TRIP12 promotes small-molecule-induced degradation through K29/K48-branched ubiquitin chains.

Targeted protein degradation is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the degradation of neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation of endogenous substrates has been extensively studied, the mechanism underlying forced degradation of neo-substrates is less well understood. We found that the ubiquitin ligase TRIP12 promotes PROTAC-induced and CRL2VHL-mediated degradation of BRD4 but is dispensable for the degradation of the endogenous CRL2VHL substrate HIF-1α. TRIP12 associates with BRD4 via CRL2VHL and specifically assembles K29-linked ubiquitin chains, facilitating the formation of K29/K48-branched ubiquitin chains and accelerating the assembly of K48 linkage by CRL2VHL. Consequently, TRIP12 promotes the PROTAC-induced apoptotic response. TRIP12 also supports the efficiency of other degraders that target CRABP2 or TRIM24 or recruit CRBN. These observations define TRIP12 and K29/K48-branched ubiquitin chains as accelerators of PROTAC-directed targeted protein degradation, revealing a cooperative mechanism of branched ubiquitin chain assembly unique to the degradation of neo-substrates.

Development of STING degrader with double covalent ligands.

Stimulator of interferon genes (STING), a homodimeric membrane receptor localized in the endoplasmic reticulum, plays a pivotal role in signaling innate immune responses. Inhibitors and proteolysis-targeting chimeras (PROTACs) targeting STING are promising compounds for addressing autoinflammatory and autoimmune disorders. In this study, we used a minimal covalent handle recently developed as the ligand portion of an E3 ligase. The engineered STING degrader with a low molecular weight compound covalently binds to STING and E3 ligase. Degrader 2 showed sustained STING degradation activity at lower concentrations (3 µM, 48 h, about 75 % degradation) compared to a reported STING PROTAC, SP23. This discovery holds significance for its potential in treating autoinflammatory and autoimmune diseases, offering promising avenues for developing more efficacious STING-targeted therapies.

Photo-regulated PROTACs: A novel tool for temporal control of targeted protein degradation.

PROTACs (Proteolysis targeting chimeras) are chimeric molecules designed to induce targeted protein degradation via the ubiquitin-proteasome system. These molecules catalytically degrade target proteins and sustainably inhibit their function. Therefore, PROTAC's unique mechanism of action is not only beneficial in medicine but also serves as a valuable tool for molecular biological analysis in fields like chemical biology, biochemistry, and drug discovery. This study presents a novel turn-off (ON-OFF) type PROTAC development strategy utilizing a photocleavable linker. The inclusion of this linker enables temporal control of the degradation activity targeting BRD4 protein upon UV light exposure. PROTAC-2 demonstrated the most potent degradation activity against BRD4 among the other synthesized PROTACs with varying linker lengths. The UV light-induced cleavage of PROTAC-2 was confirmed, leading to a reduction in its BRD4 degradation activity. Notably, this study introduces a novel linker capable of nullifying degradation activity of PROTACs which is activated by light irradiation. These findings offer a promising strategy for the development of turn-off type PROTACs, providing enhanced temporal control over protein degradation. The approach holds significant potential for applications in molecular function studies and drug discovery.

Expansion of targeted degradation by Gilteritinib-Warheaded PROTACs to ALK fusion proteins.

