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.

Discovery of novel BCR-ABL PROTACs based on the cereblon E3 ligase design, synthesis, and biological evaluation.

Protein degradation is a promising strategy for drug development. Proteolysis-targeting chimeras (PROTACs) hijacking the E3 ligase cereblon (CRBN) exhibit enormous potential and universal degradation performance due to the small molecular weight of CRBN ligands. In this study, the CRBN-recruiting PROTACs were explored on the degradation of oncogenic fusion protein BCR-ABL, which drives the pathogenesis of chronic myeloid leukemia (CML). A series of novel PROTACs were synthesized by conjugating BCR-ABL inhibitor dasatinib to the CRBN ligand including pomalidomide and lenalidomide, and the extensive structure-activity relationship (SAR) studies were performed focusing on optimization of linker parameters. Therein, we uncovered that pomalidomide-based degrader 17 (SIAIS056), possessing sulfur-substituted carbon chain linker, exhibits the most potent degradative activity in vitro and favorable pharmacokinetics in vivo. Besides, degrader 17 also degrades a variety of clinically relevant resistance-conferring mutations of BCR-ABL. Furthermore, degrader 17 induces significant tumor regression against K562 xenograft tumors. Our study indicates that 17 as an efficacious BCR-ABL degrader warrants intensive investigation for the future treatment of BCR-ABL+ leukemia.

Directed evolution of an amine transaminase for the synthesis of an Apremilast intermediate via kinetic resolution.

Apremilast is an important active pharmaceutical ingredient that relies on a resolution to produce the key chiral amine intermediate. To provide a new catalytic and enzymatic process for Apremilast, we performed the directed evolution of the amine transaminase fromVibriofluvialis. Six rounds of evolution resulted in the VF-8M-E variant with > 400-fold increase specific activity over the wildtype enzyme. A homology model of VF-8M-E was built and a molecular docking study was performed to explain the increase in activity. The purified VF-8M-E was successfully applied to produce the key chiral amine intermediate in enantiopure form and 49% conversion via a kinetic resolution, representing a new enzymatic access towards Apremilast.

The structure of isolated thalidomide as reference for its chirality-dependent biological activity: a laser-ablation rotational study.

Thalidomide is a drug that presents two enantiomers with markedly different pharmacological and toxicological activities. It is sadly famous due to its teratogenic effects mostly caused by the preferential docking of the (S)-enantiomer to the target protein cereblon (CRBN). To compare the structure of the bound CRBN thalidomide enantiomers with that of the isolated molecule, the rotational spectrum of laser-ablated thalidomide has been studied by chirp-pulsed Fourier transform microwave spectroscopy in supersonic jets complemented by theoretical computations. A new setup of the laser ablation nozzle used is presented. Two stable equatorial and axial conformers of thalidomide have been predicted corresponding to the two possible bent conformations exhibited by the glutarimide moiety. Only the most stable equatorial conformer has been detected. The comparison of its structure with those of the (S)- and (R)-enantiomers bound to CBRN shows that the bound (S) species is only slightly distorted. On the contrary, the bound (R)-enantiomer exhibits a highly distorted structure which affects the degree of puckering of the glutarimide ring and especially to the orientation of the phtalimide and glutarimide subunits. This is consistent with a less stable (R)-enantiomer and the known preference of (S)-thalidomide to bind CRBN, which starts the process leading to teratogenic effects.

Synthesis and pharmacological evaluation of pomalidomide derivatives useful for sickle cell disease treatment.

Fetal hemoglobin (HbF) induction constitutes a valuable and validated approach to treat the symptoms of sickle cell disease (SCD). Here, we synthesized pomalidomide-nitric oxide (NO) donor derivatives (3a-f) and evaluated their suitability as novel HbF inducers. All compounds demonstrated different capacities of releasing NO, ranging 0.3-30.3%. Compound 3d was the most effective HbF inducer for CD34+ cells, exhibiting an effect similar to that of hydroxyurea. We investigated the mode of action of compound 3d for HbF induction by studying the in vitro alterations in the levels of transcription factors (BCL11A, IKAROS, and LRF), inhibition of histone deacetylase enzymes (HDAC-1 and HDAC-2), and measurement of cGMP levels. Additionally, compound 3d exhibited a potent anti-inflammatory effect similar to that of pomalidomide by reducing the TNF-α levels in human mononuclear cells treated with lipopolysaccharides up to 58.6%. Chemical hydrolysis studies revealed that compound 3d was stable at pH 7.4 up to 24 h. These results suggest that compound 3d is a novel HbF inducer prototype with the potential to treat SCD symptoms.

Development of Apremilast Solid Dispersion Using TPGS and PVPVA with Enhanced Solubility and Bioavailability.

