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.

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.

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.

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.

(3+2) Cycloaddition of Heteroaromatic N-Ylides with Sulfenes.

A (3+2) cycloaddition of heteroaromatic N-ylides with sulfenes, which are generated in situ from sulfonyl chlorides, has been developed. A variety of ylides were transformed into the corresponding sulfone-embedded N-fused heterocycles in high yields. Hexafluoroisopropyl mesylate was demonstrated to be a suitable reactant for quinolinium ylides. Furthermore, this cycloaddition could be performed with an ylide prepared by a Cu-catalyzed ylide transfer reaction in a one-pot manner, extending the substrate scope to an unisolable ylide.

Sculpting Secondary Structure of a Cyclic Peptide: Conformational Analysis of a Cyclic Hexapeptide Containing a Combination of l-Leu, d-Leu, and Aib Residues.

We have previously reported that cyclo(l-Leu-d-Leu-Aib-l-Leu-d-Leu-Aib) (2), a cyclic hexapeptide consisting of heterochiral l-Leu and d-Leu (l-Leu-d-Leu) residues with achiral 2-aminoisobutyric acid (Aib) residues, forms a figure-8 conformation. In this study, we newly designed cyclo(l-Leu-d-Leu-Aib-d-Leu-l-Leu-Aib)+ (4), an epimer of 2, and examined the conformational differences between 2 and 4 by X-ray crystallographic analysis. Peptide 4 formed a planar cyclic conformation with an antiparallel ß-sheet hydrogen-bonding pattern. This investigation demonstrates the potential to manipulate the molecular conformation of cyclic peptides by simply arranging the l- and d-amino acids and emphasizes that diverse conformations can be obtained by using cyclic peptides. Harnessing cyclic peptides as platforms for distinct molecular structures is a promising approach to expanding the chemical space for various applications.

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.

Magainin 2-derived stapled peptides derived with the ability to deliver pDNA, mRNA, and siRNA into cells.

We have developed cell-penetrating stapled peptides based on the amphipathic antimicrobial peptide magainin 2 for intracellular delivery of nucleic acids such as pDNA, mRNA, and siRNA. Various types of stapled peptides with a cross-linked structure were synthesised in the hydrophobic region of the amphipathic structure, and their efficacy in intracellular delivery of pDNA was evaluated. The results showed that the stapled peptide st7-5 could deliver pDNA into cells. To improve the deliverability of st7-5, we further designed st7-5_R, in which the Lys residues were replaced by Arg residues. The peptide st7-5_R formed compact and stable complexes with pDNA and was able to efficiently transfer pDNA into the cell. In addition to pDNA, st7-5_R was also able to deliver mRNA and siRNA into the cell. Thus, st7-5_R is a novel peptide that can achieve efficient intracellular delivery of three different nucleic acids.

Structural analysis of an lysergic acid diethylamide (LSD) analogue N-methyl-N-isopropyllysergamide (MiPLA): Insights from Rotamers in NMR spectra.

Lysergic acid diethylamide (LSD) is a hallucinogenic compound that binds to and activates the serotonin 2A receptor and is classified as a controlled narcotic in Japan. Recently, MiPLA, an N-methyl-N-isopropyl derivative of LSD, has been detected in paper-sheet products in several countries. This study focuses on the synthesis of MiPLA and includes a comprehensive analysis involving structural and liquid chromatography-mass spectrometry (LC-MS). Particularly, MiPLA was synthesized in three-steps starting from ergometrine maleate, which resulted in the formation of (8S)-isomer, iso-MiPLA, as a by-product. The LC-MS results showed that LSD, MiPLA, and iso-MiPLA exhibited different retention times. Their chemical structures were determined using nuclear magnetic resonance spectroscopy, which revealed the presence of rotamers involving the N-methyl-N-isopropyl groups of tertiary amides in MiPLA and iso-MiPLA.

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.

Enhancing Chemical Stability through Structural Modification of Antimicrobial Peptides with Non-Proteinogenic Amino Acids.