Proteolysis targeting chimeras (PROTACs) induce the ubiquitination and subsequent proteasomal degradation of targeted proteins. Numerous PROTACs have emerged as promising drug candidates for various disease-related proteins. This study investigates PROTACs targeted to degrade anaplastic lymphoma kinase (ALK) fusion proteins, which are implicated in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. We recently reported the development of a gilteritinib-warheaded PROTAC to target and degrade the Fms-like tyrosine kinase 3 (FLT3) protein. Gilteritinib is a tyrosine kinase inhibitor that targets FLT3, and recent studies have revealed that it also functions as an ALK inhibitor. We conducted a structure-activity relationship (SAR) study and expanded the range of target proteins for gilteritinib-warheaded PROTACs to include echinoderm microtubule-associated protein-like 4 (EML4)-ALK and nucleophosmin (NPM)-ALK, in addition to FLT3. Our SAR study utilized three types of ligands for E3 ligase- inhibitor of apoptosis protein (IAP), cereblon (CRBN), and von Hippel-Lindau (VHL)- in the PROTAC designs and we observed varied efficacy in the degradation of target proteins. The CRBN-based PROTAC effectively reduced the protein expression of FLT3, EML4-ALK, and NPM-ALK. The IAP-based PROTAC reduced expression of both FLT3 and EML4-ALK proteins but not that of NPM-ALK, while the VHL-based PROTAC was ineffective against all target proteins. Several ALK-targeted PROTACs have already been developed using CRBN or VHL as E3 ligase, but this is the first report of an IAP-based ALK degrader. The length of the linker structure utilized in PROTAC also had a significant effect on their efficacy and activity. PROTACs formed with shorter linkers demonstrated an enhanced degradation activity to target proteins compared with those formed with longer linkers. These findings provide valuable insight for the development of effective PROTACs to target and degrade ALK fusion proteins.

Rapid Structure Determination of Ranitidine Hydrochloride API in Two Crystal Forms Using Microcrystal Electron Diffraction.

The solid-state properties of drug candidates play a crucial role in their selection. Quality control of active pharmaceutical ingredients (APIs) based on their structural information involves ensuring a consistent crystal form and controlling water and residual solvent contents. However, traditional crystallographic techniques have limitations and require high-quality single crystals for structural analysis. Microcrystal electron diffraction (microED) overcomes these challenges by analyzing difficult-to-crystallize or small-quantity samples, making it valuable for efficient drug development. In this study, microED analysis was able to rapidly determine the configuration of two crystal forms (Forms 1, 2) of the API ranitidine hydrochloride. The structures obtained with microED are consistent with previous structures determined by X-ray diffraction, indicating microED is a useful tool for rapidly analyzing molecular structures in drug development and materials science research.

Rational Design of Amphipathic Antimicrobial Peptides with Alternating L-/D-Amino Acids That Form Helical Structures.

Antimicrobial peptides (AMPs) are promising therapeutic agents against bacteria. We have previously reported an amphipathic AMP Stripe composed of cationic L-Lys and hydrophobic L-Leu/L-Ala residues, and Stripe exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria. Gramicidin A (GA), composed of repeating sequences of L- and D-amino acids, has a unique ß6.3-helix structure and exhibits broad antimicrobial activity. Inspired by the structural properties and antimicrobial activities of LD-alternating peptides such as GA, in this study, we designed Stripe derivatives with LD-alternating sequences. We found that simply alternating L- and D-amino acids in the Stripe sequence to give StripeLD caused a reduction in antimicrobial activity. In contrast, AltStripeLD, with cationic and hydrophobic amino acids rearranged to yield an amphipathic distribution when the peptide adopts a ß6.3-helix, displayed higher antimicrobial activity than AltStripe. These results suggest that alternating L-/D-cationic and L-/D-hydrophobic amino acids in accordance with the helical structure of an AMP may be a useful way to improve antimicrobial activity and develop new AMP drugs.

In Silico Prediction of N-Nitrosamine Formation Pathways of Pharmaceutical Products.

The recent discovery of N-nitrosodimethylamine (NDMA), a mutagenic N-nitrosamine, in pharmaceuticals has adversely impacted the global supply of relevant pharmaceutical products. Contamination by N-nitrosamines diverts resources and time from research and development or pharmaceutical production, representing a bottleneck in drug development. Therefore, predicting the risk of N-nitrosamine contamination is an important step in preventing pharmaceutical contamination by DNA-reactive impurities for the production of high-quality pharmaceuticals. In this study, we first predicted the degradation pathways and impurities of model pharmaceuticals, namely gliclazide and indapamide, in silico using an expert-knowledge software. Second, we verified the prediction results with a demonstration test, which confirmed that N-nitrosamines formed from the degradation of gliclazide and indapamide in the presence of hydrogen peroxide, especially under alkaline conditions. Furthermore, the pathways by which degradation products formed were determined using ranitidine, a compound previously demonstrated to generate NDMA. The prediction indicated that a ranitidine-related compound served as a potential source of nitroso groups for NDMA formation. In silico software is expected to be useful for developing methods to assess the risk of N-nitrosamine formation from pharmaceuticals.

