recognizes various neosubstrates based on the ligand shape and properties. We have identified novel CRBN inhibitors, namely DHFO and its analogs, with structural features that are slightly different from thalidomide but stronger cereblon-binding affinity. We selected indanedione and indanone derivatives from the literature to understand and compare their cereblon-mediated substrate recognition potential. METHODS: Computational investigations of possible CRBN inhibitors were investigated by molecular docking with Autodock Vina and DockThor programs. The properties of the compounds' ADME/T and drug-likeness were investigated. A molecular dynamics study was carried out for four selected molecules, and the molecular interactions were analyzed using PCA-based FEL methods. The binding affinity was calculated using the MM/PBSA method. RESULTS: We conducted computational investigations on 68 indanedione and indanone derivatives binding with cereblon. Ten molecules showed better CRBN binding affinity than thalidomide. We studied the drug-likeness properties of the selected ten molecules, and four of the most promising molecules (DHFO, THOH, DIMS, and DTIN) were chosen for molecular dynamics studies. The MM/PBSA calculations showed that the DHFO, already shown to be a 5-LOX/COX2 inhibitor, has the highest binding affinity of - 163.16 kJ/mol with cereblon. CONCLUSION: The selected CRBN inhibitor DHFO has demonstrated the highest binding affinity with cereblon protein compared to other molecules. Thalidomide and its derivatives have a new substitute in the form of DHFO, which produces an interaction hotspot on the surface of the cereblon. Ease of chemical synthesis, low toxicity, versatile therapeutic options, and pleiotropism of DHFO analogs provide an opportunity for exploring clinical alternatives with versatile therapeutic potential for a new category of indanedione molecules as novel modulators of E3 ubiquitin ligases.

Refinement of Computational Access to Molecular Physicochemical Properties: From Ro5 to bRo5.

There is a need of computational tools to rank bRo5 drug candidates in the very early phases of drug discovery when chemical matter is unavailable. In this study, we selected three compounds: (a) a Ro5 drug (Pomalidomide), (b) a bRo5 orally available drug (Saquinavir), and (c) a polar PROTAC (CMP 98) to focus on computational access to physicochemical properties. To provide a benchmark, the three compounds were first experimentally characterized for their lipophilicity, polarity, IMHBs, and chameleonicity. To reproduce the experimental information content, we generated conformer ensembles with conformational sampling and molecular dynamics in both water and nonpolar solvents. Then we calculated Rgyr, 3D PSA, and IMHB number. An innovative pool of strategies for data analysis was then provided. Overall, we report a contribution to close the gap between experimental and computational methods for characterizing bRo5 physicochemical properties.

Discovery of CRBN as a target of thalidomide: a breakthrough for progress in the development of protein degraders.

Progress in strategies aimed at breaking down therapeutic target proteins has led to a paradigm shift in drug discovery. Thalidomide and its derivatives are the only protein degraders currently used in clinical practice. Our understanding of the molecular mechanism of action of thalidomide and its derivatives has advanced dramatically since the identification of cereblon (CRBN) as their direct target. The binding of thalidomide derivatives to CRBN, a substrate recognition receptor for Cullin 4 RING E3 ubiquitin ligase (CRL4), induces the recruitment of non-native substrates to CRL4CRBN and their subsequent degradation. This discovery was a breakthrough in the current rapid development of protein-degrading agents because clarification of the mechanism of action of thalidomide derivatives has demonstrated the clinical value of these compounds. This review provides an overview of the mechanism of action of thalidomide and its derivatives and describes perspectives for protein degraders.

A novel Cereblon E3 ligase modulator with antitumor activity in gastrointestinal cancer.

Targeted protein degradation offers new opportunities to inactivate cancer drivers and has successfully entered the clinic. Ways to induce selective protein degradation include proteolysis targeting chimera (PROTAC) technology and immunomodulatory (IMiDs) / next-generation Cereblon (CRBN) E3 ligase modulating drugs (CELMoDs). Here, we aimed to develop a MYC PROTAC based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and Thalidomide, called MDEG-541. We show that a subgroup of gastrointestinal cancer cell lines and primary patient-derived organoids are MDEG-541 sensitive. Although MYC expression was regulated in a CRBN-, proteasome- and ubiquitin-dependent manner, we provide evidence that MDEG-541 induced the degradation of CRBN neosubstrates, including G1 to S phase transition 1/2 (GSPT1/2) and the Polo-like kinase 1 (PLK1). In sum, we have established a CRBN-dependent degrader of relevant cancer targets with activity in gastrointestinal cancers.

Discovery of KRas G12C-IN-3 and Pomalidomide-based PROTACs as degraders of endogenous KRAS G12C with potent anticancer activity.

A series of KRAS G12C-targeting PROTACs (PROteolysis TArgeting Chimeras) were designed and synthesized based on KRas G12C-IN-3 (a KRAS G12C inhibitor) and pomalidomide as degraders of KRAS G12C with a molecular weight of < 900. Among them, compound KP-14 (m.w. = 852.16; tPSA = 174.53) showed the highest KRAS G12C-degrading capability in NCI-H358 cancer cells (DC50≈1.25 µM). KP-14 bound to KRAS G12C through the acrylamide warhead and recruited the E3 ligase CRBN, causing rapid and sustained KRAS G12C degradation which led to suppression of MAPK signaling pathway in NCI-H358 cells. In addition, KP-14 selectively induced the degradation of KRAS G12C but not other KRAS isoforms such as G13D via PROTAC mechanism. Furthermore, KP-14 exhibited potent antiproliferative activity against NCI-H358 cancer cells and was able to suppress the formation of NCI-H358 tumor colonies. Collectively, this work suggests that KP-14 may serve as a tool compound for exploring the degradation of KRAS G12C by PROTAC strategy and deserve further investigation as a potential anticancer agent.