Multidrug-resistant bacteria (MDRB) remain a significant threat to humanity, resulting in over 1.2 million deaths per year. To combat this problem effectively, the development of therapeutic agents with diverse mechanisms of action is crucial. Antimicrobial peptides (AMPs) have emerged as promising next-generation therapeutics to combat infectious diseases, particularly MDRB. By targeting microbial membranes and inducing lysis, AMPs can effectively inhibit microbial growth, making them less susceptible to the development of resistance. Numerous structural advancements have been made to optimize the efficacy of AMPs. Previously, we developed 17KKV-Aib, a derivative of the Magainin 2 (Mag2) peptide, by incorporating a,a-disubstituted amino acids (dAAs) to modulate its secondary structure. 17KKV-Aib demonstrated potent antimicrobial activity against Gram-positive and Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa (MDRP), with minimal hemolytic activity against human red blood cells. However, 17KKV-Aib faces challenges regarding its susceptibility to digestive enzymes, hindering its potential as an antimicrobial agent. In this study, we designed and synthesized derivatives of 17KKV-Aib, replacing Lys residues with 4-aminopiperidine-4-carboxylic acid (Api), which is a cyclized dAA residue possessing cationic properties on its side chain. We investigated the impact of Api substitution on the secondary structure, antimicrobial activity, hemolytic activity, and resistance to digestive enzymes. Our findings revealed that introducing Api residues preserved the helical structure and antimicrobial activity and enhanced resistance to digestive enzymes, with a slight increase in hemolytic activity.

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.

Structure-Activity Relationship Study of Helix-Stabilized Antimicrobial Peptides Containing Nonproteinogenic Amino Acids.

Helical amphipathic peptides containing cationic and hydrophobic amino acid residues can possess potent antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this study, several amphipathic peptides with enhanced helical structures containing nonproteinogenic amino acids were designed, and the relationships between the antimicrobial activity, hemolytic activity, and cytotoxicity were evaluated. In particular, the effect on the antimicrobial activity and cytotoxicity of the number and position of stapling structures introduced into the sequence was investigated. Peptide stp1 containing α,α-disubstituted amino acids showed potent antimicrobial activity against multidrug-resistant bacteria (MDRP, SP45, and Staphylococcus aureus) without causing appreciable hemolytic activity or cytotoxicity. The cytotoxicity was found to be somewhat correlated to the hydrophobicity of the peptides.

Identification of the Stapled α-Helical Peptide ATSP-7041 as a Substrate and Strong Inhibitor of OATP1B1 In Vitro.

ATSP-7041, a stapled α-helical peptide that inhibits murine double minute-2 (MDM2) and MDMX activities, is a promising modality targeting protein-protein interactions. As peptides of molecular weights over 1000 Da are not usually evaluated, data on the drug-drug interaction (DDI) potential of stapled α-helical peptides remain scarce. Here, we evaluate the interaction of ATSP-7041 with hepatic cytochrome P450s (CYPs; CYP1A2, CYP2C9, CYP2C19, CYP3A4, and CYP2D6) and transporters (organic anion transporting polypeptides (OATPs; OATP1B1 and OATP1B3), P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP)). ATSP-7041 demonstrated negligible metabolism in human liver S9 fraction and a limited inhibition of CYP activities in yeast microsomes or S9 fractions. On the contrary, a substantial uptake by OATPs in HEK 293 cells, a strong inhibition of OATP activities in the cells, and an inhibition of P-gp and BCRP activities in reversed membrane vesicles were observed for ATSP-7041. A recent report describes that ALRN-6924, an ATSP-7041 analog, inhibited OATP activities in vivo; therefore, we focused on the interaction between ATSP-7041 and OATP1B1 to demonstrate that ATSP-7041, as a higher molecular weight stapled peptide, is a substrate and strong inhibitor of OATP1B1 activity. Our findings demonstrated the possibility of transporter-mediated DDI potential by high molecular weight stapled peptides and the necessity of their evaluation for drug development.

Development of DNA Aptamer-Based PROTACs That Degrade the Estrogen Receptor.

Targeted protein degradation (TPD), using chimeric molecules such as proteolysis-targeting chimeras (PROTACs), has attracted attention as a strategy for selective degradation of intracellular proteins by hijacking the ubiquitin-proteasome system (UPS). However, it is often difficult to develop such degraders due to the absence of appropriate ligands for target proteins. In targeting proteins for degradation, the application of nucleic acid aptamers is considered to be effective because these can be explored using systematic evolution of ligand by exponential enrichment (SELEX) methods. In this study, we constructed chimeric molecules in which nucleic acid aptamers capable of binding to the estrogen receptor α (ERα) and E3 ubiquitin ligase ligands were linked via a linker. ERα aptamer-based PROTACs were found to degrade ERα via the UPS. These findings represent the development of novel aptamer-based PROTACs that target intracellular proteins and are potentially applicable to other proteins.

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.

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.

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.