Structural Optimization of Decoy Oligonucleotide-Based PROTAC That Degrades the Estrogen Receptor.

Proteolysis-targeting chimeras (PROTACs) have attracted attention as a chemical method of protein knockdown via the ubiquitin-proteasome system. Some oligonucleotide-based PROTACs have recently been developed for disease-related proteins that do not have optimal small-molecule ligands such as transcription factors. We have previously developed the PROTAC LCL-ER(dec), which uses a decoy oligonucleotide as a target ligand for estrogen receptor α (ERα) as a model transcription factor. However, LCL-ER(dec) has a low intracellular stability because it comprises natural double-stranded DNA sequences. In the present study, we developed PROTACs containing chemically modified decoys to address this issue. Specifically, we introduced phosphorothioate modifications and hairpin structures into LCL-ER(dec). Among the newly designed PROTACs, LCL-ER(dec)-H46, with a T4 loop structure at the end of the decoy, showed long-term ERα degradation activity while acquiring enzyme tolerance. These findings suggest that the introduction of hairpin structures is a useful modification of oligonucleotides in decoy oligonucleotide-based PROTACs.

Synthesis and properties of PNA containing a dicationic nucleobase based on N4-benzoylated cytosine.

We report the synthesis of a peptide nucleic acid (PNA) monomer containing N4-bis(aminomethyl)benzoylated cytosine (BzC2+ base). The BzC2+ monomer was incorporated into PNA oligomers using Fmoc-based solid-phase synthesis. The BzC2+ base in PNA had two positive charges and exhibited greater affinity for DNA G base than the natural C base. The BzC2+ base stabilized PNA-DNA heteroduplexes through electrostatic attractions, even in high salt conditions. The two positive charges on the BzC2+ residue did not compromise the sequence specificity of PNA oligomers. These insights will aid the future design of cationic nucleobases.

CRBN ligand expansion for hematopoietic prostaglandin D2 synthase (H-PGDS) targeting PROTAC design and their in vitro ADME profiles.

An increasing number of research reports are describing modifications of the E3 ligand, in particular, cereblon (CRBN) ligands, to improve the chemical and metabolic stabilities as well as the physical properties of PROTACs. In this study, phenyl-glutarimide (PG) and 6-fluoropomalidomide (6-F-POM), recently used as CRBN ligands for PROTAC design, were applied to hematopoietic prostaglandin D2 synthase (H-PGDS)-targeted PROTACs. Both PROTAC-5 containing PG and PROTAC-6 containing 6-F-POM were found to have potent activities to induce H-PGDS degradation. Furthermore, we obtained in vitro ADME data on the newly designed PROTACS as well as our previously reported PROTACs(H-PGDS) series. Although all PROTACs(H-PGDS) are relatively stable toward metabolism, they had poor PAMPA values. Nevertheless, PROTAC-5 showed Papp values similar to TAS-205, which is in Phase 3 clinical trials, and is expected to be the key to improving the pharmacokinetics of PROTACs.

In silico optimization of peptides that inhibit Wnt/ß-catenin signaling.