Apremilast (APST) is an effective inhibitor of phosphodieasterase 4 (PDE4) which is the first oral drug for the treatment of adult patients with active psoriatic arthritis. However, Apremilast's low solubility restricts its dissolution and bioavailability. In this study, APST solid dispersion with D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and Poly(1-vinylpyrrolidone-co-vinyl acetate) (PVPVA) was developed to improve the dissolution and bioavailability of APST by spray drying. A series of TPGS were synthesized to elucidate the effect of the ratio of monoester to diester on solubilizing capacity. X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), and Fourier transform infrared spectrophotometry (FT-IR) were used to characterize the solid dispersion, and the results showed that APST was amorphous in solid dispersion. In vitro dissolution study showed that the dissolution rate of solid dispersion in phosphate buffered saline (pH 6.8) was remarkably increased, reaching a release of 90% within 10 min. Moreover, in vivo pharmacokinetics study revealed that the bioavailability of solid dispersion in rats had significant improvement. In particular, its Cmax and AUClast were nearly 22- and 12.9-fold greater as compared to APST form B, respectively. In conclusion, APST solid dispersion with TPGS and PVPVA is an alternative drug delivery system to improve the solubility and oral bioavailability of APST.

Chemical Degradation of Androgen Receptor (AR) Using Bicalutamide Analog-Thalidomide PROTACs.

A series of PROTACs (PROteolysis-TArgeting Chimeras) consisting of bicalutamide analogs and thalidomides were designed, synthesized, and biologically evaluated as novel androgen receptor (AR) degraders. In particular, we found that PROTAC compound 13b could successfully demonstrate a targeted degradation of AR in AR-positive cancer cells and might be a useful chemical probe for the investigation of AR-dependent cancer cells, as well as a potential therapeutic candidate for prostate cancers.

Design and linkage optimization of ursane-thalidomide-based PROTACs and identification of their targeted-degradation properties to MDM2 protein.

Ursolic acid (UA) is an accessible triterpenoid, widely applied in the design and synthesis of antitumor compounds. However, the mechanism of its anti-tumor effect is still unclear. To verify the molecular mechanism of its biological activity, based on the bifunctional activity of ubiquitination and subsequent proteasomal degradation of the target protein of the proteolysis-targeting chimeras (PROTACs) strategy, here we report the design, synthesis and cellular activity of six UA PROTAC hydrochloride compounds 1A-1F, in which UA acts as the binding ligand of the PROTAC and is linked to thalidomide (E3 ligand) through a series of synthetic linkers. The results revealed that compound 1B, connected with a POE-3 (3-Polyoxyether) possessed remarkable in vitro antitumor activity (with the IC50 value of 0.23 ~ 0.39 µM against A549, Huh7, HepG2). WB results demonstrated that the administration of compound 1B induced significant degradation of MDM2 (only 25% to that of SM1), and promoted the expression of P21 and PUMA proteins, and thus inhibited the proliferation (77.67% of 1B vs 60.37% of CON in G1 phase) and promoted the apoptosis (26.74% of 1B vs 3.35% of CON) of A549 cells. This work demonstrated proof of designing the efficient target protein degradation by UA PROTACs with the POE linkers. In addition, we confirmed that UA possess the characteristic of targeted-binding the protein of murine double minute-2 protein (MDM2). This will lay a foundation for the comprehensive utilization of forest natural compound UA.

Discovery of thalidomide-based PROTAC small molecules as the highly efficient SHP2 degraders.

SHP2, a non-receptor tyrosine phosphatase, plays a pivotal role in numerous oncogenic cell-signaling cascades like RAS-ERK, PI3K-AKT and JAK-STAT. On the other hand, proteolysis targeting chimera (PROTAC) has emerged as a promising strategy for the degradation of disease-related protein of interest (POI). SHP2 degradation via the PROTAC strategy will provide an alternative startegy for SHP2-mediated cancer therapy. Herein we described the design, synthesis and evaluation of a series of thalidomide-based heterobifunctional molecules and identified 11(ZB-S-29) as the highly efficient SHP2 degrader with a DC50 of 6.02 nM. Further mechanism investigation illustrated that 11 came into function through targeted SHP2 protein degradation.

Cereblon modulator CC-885 induces CRBN-dependent ubiquitination and degradation of CDK4 in multiple myeloma.

Molecular glue degraders that hijack cellular E3 ubiquitin ligases to target disease-driven proteins for proteosome-dependent degradation are emerging as a promising treatment. Immunomodulatory drugs are classical molecular glue that bind to cereblon (CRBN) to repurpose the function of the CRL4(CRBN) E3 ubiquitin ligase and developed to treat various hematological malignancies. Recently, a novel cereblon modulator CC-885 was developed to elicit broad antitumor activity. Although the degradation of GSPT1 is essential for the broad in vitro antitumor activity of CC-885, it is unclear whether other neosubstrates also contribute to the pharmacological effects of CC-885, especially in multiple myeloma (MM). Here, we show that CC-885 treatment caused growth retardant of MM cells via impairment of cell cycle progression and cell death both in vitro and in vivo. Mechanically, CC-885 selectively induced the ubiquitination and degradation of CDK4 in MM cells in a CRBN-dependent manner. CC-885-mediated CDK4 destruction decreased the phosphorylation of the tumor suppressor retinoblastoma (RB) and prevented the expression of E2F downstream genes. Importantly, genetic ablation or pharmacological inhibition of CDK4 enhances CC-885-induced cytotoxicity in MM cells, suggesting CDK4 destruction contributed to the cytotoxicity of CC-885 in MM cells.