The Wnt/ß-catenin signaling pathway causes transcriptional activation through the interaction between ß-catenin and T cell-specific transcription factor (TCF) and regulates a wide variety of cellular responses, including proliferation, differentiation and cell motility. Excessive transcriptional activation of the Wnt/ß-catenin pathway is implicated in developing or exacerbating various cancers. We have recently reported that liver receptor homolog-1 (LRH-1)-derived peptides inhibit the ß-catenin/TCF interaction. In addition, we developed a cell-penetrating peptide (CPP)-conjugated LRH-1-derived peptide that inhibits the growth of colon cancer cells and specifically inhibits the Wnt/ß-catenin pathway. Nonetheless, the inhibitory activity of the CPP-conjugated LRH-1-derived peptide was unsatisfactory (ca. 20 µM), and improving the bioactivity of peptide inhibitors is required for their in vivo applications. In this study, we optimized the LRH-1-derived peptide using in silico design to enhance its activity further. The newly designed peptides showed binding affinity toward ß-catenin comparable to the parent peptide. In addition, the CPP-conjugated stapled peptide, Penetratin-st6, showed excellent inhibition (ca. 5 µM). Thus, the combination of in silico design by MOE and MD calculations has revealed that logical molecular design of PPI inhibitory peptides targeting ß-catenin is possible. This method can be also applied to the rational design of peptide-based inhibitors targeting other proteins.

Development of decoy oligonucleotide-warheaded chimeric molecules targeting STAT3.

Proteolysis-targeting chimera (PROTAC) technology is a disruptive innovation in the drug development community, and over 20 PROTAC molecules are currently under clinical evaluation. These PROTAC molecules contain small-molecule warheads that bind to target proteins. Recently, oligonucleotide-warheaded PROTACs have emerged as a promising new tool to degrade DNA-binding proteins such as transcription factors. In this study, we applied an oligonucleotide-warheaded PROTAC technology to induce the degradation of signal transducer and activator of transcription 3 (STAT3), which is a hard-to-target protein. A double-stranded decoy oligonucleotide specific to STAT3 was conjugated to E3 binders (pomalidomide, VH032, and LCL161) to generate PROTAC molecules that recruited different E3 ubiquitin ligases cereblon (CRBN), von Hippel-Lindau (VHL), and inhibitor of apoptosis protein (IAP), respectively. One of the resulting PROTAC molecules, POM-STAT3, which recruits CRBN, potently induces STAT3 degradation. STAT3 degradation by POM-STAT3 was abolished by scrambling the oligonucleotide sequences of POM-STAT3 and by adding a double-stranded decoy oligonucleotide against STAT3 in a competitive manner, suggesting the significance of oligonucleotide sequences in STAT3 degradation. Moreover, POM-STAT3-induced STAT3 degradation was suppressed by the CRBN binder thalidomide, proteasome inhibitor bortezomib, E1 inhibitor MLN7243, and siRNA-mediated depletion of CRBN, indicating that STAT3 degradation is mediated by the ubiquitin-proteasome system, which involves CRBN as the responsible E3 ubiquitin ligase. Consistent with STAT3 degradation, NCI-H2087 cell viability was severely reduced following POM-STAT3 treatment. Thus, POM-STAT3 is a STAT3 degrader that potentially has cytocidal activity against cancer cells that are highly dependent on STAT3 signaling, which implies that inducing protein degradation by decoy oligonucleotide-warheaded PROTAC molecules could be harnessed to be therapeutic against oncogenic transcription factors.

Development of versatile solid-phase methods for syntheses of PROTACs with diverse E3 ligands.

Developing highly active proteolysis-targeting chimeras (PROTACs) requires investigating a variety of ubiquitin ligase (E3 ligase) ligands and linker structures as well as their lengths. In this study, we developed a solid-phase synthesis method that affords PROTAC design diversity. We expanded the E3 ligand range to include Von Hippel-Lindau (VHL) and inhibitor of apoptosis protein (IAP) ligands because only the cereblon (CRBN) ligand thalidomide and its derivatives have been investigated for solid-phase synthesis of PROTACs. Moreover, we examined the suitability of a polyethylene glycol (PEG) rather than an alkyl linker used in our previous study for synthesizing PROTACs. Facile and rapid solid-phase synthesis methods using the above E3 ligands for developing PROTACs targeting bromodomain-containing protein 4 (BRD4) were accomplished. Western blotting analysis revealed that minor differences in the E3 ligand and linker type significantly affected the activity of the synthesized PROTACs. Our solid-phase PROTAC synthesis methods enable rapid synthesis of multiple PROTACs with various combinations of ligands for the protein-of-interest and E3 ligands and linkers that connect these ligands.

Advanced Solid-State NMR Analysis of Two Crystal Forms of Ranitidine Hydrochloride: Detection of 1H-14N Intra-/Intermolecular Correlations.

Understanding the characteristics of crystal polymorphism of active pharmaceutical ingredients and analyzing them with high sensitivity is important for quality of drug products, appropriate characterization strategies, and appropriate screening and selection processes. However, there are few methods to measure intra- and intermolecular correlations in crystals other than X-ray crystallography, for which it is sometimes difficult to obtain suitable single crystals. Recently, solid-state NMR has been recognized as a straightforward method for measuring molecular correlations. In this study, we selected ranitidine hydrochloride, which is known to exist in two forms, 1 and 2, as the model drug and investigated each form using solid-state NMR. In conducting the analysis, rotating the sample tube, which had a 1-mm inner diameter, increased the solid-state NMR resolution at 70 kHz. The 1H-14N dipolar-based heteronuclear multiple quantum coherence (D-HMQC) analysis revealed the intermolecular correlation of Form 1 between the N atom of the nitro group and a proton of the furan moiety, which were closer than those of the intramolecular correlation reported using single X-ray crystal analysis. Thus, 1H-14N D-HMQC analysis could be useful for characterizing intermolecular interaction in ranitidine hydrochloride crystals. In addition, we reassigned the 13C solid-state NMR signals of ranitidine hydrochloride according to the liquid-state and multiple solid-state NMR experiments.

Development of Hydrophobic Cell-Penetrating Stapled Peptides as Drug Carriers.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of a variety of cargo molecules, including small molecules, peptides, nucleic acids, and proteins. Many cationic and amphiphilic CPPs have been developed; however, there have been few reports regarding hydrophobic CPPs. Herein, we have developed stapled hydrophobic CPPs based on the hydrophobic CPP, TP10, by introducing an aliphatic carbon side chain on the hydrophobic face of TP10. This side chain maintained the hydrophobicity of TP10 and enhanced the helicity and cell penetrating efficiency. We evaluated the preferred secondary structures, and the ability to deliver 5(6)-carboxyfluorescein (CF) as a model small molecule and plasmid DNA (pDNA) as a model nucleotide. The stapled peptide F-3 with CF, in which the stapling structure was introduced at Gly residues, formed a stable α-helical structure and the highest cell-membrane permeability via an endocytosis process. Meanwhile, peptide F-4 demonstrated remarkable stability when forming a complex with pDNA, making it the optimal choice for the efficient intracellular delivery of pDNA. The results showed that stapled hydrophobic CPPs were able to deliver small molecules and pDNA into cells, and that different stapling positions in hydrophobic CPPs can control the efficiency of the cargo delivery.

Helix-Stabilized Cell-Penetrating Peptides for Delivery of Antisense Morpholino Oligomers: Relationships among Helicity, Cellular Uptake, and Antisense Activity.

The secondary structures of cell-penetrating peptides (CPPs) influence their properties including their cell-membrane permeability, tolerability to proteases, and intracellular distribution. Herein, we developed helix-stabilized arginine-rich peptides containing α,α-disubstituted α-amino acids and their conjugates with antisense phosphorodiamidate morpholino oligomers (PMOs), to investigate the relationships among the helicity of the peptides, cellular uptake, and antisense activity of the peptide-conjugated PMOs. We demonstrated that helical CPPs can efficiently deliver the conjugated PMO into cells compared with nonhelical CPPs and that their antisense activities are synergistically enhanced in the presence of an endosomolytic reagent or an endosomal escape domain peptide.

Copper-Catalyzed Asymmetric Oxidative Desymmetrization of 2-Substituted 1,2,3-Triols.

Asymmetric oxidative desymmetrization of 2-substituted glycerols has been achieved by using a new chiral bisoxazoline ligand/copper catalyst system with 1,3-dibromo-5,5-dimethylhydantoin and MeOH. The present transformation smoothly proceeds with readily accessible 2-(hetero)aryl- and alkyl-substituted glycerols and provides straightforward access toward various glycerate derivatives in good to high yields with high enantioselectivities. The synthetic utility of the present protocol was demonstrated by the transformation of the optically active glycerol into a glyceraldehyde derivative.

Fcγ Receptor-Dependent Internalization and Off-Target Cytotoxicity of Antibody-Drug Conjugate Aggregates.

PURPOSE: Antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) conjugated with highly toxic payloads, achieve high tumor killing efficacy due to the specific delivery of payloads in accordance with mAbs' function. On the other hand, the conjugation of payloads often increases the hydrophobicity of mAbs, resulting in reduced stability and increased aggregation. It is considered that mAb aggregates have potential risk for activating Fcγ receptors (FcγRs) on immune cells, and are internalized into cells via FcγRs. Based on the mechanism of action of ADCs, the internalization of ADCs into target-negative cells may cause the off-target toxicity. However, the impacts of aggregation on the safety of ADCs including off-target cytotoxicity have been unclear. In this study, we investigated the cytotoxicity of ADC aggregates in target-negative cells. METHODS: The ADC aggregates were generated by stirring stress or thermal stress. The off-target cytotoxicity of ADC aggregates was evaluated in several target-negative cell lines, and FcγR-activation properties of ADC aggregates were characterized using a reporter cell assay. RESULTS: Aggregation of ADCs enhanced the off-target cytotoxicity in several target-negative cell lines compared with non-stressed ADCs. Notably, ADC aggregates with FcγR-activation properties showed dramatically enhanced cytotoxicity in FcγR-expressing cells. The FcγR-mediated off-target cytotoxicity of ADC aggregates was reduced by using a FcγR-blocking antibody or Fc-engineering for silencing Fc-mediated effector functions. CONCLUSIONS: These results indicated that FcγRs play an important role for internalization of ADC aggregates into non-target cells, and the aggregation of ADCs increases the potential risk for off-target toxicity.

Development of delivery carriers for plasmid DNA by conjugation of a helical template to oligoarginine.

Arginine (Arg)-rich peptides can penetrate the cell membrane and deliver nucleic acid-based therapeutics into cells. In this study, a helical template designed with a repeating sequence composed of two l-leucines (l-Leu) and a 2-aminoisobutyric acid (Aib) (l-Leu-l-Leu-Aib) was conjugated to nona-arginine on either the C- or N- terminus, designated as Block 1 and Block 2. Each terminal modification induced helical structure formation and improved the physicochemical properties of peptide/plasmid DNA (pDNA) complexes, resulting in efficient intracellular pDNA delivery. The introduction of a helical template may be effective for the endosomal escape of pDNA and pDNA release from complexes in cells. These results emphasized the potency of a helical template for the development of novel cell-penetrating peptides for pDNA delivery.

Development of a penetratin-conjugated stapled peptide that inhibits Wnt/ß-catenin signaling.

Wnt/ß-catenin pathway triggers the formation of a complex between ß-catenin and T cell-specific transcription factor (TCF), which induces transcriptional activation. Excessive transcriptional activation of this pathway is associated with the development, cause, and deterioration of various cancers. Therefore, the Wnt/ß-catenin pathway is an attractive drug target for cancer therapeutics and small molecule- and peptide-based protein-protein interaction (PPI) inhibitors have been developed. However, peptide-based PPI inhibitors generally have low cell-membrane permeability because of their large molecular size. To improve cell-membrane permeability, conjugating cell-penetrating peptides (CPPs) to PPI-inhibiting peptides is a useful method for developing intracellularly targeted PPI inhibitors. In this study, we focused on the interaction between ß-catenin and liver receptor homologue-1 (LRH-1) and designed and synthesized a series of LRH-1-derived peptides to develop inhibitors against Wnt/ß-catenin signaling. The results showed that a penetratin-conjugated LRH-1-derived peptide (Penetratin-st7) predominantly inhibited DLD-1 cell growth at 20 µM treatment via inhibition of the Wnt signaling pathway. This result suggests that Penetratin-st7 is one of promising PPI inhibitors between TCF and ß-catenin.